4 o54 PAS wt dua! Gt vet es ee Hiv? bP 9 i Medy'd-aeea'y r » Vian eae ear oer te 235 No. 2. July, 1921. Eppy, WavTeR H., Herr, Harrie L., Stevenson, HELEN C., and Jonn- son, Rutru. Studies in the vitamine content. II. The yeast test asa mefsure of ‘vitamine B. es eects ee noe - i ei Baitey, Cameron V. Notes on apparatus used in determining the respiratory exchange inman. I. An adaptation of the French gas mask for use In: respiratoryawoukeyeee ey eee ens... eee 277 Bartey, Cameron V. Notes on apparatus used in determining the respiratory exchange in man. II. A sampling bottle for gas hy: |b) |. Ea oR cw son RR Mee ear WatTeRMAN, Henry C., and Jones, D. BreEsE. Studies on the digesti- bility of proteins in vitro. II. The relative digestibility of various preparations of the proteins from the Chinese and Georgia velvet D@ANS, «ooo viccils cociels 2s 2c ge EE ee ee ee eee 285 SHERWIN, Cart P., and Hynes, WatrerR A. The metabolism of nitro- benzaldehydes and nitrophenylacetaldehyde..................... 297 STeENBOCK, H., Sevy, Marana T., and Boutwetu, P.W. Fat-soluble vitamine. VIII. The fat-soluble vitamine content of peas in rela- tion to their pigmentation. .... svcc< ince) o ce eee eee 3038 Cuapin, Ropert M. The determination of cresol by the phenol reagent of Folin and Denis.......... 7 eb oce ee eee 309 STEHLE, Raymonp L., and McCarty, ArtHuRC. The effect of hydro- chloric acid ingestion upon the composition of the urine inman..... 315 Jones, Martua R., and Nyg, Lituran L. The distribution of calcium and phosphoric acid in the blood of normal children............. 321 Gipson, CHARLES A., UMBREIT, FrREDA, and BRADLEY, H.C. Studies oLautolysis. VII. Autolysis of brainse-gs- eho 70. ee 333 Morcttis, Sercius. A study of the catalase reaction............... 341 Dotsy, Epwarp A., and Eaton, Emity P. - The relation of the migra- tion of ions between cells and plasma to the transport of carbon IOXIGE 2. eo se ow ecu oo pores Ee moe 377 Hess, A. F., McCann, G. F., and PAppENnHEIMER, A.M. Experimental rickets in rats. II. The failure of rats to develop rickets on a diet —. deficient in yitamine A. Plates. itoise 7-50 eee as eee 395 McCuienpon, J. F. Methods of extracting and concentrating vita- mines A, B, and C, together with an apparatus for reducing milk, fruit juices, and other fluids to a powder without destruction of WICAMINES a. 37 oitges 5, 's.< > « » 402 £0 vt eae i . ier Fy vouly . i% ott Reger Vi ae ae ay i: | wd c se Ne < a a : Loli t Vag Be / ) a) ir Tae * ae dc) i. Faeyr we, meen 2 Bi Ss / ~wibegs aaa an aise rv)? di is . Ware giana 7, ote “1s Gir a AiO? Hits 2 ‘eb, kth (ROW? a SR : tye Anis? feeble . vere: een ASI ey Lap yee ag ae iGO yn @: 01D aie Tee te aaTeys yer 2 \ F ori a Ay 5 ? al =) ahi if SK at, a Ms, ee San 4 ‘es -& iH ‘} a 4 _ a4 oad "el ae ; . igs nA A y bs ete eas nod, ‘4 or. oa. i Tata tl) > 4 NOTE ON THE GASOMETRIC DETERMINATION OF NITROGEN. By RAYMOND L. STEHLE. (From the Laboratory of Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia.) (Received for publication, April 28, 1921.) Ina recent communication on this subject! it was observed that oxygen was liberated to some extent along with nitrogen when the Kjeldahl digest was treated with sodium hypobromite. The procedure there described called for its absorption with alka- line pyrogallate before reading the gas volume. An investigation as to the cause of the liberation of oxygen has shown that the copper sulfate added to hasten the digestion is responsible. Hence, if this substance is omitted, nitrogen only is liberated and there is no need for the pyrogallate treatment. Besides simplifying the manipulation, the gas volume can then be read with more accuracy, due to the fact that one is dealing with a water-clear solution instead of a highly colored one. 1 Stehle, R. L., J. Biol. Chem., 1920-21, xlv, 223. 11 i a - e — - | — : ~ . 4 “ can = - t ¢ i] *» Wiebe } - fy o : ° =) ms x, » va | * NL, ~ By ; WAT Ta; «italia? Ha dse's . ORT? me at? 7) ee \ wt ! ay eee 1h | ALS aT 4 ALAS me es | ‘ i . gant sea) lan hein. =e ond } conan A hag P hick. sia Mes Ske y tome hi hare hei, ae mae alti) , UP aha ae . [ 1 =a a meg j ray PR if aodalia or . ro lal ve a ). . a bert ad ‘v \ iL a =) i re oo, THE GASOMETRIC DETERMINATION OF UREA IN URINE. By RAYMOND L. STEHLE. (From the Laboratory of Physiological Chemistry, School of Medicine, University of Pennsylvania.) (Received for publication, April 29, 1921.) Several months ago! in a communication describing a gas- omotric method for the determination of total nitrogen, mention was made of the fact that when carried out in vacuo the reaction between sodium hypobromite and urea results in the liberation of the theoretical volume of nitrogen. The necessary vacuum is readily obtainable with the Van Slyke apparatus for determining the carbon dioxide content of blood plasma. Some effort was spent at the time in attempting to utilize the reaction for a blood urea method but it became evident that the error introduced by other nitrogenous constituents of blood was greater than permissible. Recently Youngburg? has shown that the urease method for urinary urea may be somewhat simplified by first removing the ammonium salts and then carrying out the urease decomposition and aeration in the manner described by Van Slyke and Cullen.’ The ammonium salts are removed by shaking the urine with permutit. The idea immediately suggested itself that here was a place to apply the hypobromite reaction. In the older hy- pobromite methods the results were unsatisfactory because the reaction (using pure urea solutions) was found to give less than the theoretical amount of nitrogen even when allowed to continue for hours, and in addition ammonium salts and other urinary constituents also yielded nitrogen when subjected to the action of hypobromite. With the aid of permutit the ammonium salts 1Stehle, R. L., J. Biol. Chem., 1920-21, xlv, 223. 2 Youngburg, G. E., J. Biol. Chem., 1920-21, xlv, 391. 3 Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1914, xix, 211. 13 14 Urea in Urine may now be eliminated as sources of error. It remained, there- fore, to determine the error which might be introduced by other substances present in urine. Assuming for the moment that an average 24 hour specimen of urine contains 15 gm. of urea nitrogen, 0.300 gm. of creati- nine nitrogen, and 0.250 gm. uric acid nitrogen, it is evident that if all the nitrogen of the two latter constituents was liberated quantitatively the error.introduced would be about 3.7 per cent. In order to determine the actual quantities liberated these two compounds were subjected to the action of hypobromite. It was found that creatinine yielded about one-seventh of its ni- TABLE I. Urea Nitrogen, per 1 Cc. of Urine. Hypdbromite. Van Slyke and Cullen. Youngburg. 7.60 7.48 538) 1.58 MEY 7.62 (ear Tas 1.15 Chat hs: 7.56 7.56 6.43 6.43 6.46 6.40 6.44 5.16 5.12 5.18 5.26 6.89 6.89 6.83 6.89 6.78 6.88 6.66 6.71 6.67 6.73 6.60 6.72 4.12 4.72 4.61 4.72 4.70 4.87 14.4* 14.5 14.2 14.2 14.3 14.6 T1207 10.9 10.9 10.8 10.9 * Dog urine. trogen while uric acid yielded about one-half. In the case of the latter, however, the evolution of nitrogen takes place slowly and in consequence, by limiting the reaction time to that required for the urea reaction to go to completion, the error need not exceed about 0.3 per cent. Conceivably, hippuric acid and amino-acids might be sources of error. However, the first does not yield any nitrogen with hypobromite. Glycocoll yields about 3 per cent of its N. In- asmuch as the amount of amino-acids present in urine is very small to begin witht and provided that other amino-acids con- duct themselves similarly to glycocoll, it is evident that the the error from this direction is negligible. Creatine, which is * Levene, P. A., and Van Slyke, D. D., J. Biol. Chem., 1912, xii, 301. Wren Se | R. L. Stehle 15 sometimes a constituent of urine, liberates 2 of its 3 nitrogen atoms under the conditions of the experiment. Since it is us- ually absent or present only in small amounts this source of nitrogen may In general be neglected. Allantoin conducts it- self very similarly to uric acid. Table I contains some results obtained by the hypobromite method, and the urease method. In the case of the latter, de- terminations were made by Youngburg’s modification as well as by the usual procedure of Van Slyke and Cullen. In view of the general satisfaction given by the urease method the present method may seem superfluous. It is fitting, therefore, that its advantages be stated. They are: (1) Rapid- ity. Starting with a sample of urine the urea content may be known in 10 minutes. (2) Standard solutions are not em- ployed. (3) Reagents necessary are simple and easily prepared. (4) There is practically no opportunity in the procedure for things to go awry as there are opportunities in the urease method. For example, the keeping qualities of dilute standard solutions and of urease solutions are not matters of concern. Neither is there any question of foaming nor about how long and at what rate to aerate. Procedure. 25 cc. of diluted urine (diluted in the ratio of 1:10) are shaken with 4 gm. of permutit for 4 minutes. The mixture is then centrifuged or filtered. 1 cc. of the NH;-free urine is introduced into the Van Slyke CO, apparatus, the last portion being rinsed in with 1 ce. of water followed by 1 cc. of sodium hypobromite solution. (This is made by mixing equal volumes of two so- lutions, one containing 12.5 gm. of sodium bromide and 12.5 gm. of bromide per 100 ce. and the other 28 gm. of sodium hydrox- ide per 100 cc.) The mercury is lowered to the 50 cc. mark and the apparatus is then shaken vigorously for about half a minute. The aqueous solution is collected in the proper chamber below the lower stop-cock, mercury is admitted to the 50 ce. chamber, and after adjusting the pressure the volume of nitro- gen is read. Correction is then made for the dissolved air con- 16 Urea in Urine tained in the diluted urine, the rinse water, and the hypobromite solution.® It may be assumed with reasonable accuracy that the solu- bility of air in the diluted urine is the same as in pure water. For temperatures between 15 and 30°C. and a pressure of 1 atmosphere the solubilities are as given in Table II. The volumes are those which the gas would occupy at 760 mm. and the temperature in question and may, therefore, be subtracted from the gas volume as read. Determinations of the air dis- solved in the hypobromite solution showed that between 15 and 20°C. this amounts to 0.006 ec. and between 21 and 25° to 0.005 ee. TABLE II. Ce. of Air Measured at 760 mm. of Mercury and the Temperature in Question per 1 Ce. of Water. Temperature. | Volume. Temperature. Volume. Temperature. Volume. BOs cc. °C. ce. “Gi. Co: 15 0.0216 21 0.0198 27 0.0184 16 0.0212 22 0.0196 28 0.0182 17 0.0209 23 0.0193 29 0.0180 18 0.0206 24 - 0.0191 30 0.0178 19 | 0.0203 25 0.0188 20 0.0201 26 0.0186 The corrected volume is then reduced to standard conditions (0° and 760 mm. mercury) by means of the following formula. Pe Po —h z (1 + 0.00367 t) 760 where V = gas volume as measured. Po= corrected barometric pressure. h = aqueous tension. ¢t = temperature at which gas was measured. To facilitate the calculation it will be found advantageous to refer to almost any of the compilations of physical and chem- ical data for useful gas reduction tables. Once the volume of nitrogen measured at 0° and 760 mm. of Hg has been determined the weight is determined by mal- ° The correction for the air content of the diluted urine and rinse water can be eliminated by extracting the two in the apparatus and expelling the extracted gases before adding the hypobromite. : jn ti! ie. ag: R. L. Stehle il tiplying by 0.0012507, the weight of 1 ce. of nitrogen. Taking the dilution and amount of sample into consideration the amount of urea nitrogen per 1 cc. of urine is easily found. Some consideration was given to the adaptability of the hy- pobromite reaction to the determination of ammonium salts in urine as well as to the determination of urea. Theoretically there are no difficulties. The difference between the quantities of nitrogen liberated by diluted whole urine and by urine treated with permutit represents ammonia N and that only. The diffi- culty is in the measurement of the two quantities with sufficient accuracy to make it possible to express the ammonia N concen- tration with at least two significant figures. The ordinary Van Slyke apparatus cannot be read without a possible error of at least 0.003 of a cc. in the opinion of the writer. In addition no protection is provided against slight differences between the temperatures of the gas itself and the air registered by the ther- mometer close by. Consequently since the ammonia N is usually less than one-tenth of the sum of the ammonia and urea nitrogen and therefore occupies less than one-tenth of a cc. in the appa- ratus, its volume cannot be expressed accurately in thousandths of a ce. as is necessary if the result is to be expressible with two significant figures. If an approximate relation between the urea N and ammonia N is desired this can be very readily obtained. SUMMARY. A method for determining urea in urine is described which is both brief and accurate. Ammonium salts are removed by treating the urine with permutit and the ammonium-free solu- tion is then subjected to the action of sodium hypobromite in the vacuum obtained with a Van Slyke CO: apparatus. Nitrogen is liberated quantitatively from the urea but to an entirely negligible extent from other urinary constituents. Comparative analyses obtained with the urease and hypo- bromite procedures demonstrate the accuracy of the method. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 =i > , . A cn i 7 “ FS iat " i 4 > ne = ia : mAL , 7 ' : 1h “iF he j ‘i ee ” : oe ‘+ 7 pS p ” - 7 | ' - | _ a ay NY ‘so ; ovis tar OE . 1 ce - / an ‘4 ‘e ie ior ae Wee Pad Pr hig Sats ahi raf ue iLfus ‘inet oy age cvs: re aa vl ‘eee ve teu nlite ee aS » ae weit ilpt oa ad alletokal \\ 7: Sey ies) rage at ttf EE PR Web) i ge 0 O44) hoi y 4A Ta ah Qe pet's ue ’ nw esas’ were itd i f Trae tha nt ‘uM ae a i igsest ee bi jnip ieee, 16 hele eh 2 ame Ath ‘ iu #/ nS ow) v4 (he AEs tity pare, tk fae j a = aT, ® - f . ' tie J if ty ‘TH FURTHER IMPROVEMENTS IN THE NEPHELOMETER- COLORIMETER. By PHILIP ADOLPH KOBER anp ROBERT E. KLETT. (From the Laboratories of the Kober Chemical Company, Inc., Nepera Park, N.Y., and the Klett Manufacturing Company, Inc., New York.) (Received for publication, March 29, 1921.) The colorimeter described by Kober! has been improved in certain details which have resulted in the instrument described in the present paper. The first departure in construction from the Duboseq model was made by Kober! in 1915. It consisted of a 100 mm. scale, tiltable stand, adjustable verniers, fused black glass plungers, a rack, and a pinion. In 1917 this model was abandoned for the present one,? which has incorporated in it a fine screw arrangement in back of the instrument, away from possible contact with corroding liquids overflowing from the cups, in place of the rack and pinion exposed to such accidental overflowing by virtue of its position, retaining the fused black glass plungers with clear glass bottoms, but also having fused cups made of different colored glass which, in con- junction with the black plungers, made the transformation of the colorim- eter into a nephelometer a matter of simply changing the cups. This 1917 model also possessed an ingenious device for adjusting the verniers to the zero point. Besides these changes there was added a set of split reflectors for the regulation and adjustment of light reflection. The hollow black plungers having fused optical ends, have been found to pos- sess a special advantage in hydrogen ion determinations by Duggar.? He found that by putting shield solutions into the plungers as well as into the cups, a comparator for hydrogen ion and other work of great accuracy resulted. The split reflectors have also been found of special use by Field.4 Where an unknown solution has a slight turbidity which absorbs light, thereby darkening the transmitted light, Field operates the reflector underneath the standard solution so that equal darkening is obtained. Bock and Benedict’ in 1918 described an instrument that is in reality a half plunger and a half cell instrument. This instrument has one advan- tage over most instruments of the plunger type; namely, that the scale 1 Kober, P. A., and Graves, 8.S8., J. Ind. and Eng. Chem., 1915, vil, 843. 2Kober, P. A., J. Biol. 'Chem., 1917, xxix, 155. * Duggar, B. M., Annals of the Missouri Botanical Gardens, 1919, vi, 179. 4 Field, C. W., personal communication. 5 Bock, J. C., and Benedict, S. R., J. Biol. Chem., 1918, xxxv, 227. 19 20 Nephelometer-Colorimeter and eyepiece can be viewed from one position of the observer, usually while sitting. The disadvantage of this type of instrument is the lack of sym- metry or lack of interchangeability of the two sides of the instrument and of the two paths of light. Where only one or two heights of standard solu- tion are necessary as in certain routine work this instrument is suitable, but this lack of flexibility in height of standard solution and the limited scale (40 mm.) make it less suitable for research and scientific work. In 1918-19 Leitz® made a decided change in construction in one of their colorimeters, by substituting a lever arrangement for the rack and pinion. This departure from the rack and pinion (Duboseq) or from the fine screw arrangement (IXober) which is inherent in accurate measuring devices, is obviously a step backwards in colorimeter construction. Another objec- tion to levers is that they are apt to move from a fixed position through jars or accidental touch, no matter how slight. In 1920 Bausch and Lomb? with the aid of Folin brought out a colori- meter of the Duboseq type but which has a number of changes from the French model. Four marked changes are apparent: (1) The base and up- right frame are of very heavy castings, ‘‘to provide stability and perma- nent alignment of the opties.’’? (2) The rack and pinion are so changed that the operating heads are always in a fixed position, so that the ob- server’s readings are not influenced by the position of the pinion heads. (3) The colorimeter cups used are of ground glass cylinders and plates, incased in heavy metal. (4) The adjustable verniers of the Kober instru- ment are adopted. The heavy metal and construction provide an instru- ment resistant to rough handling, and the fixed position of the milled heads seems to be an improvement over the French model, but the extra massiveness of the parts plays no role in its accuracy. The instrument makers furnish attachments which make it possible to convert it into a nephelometer, after disconnecting plungers and adding these parts. This transformation while not a very convenient one, is of value. The means used to eliminate the meniscus from the nephelometric tubes or vials, and the care taken to eliminate the glare from the light source, which is designed to give parallel rays, are commendable features of this instrument. During the routine tests of the Kober instruments it has be- come evident that the physical condition of the operator is an important factor in maintaining a high average of accuracy in adjusting the optical arrangement. It was found that fre- quent stooping down, in order to read the scale after having matched the colors or light in the eyepiece, induced fatigue very quickly. This stooping is necessary when using any of the Du- boseq type instruments. This condition of fatigue was ag- 6 Advertised by E. Leitz, in Science, 1919. 7 Advertised by Bausch and Lomb, in J. Ind. and Eng. Chem., 1920. P. A. Kober and R. E. Klett 21. gravated by the difference between the short focal distance in the eyepiece and the longer focus necessary to observe the scale, the result being greater eye fatigue. Another source of temporary fatigue which even if it is not so pronounced yet is a factor in accurate work, is the holding or supporting of the arms while adjusting or turning milled heads, to operate the plungers or cups. As long as the nephelometer- colorimeter was only used occasionally, these defects of course were not noticeable, but since their use has become daily and in many cases almost continuous, these factors have become im- portant. Many experiments and models were made to eliminate the sources of annoyance and fatigue inherent in the Duboscq type, and although several models achieved the final result, the one described in this paper was adopted as the most satis- factory from all points of view especially since there is no in- direct transmission in changing the vertical scale to a horizontal one. The improvements, which eliminate these defects are: (1) The milled heads, formerly at the top of the instrument, are placed at the bottom, which allows the hands to rest on the table or other support and the adjustments to be made with the fingers (shown in Fig. 1). (2) An auxiliary scale is provided at the top of the instrument consisting of: two scales engraved upon the side away from the operator, fastened to the movable stages, so that when the stage is being moved up or down, the scales move with it; a stationary vernier, protruding beyond the top plate, also engraved upon the side away from the operator, fastened to the top of the instrument. A mirror facing the operator at an angle of 45° is placed in front of the protruding scale and verni- er, so that an image of the two is reflected vertically. A mag- nifying glass of the same focal distance as the telescope, serving as a second eyepiece, has been placed close beside the regular eye- piece, directly above the mirror, showing the image of the scale enlarged in good light. Fig. 1 shows the entire instrument without the lamp house.® 8 This instrument is manufactured by Klett Manufacturing Company, ~ Inc., 202 East 46th Street, New York. ANNAN ANALY AYA WSS AAA S 22 22 Z2 22 zg z 22 Z2 Z2 24 22 22 22 23 Z2 22 24 22 Z2 Ze 24 24 22 z | 24 Z3 Zz NAAN AAAAAAA AANA RRA RRND AY Fic. 1. View of the instrument without the lamp house, showing (a) the two eyepieces, the scale, and the mirror at the top of the instrument; (b) the micrometer adjustment of the zero point; (c) the milled heads operating the cups at the bottom of the instrument. P. A. Kober and: R. E. Klett 23 In Fig. 2 are shown the fields that are observed through the two eyepieces. The ease of reading the scale is apparent. Fia, 2. The two fields as seen through the two eyepieces. The upper field shows the two semicircular fields; the lower field shows the stationary vernier and the two adjustable scales. In Fig. 3 is shown the construction of the auxiliary scale. This auxiliary scale is engraved to 60 mm. but with the vernier is readable to only 50 mm., which is ample for most work. If heights above 50 mm, are to be measured the original vernier can be used. The setting of the zero point is easily and accur- ately accomplished with a micrometer arrangement, as may be seen at A by a milled head working against a spring. This con- venient method of zero point adjustment, together with the very simple method of using the instrument, the method of Lamb, Carleton, and Meldrum,? where the height of the standard solution (S) is kept constant, makes the operation of the instrument and the calculation of results extremely simple and easy without, however, sacrificing accuracy or deviating from the fundamental basis of either colorimetry or nephelometry. *Lamb, A. B., Carleton, P. W., and Meldrum, W. B., J. Am. Chem. Soc., 1920, xlii, 252. a ts ne ANN iF UMMM mM dt ANY YUU »|= \\eees ey. TINUGTOREEEES : lic. 3. Diagrammatic sketch of the rear, of the instrument. A, magni- fying lens for the horizontal scale; B, mirror at an angle of 45°; C, mo rable scale; D, serew-threaded rod; E, vernier for 50 to 100 mm. seale; F, lock nut for the zero spc iatbehd G, movable cup carrier; H, micrometer for ZETO adjustment; J, scale from 50 to 100 mm.; K, knurled thumb screw lor rapid movement; L, knurled thumb screw for fine adjustment. 24 P. A. Kober and R. E. Klett 25 In Fig. 4 is shown the instrument attached to a lamp house. Fic. 4. The instrument and lamp house showing the split reflectors as well as the front of the instrument illuminated by the light from the lamp house. SUMMARY. The advantages of the new improvements are: (1) the elimina- tion of the fatigue and annoyance, due to stooping to read the scale of Duboseq instruments; (2) an enlarged and well illumi- nated scale read through an eyepiece of the same focal length as the telescope; (3) a more convenient position for the milled heads operating the stages, allowing for resting of the operator’s arms; and (4) a micrometer arrangement for setting the zero point which can be locked in any position. ON THE SUBSTITUTION OF TURBIDIMETRY FOR NEPHELOMETRY IN CERTAIN BIOCHEMICAL METHODS OF ANALYSIS. By W. DENIS. (From the Laboratory of Physiological Chemistry, Tulane University Medical School, New Orleans.) (Received for publication, April 25, 1921.) Within the past decade nephelometric methods of analysis have become increasingly popular in biochemical work, so that at present a nephelometer has come to be counted an indispen- sable piece of apparatus in every well equipped laboratory. Nephelometry has however the serious disadvantage that, as was first pointed out by Richards and Wells,! the amount of light reflected is not strictly proportional to the weight of the pre- cipitate under observation, but seems to be influenced by a variety of factors. ‘To overcome this defect Richards and Wells adjusted the volume of their solutions so that standard and unknown contained about the same concentration... Kober? has suggested a mathematical formula by means of which he has obtained excellent results, and Bloor? recommends that a standard be selected which is of such strength that it varies not more than 25 per cent from the unknown. During the past 5 years the author has had occasion, in connec- tion with studies on blood and milk, to make extensive use of nephelometric methods, and as a result of this experience has come to appreciate more clearly the relatively large error which may be introduced in an analysis unless the strength of the standard and of the unknown are adjusted to within at least 10 per cent, a requirement which means that for a single deter- mination it may be necessary to provide from three to six stan- 1 Richards, T. W., and Wells, R. C., Am. Chem. J., 1904, xxxi, 235. 2 Kober, P. A., J. Biol. Chem., 1912~13, xiii, 485. 3 Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 33. 27 28 Turbidimetry for Nephelometry dards of varying degrees of concentration. The irksomeness of preparing large numbers of standards has led to an inves- tigation concerning the possibility of utilizing the turbidimeter in place of the nephelometer in several biochemical methods of analysis. Turbidimetric methods of analysis are extensively used in technical work, as for example in connection with the determin- ation of suspended matter in water, of sulfur in coal, etc., but so far but little use has been made of this principle in the solution of biochemical problems. Some years ago Folin and Denis* made use of this technique for the determination of albumin in urine, and recently Denis and Ayer® have employed a similar method in the analysis of cerebrospinal fluid. In connection with the above work, and as the result of a series of readings made on standard solutions of widely varying concentrations we felt justified in believing that turbidity determinations made on the precipitate obtained by the action of sulfosalicylic acid on protein give comparable results even when the concentration of the unknown and of the standard vary as much as 50 per cent. If it could be proved that this finding also applies to the measure- ment of suspensions other than protein, the usefulness of the tur- bidimeter in biochemical work becomes immediately apparent. Although several types of turbidimeters have been suggested, I have continued the use of the Duboseq colorimeter for the measurement of my suspensions. To obtain the best results all readings should be made in a dark or semidark room, and as a preliminary to any series of readings the position of the color- imeter should be so adjusted with relation to the source of light, that exactly the same illumination is obtained on both sides when both cups are filled with the standard suspension amd both scales are set at the same point. It has been my experience that the most accurate results are secured when the mirror is adjusted to give the maximum illumination. In this paper I wish to present the results of a study which has for its basis the attempt to substitute turbidimetry for nephel- ometry in three analytical procedures; v7z., the determination of * Folin, O., and Denis, W., J. Biol. Chem., 1914, xviii, 273. * Denis, W., and Ayer, J. B., Arch. Int. Med., 1920, xxvi, 436. W. Denis 29 calcium in blood by Lyman’s method® (in which the blood cal- cium in the form of colloidal calcium stearate is measured by comparison with a standard calcium stearate suspension); the determination of fat in blood and milk by the method of Bloor? (which involves the use of colloidal suspensions of fatty acids); and of phosphates by the strychnine molybdate precipitate of Pouget and Chouchak* as modified by Kober and Egerer? and by Bloor.’ The results obtained with calcium stearate suspensions in- dicate that it is easily possible to obtain quantitative results with suspensions varying in concentration by as much as 50 per cent, provided these suspensions are of such concentration that they contain between 0.75 and 0.35 mg..of calcium in a final volume of 100 cc.; with greater concentration precipitation fre- quently occurs; while if the amount of calcium is less than 0.35 mg. per 100 cc. turbidimetric readings can no longer be made with precision. TABLE I. Comparison of Results Obtained by the Use of the Nephelometer and the Colorimeter in the Determination of Calcium in Milk. Ca per 100 cc. of milk. Observation No. By nephelometry. By turbidimetry. mg. mg 210 18.6 18.6 237 22. 1 22.9 239 20.0 20.0 235 19.2 19.4 240 33.3 Soul 269 21.6 21.0 Zoe Nez 17.4 In Table 1 are presented the results obtained in a series of determinations of calcium in milk by Lyman’s method in which parallel readings were made by the turbidimeter and by the nephelometer. 6 Lyman, H., J. Biol. Chem., 1917, xxix, 169. 7Bloor, W. R., J. Biol. Chem., 1914, xvii, 377. 8 Pouget, I., and Chouchak, D., Bull. Soc. chim., 1909, v, series 4, 104. ® Kober, P. A., and Egerer, G., J. Am. Chem. Soc., 1915, xxxvii, 2373. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 30 Turbidimetry for Nephelometry With fatty acid suspensions quantitative readings can be made when the standard and unknown vary by 60 per cent provided the concentrations of fatty acid lie within the range of 8 to 2 mg. per 100 ce. In Table If comparison is made of analyses of milk fat made by the Babeock method, and by Bloor’s method, readings having been made by means of the turbidimeter instead of the nephelometer. TABLE I. . Comparison of Results Obtained by Determinations of Fat in Milk by the Babcock Procedure and by the Use of the Colorimeter in Bloor’s Micro Fat Method. Fat. Observation No. ' By Babcock method. By turbidity. per cent per cent 1 3.2 3.2 2 3.1 3.0 3 3.5 3.6 4 4.3 4.4 5 4.7 4.4 6 5.0 5.1 TABLE III. Comparison of Results Obtained by the Use of the Nephelometer and the Colorimeter in the Determination of Inorganic Phosphates in Blood Plasma. P per 100 cc. of plasma. Observation No. By nephelometer. By colorimeter. g oe 143 23 25 144 2.6 247 163 3.0 3.0 162 3.8 2.6 160 5.0 5.9 6.2 6.0 Results, essentially similar to those reported above, were obtained with strychnine phosphomolybdate suspensions. The optimum concentrations for turbidimetric work with this material were found to be 0.12 to 1.2 mg. H;PO, per 100 ce. W. Denis 31 In Table III are collected the results of a series of determi- nations of the inorganic phosphates of blood plasma in which parallel readings were made by the nephelometer and by the turbidimeter. ; SUMMARY. The suggestion is made that determinations of turbidity, made by means of a colorimeter, may with advantage be substituted for nephelometric readings in several analytical processes. The advantages of this procedure are twofold: first, as turbidimetric readings give quantitative results with large variations in con- centration between standard and unknown it is possible to omit the preparation of the large number of standards which have been found necessary in nephelometric work; second, it presents an additional use for the now universally owned colorimeter and in the case of many small laboratories would probably make the possession of a nephelometer unnecessary. CREATINURIA. II. ARGININE AND CYSTINE AS PRECURSORS OF CREATINE.* By E. G. GROSS ann H. STEENBOCK. (From the Laboratory of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, April 20, 1921.) In a previous publication (1) we have analyzed the various factors which may be operative in the production and prevention of creatinuria. This analysis was suggested by the possibility that the anomalies observed in the appearance of creatinuria could very well owe their causation to the variation in the utili- zation of amino-acid precursors of creatine with different phy- siological states or functions. Processes of growth, fetus building, and lactation would have their influence on creatinuria in modi- fying the intake and utilization of amino-acids and_ therefore varying the residue left to be catabolized into creatine. Crea- tine excretion could thus be indicated, not only as a result of excessive ingestion of its precursors, but also as a result of defi- cient ingestion of the complements of the precursors necessary to make protein building possible. Excessive catabolism such as obtains during fasting, fever, acidosis, and phosphorus poi- soning would then similarily owe its creatinuria to the excessive liberation of creatine precursors although in part some of the creatine excreted would originate from the creatine normally found as a constituent of muscle. Our previous investigations designed to correlate some of these factors have shown that creatinuria can always be induced in the pig by feeding sufficient protein. This was found to be the case irrespective of the sex of the animal, or the acidity or alkalinity of the diet. As a result we have continued our study of the problem in an attempt to define further its origin. * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 33 34 Creatinuria. II In the search for possible precursors of creatine, arginine has attracted the most attention because, like creatine, it is a deriva- tive of guanidine and because it is contained in all the known proteins. In fact it is the only cleavage product of protein so far isolated which contains the guanidine nucleus. While it is now generally accepted that creatine is derived from proteins, such a difference of opinion exists in the literature as to the possibility of arginine being the precursor of creatine, that no definite conclusion as to the exact state of affairs can be drawn. Direct experimentation with the feeding of arginine has been rather limited but considerable work has been done by transfusion and injection. Inouye (2) found an increase in crea- tine upon adding arginine to liver extract, and also when argin- ine was perfused through the surviving liver. Myers and Fine (3) in feeding rats with edestin, which contains 14 per cent of arginine, and casein, which contains 4 per cent of arginine, found a slight increase in the muscle creatine with the former, but they draw no conclusions. Thompson (4) reported an increase in urinary creatine following injection of arginine carbonate, but failed to obtain an increase by oral administration. Jaffé (5) found no increase in urinary creatine following the feeding of arginine to the rabbit. Recently Harding and Young (6) have suggested that cystine might also be metabolized into creatine “‘through the inter- mediate stages of taurine and amino ethyl alcohol, followed by methylation, combination with urea, and oxidation.” Using dogs for the experiments they obtained increases with cystine but not with arginine. It appeared significant to us that in their report they state that the endogenous creatine was affected as well as the exogenous. This suggested that possibly what was inter- preted by them as a direct affect of cystine was in reality an indirect effect—the cystine stimulating catabolism by means of the sulfuric acid formed by its oxidation. Of their failure with arginine we will have more to say later. In this paper there will be presented data showing how arginine and cystine may function in inducing or augmenting creatinuria. ; E. G. Gross and H. Steenbock 35 EXPERIMENTAL. In the following experiments as in the past, the pig was chosen as the experimental animal as it stands confinement well and can be fed a nitrogen-free diet over a long period of time. Such a diet offers especial advantages in that it simplifies the conditions of experimentation and allows more accurate interpretation of the data. Methods of analysis and the experimental procedure used were the same as before (1). Total nitrogen and creatinine determinations were made to serve as an index of general excretory activity. The arginine used was prepared according to Kossel’s method from the protein arachin. As a check on its purity it was ana- lyzed according to Van Slyke. To remove traces of barium which always tend to be carried along in the preparation, sodium sul- fate was added in excess. In later experiments, Tables IV and V, however, the barium was removed quantitatively with sulfuric acid as it was feared that the sodium carbonate formed in the other precedure might affect the results. The arginine was usually given by stomach tube. In the experiments of Table IX it was given in gelatin capsules. Usu- ally some difficulty was experienced in getting the pig to retain it as it appeared to be nauseating. Some success was had in neutralizing or acidifying it with acetic or lactic acid but even with this precaution the largest amount that could be given without always causing vomiting or diarrhea was the equivalent of 100 gm. of casein. The cystine given was prepared from human hair by acid hydrolysis. Analyses showed it to contain 26.63 per cent sulfur, the theoretical being 26.69 per cent. It was administered in water suspension by stomach tube. Not all the pigs were found suitable for the demonstration of the effect of these amino-acids. In some the production of crea- tine was so low that excessive casein, 300 to 400 gm. had to be fed before the threshold for creatine excretion was reached. As the arginine equivalent of such amounts of casein could not be successfully given these animals were discarded. With all pigs, preliminary to the amino-acid administrations, casein was given for purposes of comparison as to the amounts of creatine produced. 36 Creatinuria. II Experiments with Arginine. TABLE I, Pig, male, weight 28 kilos. Energy intake, 75 calories per kilo. Day of |Volume|Urinary| -Pte- | ,Crea- starch a eee: creati- | creati- ceding.) urine. | gen. | nine. | nine. | fm | mm cs. gm. gm gm 1k 1,550) 4.40*| 0.857) 0.068) Starch 500 gm. 12 1,400) 2.57 | 0.945) 0.044 Te at AO 13 1 225) 3.62 | 0.772) 0.168 500 “ arginine 4.08 gm. 14 1,575) 2.52 | 0.849) 0.000 3 500. .** 15 1,300) 2.10 | 0.819) 0.084 ts 500. “ 16 1,450) 2.32 | 0.804) 0.073 = 500 “ * The high urinary nitrogen represents in part residual nitrogen from an attempt to feed 200 gm. of casein 2 days previously. In Table 1 is shown the effect of arginine on creatine production. Previous to the collection of the data here presented, observa- tions had been taken on the effect of casein administration. When 100 gm. of casein were given, after 4 days maintenance on a starch diet, the creatine increased from an average value of 60 to 103 mg. An attempt to further increase the excretion by giving 200 gm. of casein resulted in partial anorexia. The nitrogen excretion on the first day bears evidence, however, as shown in the table, that the casein had been partially con- ~ sumed. TABLE I. Pig, female, weight 24 kilos. Energy intake, 75 calories per kilo. Day of Volume|Oripary| orm tine ae | the feeding. une. | gen. Sg nee ce. | gm gm. gm, 8 1,450, 1.94 | 0.460) 0.053) Starch 400 gm. 9 | 2,200) 2.20 | 0.3441 0.073) “ 400 “ 10 1,200) 5.54 | 0.342) 0.163 “300 “casein 100 gm. 11 | 1,200) 2.80 | 0.330] 0.065) “ 400 “ 12 | 1,250] 1.85 | 0.485] 0.075) “ 400 “ 13. | 1,450) 2.92 | 0.468) 0.087, “ 400 “ arginine 4.08 gm. 14 | 1,550) 1.92 | 0.451) 0.1211 “ 400 “ 15 | 1,650| 1.88 | 0.478] 0.051, “ 400 “ 16 | 1,500) 1.68 | 0.505) 0.085) “ 400 “ _ E. G. Gross and H. Steenbock ot In Table II is brought out the effect of casein and arginine in creatine production. In both cases the creatine excretion was increased, although after arginine feeding, the increase was not observed until the second day. This retardation in excretion has also been noted after casein feeding, even though the major portion of the nitrogen was excreted on the first day. We be- lieve this is indicative of the fact that we are dealing with a special form of amino-acid metabolism which is independent, at least to a considerable extent, of the major processes of protein metabolism. TABLE III. Pig, male, weight 25 kilos. Energy intake, 65 calories per kilo. % ; Pre- | Crea- starch |" of | nitro: | formed] tine as Diet. feeding.| urine.| gen. eee ce. gm, gm. gm. 20 1,850) 1.59 | 0.410} 0.121 Starch 400 gm. 21 1,800) 1.33 | 0.427} 0.090 SS 400 “ ae, 1,850} 3.95 | 0.415] 0.238 es 300 ‘ casein 100 gm. 20 1,800} 2.48 | 0.382) 0.122 S Ae 24. 2,000) 1.76 | 0.384) 0.112 sf ALT) 25 | 1,750] 3.11 | 0.398] 0.162} “ 400 “ arginine 4.08 gm. 26 | 1,800) 1.40 | 0.382] 0.122 mF 4900 “ 27 2,000] 1.52 | 0.368} 0.100 ss 400 “ 28 1,850) 3.14 | 0.415) 0.278 i: 300 “ casein 100 gm. 29 2,350} 2.39 | 0.305] 0.103 e 400 “‘ 30 1,850] 1.36 | 0.360) 0.125 re 400 “‘ Table III again shows a distinct, though small, rise in creatine excretion after arginine administration. It is not comparable to the rise obtained on casein of equal arginine content. In fact, this is what is generally observed. The arginine given in this experiment was neutralized with lactic acid, instead of ace- tic acid, after sodium sulfate had been added to remove the barium. Tables IV and V also bear testimony to the fact that arginine as well as casein can increase the creatinuria. These tables are presented as examples of what has been obtained time and again, none of them showing any great uniformity in creatine produc- tion. Sometimes a rise of 30 mg. was obtained, then again one 38 Creatinuria. II of 115 mg.,:and occasionally none at all. This latter observation is not to be taken as one which invalidates our contention that arginine is a precursor of creatine as in such instances a drop TABLE IV. Pig, male, weight 28 kilos. Energy intake, 75 calories per kilo. Day of [Volume|Urinary|grmea} ene a sigh feeding.) urine. gen. nisin Ay nee ce, gm qm, gm 5 1,600, 1.76 | 0.507) 0.052) Starch 500 gm. 6 1,800) 1.43 | 0.534) 0.036 ‘i 500 7 1,750, 1.57 | 0.585) 0.055 Po) Se 8 2,000, 2.25 | 0.595) 0.045 ines | Miia 9 1,550) 3.13 | 0.541) 0.192 « 400 “ casein 105 gm.* 10 | 2,000| 2.40 | 0.695] 0.115“ 500 « 11 2,100, 1.34 | 0.598) 0.086 ds BOO) 12 1,850) 2.59 | 0.654) 0.154 “500 “ arginine 4.08 gm. 13 1,850) 1.83 | 0.675, 0.058 $6) OO 90 * 105 gm. of this casein was required to furnish the same amount of nitrogen as 100 gm. before. TABLE V. Pig, male, weight 29 kilos. Energy intake, 70 calories per kilo. S res | Pre- | Crea- Da (um aoe i Diet. | i) (ed a nine. | nine. ce gm. gm. qm. 9 2,100) 1.47 | 0.430) 0.042) Starch 500 gm. 10 | 2,000 1.72 | 0.505] 0.070) “ 500 “ 11 | 2,200) 2.44 | 0.533) 0.182} “ 400 “ casein 105 gm. 12 | 1,650) 2.31 | 0.539} 0.000) - “ 500 “ 13 1,700; 2.72 | 0.489) 0.131 “500 “ — arginine 4.08 gm. 14 | 2,300) 2.34 | 0.517) 0.195) “ 500 “ 15 | 2,700] 1.72 | 0.561] 0.060) “ 500 “ 16 | 2,200 1.64 | 0.616) 0.066, “ 500 “ 17 | 2,500) 2.20 | 0.415] 0.205, “ 500 “ arginine 4.08 gm. 18 | 2,400] 1.92 | 0.479] 0.143) “ 500 “ 19 | 1,800] 1.76 | 0.468} 0.072) “ 500 “ in the excretion independent of dietary influences may have occurred and the arginine administered merely counterbalanced this decrement. Such changes, irrespective of dietary modifi- E. G. Gross and H. Steenbock 39 cations, are very common, in fact, in some pigs the creatinuria may disappear entirely only to reappear upon the administration of very small amounts of protein. Apparently the production of creatine in these cases has merely fallen below the threshold of its excretion. As a general occurrence, more creatine was excreted after casein feeding than after the administration of its arginine equiv- alent—the former exceeding the latter by about 25 mg. or 25 per cent. This is hardly surprising when we take into con- sideration the fact, that acids, such as phosphoric acid which is liberated in the metabolism of casein, stimulate the production of creatinuria, but two other possibilities also suggest them- selves. In the first place, the casein molecule may carry still other precursors of creatine to augment its excretion, and in the second place, it is possible that free arginine may be metab- olized to urea faster—and thus circumvent its formation into creatine—than the arginine as absorbed with the products of digestion. These are mere hypotheses of which only the first one can derive any support from facts now available. Experiments with Cystine. In the studies of the production or augmentation of creatin- urla by cystine feeding, analyses of the urines for total sulfur and sulfates as well as for total nitrogen, creatine, and creatinine were made. Total sulfur was determined by Benedict’s method and total sulfates by Folin’s method. 4.08 gm. of cystine—the same amount as arginine previously—were given to the pig in each trial. In Tables VI and VII it is seen that cystine feeding increased the creatinuria to about the same degree as, or possibly slightly more, than the same amount of arginine. In Table VI after the first cystine administration the rise in creatine was delayed 1 day. This is similar to what we have seen with arginine and possibly indicates that the creatine is subject to special laws of excretion in comparison with the total nitrogen, sulfur, and other compounds. The cystine sulfur makes its appearance in the -_ urine vary largely in the oxidized form, showing a tendency to a disturbance of the acid-base balance. Acidity determinations were not made. 40 II Creatinuria. TABLE VI. This record is a continuation of that shown in Table IV, the same pig being used throughout. 500 gm. of starch were fed daily. Day of|Volume Urinary) ¢,, Pre- Crea- Total earch | of | mito- | Greate erent |oulfur. [SUS] Sulfur] Dietary addition ce. gm. gm. gm. gm. gm. gm. 14 2,200), 1.27 | 0.533) 0.094) 0.475) 0.255) 0.220 15 2,200) 1.45 | 0.550) 0.027) 0.446) 0.279) 0.166 16 | 1,800) 1.40 | 0.575) 0.052) 0.399) 0.234) 0.162 17 | 2,450) 2.53 | 0.587} 0.038} 1.029) 0.718) 0.310) Cystine 4.08 gm. 18 | 2,150) 1.41 | 0.596} 0.150) 0.627) 0.462) 0.165 19 1,950) 1.48 | 0.472) 0.039) 0.764) 0.580) 0.184 20 1,950) 2.34 | 0.589) 0.087| 0.593) 0.378) 0.215 21 | 2,300) 2.85 | 0.644) 0.132) 1.110) 0.668) 0.442) Cystine 4.08 gm. 22 | 2,150) 1.72 | 0.612) 0.039) 0.602) 0.385} 0.217 23 1,950} 1.82 0.560) 0.062 TABLE VII. This record is a continuation of that shown in Table V, 3 days’ collec- tion being omitted. 500 gm. of starch were fed daily. Day of|Volumne| Urinary| oe atk Total | Total |Neutral ce oe | ase |e [ee | lus SSSR] Dietary aati ce. qm. gm. gm. gm. gm. gm. 23 | 2,100) 1.72 | 0.582) 0.053) 0.359) 0.310) 0.048 24 2,100} 1.93 | 0.619) 0.063) 0.462) 0.277) 0.184 25 | 2,150) 2.66 | 0.541) 0.155) 0.673) 0.364] 0.309) Cystine 4.08 gm. 26 | 2,200) 2.24 | 0.632) 0.083) 0.712) 0.495) 0.217 27 2,000) 2.04 | 0.650) 0.100) 0.535) 0.351) 0.184 28 2,200) 1.76 | 0.577; 0.066) 0.485) 0.323) 0.162 29 | 2,150) 2.15 | 0.617] 0.129] 0.939] 0.558] 0.384] Cystine 4.08 gm. 30 | 2,400) 2.18 | 0.538) 0.089) 0.683 31 | 2,300) 1.93 | 0.542) 0.058 0.251 : ‘ 54: 58) 0.618) 0.367) E. G. Gross and H. Steenboek 41 Table VIII shows how creatinuria is influenced by acidosis. ‘Normally on a starch diet the urine of a pig is always acid. Under ‘such conditions, with this particular animal creatine was always excreted. When the urine was changed to an alkaline reaction by sodium acetate administration the creatine promptly disap- TABLE; VIII. Pig, male, weight 40 kilos. Energy intake in the form of starch, 75 calories per kilo except when casein was given; then an equivalent isody- namic reduction in starch intake was made. Z| _ on oS a |e pee les a, Facil SUC GEE ie ne Es as ss 38 E oe 's 3 Dietary addition. popes ee |p eyoe | = | Se | 32 a > =) Ay S) B |e Zz ce. gm. gm gm. gm gm gm 14 |2,300) 2.25}1.177/0.101 15 |1,050) 1.98)1.344/0. 115|0.424|0.3290.095) Sodium acetate 50 gm. 16 |1,000; 2.32)1.470,0.000\0.508 oe ee Ses 17 |2,000) 2.72)1.200;0.000/0.560)0.320\0. 240 - ees 18 |2,250) 2.92/1.327/0.000)1.417/0.732/0.685 ey “ 50. eys= tine 4.08 gm. 19 2,150) 2.27/1.128|0.000)0. 691)/0.645|0.046; Sodium acetate 20 gm. 20 |1,750| 5.32|1.235|/0.222/0.937/0.576|/0.361 a cs Oe Ee anes sein 165 gm. 21 {1,800} 2.11)1.151/0.000\0.335)0.343/0.191| Sodium acetate 25 gm. 22 |2,800) 1.48/1.109/0.000,0.806|0.761/0.045 cS ae 2b ies 23 |2,800| 3.81)1.299|0.000/0.576/0.375)0. 201 ie Ye 50 “ cys- tine 4.08 gm. 24 |2,700| 2.43|/1.296/0.000/0.475|0. 278|0.197) Sodium acetate 25 gm. 25 |2,100} 2.56/1.207/0.000|1.032|0.777|0. 265 ‘3 c 50 “ ‘eys- tine 4.08 gm. 26 |3,100} 2.70/1.010)/0.000)/0. 750)0.540|0.210| Sodium acetate 25 gm. 27 |2,400) 2.44/1.190/0.000)1.190)1.040/0. 150 § pe 50 “ cys- tine 4.08 gm. 28 |3 ,000} 2.10/1.080/0.000/0.422/0.308|0.014| Sodium acetate 25 gm. 29 |1,800} 5.18/1.212/0. 1680. 765|0. 582)/0. 183 ve ‘ 50“ ca- sein 165 gm. 30 (3 ,000| 6.06/1.033)/0.080)/0.525|0.315,0.210) Sodium acetate 25 gm. ‘peared. Under these conditions cystine feeding was without -effect in each of the four individual trials attempted; casein, on the other hand, was effective as usual. This seems to prove beyond a question that cystine creatinuria owes its origin to other factors than those operative when casein is fed. 42 Creatinuria. II TABLE IX. A continuation of Table VIII with the same dietary régime but with other additions, comparing the effect of arginine with cystine adminis- trations on creatinuria production. | Crea- ee | [Vatume gins | 3 formed sec Dietary additions. ail urine. | gen. | pay Wart 9 ce. gm. gm. gm, 31 2,150 3.27 | 1.1310.000 | Sodium acetate 25 gm. 32 | 2,150] 3.39 | 1.13910.140| “ “ 25 “ arginine 4.08 gm. 33 2,950 3.00 | 1.1300.000 | Sodium acetate 25 gm. 34 2,500, 3.50 | 1.060,0.090 “ “ 25% arginine. 4.05 gm. 35 3,000, 3.12 | 1.110.0.040 | Sodium acetate 25 gm. 36 =| 3,100) 3.03 | 1.187|0.000 2 fee Doren 37 3,600) 2.52 | 1.034,0.000 “« 50 “ cystine 4.08 gm. 38 | 3,850) 3.08 | 1.155)0.000 39 | 3,000 2.64 | 1.010)0.000* 40 | 3,050) 2.44 | 1.097|0.000F 41 2,800) 2.63 | 1.065)0.071T 42 | 3,400) 2.65 | 1.144/0.067 43 | 3,300) 2.64 | 1.056/0.090 44 | 3,500) 3.57 | 1.0380. 160 Cystine 4.08 gm. 45 | 2,200) 2.11 | 0.990/0.000 * The urine, since the first administration of sodium acetate (Table VIII) had always been kept alkaline to phenolphthalein. On this day with the cessation of sodium acetate feeding it was acid to phenolphthalein but alkaline to litmus. + Urine was neutral to litmus. t Urine was acid to litmus on this day and subsequently. The relations brought out in Table VIII are emphasized in their significance by the data presented in Table IX where crea- tinuria, when induced by arginine feeding is also shown not to be affected by sodium acetate administration. Under such con- ditions cystine was again shown to be unable to produce crea- tinuria as long as the urine remained alkaline. After the urine had become acid to litmus again creatine reappeared and increased in amount with cystine administration. Failure of its continued appearance is left unexplained as the experiment was termin- ated, but probably represents one of the variations in creatine excretion called attention to before. E. G. Gross and H. Steenbock 43 SUMMARY. Arginine administered orally in sufficient amounts augments creatine excretion in the pig. Creatinuria induced by casein feeding appears to have its origin in large part in the formation of creatine from arginine, but the acidity of the phosphoric acid split off no doubt also | contributes to the creatinuria as a result of the stimulation of endogenous metabolism. Cystine feeding causes creatinuria only when the sulfuric acid formed by the oxidation of its sulfur is left unneutralized; when neutralized the creatinuria promptly disappears. Neutralization of acidity does not prevent the creatinuria called forth by casein or arginine feeding. BIBLIOGRAPHY. . Steenbock, H., and Gross, E. G., J. Biol. Chem., 1918, xxxvi, 265. . Inouye, K., Z. physiol. Chem., 1912, Ixxxi, 71. . Myers, V. C., and Fine, M.S., J. Biol. Chem., 1915, xxi, 389. . Thompson, W. H., J. Physiol., 1917, li, 111. . Jaffé, M., Z. physiol. Chem., 1906, xlviii, 430. . Harding, V. J., and Young, E. G., J. Biol. Chem., 1920, xh, p. xxxvi. Ook WN CREATINURIA. III.) THE EFFECT OF THYROID FEEDING UPON CREATINURIA.* By E. G. GROSS ann H. STEENBOCK. (From the Department of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, April 20, 1921.) The study of any reaction in the animal body, where only the excretory end-products are available to indicate the course of the whole, is bound to reveal many apparent inconsistencies. Thus in attributing the origin of creatine to certain processes of protein metabolism, investigators are confronted with the unde- niable fact that the most drastic attempts to induce creatinuria in men by protein feeding have met with failure. With women no such difficulty has been encountered. In our experiments (1) carried out with the pig no difficulty in the production of creatin- uria by protein feeding has manifested itself, irrespective of the sex of the animal. We have, however, observed certain var- iations among individuals. For instance, some pigs do not ex- crete creatine in their urine when fed a starch diet or when on a normal ration, but when fasted for a number of weeks such in- dividuals may or may not show the presence of creatine. We have also observed a great variation in the amount of casein necessary to produce creatinuria in the animals. Some require 100 gm., and others require as much as 300 gm. or more. Differ- ences in behavior might be taken as an index of the nearness of approach of endogenous creatine production—though the ex- ogenous would present similar relations—to the threshold level _of its excretion. Yet even this does not tell the whole story as animals already showing creatinuria on a nitrogen-free diet often require more than 100 gm. of casein before any demonstrable increase in creatinuria results. Creatine precursors of exogenous origin evidently are submitted to a different array of metabolic processes from those originating endogenously. Otherwise, very * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 45 46 Creatinuria. III small amounts of casein should suffice to increase a creatinuria already in existence. As a matter of fact Thompson (2) actually found that while he was unable to increase creatinuria in certain dogs by feeding arginine, upon injecting it subcutaneously 9 per cent was recoverable as creatine. Normally we believe that the great bulk of creatine formed is metabolized into creatinine and thus kept from accumulating in the blood stream to the level at which its excretion would become possible, but as this reaction is relatively constant among individuals and is not modified except by the administration of tremendous amounts of creatine it undoubtedly is a factor of little importance in the problem under consideration. The main: possibility demanding our attention is the fact that creatine is formed or not formed in direct proportion to the balance that obtains between the arginase system, destructive as far as creatine formation is con- cerned, and the oxidative system whereby the guanidine grouping is left intact. A change in the velocity of either would imme- diately affect the end-result. Under ordinary conditions the arginase reaction appears to be very prominent as the administration of arginine results in creatine formation to the extent of only 3 or 4 per cent of the theoretical possibility as shown in our experiments (3). Of the nature of the other mechanism practically nothing is known. Denis (4) in 1917 reported the production of creatinuria, in a man afflicted with Graves disease, by protein feeding. As it is well known that in this malady the rate of metabolism is enor- mously increased it suggested itself to us that possibly the thyroid mechanism might be responsible for the differences in the result of protein feeding to normal-men as compared with women. As the active principle, thyroxin, of the thyroid gland functions in oxidative reactions it appears possible that it may take part in influencing creatinuria in oxidizing arginine, thus removing it from the sphere of activity of arginase and increasing the amount of creatine formed. On the basis of these hypotheses an attempt was made to study the effect of feeding thyroid preparations on creatinuria of both exogenous and endogenous origin. While a vast amount of information, relative to oxygen con- sumption and carbon dioxide production under thyroid influences, E. G. Gross and H. Steenbock 47 has been collected, surprisingly little work appears to have been done on the effect of thyroid secretion or preparations on ni- trogen metabolism beyond establishing an increased nitrogen excretion. This has been observed by Magnus-Levy (5) and Andersson and Bergman (6) on feeding thyroid to normal men. Sch6ndorff (7), Gluzinski and Lemberger (8), and Voit (9) obser- ved it with dogs and Farrant (10) with cats and rabbits. Under- hill and Saiki (11) found but a slight increase in urinary nitrogen as a result of thyroid feeding with no change in urinary nitrogen distribution. Cramer and Krause (12), on the other hand, ob- tained an increased creatinuria with both men and dogs as the result of artificially induced hyperthyroidism. TABLE I. Pig, male, weight 31.5 kilos. Energy intake as starch, 65 calories per kilo. f i f i . ape es en eee, | ees | Seer | nearaarene cc. gm. gm. gm. 5 1,650 3.63 1.012 0.033 6 1775 2.48 0.918 0.047 1 gm. thyroid. a 1,800 2.66 0.990 0.084 NS sy 8 1,800 2.91 1.023 0.324 Shales ze 9 1,650 2.57 0.957 0.264 10 1 ,450 2.95 1.050 0.058 EXPERIMENTAL. In the following experiments desiccated sheep’s thyroid pre- pared by Armour and Company was given to pigs after their nitrogen metabolism had been reduced to the endogenous type by starch feeding. The sheep’s thyroid contained 0.14 per cent of iodine. . It was administered, suspended in water, by stomach tube. Analyses for total nitrogen creatine and creatinine were made daily. The data in Table I show that small amounts of thyroid are ineffective in changing the nitrogen metabolism. Not until 3 gm. were given was the creatine excretion increased. As the prep- aration contained only 3.6 mg. of creatine per gm. the increase could not be accounted for as being derived from exogenous 48 Creatinuria. III sources. It is significant that the total nitrogen and creatinine remained constant as far as our technique allowed us to determine. Tables II and III show the discrepancy that obtains between. the creatine excretion induced by thyroid feeding and the increase in total nitrogen. Creatinine excretion was not increased which may not be so evident from Table III alone but becomes evident when the creatinine level shown here is compared with that shown TABLE Il. Pig, male, weight 28 kilos. Energy intake as starch, 74 calories per kilo. Dey of ptarch| Volume of | Urinary | ecsueke. | creatinine, | Dietary additions ce. qm. gm. gm. 7 | 1,450 PSY | 0.449 0.142 8 / 1,400 2.88 0.488 0.150 4 gm. thyroid. 9 | 1,700 3.29 0.441 0.456 10 1,200 2.49 | 0.393 0.376 ll | 1,300 2.13 0.421 0.105 TABLE III. Pig, male, weight 29 kilos. Energy intake as starch, 70 calories per kilo. This record is a continuation of that shown in Table IV. Day_cfmarch| Volume of | Uiroges. | ercatiuinc | creatinine, | Dietary additions ae ce. gm. gm. eae 21 | 1,500 | 1.65 0.702 | 0.160 22 | 1,500 | 1.89 0.727 0.165 4 gm. thyroid. 2 | 013700 2.89 0.680 0.707 24 | 1,550 2.35 | 0.737 0.707 25 1,500 | 2.37 0.630 0.412 26 1,500 | 1.50 0.588 0.169 on the same animal in Table IV. This again emphasizes the marked limitation of the animals ability to change creatine into creatinine, otherwise with the large increase in creatine to the point where it even exceeds the creatinine excretion, the latter would also have been increased. While the toval nitrogen increase does not appear to be commen- surate with the creatine increase, which amounts to a three- to fourfold miltiplication when compared with total creatinine, com- parisons appear warranted. E. G. Gross and H. Steenbock 49 In Table II the total creatinine nitrogen ranges approximately from 8 to 11 per cent of the total nitrogen and in Table III from 16 to 22 per cent of the total nitrogen on different days. If these values could be accepted as indicating a relative constancy of relations in the individual it would lend support to our hypoth- eses that creatinine is formed from creatine, both having a common origin. If on the other hand they are to be considered inconstant it would tend to prove that thyroid medication ex- erted a special influence on the direction of protein catabolism in relation to the guanidine nucleus. Our data are hardly ex- tensive enough to warrant either conclusion especially as we know that the urinary constituents obey different laws of excretion (13) and therefore changes in nitrogen distribution subsequent to thyroid administration for only 1 day could be expected to re- veal little of importance in these guanidine relations. It is noteworthy that the effect of thyroid feeding did not become evident until the second day. This was usually though not invariably observed as a few records showed a response on the first day. For the determination of the effect of thyroid feeding on crea- tinuria when creatine precursors from exogenous sources were available we used pigs which did not show any increase in crea- tinuria after moderate casein feeding. When this fact had been established with an animal we gave it the thyroid with casein and later thyroid alone. The casein was always given 1 day after the thyroid as our previous results had shown that the thyroid effect was usually delayed 1 day. The results of these trials are shown in Tables IV, V, and VI. In Table IV, taking the average of 3 days, there were produced 166 mg. more creatine when casein plus thyroid was fed than when thyroid was fed alone. In Table V the increase was 400 mg. daily for 2 days but in Table VI there is a balance of 60 mg. for 3 days in the opposite direction. The latter is too small to have any contradictory significance in our conclusion that thy- roid medication may affect the exogenous as well as the endoge- nous arginine metabolism, yet it does show that the effect is not of such great magnitude that it is always manifested. 50 Creatinuria. TABLE IV. Ill Pig, male, same individual as used in experiment shown in Table ITI. Energy intake as starch or its equivalent, 70 calories per kilo. Day of starch); Volume of Urinary i nitrogen. eeding. 9 10 1] 12 13 14 15 16 17 18 19 20 urine, Io Ww im pt RAGS sS OOO or De ee PELL ' t 3s = Bg | Preformed | Creatine as creatinine. creatinine. gm, em bo NNR RR bb Ee OR eR ie ee 35 30 50 gm. 0.658 0.653 0.652 0.660 0.693 0.714 0.630 0.762 0.751 0.651 0.732 0.666 0.702 0.727 0.680 0.630 0.588 184 Bee SIR. oS 412 oosossossoSsHrssosessse Dietary changes. 105 gm. casein. 4 gm. thyroid. 105 gm. casein. 4 gm. thyroid. * Animal had diarrhea, but not severe enough to interfere with the urine analyses. Pig, male, weight 36 kilos. 68 calories per kilo. Day of starch eeding. Volume of urine. ee te ee ~ BZSSESaaE ; NONNN NK NOK tO Urinary nitrogen. S _ NWN WDON WOW bo ERESSSr Se een e+ | bo b& bo TABLE V. Energy intake as starch or its equivalent, Preformed | Creatine as creatinine. creatinine. Dietary changes. 210 gm. casein. 2 gm. thyroid. 210 gm. casein. 2 gm. thyroid. E. G. Gross and H. Steenboeck Sil TABLE VI. Pig, male, weight 28 kilos. Energy intake as starch or its equivalent, 68 calories per kilo. Day of starch} Volume of Urinary Preformed | Creatine as Dietary clades feeding. urine. nitrogen. creatinine. | creatinine. ce. gm. gm gm. 7 1,300 1.50 0.486 0.144 8 1,500 1.32 0.450 0.145 9 1,000 2.10 0.450 0.145 105 gm. casein. 10 1,300 2.60 0.437 0.134 11 1,100 1.82 0.407 0.115 12 1,350 2.56 0.4387 0.128 4 em. thyroid. 13 1,250 5.20 0.470 0.460 105 gm. casein. 14 1,550 4.06 0.490 0.254 15 1,500 2.70 0.410 0.178 16 1 ,550 2.32 0.428 0.123 17 1,400 1.93 0.487 0.134 18 1,450 EY | 0.449 0.142 19 1,400 2.88 0.488 0.150 4 gm. thyroid. 20 1,700 3.29 0.441 0.456 21 1,550 3.03 0.485 0.376 22 1,200 2.49 0.393 0.243 23 1,300 2.13 0.421 0.105 SUMMARY. The feeding of sheep’s thyroid to a pig on a nitrogen-free diet ealls forth a marked stimulation of creatine formation. This is accentuated when creatine precursors from exogenous sources are available. It is suggested that creatine formation is_ pri- marily dependent upon the balance that obtains between the arginase and oxidative systems whereby arginine is destroyed. On these premises arginine from exogenous sources is not me- tabolized into creatine in the same proportions as arginine from endogenous sources because this balance varies in different organs. Furthermore, it is suggested that the thyroid principle may be active in causing creatine formation by accelerating the oxi- dative system of arginine destruction at the expense of the effect of arginase and that in the thyroid mechanism is to be sought the variable responsible for the difference in reaction of men and women to protein feeding. 52 Creatinuria. III Creatinuria is looked upon as the result of the accumulation of creatine up to and beyond the threshold of its excretion. Usu- ally this is prevented by the prevalent rate of conversion of creatine into creatinine which appears to be an invariable reaction. BIBLIOGRAPHY. . Steenbock, H., and Gross, E. G., J. Biol. Chem., 1918, xxxvi, 265, and unpublished data. . Thompson, W. H., J. Physiol., 1915, li, 111. . Gross, E. G., and Steenbock, H., J. Biol. Chem., 1921, xlvii, 33. . Denis, W., J. Biol. Chem., 1917, xxx, 47. . Magnus-Levy, A., Z. klin. Med., 1897, xxxiil, 269. . Andersson, J. A., and Bergman, P., Skand. Arch. Physiol., 1898, viii, 326. . Schéndorff, B., Arch. Physiol., 1897, Ixvii, 395. . Gluzinski, L. A., and Lemberger, I., Centr. inn. Med., 1897, xviii, 89. . Voit, F., Z. Biol., 1897, xxxv, 116. . Farrant, R., Brit. Med. J., 1918, ii, 1363. . Underhill, F. P., and Saiki, T., J. Biol. Chem., 1908-09, v, 225. . Cramer, W., and Krause, R. A., Proc. Roy. Soc. London, Series B, 1912-13, Ixxxvi, 550. . Marshall, E. K., Jr., J. Pharm. and Exp. Therap., 1920, xvi, 141. a a eee THE DISTRIBUTION OF PHOSPHORIC ACID IN THE BLOOD OF NORMAL INFANTS. By G. M. McKELLIPS, I. M. De YOUNG, ann W. R. BLOOR. (From the Depariments of Biochemistry and Pediatrics, University of Cali- fornia, Berkeley.) (Received for publication, April 7, 1921.) As part of an investigation of the factors influencing the occur- rence of anemia in infants and young children it was thought de- sirable to make a study of the various combinations of phosphoric acid in the blood, since, as has been shown,'one of these compounds (the organic phosphorus) is a quantitatively important constituent of the red blood corpuscles, while another (the lecithin) also pres- ent in fairly large amounts, is commonly believed to predispose to the destruction of these cells. The work was further desirable as a contribution to the comparative physiology of these com- pounds which is being investigated in this laboratory for the purpose of getting information regarding their functions, and especially that of the unknown organic phosphorus compound present in large amounts in the corpuscles. The present report consists of the results of an examination of the blood of normal infants from birth up to 2 weeks of age. For the determination 15 cc. of blood were drawn from the supe- rior sagittal sinus, prevented from clotting by the addition of minimal amounts of oxalate or citrate, and delivered at the labo- ratory as soon as possible afterwards—generally in 2 to 3 hours. It was then centrifuged in graduated centrifuge tubes for 10 min- utes at about 4,000 r. p. m. and the levels of total blood and cor- puscles read off for the purpose of determining the ratio of cor- puscles to plasma. Hemolyzed blood was rejected because of the probability that significant amounts of corpuscle phosphorus had passed into the plasma. After separation of the plasma from the corpuscles, determinations of the various phosphoric acid compounds were made in each, using the methods previously described.? The results of the examination are given in Table I. 1 Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 49. 2 Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 33. 53 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 in Blood of Infants Phosphoric Aci ot 9° LF O°O6T | €°0FZ | 8'L | 6ST | SOT | O'ST | O' FE; 0880'S 6 1P OsT's | 0°8¢ O'91Z | 2°S22 | OFT | 2ST | 96 | 98S) 0°SE| 02'S | 2'eP 006°¢ €°Shl | OSS | L°92% | O'SLT | O' FES 0OL6°S | 2°TS | | / 00%" S SHI | SFL | SST | O'SZT | 0'092 | G'S | TOT} 2°24 | 2 EL | FEZ} O8e'E | O'SF OOPS | Z'O1Z | 8°69 | SOL | O'les | O'18e | St | 201} G2 | 88 | Sst] 06F€s | 9'0F | 989'¢ G'¥02 | $69 | %'6 | O' FIZ | O' F892) FS | GIT} 99 | O'OT | L412) Sé6c'e | OF GSr'e €°221 | $29 | LPL | O'LET | & 806 9°91 Zo EI | $66] OME “Ou “Ou “Ou “Bu “Ou “Bu Ou “pu “Du “Ou ud juao sad ormesiG | prodiy Shor: ui poy [MOL RNs ‘prodry men bal SN sopsndiog ‘RUISUL yymyom | | “oy0 “09 001 4od 'Od*H | uLowunyg -sndsoy a> €I OL sivp ‘omy ‘syupfuy fo poojg 94} ur spunodmoy pry o4oydsoyd 1 Wav nV nouSane cn Hees ig *sAOG ‘OUIUN PI rea 59 MeKellips, DeYoung, and Bloor 9°9ZT 9°8ZT 8°91 G'I3l €°OLT Test 0°82 9°9¢ 9 9S 8° OF 0° 6P ‘OSVIOAG UL PopnpouL JON x OFE'S O'S9T | O' FEZ (Weyl ler OL OL Same Oselelee cau) sOGpae. | C20 Pulp Gila same tear Wek 9 O1g's S°eT | O'SET |.0' 2PE rou! SZ 922e | OVS: | G:OF |) C1 Dp ie a ING BSI] Fe *S[ILD GTI G'06I F' 8G $9 0°ST 0'6 6 FI 8'°8z Gop eve enee teres s dal eue. @le%ele eis9 ave dSVIDAY Cres GuLLaOsvOGel PROGGHIRe IP LG eeOL WG PE Gros) GER ONGPal (Glo lps ame Gr aaa al ea fol" #e | f 0so's €°S6L | 0' OFZ GelLale2 O10 00g | 000‘E | el aaa ey ee EE Ketan we Al 09F'S CROAT OLOSS. GS. |S SLs Oo St | Sale eOOr a eee O00 Maecenas: ae ay) V'W| SI Phosphorie Acid in Blood of Infants 56 "99 OOT 404 'Od*H 9° 271 | 0°2S | 6 FT | S291 | FSS | FO | TFL | SIT | PLT | SOE I SP GPS e SSSI | och | O'ZT | S°SZT | 0°092 | 22 | OIL | O'OL | oct | FEZ) SI8'e | OFS | 09'S | Z OSI | S°SS | SSI |.0°SFT:| FSG | FS | 9°OT | STL | SZT | O Fe 029'S =| O'SF | $90‘F G°6ZI | 2°2r | SSL | O'SFT | GOST | 9F | 6CL| GOT | 2ST | Se} OZ‘ | 3°SF Gols | GSCI | 0'0S | IST | 9'EZT | FES | FIT | GAT | OIL | OSS | T&F C2908 |GoZ8 | | | | 06's | P'SEl | 1th | 8 Sr | ost | 0'802)| SF | 9'°9OT | 6'CT | SLI #60, 029° | F'98 “Du “Dut “Ou . “pu “Du “Out “Sut “Pua “fut “Ou | “mb uoo sud copuuatag |-prodyy [PNPM “OUINIOS | mot | LG” | Prod aaa fey | Peo *soposndiog “BUISUL | arate aise) N LT II sivp ‘ony Siw Cte piers 1n.9 BPS Sree TO A SAR OL aah oedctal Saeed" rea G 2S Susierpis lesa 5.87618) e SN pt 14 IZ ays carey Bue are ae in 0% bites oe Bree REA Se ery 6I ‘uN om) *papnpouo pt WAV McKellips, DeYoung, and Bloor 57 DISCUSSION. Influence of Sex.—Very little difference can be noted in the values as characteristic of the sexes. In the plasma the values for in- organic phosphorus are markedly lower in the. boys than in the girls while the lipoid phosphorus is slightly higher but not sig- nificantly so. The values for organic phosphorus are the same in both and show the same wide variations. In the corpuscles the only striking difference is in the organic phosphorus which is considerably higher in the boys. Since, however, relatively few determinations are available much emphasis cannot be laid on these differences. ; TABLE II. Averages and Variations in the Phosphoric Acid Compounds in the Blood of Infants and of Adults. Plasma. Corpuscles Aa Vinee 0 (Sa ian a fie Bie |) aed dle o =< ua) = oO HH =< i 4 S) 7 ‘Adult.....| 27.6] 8.4] 7.0] 17.5] 0.2/200.01150.0| 10.9] 45.0/128.0 Infant....| 18.5| 8.8] 3.7/ 10.1) 1.3/186.5|135.0| 9.4] 42.7|121.5 Aver- {Adult..... 34.0] 11.4| 9.9] 23.5] 1.5/248.0|187.0] 17.2] 57.0|169.5 age \Infant....| 29.7/ 16.5] 9.6] 14.8) 7.0|241 Thee 14.3] 56.0/156.0 peed pAdalt:.... 42.2} 14 | 14 | 26.2} 4.0/295.0/228.0| 26.8) 64.0/213.0 18" \Infant....| 43.0] 25 | 13.9] 21.4] 11 4|284.0|221.0| 26.7) 74.8/204.5 Influence of Weight Changes.—Most of the infants were losing weight at the time the samples were taken so that little can be said with regard to the effect of gaining or losing weight on the blood phosphates. As far as can be seen, however, in infants gaining weight the values are higher than the average in the plasma and somewhat lower in the corpuscles, while in those losing weight the reverse is the case although the difference is less noticeable. Comparison with Adults —For convenience in comparison there are given in Table II the averages and variations in the phosphoric acid compounds of infants and adults. \ _ 58 Phosphoric Acid in Blood of Infants Corpuscles.—The average values for total and lipoid phosphorus in the corpuscles of adult and infant agree very closely while the average inorganic value is considerably, and the organic some- what, lower in the infant. The low values are lower in the in- fant than in the adult as is also the case with the high values with the exception of the lipoid phosphorus which reaches higher levels than are normally found in the adult. The latter may be due to the almost continuous absorption of fat in infants of this age, since high values for fat in blood have been found to bring about increases of lipoid phosphorus in the corpuscles. The corpuscle values as a whole are remarkably similar in the infant and the adult and indicate that there is little if any change in the composition of the red cells after birth—a finding which is in marked contrast to that in cattle in which Meigs* found much higher values (nearly twice in some cases) for total phosphorus in the corpuscles of young calves than in those of cows. Plasma.—The organic phosphorus is regularly much higher in the infant than in the adult, resulting also in a higher acid-soluble fraction. Lipoid phosphorus is much lower throughout in the infant than in the adult, in this respect agreeing with the findings of Meigs* with cattle in which the lipoid phosphorus of the plasma of calves is found very low, increasing up to the age of about 1 year. * Meigs, E. B., Blatherwick, N. R., and Cary, C. A., J. Biol. Chem., 1919, xxxvil, l. THE DETERMINATION OF INORGANIC SULFATE, TOTAL, SULFATE, AND TOTAL SULFUR IN URINE BY THE BENZIDINE METHOD. By CYRUS H. FISKE. (From the Biochemical Laboratory, Harvard Medical School, Boston.) (Received for publication, April 28, 1921.) As far as its use for the analysis of urine is concerned, the benzidine method for the determination of sulfur is subject to two sources of error that may under certain conditions be serious. One of these, the contamination of the benzidine sulfate pre- cipitate by phosphate, has been recognized before, and can be eliminated by adding acid before precipitating with benzidine. For this purpose Rosenheim and Drummond! recommend the addition of hydrochloric acid to the urine until Congo red paper shows an acid reaction. Gauvin and Skarzynski,? on the other hand, add the same quantity of hydrochloric acid in every in- stance, without the use of an indicator, and this scheme has also been adopted by Drummond? in a more recent modification of the Rosenheim and Drummond method on a smaller scale. In the methods for inorganic sulfate proposed by all these writers, the concentration of benzidine during the precipitation is about the same ( 0.017 to 0.02 N ), but the concentration of hydro- chloric acid is much less uniform. In Table I are collected data showing the concentrations of sulfuric acid, benzidine, and hydro- chloric acid existing during the precipitation from sulfate solu- tions by these three methods. The figure for hydrochloric acid given in the table for Rosenheim and Drummond’s method holds also with urine, since in this method the urine is acidified before adding the benzidine reagent. In the other two methods, in- 1 Rosenheim, O., and Drummond, J. C., Biochem. J., 1914-15, viii, 143. 2 Gauvin, R., and Skarzynski, V., Bull. Soc. chim., 1913, 4th series,, oie WA 3’ Drummond, J. C., Biochem. J., 1915, ix, 492. 59 60 Determination of Sulfur in Urine | asmuch as this step is omitted, the concentration of hydrochloric acid during the precipitation of benzidine sulfate from urine is less than stated by an amount that varies with the character of the urine. In spite of these differences, all three methods have given re- sults that agree fairly well with the gravimetric method, but they have done so only because the comparisons have been con- fined to urines that are very much alike with respect to the factors that affect the accuracy of the determination. As long as their use is restricted to ordinary 24 hour urines, any one of a great many possible modifications would suffice to avoid trouble due to the presence of phosphate, simply because in such urines there is never a very large amount of phosphate in proportion to the sulfate content. But the much more exacting requirements of TABLE I..- Hydro- Method. Sulfurie acid. | Benzidine.| chloric acid. N N N Rosenheim and Drummond............. 0.004-0.007 | 0.017 0.024 iB rvs Vealoyi\o bea Sea wees Ape rer eee nme e Ae Laney, oe 0.003-0.010 | 0.017 0.084 Gauvin and Skarzynski................. 0.002-0.003 | 0.020 | 0.031 the less uniform urines obtained in short period metabolism ex- periments are satisfactorily met neither by any modification of the benzidine method that has been suggested so far, nor by any that I have been able to find that does not involve the pre- liminary removal of the phosphate. If phosphate were the only source of trouble, it would be easy to devise a method that would do for all circumstances that are likely to arise, although such a method would call for a more precise adjustment of the acidity than has heretofore been thought necessary. But unfortunately there is a second cause of error, in some respects even more troublesome. This is the increase in solubility of benzidine sulfate due to the presence of chloride, which often exists in urine in sufficient concentration to be in- jurious in this way. That this may rarely or never be the case in 24 hour urines presumably accounts for the fact that the effect of chloride has previously escaped notice in connection with urine analysis. C. H. Fiske 61! In 1 hour urines, on the other hand, for each mg. of sulfur in the form of inorganic sulfate, there may be as much as 5 mg. of inorganic phosphorus and 30 mg. of chlorine, and even more under conditions that can hardly be called unusual, although they may not be especially frequent. The influence of the addi- tion of sodium chloride and disodium phosphate on the anal- ysis of sulfate solutions by Drummond’s method will be seen from the figures recorded in Table II. That the same factors TABLE II. Analysis of Sodium Sulfate Solution by Drummond’s Method. Composition of solution. Sulfur found. Sulfur. Phosphorus. Chlorine. mg. mg. mg. mig. 1 0 0 1.00 1 4) 0 1.04 1 0 30 0.97 1 0) 60 0.91 , < TABLE III. Analysis of Urine by Drummond’s Method. ee euucnt Wee: eae Gilarine: Thongenie sulfate sulfur cc. mg. mg. mg. mg. per 100 cc. 1 il 0.66 14.8 0.674 67.4 2 1 3.36 14.8 0.678 67.8 3 1 0.66 34.8 0.642 64.2 4 2 P32 29.6 1.39 69.5 5 2 6.72 29.6 1.44 | 72.0 are not without considerable effect on sulfate determinations in urine is evident from the results in Table ITI, all obtained with one sample of urine, which was analyzed alone (Experiments 1 and 4) and after the addition of disodium phosphate (Experi- ments 2 and 5) and of sodium chloride (Experiment 3). De- pending upon the amount of phosphate and chloride present (and the conditions in this respect are not extreme for 1 hour periods) the figures vary by more than 10 per cent. A considerable experience with short period metabolism ex- periments has led me to the conclusion that any sulfur method, 62 Determination of Sulfur in Urine to be safe for such work, must give accurate results in the pres- ence of 10 mg. of phosphorus or 60 mg. of chlorine for each mg. of sulfur in the form of inorganic sulfate. Many different ben- zidine reagents have been tried, under all sorts of conditions, but none has been found equal to these requirements, and it is apparently necessary to accept the fact that both these difficul- ties cannot be successfully contended with at the sdme time. Once the phosphate has been removed, the situation is much simplified, as it is then possible to avoid trouble from the pres- ence of chloride by precipitating the benzidine sulfate at a much lower acidity than would otherwise be permissible. Removal of Phosphate. For the removal of phosphate from urine as a preliminary to the precipitation of sulfate with benzidine, nothing has been found equal in effectiveness to magnesia mixture in some form, amd it is fortunately possible to precipitate the phosphate nearly quantitatively as magnesium ammonium phosphate without the introduction of injurious quantities of electrolytes, which, like sodium chloride, would prevent the complete precipitation of ben- zidine sulfate. This is accomplished by shaking the urine (pre- viously neutralized with ammonia) with basic magnesium car- bonate in the presence of a small amount of ammonium chloride. The whole process of precipitation and filtration requires only a very few minutes. If the first 15 or 20 cc. of filtrate are poured back on the paper and filtered again, the solution will percolate through a layer of magnesium carbonate mixed with triple phosphate crystals, and this is a particularly effective way of removing phosphate. The final filtrate should then con- tain less then 0.1 mg. of inorganic phosphorus in 5 ce. The urine must be fairly dilute before the phosphate is removed, for magnesium ammonium phosphate crystallizes with 8 mol- ecules of water, and the removal in this way of more than 0.2 per cent of phosphorus would appreciably alter the concentration _ of sulfate in the filtrate. In the following directions for preparing the essentially phos- phate-free filtrate, the quantities prescribed are sufficient for: duplicate determinations of all three forms of sulfur (inorganic sulfate, total sulfate, and total sulfur). C3. Riske 63 Transfer to a 50 cc. volumetric flask sufficient urine to contain between 5 and 10 mg. of sulfur in the form of inorganic sulfate, and dilute to about 25 cc. with water. Add 1 drop of phenol- phthalein solution and 1 drop of concentrated ammonium hy- droxide (or as much as is necessary to make the solution faintly pink), followed by 5 ce. of a 5 per cent solution of ammonium chloride. Make up to the mark, mix, and pour the solution into a dry Erlenmeyer flask containing about 0.65 gm. of finely powdered basic magnesium carbonate.* Shake for 1 minute, and transfer to a 9 em. filter paper enough of the suspension to fill the paper nearly to the top. Allow this first filtrate to drain back into the Erlenmeyer flask, and then filter the entire sus- pension through the same paper into a dry container. In case the urine is already extremely dilute, the phosphate ean be precipitated without appreciably altering the concen- tration by using solid ammonium chloride (0.25 gm.) instead of a solution. Urines obtained in short period experiments are sometimes so dilute as to make this modification necessary. The filtrate, prepared as described above, is now used for all three sulfur determinations. Determination of Sulfur in the Phosphate-Free Filtrate. Inorganic Sulfate—Pipette 5 cc. of the filtrate into a 100 ce. beaker. Add 2 drops of a 0.04 per cent alcoholic solution of brom-phenol blue® and 5 cc. of water. Then add approximately N HCl, drop by drop, until the solution is yellow without a trace of blue. Run in, from a pipette, 2 cc. of benzidine reagent,® and let stand for 2 minutes. Finally, add 4 ec. of 95 per cent acetone, and let stand for 10 minutes more. Filter through a mat of paper pulp in a special filtration tube (described below). Wash the beaker and the filter, first with three 1 cc. portions of 95 per cent acetone, and then once with 5 cc. Transfer about 2 cc. of water to the filtration tube, and poke the precipitate 4 This reagent must obviously be free from sulfate. Baker’s analyzed magnesium carbonate has proved satisfactory. 5 Clark, W. M., The determination of hydrogen ions, Baltimore, 1920, 63. 6 Suspend 4 gm. of benzidine in about 150 cc. of water in a 250 ce. volu- metric flask. Add 50 cc. of nN HCl (standardized). Shake until dissolved, and make up to volume. Filter if necessary. 64 Determination of Sulfur in Urine and mat through the hole in the lower end into a large Pyrex test-tube (200 by 20 mm.), using a sharpened nichrome wire. Rinse off the wire with a few drops of water, and heat the contents of the test-tube just to boiling, leaving the filtration tube sus- pended in the mouth of the test-tube. Add 2 drops of a 0.05 per cent aqueous solution of phenol red (monosodium salt),° and run in from a micro-burette,”® through the filtration tube, about 1 ec. of 0.02 N NaOH. Rinse down the wall of the filtra- tion tube with 2 or 83 ce. of water from a wash bottle, heat again to boiling until steam escapes actively from the test-tube, and rinse a second time with sufficient water to bring the total volume up to about 10 cc. This treatment should suffice to remove all traces of precipitate from the filtration tube, which may now be removed, and the titration with 0.62 n NaOH continued. When the color begins to change from yellow to red, again heat to boiling, and pour the hot solution into the beaker (in which the precipitation took place) and back.® This will decompose any trace of precipitate that may have adhered to the wall of the beaker. From this point on the standard alkali should be added, not more than 0.02 cc. at a time, until the solution acquires a definite pink color, which further boiling does not discharge. Total Sulfate-—To 5 cc. of the filtrate in a 100 ec. beaker add 1 cc. of 3 N HCl (approximate). Heat on the water bath until the solution has evaporated to dryness, and for 10 minutes longer. Immediately add 10 cc. of water, and break up the residue by rotating the beaker. Add 2 ec. of the benzidine reagent and (2 minutes later) 4 ec. of acetone, exactly as in the method for inorganic sulfate, and complete the determination as described above. Total Sulfur.—Transfer 0.25 cc. of Benedict’s total sulfur reagent’ to a 6 cm. evaporating dish, and add 5 ec. of the urine 7 Folin, O., and Peck, E. C., J. Biol. Chem., 1919, xxxviii, 289. § Fiske, C. H., J. Biol. Chem., 1921, xlvi, 285. * This step may be avoided by conducting both precipitation and titra- tion in a large lipped test-tube, but except for the inorganic sulfate deter- - mination a beaker is on the whole more convenient. ‘° Benedict, 8. R., J. Biol. Chem., 1909, vi, 363. The reagent contains 20 gm. of copper nitrate crystals and 5 gm. of potassium chlorate per 100 ce. A blank (gravimetric) must, of course, be run on the reagent unless the copper nitrate is free from sulfate. C. H. Fiske 65 filtrate. Evaporate to dryness, preferably on an electric hot plate at low heat.. When the mixture has become dry, increase the heat by steps to the maximum, and finish the ignition with a microburner, allowing 2 minutes at red heat after the contents of the dish have become thoroughly black. Cool for 5 minutes. Add 1 cc. of 3 N HCl, and evaporate to dryness on the hot plate (low heat). When the residue is thoroughly dry, dissolve and wash into a 100 cc. beaker with five 2 ce. portions of water. Add 1 drop of n HCl, and precipitate with the benzidine reagent and acetone as in the other two methods. The rest of the deter- mination is likewise the same as before, with the single excep- tion that 2 cc. of 50 per cent acetone should be used in place of the first of the three 1 cc. portions of 95 per cent acetone, otherwise it will be impossible to wash the filter free from copper. The amount of sulfur in the 5 ce. of filtrate analyzed is in each case obtained (in mg.) by multiplying the titration figure by 0.32. DISCUSSION. Use of Acetone.—At a time when there was still some hope of avoiding the necessity of removing the phosphate, the addi- tion of acetone during the precipitation with benzidine was introduced for the purpose of diminishing the solubility of the precipitate. Although this may not be altegether necessary under the conditions finally adopted, the modification has been retained, ’ and the same liquid used for washing the precipitate, for various reasons. Washing with a saturated solution of the precipitate is a thing to be avoided whenever possible, and acetone is to be preferred on that account. Since acetone wets glass more readily than does water, it does not collect in drops on the wall of the beaker, a matter of some consequence when a fairly large surface must be washed with a small volume of liquid. But the most important consideration of all is that the use of acetone altogether prevents the benzidine sulfate from assuming the form of large flakes, which can be decomposed only by prolonged boil- ing at the end of the titration, and on account of which Rosenheim and Drummond,! who wash the precipitate with a saturated solution of benzidine sulfate, have been obliged to recommend that the filter never be allowed to be sucked dry. 66 Determination of Sulfur in Urine Indicator.—Boiling aqueous solutions of purified benzidine, at concentrations corresponding with the conditions at the end- point of the titration described above, give a brownish color with phenol red, intermediate between yellow and red. The addition of less than 1 per cent of one equivalent of sodium hydroxide is sufficient to change the color to a definite pink, whereas phenolphthalein under these conditions is still color- less. Since phenol red is besides a much more brilliant indicator, it is to be preferred on all counts. . Filtration Tube.—This is, in principle, the same as the tube recently described in connection with a method for the determi- nation of inorganic phosphate in urine. The narrow tube recom- mended for the filtration of magnesium ammonium phosphate v Fig. 1. Filtration tube (one-half natural size). is readily clogged by the much more compact benzidine sulfate precipitate, and for sulfate determinations it should be replaced by one considerably larger in diameter. The tubes that have been found most convenient are made from glass tubing" 15 mm. in internal diameter, shrunken at one end so as to leave a hole 3 mm. in diameter, cut to a length of 70 mm., and flanged at the cut end. The somewhat elongated tip shown in Fig. 1 gives the best results. In using this tube, only enough paper pulp should be intro- duced to form a thin cup-shaped mat lining the constricted tip. It is neither necessary nor desirable to fill the tip with pulp. The mat having been prepared, the tube should be filled with the ‘t Pyrex tubing is preferable. Since the tube is subjected to the action of steam during the titration, soft glass should not be used. C. H. Fiske 67 solution to be filtered before starting the suction, and the suction, when it is started, should be very gentle. Any attempt to has- ten the filtration by applying strong suction is almost certain to produce the opposite result by packing down the precipitate. Adjustment of Acidity—The purpose of the preliminary acid- ification ( to brom-phenol blue ) in the determination of inorganic sulfate is to neutralize any basic substances that may be present and to liberate weak acids from their salts. No appreciable TABLE IV. ie iZ 2 Sulfur per hr. 2 |g] Meine 2 Ale Z E Tnorganic Total sulfate. Total sulfur. s | Se | alg = = a = = Giavi-| Titra- | Gravi-| Titra- | Gravi-| Titra- = = = metric.| tion. | metric.| tion. | metric.| tion. "66 ~~ mg. mg. mg. mg. mg. mg. 1 (a) 21.0 1.8 14 HHO || Wesses | UZ! | are || PAL TL |) Pali (b) 10.0 14 15.4 io! 20.9 (c) 1.8 60 15.3 lide PAE IL 2 (a) 9.5 0.9 13 iT || 2B |) PRES PAO eey4G || BE (b) 10.0 13 22.8 25.6 (c) 0.9 60 22.4 25.93 32.0 3 (a) 8.5 1.0 18 TAS lA Ss It died Nlidece ecole aan f (b) 10.0 18 14.7 17.9 22.3 (c) 10 60 ab 76 18.0 2263 4 (a) 740). 0.8 9 Be BAC BY/ca) | Bloc |) 2P4c0) |) Zalee/ (b) 10.0 9 34.8 Bhat 41.8 (c) 0.8 60 34.3 BYladh 42.2 amount of the hydrochloric acid in the benzidine reagent. will then be neutralized by constituents of the urine filtrate. In the method for total sulfate, this adjustment is made automat- ically by evaporating the urine with hydrochloric acid. Hydrolysis of Ethereal Sulfate—Evaporation with hydrochloric acid is substituted for the usual boiling mainly because the additional acid required for hydrolysis would be enough to cause low results in the presence of large amounts of chloride. 68 Determination of Sulfur in Urine Total Sulfur Method.—The only special modification introduced in this determination is the removal of the excess hydrochloric acid, after dissolving the residue of copper oxide, by evaporation to dryness instead of by neutralization with alkali. Neutral- ization would introduce more sodium ehloride, which is unde- sirable for reasons that have been mentioned. After the excess acid has been removed by evaporation, the residue is at times so nearly neutral that a drop of dilute acid should be added (as stated) before running in the benzidine reagent. Results. The method as described gives satisfactory results in the pres- ence of 10 mg. of inorganic phosphorus or 60 mg. of chlorine for each mg. of sulfur in the form of inorganic sulfate. A series of analyses is given in Table IV. Each of the urines was ana- lyzed first in the manner described above (a), secondly after add- ing sufficient disodium phosphate to bring the inorganic phos- phate content up to the stated figure (b), and finally after the addition of enough sodium chloride to make the ratio of chlorine to inorganic sulfate S equal to 60 (c). . For comparison the same urines were analyzed gravimetrically (Folin’s” method for inor- ganie and total sulfate; Benedict’s!® method for total sulfur). Differences greater than about 1 per cent sometimes occur in the determination of inorganic sulfate, especially when the sulfur excretion is small, however closely duplicates by each method may agree. In en cases there is apparently no way of deciding which method is the more accurate. Discrepancies of this nature have not been observed in | the determination of total sulfate or total sulfur. 12 Folin, O., J. Biol. Chem., 1905, i, 131. BASAL METABOLISM OF NORMAL WOMEN.* By KATHARINE BLUNT anp MARIE DYE. (From-the Department of Home Economics, University of Chicago, Chicago.) (Received for publication, April 9, 1921.) The primary purpose of this investigation was to find out whether there is any regular periodic variation in the basal me- tabolism of normal women during menstruation. The figures obtained show the lack of such periodicity. They bring out also the marked day by day variation in basal metabolism which may be expected at any time, and the 216 observations add to the already accumulated data on standards for women. These three points will be taken up successively. Procedure for Determining Metabolism. Method. The Benedict portable respiration apparatus was used to de- termine the basal metabolism. In general, the procedure of the Nutrition Laboratory for determining oxygen consumption was followed (1, 2). The subject, in a postadsorptive condition, took a 30 minute rest period in a quiet room preceding the two 10 minute observations. The initial reading for the volume of oxy- gen was taken after the subject was connected with the air cur- rent at the end of a series of regular expirations, and at the same instant a stop-watch was started. With a second stop-watch another reading was taken so that duplicate records were made for each 10 minute period. The day’s basal metabolism was taken as the average of the duplicates of the two 10 minute periods. If for any subject checks were not obtained on the first two periods, a third period was observed. Very occasionally * The work reported in this paper will form part of the thesis to be submitted by Marie Dye for the degree of Doctor of Philosophy, University of Chicago. 69 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 70 Metabolism of Women a whole day had to be discarded because of failure to obtain satisfactory duplicates. The results are given only when dupli- cate readings agree within 5 per cent. No attempt was made to determine the carbon dioxide. Benedict (1) has found that the error, made in assuming a constant respiratory quotient, when the subject has been 12 or more hours without food, is slight. We have assumed the customary respiratory quotient of 0.82 and the factor 4.825 calories per liter of oxygen. Subjects—Our seventeen subjects were faculty members or students at the University of Chicago from 21 to 44 years old. All were in normal physical condition, and continued their usual academic work during the menses, four of them stating that they felt absolutely no discomfort or disturbance of any kind (Nos. 12, 13, 14, and 15) and the others experiencing more or less fatigue or lassitude. From one to three complete periods were studied with pre- and intermenstrual observations on each of fourteen of the subjects. One case of amenorrhea (No.1) was observed for 26 almost successive days. Two intermenstrual studies of 10 days each were made (Nos. 3 and 8). Experimental Data. The detailed results of all observations are given in Table VI, listing the subjects according to age. The age is that at the nearest birthday, the weight is taken nude, and the surface area is read from the DuBois height-weight chart (3). Comparison of Metabolism during Menstrual and Intermenstrual Phases. Averages for Menstrual and Intermenstrual Phases—In Table I are brought together the averages of all the observations made on the fourteen women during the menstrual and the intermen- strual periods. The figures show no consistent variation. In only six of the subjects is the difference between averages for the two phases greater than 3 per cent, and of these six, some have the higher metabolism during menstruation and some in the intermediate period. The average of all the subjects gives a 1.6 per cent lower metabolism during menstruation than at other times and practically the same decrease when only the be ie ane ty Mea eS K. Blunt and M. Dye Tt first 2 menstrual days are included. This difference is too small to be considered significant. Periodicity of Metabolism during Premenstrual and Menstrual Phases—There also seems to be no regular, rhythmic, day by day variation. The curve of the daily observations is far from smooth. Table II, which lists the daily metabolism immedi- ately before and during menstruation, gives a confused sense of TABLE I. Comparison of Average Heat Production during Menstrual and Intermenstrual Phases. ana, | (, Mensinaat, og | tee ene Subj g a || | 8 Be asi (iia Sa Subject. ; : z S ao 2 ae Ee 2 ae Es , | aie |S eel ee | ee] Ba | be | 2s | ee | ee & 3 3 2 o| qa aa 3a E ia mega ia ls (eo 1o \ |e hoes cm kg. |sq.m.| No No ee No. oat 2 24 | 152 | 66.7) 1.63) 3 |1,407| 6 |1,445) 2.7; 2 |1,430) 1.6 t 25 | 164 | 64.1) 1.70) 2 |1,277|) 3 1,332) 4.3) 2 |1,357] 6.3 5 26 | 161 | 50.1) 1.51) 5 {1,281] 11-]1,231/—3.9] 6 |1,229|—4.0 6 28 | 148 | 53.0) 1.44) 4 |1,137| 8 |1,132/—0.4| 3 |1,170| 2.9 7 28 | 160.| 61.0) 1.63) 3 |1,353) 6 |1,318)/—2.6| 4 |1,322)/—2.3 i) 29 | 149 | 51.0) 1.43) 3 |1,238) 6 |1,272) 2.7| 4 |1,281| 3.5 10 30 | 170 | 61.7] 1.70) 2 11,537; 7 |1,468)—4.5| 3 |1,505|—2.1 11 32 | 151 | 43.0) 1.34) 3 /1,135) 3 |1,068/—5.9) 2 |1,052/—7.3 12 33 | 168 | 62.0) 1.69] 2 |1,360| 3 |1,337/—1.7| 2 |1,315|—3.3 13 33 | 173 | 79.0) 1.93] 3 |1,633] 8 |1,551/—5.0| 2 |1,562)—4.3 14 37 | 162 | 75.5) 1.80] 2 |1,500} 5 |1,454;—3.1| 4 |1,454)/—-3.1 15 40 | 166 | 66.6) 1.74) 2 |1,510| 3 |1,437|/—4.8| 2 |1,442|/—4.5 16 41 | 158 | 70.0) 1.71] 2 |1,347| 4 |1,329|—1.3) 2 |1,310|—2.7 17 44 | 162 | 49.1) 1.49) 7 |1,203} 11 |1,224) 1.7) 5 {1,210} 0.5 Average. 1,351 il ,328|—1.6 1 ,331|—1.3 irregularity rather than of periodicity. With a number of sub- jects a high metabolism day can be found just before or at the beginning of menstruation, but so also can a low metabolism day. Moreover, the high day connected with menstruation is only sometimes higher than other days observed. The only possible conclusion seems to be that the variation connected with the menstrual cycle is irregular and no greater than at other times. 72 Metabolism of Women We have been able to find in the literature only three other series of observations on basal metabolism during menstruation, a long series by Zuntz (4) in 1906, two observations by Gephart and Du Bois (5) in 1916, and a preliminary report by Ford in a TABLE II. Daily Variations during Premenstrual amd Menstrual Phases in Calories per 24 Hours. 2 days Day Day of menstruation. before | before Subject. MONS U=|MENKWUs| as ae eae | eee Tae a. ation. | ation. Ist 2nd 3rd 4th 5th 6th 2 1,390*| 1,355 | 1,500 | 1,360 | 1,440 | 1,440 | 1,440 | 1,490 4 1 ,375* 1,355 | 1,360 | 1,280 5 (I) 1,375*| 1,375 | 1,300 | 1,390 | 1,330 | 1,230 5 (II) 1,370 | 1,235 | 1,195 | 1,195 | 1,330 5 (III) | 1,100 | 1,135 | 1,110 | 1,145 | 1,080 6 (I) 1,200 1,090 | 1,070 | 1,125 6 (II) | 1,200 | 1,145 | 1,210 | 1,100 | 1,140 | 1,125 7 (1) 1,390 | 1,310 | 1,310 7 (II). | 1,320 | 1,320 | 1,300 | 1,290 | 1,310 9 (I) 1,250 | 1,310 | 1,260 | 1,270 9 (II) 1,230 | 1,265 | 1,290 | 1,235 10 (1) 1,540 | 1,515 | 1,490 | 1,425 | 1,465 | 1,475 10 (II) 1,585 | 1,425 | 1,600 1,420 | 1,400 11 1,125 | 1,105 | 1,085 | 1,020 | 1,105 12 1,320 | 1,310 | 1,380 13 (I) 1,650 | 1,620 | 1,520 | 1,620 | 1,515 13 (II) 1,585 | 1,575 | 1,535 | 1,535 | 1,490 14 (1) 1,390 | 1,455 | 1,390 | 1,455 14 (II) 1,460 | 1,480 | 1,490 / 15 1,410 1,425 | 1,460 | 1,425 16 1,290 | 1,360 | 1,370 | 1,250 | 1,320 | 1,375 17 (I) 1,145 | 1,315 1,270 | 1,300 17 (II) 1,080 | 1,140 | 1,140 1,220 | 1,235 17 (III) | 1,260 | 1,190 | 1,235 | 1,220 | 1,220 | 1,175 * The actual observations of ce. of oxygen per minute from which these figures were calculated were for No. 1: 202, 202, 200, and 197; for No. 2: 193, 198, 200, and 200; and for No. 3: 197, 198, 200, and 199. These may be taken as typical of the range in our duplicate observations. recent paper by Snell, Ford, and Rowntree (6). Zuntz deter- mined the carbon dioxide output and part of the time the oxy- gen consumption for two women for 97 days almost without a break, including three menstrual periods with inter-, pre-, and K. Blunt and M. Dye 73 postmenstrual observations. His conclusions, and also those of Gephart and Du Bois, are in line with ours—that there is no regularity of variation. Snell, Ford, and Rowntree, on the other hand, conclude that menstruation ‘‘does affect the basal metabolic rate of women at times in health and disease.’’ Ford concluded for eight cases out of ten studied that a rather constant rise occurs during men- struation or in the premenstrual period, the rise being followed by a postmenstrual fall. Two of the ten cases showed a drop rather than a rise. The details of this work have not yet been published. We have no information of our subjects in regard to postmenstrual metabolism, but we do not find regularly the menstrual or premenstrual rise that Ford does. Daily Variation in Metabolism. As already pointed out in the discussion of menstrual vari- ation, the extremes of metabolism of these subjects on different days show a wide range. Since this is as marked during the in- termenstrual as the premenstrual and menstrual phases, no dis- tinctions of phases are made in this part of the discussion. The maximum and the minimum metabolism observed at any time for each subject, including the three subjects observed without menstrual phase, are brought together in. Table III. The per- centage of the maximum above the minimum is very high in many cases. The highest difference is 28.8 per cent, the lowest 7.4 per cent, and the average 13.2 per cent. Only six of the seventeen subjects show a range lower than 10 per cent which the clinician often uses as his criterion of normality as compared with a standard. Even if the variations are calculated from the average for the individual, instead of by comparison of max- imum and minimum values, three of the subjects still have a greater range than 10 per cent.!. Our observations, therefore, emphasize the importance of taking several days’ rather than 1Compare the recommendation of Boothby (7) in a recent article: ‘‘all patients having a rate ranging between +10 and +20 per cent. should have the test repeated on a subsequent day.”’ 74 Metabolism of Women a single day’s observation to arrive at what is average for an individual.” These observations on the high variation which may be ex- pected for an individual on different days merely confirm those given by Benedict in 1915 (8). At that time he listed the ex- TABLE III. Extreme Variations. | | Total Heat production per day. No. of Variation of Extreme variation Sobiect. lanys ob-| ai | Mini- Javerage| minimum.” | fromaversge. ; 7 calories | calories | calories | calories | per cent | calories | per cent 1 | 26.-| 1.390 |. 1.725 .)-46280/) 196° ),017.3-)) 1 ee 2 14 | 1,500 | 1,355 | 1,413 | 145 10.7 Be 6.2 3 10 | 1,345 | 1,125 | 1,205 | 220 | 19.6 140 11.6 4 | 6 | 1,875 | 1,260] 1,321} 115 9.1| —61| —4.6 5 25 | 1,390 | 1,080 | 1,254} 310 | 28.8] —174 | —14.0 6 14 | 1,210 | 1,070 | 1,140} 140 13.1} —70| —6.1 7 13 | 1,390 | 1,290 | 1,335 | 100 Go8 55 4.1 8 10 | 1,495 | 1,340 | 1,396 | 155 11.6 99 7H 9 APs py 0 a ee 54 4.3 10 | 13 | 1,600 | 1,400 | 1,493 | 200 14.2 107 (he: ll | 8 | 1,170 | 1,020| 1,105 | 150 14.7| —85| —7.7 12 | 5 | 1,430 | 1,290 | 1,346] 140 | 10.8 84 6.2 13 13. | 1,660 | 1,490 | 1,580 | 170 | 11.4} -—90| —5.7 14 1) 9. P4510) 1,390: |"1, 457"). 120 8.6 | —67| —4.6 15 7 | 1,520 | 1,410 | 1,457] 110 ies) 63 4.3 16 9 | 1,375 | 1,250 | 1,331 | 115 9.5| —81| —6.1 17 2% | 1315 | 1,080 |-1,218 | 235 | 21.8 | —138°|— Tee Average. . 13.2 treme variations for a large number of his subjects who had been studied 5 days ormore. His greatest variation above this mini- mum was 31.3 per cent, his lowest 3.5 per cent, and his average 13.9 per cent. We have no explanation to offer for the wide variation, with the possible exception of the subject showing the widest range— * If the 1 exceptionally high day for Subject 3 is omitted (discussed below in connection with pulse rate), her average becomes 1,189 calories per 24 hours, her variation of the maximum above the minimum 125 calories, or 11.1 per cent, and her greatest variation from her average—64 calories, or 5.4 per cent. K. Blunt and M. Dye 75 Subject 5 with 28.8 per cent. Her average metabolism in winter (February and March) was 1,302, distinctly higher than in the summer (July) 1,145. Yet Subject 10, the only other observed both winter (early March) and summer (June), averaged the same at the two seasons (1,486 and 1,487). Another possible though not completely satisfactory reason for the differences. in Subject 5 was that she was observed very early in the morning, 6.30 or 7.00, at the time when her metabolism was running lowest, and sometimes later in the day, about 11 a. m., when it ran high. She had been fasting, of course, since the night before, and had the usual rest period, but at the later date she had been attending classes and doing laboratory work before coming for the meas- urement of her metabolism. The late figures, however, are by no means always higher. With only a very few exceptions, all observations on other subjects were made early in the morning, usually from 7.00 to 8.30. The diet of the subjects was not controlled, but it is not thought that there were any marked changes in it during the experiments. Neither were there marked changes of occupation nor of genera! health. | We wish to call special attention to the series of observations on Subject 1 (see Table VI), a young Chinese woman of excellent mentality and apparently good general health in spite of a tend- ency to amenorrhea. Her basal metabolism was observed daily for 26 almost consecutive days, without menstrual period. As far as we know, this is the longest series made on a woman, with the exception of Zuntz’s observations. She showed much day by day variation with sudden rises and falls. There are only three subjects of the total seventeen who show a wider range than hers. The wide range is also shown in two other women for whom we have a fairly long series of consecutive observations, Nos. 3 and 8. For No. 8 with 10 consecutive days, the variation is 11.6 per cent, and for No. 3 with 7 consecutive days, 11.1 per cent, or 19.6 per cent if the high observation of the previous month is included. Comparison with Normal Standards for Metabolism. The two commonly used series of ‘“‘normal’’ standards for the basal metabolism of women are Benedict’s and Du Bois’. Bene- 76 Metabolism of Women dict’s standards, published as ‘‘multiple prediction tables’ ’(9) are based on observations of 103 women made in the Nutrition Laboratory. They give the basal metabolism in calories per — 24 hours for different ages, heights, and weights. Du Bois’ (10) standards are expressed in calories per square meter per hour, and are computed from those for men on the assumption that TABLE IV. Comparison of Observed Metabolism with that Predicted by Benedict and by Dw Bois. Heat production per day. Heat production per sq. m. per hr.* Subject. an | Bene-'| Actual | 7; All |Du Bois’| Actual | 7. observa- Cee less cal- Dee, observa-| predic- | less cal- Differ- tions. Pon. culated.| ©?°°: tions. tion. |culated.| ©? calories | calories | calories | per cent | calories | calories | calories | per cent 1 1,239 | 1,412 | —173 | —12.2) 31.9 | 37.0 |-—5.1.| =i 2 1,413 | 1,466| —53| —3.6] 36.2] 37.0] —0.8| —2.2 3 1,205 | 1,305 | —100| —7.6| 34.2] 37.0| —2.8| —7.6 4 1,321 | 1,459 | —138| —9.5| 32.3| 387.0 | —4.7 | —12.7 5 1 .954'|.1.311 | —57)| —4.3| 34.7 | 37-0'| —2.3 [Se 6 1,140 | 1,309 | —169 | —12.9| 33.1] 37.0] —3.9 | —10.5 7 1,335 | 1,401} —66| —4.7| 34:2], 37.0 | —2:3'| =76 8 | 1,396 | 1,392 4 0.2) <35.7:| 37:0)):—1.30le ee 9 1,256 | 1,287 | —31| —2.3| 36.6] 37.0] —0.4| —1.1 10 | 1,493 | 1,416 77 5.5| 36.4] 36.5 | —0.1| —0.3 11 1,105 | 1,196 | —91| —7.7| 34.3] 36.5] —2.2] -—6.0 12 | 1,346 | 1,405 | —59] —4.3| 33.1| 36.5] —3.4| —9.3 13 1,580 | 1,576 4 0-1) 34.1.) 36.5 | —2940) 5 eee 14 | 1,457 | 1,513 | —56| —3.8] 33.7] 36.0| —2.3| -—6.6 15 1,457 | 1,417 40 4:2), 34.9 | (36.0 | —1,1 |) aa 16 1,331 | 1,423 | —92]. —6.7| 32.5] 36.0] —3.5| —9.7 17 1 1218) 1218 0 0.0) 34.2 | 36.0 |: —1/8 )> aes Average.. ae —6.5 * Surface area by the Du Bois height-weight chart. the basal metabolism of women is 7 per cent lower than that of men. As the height and weight variables are included in the surface area these standards of Du Bois vary only with age, ranging for our group from 37.0 calories for 20 to 30 years to 36.0 calories for 40 to 50 years. In Table IV the averages of all observations on our subjects are compared with these two standards. Two of our subjects K. Blunt and M. Dye Vi average higher than Benedict’s standards, three almost exactly the same, and twelve lower. The general average is distinctly lower—4.1 per cent. In comparison with the Du Bois standard all our subjects without exception are low, ranging from —0.3 per cent to —13.8 per cent. Two are more than 10 per cent be- low the Benedict standard and three more than 10 per cent below the Du Bois. We have no explanation of the iow metabolism of our women. Most of.them were leading fairly active lives, doing laboratory work, and some of them a limited amount of housework. They were probably not quite so active as the group of. nurses studied by Harris and Benedict (9) and Palmer, Means, and Gamble (11) who also showed a metabolism below the standard. It is inter- esting, though of course not conclusive, that a number of people who were asked which of our group they considered most mus- cular all mentioned first the two individuals whose metabolism is above Benedict’s standard. Pulse Rate. In connection with the metabolism determinations, data were collected on the pulse rate of the seventeen subjects, a total of 186 observations. They are included in Table VI and summar- ized in Table V. The counts were made during the experimental metabolism period after the subject had been lying down 35 minutes or more and may therefore be taken as minimum values. The averages for the different women range from 60 to 79. The average of these individual averages is 68.9, which is in rather surprisingly close agreement with that for Benedict’s (9) 90 women—68.67, thus confirming his conclusion that pulse rate is higher in women than in men (Benedict’s average 61.26) although basal metabolism is lower. The range of our single observations is from 54 to 100, or omitting this one extremely high case, from 54 to 88. Benedict’s range is from 51 to 92. - There is no relation between pulse rate and basal metabolism for our different women (a lack of correlation also observed by Benedict), and, more surprising, there is no relation between the daily variation in basal metabolism and pulse rate for the single in- dividual (a confirmation of Zuntz’s (4) observations). The higher 78 Metabolism of Women pulse rates: do not occur on the same days as the higher basal metabolism nor the lower pulse rates as the lower metabolism. The correlation between pulse rate and metabolism which is often spoken of is their similar increase above minimum values brought about, for instance, in exercise (12), rather than the day by day variation in minimum. We have one exception to this statement of lack of agreement—the case of Subject 3, who in one day showed the exceptional pulse rate of 100, which TABLE V. Pulse. Subject. No. of observations. Range. Average. 1 25 54- 68 62 2 12 64- 78 68 3 7 64-100 74 4 5 72- 84 79 5 22 64- 84 71 6 10 ; 56- 78 66 M 13 68- 88 79 8 10 56- 72 65 9 11 68- 80 75 10 12 64- 78 72 11 8 62- 66 65 12 5 60- 68 65 13 10 56- 80 67 14 9 60- 68 65 15 1 60 60 16 a 68- 78 72 17 19 64- 72 67 Total. . .186 Average. ..68.9 is 30 beats above her next count, and also her highest basal metabolism, which was 11 per cent above her average and 19.6 per cent above her minimum. This 1 day thus shows an asso- ciation of tachycardia and increased metabolism similar to that recently observed by Sturgis and Tompkins (13) in hyperthyroid- ism. It must be remarked, however, that even this maximum metabolism of our subject is only 3.1 per cent above Benedict’s prediction for her. It is to be regretted that the body tempera- K. Blunt and M. Dye TABLE VI. Fundamental Data. 73) Heat production. Per day. Per kilo|Pe?_84- per hr. calories 1,220 1,280 1,270 1,315 1,285 fat bo 5 bow Or Or Wwndnbee Dy y eh o Ww oO oe oO oo © or © oro ee ee ee ee ee ee ee (=) ~_ & Nae Gate sate Une nal ne ~_ ~ No nw w oe) Ou calories 0.871 0.909 0.906 0.945 0.915 0.902 0.856 0.909 0.877 0.906 0.856 0.880 0.802 0.880 0.865 0.877 0.856 0.822 0.840 0.871 0.880 0.932 0.856 calories 31.4 32.8 32.7 33.8 33.0 Subject. Date. Phase. Habe 1920 1 Jan. 27| Intermenstrual. 54 21 yrs. Feb. 3 im 60 160 cm. re 4 s 62 58.5 kg. rs 5 ¢ 62 1.62 sq. m. sf 6 a 60 cc 7 ce 58 (<4 8 “ce 58 cc 9 “ce 60 “ 10 “ 60 ot ple es 62 pan oe 70 co aos . 2s < 72 | 196 | 1,360} 0.811) 33.2 “« 24) Menstrual. 197 | 1,370] 0.814] 33.4 Se tp “ 72 | 180 | 1,250) 0.745) 30.5 a = 72 | 190 | 1,320) 0.789) 32.3 164 | 1,140} 0.975) 32.1 eal He 68 | 176 | 1,220] 1.045) 34.4 EUS. 5] ¥ 72 | 178 | 1,235) 1.057) 34.8 49.5 kg. Apr. 7| Premenstrual. 70 | 176 | 1,220} 1.028) 33.9 feoOrsgi m=: |“ 8 ‘ 180 | 1,250} 1.061| 34.7 9 x 181 | 1,260} 1.058) 34.9 eames (5) ey 60 | 171 | 1,190} 1.000) 33.0 “~~ 11} Menstrual. 64 | 178 | 1,235] 1.041] 34.4 bane) (y. ze 176 | 1,220) 1.028] 33.9 ey 13 . 176 | 1,220] 1.028} 33.9 Si) eat i: 169 | 1,175] 0.987} 32.6 “ 27) Intermenstrual. 72 | 171 | 1,190} 1.000} 33.0 aol ee i 64 | 169 | 1,175) 0.987) 32.6 1S SO i 64 | 181 | 1,260) 1.058) 34.9 May 1 * 169 | 1,175} 0.987) 32.6 Average.... 67 1,218 34.2 ture was not determined on this day. None of our other pulse rates is within the range of pathological significance. The variation of pulse rate during menstruation is almost as irregular as the basal metabolism. In three cases Subjects 2, T (first period observed), and 10 (first period observed), there is 86 Metabolism of Women a slightly higher pulse rate the first of the menstrual period than later, an observation in agreement with King (14), who found that pulse rate and temperature follow the rhythmical movement in life processes, while blood pressure, systolic and diastolic, and pulse pressure varied irregularly. In most of our subjects, however, no regular variation was observed. SUMMARY. 1. Series of 216 observations are given on the basal metabolism of 17 women, 14 of them including 1 or more menstrual cycles, and 1 being observed for 26 almost consecutive days. 2. There is no definite change in the basal metabolism during menstruation. This is seen from the facts that the average of the intermenstrual and menstrual observations is almost the same, and that no rhythmical periodic variation in metabolism can be noted. 3. The daily variation for each subject is great, ranging from 7.4 to 28.8 per cent, or an average of 13.2 per cent. This is slightly less than the average which Benedict found, 13.9 per cent, with a group of individuals observed 5 days or more. Erroneous conclusions can easily be drawn from metabolism observations unless measurements are made on more than 1 day. 4, Most of our subjects show a somewhat lower basal metab- olism than that calculated for them from the Benedict or the Du Bois standards. 5. There is no relation between minimum pulse rate and basal metabolism in our subjects, except in one case for 1 day where the pulse rate increased to an extent which may be considered pathological. Neither is there definite constant change in the pulse rate during menstruation. The authors wish to express their thanks to Dr. F. G. Benedict for reading and criticizing the manuscript of this paper, to Dr. T. M. Carpenter for helpful suggestions at the beginning of the work, and to the women who served as subjects, through whose interest and cooperation the investigation was made _ possible. oo 14. K. Blunt and M. Dye 87 BIBLIOGRAPHY. . Benedict, F. G., A portable respiration apparatus for clinical use, Boston Med. and Surg. J., 1918, elxxviii, 667. . Benedict, F. G., Notes on the use of the portable respiration appa- ratus, Boston Med. and Surg. J., 1920, elxxxii, 243. . Du Bois, D., and Du Bois, E. F., Clinical calorimetry. X. A formula to estimate the approximate surface area if height and weight be known, Arch. Int. Med., 1916, xvii, 863. . Zuntz, L., Untersuchungen iiber den Einfluss der Ovarien auf den Stoffwechsel, Arch. Gyndk., 1906, xxviii, 106. . Gephart, F. C., and Du Bois, E. F., Clinical calorimetry. XIII. The basal metabolism of normal adults with special reference to surface area, Arch. Int. Med., 1916, xvii, 902. . Snell, A. M., Ford, F., and Rowntree, L. G., Studies in basal metab- olism, J. Am. Med. Assn., 1920, Ixxv, 515. . Boothby, W. M., The fundamental classification of disease by the basal metabolic rate, J. Am. Med. Assn., 1921, Ixxvi, 84. . Benedict, F. G., Factors affecting basal metabolism, J. Biol. Chem., 1915, xx, 263. . Harris, J. A., and Benedict, F. G., A biometric study of basal met- abolism in man, Carnegie Inst. Washington, Pub. 279, 1919. . Aub, J. C., and Du Bois, E. F., The basal metabolism of old men, Arch. Int. Med., 1917, xix, 823. . Palmer, W. W., Means, J. H., and Gamble, J. L., Basal metabolism and creatinine elimination, J. Biol. Chem., 1914, xix, 239. . Benedict, F. G., and Murchhauser, H., Energy transformations during horizontal walking, Carnegie Inst. Washington, Pub. 231, 1915. . Sturgis, C. C.; and Tompkins, E. H., A study of the correlation of the basal metabolism and pulse rate in patients with hyperthyroidism, Arch. Int. Med., 1920, xxvi, 467. King, J. L., Concerning the periodic cardiovascular and temperature variations in women, Am. J. Physiol., 1914, xxxiv, 203. FAT-SOLUBLE VITAMINE. VII. THE FAT-SOLUBLE VITAMINE AND YELLOW PIGMENTATION IN ANIMAL FATS WITH SOME OBSERVATIONS ON ITS STABILITY TO SAPONIFICATION.* By H. STEENBOCK, MARIANA T. SELL, anp MARY V. BUELL. (From the Department of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, April 12, 1921.) When in the early part of 1919, we (1, 2) formulated the working hypotheses, in our fat-soluble vitamine investigation, that the vitamine might be identical with, or closely related to certain yellow pigments of the carotinoid type, we made the attempt to correlate its occurrence on this basis, to formulate a procedure for its isolation, and to collect information as to its possible chemical nature. In these objects we have been substantially aided by our hypotheses, for while all the possibilities antici- pated have not materialized yet there has been given a direction to our experimental efforts in this field, and apparently to those of others, which has lead to great centralization of effort. It was obvious that, in spite of the numerous instances of asso- ciation of the physiological growth-promoting property which is attributed to the presence of fat-soluble vitamine and yellow plant pigments, the two would not necessarily have to be iden- tical, in fact there need be no material relationship in compos- ition or structure, as their coincident occurrence in nature might be due to physiological determination, pure and simple. In this event, with the diversification of metabolic processes which obtain in the plant and animal kingdom, it was to be expected that sooner or later the fat-soluble vitamine would be found to be present in a menstruum entirely free from pigments of the carotinoid type. To run across such an instance, appears to * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 89 90 Fat-Soluble Vitamine. VII have been the good fortune of Palmer and Kempster (3), who demonstrated that pork liver, rich in the vitamine, contained no pigments of the aforementioned character. Gross of this laboratory has obtained similar results! but Rosenheim and Drummond (4) purport to have shown that while carotin and xanthophyll are absent, another pigment of this type is pres- ent. It may still be said that in such instances where the fat-soluble vitamine is found to occur in the absence of color the pigment is present in the leuco form (2). While this can be accepted as a possibility it is at present not worthy of serious consideration because nothing is known of the structure of the carotinoid molecules and therefore nothing is known as to the probability of the existence of leuco compounds in the series. Nevertheless, as far as studies in this domain have been pur- sued, both in regard to distribution of vitamine and pigment and in regard to their physical and chemical properties, there is left no doubt but that chemically and physiologically they are related. These relations will be discussed in succeeding papers in which our results will be correlated with those of others who have been active in this field of investigation. In this paper it is desired to present data on the fat-soluble vitamine content of various animal fats as correlated with yellow pigmentation, and to record a few observations made on its stability to sapon- ification. EXPERIMENTAL. For the laboratory technique employed in these experiments the reader is referred to previous papers of this series (5-10), as the experimental methods employed were essentially the same. In no instance has it been deemed permissible to make any selection of data. Four animals, usually two males and two females, were started in each group and continued until death or until the differences among the groups became so pronounced as to make further continuation superfluous. In a few instances where data were purely confirmatory we have felt at liberty to to present merely representative illustrations. Failure of growth alone has not been accepted by us as.a good criterion to use in establishing the lack of the fat-soluble vitamine ‘ Unpublished data. H. Steenbock, M. T. Sell, and M. V. Buell 91 and this for very obvious reasons. Emphasis was, therefore, always placed upon the appearance of the eyes of the rats, as an inflamed condition, an ophthalmia variously termed a con- junctivitis, a xerophthalmia, and a keratomalacia, makes its appearance in the vast majority of animals on a fat-soluble vita- mine-poor diet as first pointed out by Osborne and Mendel (11). Though the exact condition which is incident when the eyes become infected is not described accurately by these terms we have used them rather indiscriminately in the past hoping that pathologists would soon describe the condition in detail and designate it accurately.2, Stephenson and Clark (12) introduced the term keratomalacia in preference to xerophthalmia, appar- ently because in these inflammatory reactions a softening of the cornea with loss of the lens frequently results. In a certain sense each of these terms has its proper application in the various types or stages of the ophthalmitis observed. Generally a con- junctivitis with an erythema and edema of the eyelids appears first, later a keratitis or inflammation of the cornea results, and ultimately, if the diet is not corrected or if resistance to the infection does not develop of its own accord especially in se- verely purulent inflammatory reactions, a dryness or xerosis of the eye—a xerophthalmia—marks the culmination of the eye symptoms. As usually observed and especially in the primary stages the condition is marked by an excessive secretion and is certainly not a xerophthalmia. We believe that, in most instances when the inflammation is not confined to the con- junctiva, the infection is most accurately designated as a ker- atoconjunctivitis. Only occasionally will we have reason to speak of a xerophthalmia because true xerophthalmia has rarely made its appearance in our colony. When the inflammatory reaction is so severe as to cause permanent injury and the diet is not corrected, death results rapidly; if it is transitory, making its appearance now and then—indicative of only a partial de- ficiency of the fat-soluble vitamine—then practically complete recovery has always been observed. The recovery cannot be considered entirely complete because an inflamed condition of 2 Such studies have recently been published by Wason (Wason, I. M., J. Am. Med. Assn., 1921, lxxvi, 908), since the preparation of this manu- script. 92 Fat-Soluble Vitamine. VII the eyes, beyond a mere infiltration of fluid—an edema of the con— junctiva and eyelids—leaves its permanent mark in the destruc- tion of the hair follicles on the lids. This “bare eyed’’ con- dition always arouses our suspicions of a fat-soluble vitamine deficiency, suggesting that a transitory reaction has escaped. our notice if the inflammation was not actually observed. In addition to the inflammation of the eyes, we have observed in many animals, large and small, an apparent resistance to infec- tion of the eyes even though continually exposed by contact to severely infected animals, all on a fat-soluble vitamine-free ration. In such individuals an enophthalmia or a “‘small eyed condition” is frequently observed. The eyeballs are not ‘‘beaded’’ as in normal rats but appear small and sunken in the orbital cavity. Many of these individuals succumb to respiratory infections, in fact we have been led to think that possibly a certain immunity to infection of the eyes is thus conferred. The incidence of respiratory infections as part of the syndrome induced by fat-soluble vitamine deficiency was described by McCollum (13) in his early work. It may consist, as we have observed, of a nasal or bronchial catarrh or even pulmonary infection with mucous or purulent exudate, at times even result- ingin hemorrhage. Animals thus afflicted in the early stages of the disease sneeze and cough violently but later as the inflammation becomes confined more to the lungs the cough subsides and dyspnea becomes very pronounced with the slightest activity. Such animals fail very rapidly and even with the introduction of fat-soluble vitamine in the ration rarely show normal growth subsequently. A fat-soluble vitamine deficiency is also far from being con- ducive to normal cutaneous nutrition so that very often— especially after an age of 4 months has been reached—evidence of dermal malnutrition makes its appearance. The fur appears bushy and thin, cutaneous growths occur on the tail, ears, and nose, and finally sores, which heal with difficulty, appear on the feet, limbs, and body; all bear testimony to this state of mal- nutrition. All of the aforementioned symptoms and conditions have been: carefully watched and noted at the time of the weekly weigh- ings of our animals unless their condition or the nature of the H. Steenbock, M. T. Sell, and M. V. Buell 93 experiment made examinations at shorter time intervals of mo- ment. Judgment was based on the sum total of indications. Fat-Soluble Vitamine Content of Cod Liver Oil. Since Osborne and Mendel (14) in their pioneering studies on the fat-soluble vitamine found cod liver oil to contain this dietary essential surprisingly little experimental endeavor has been made to study it in its quantitative relations though repeated emphasis has been placed on the efficacy with which it can be used as a therapeutic agent in perverted metabolism of the osteoid tissues. As far as known to the writers, Zilva and Miura (15) are the only investigators who have studied this problem. They have recently published a preliminary note on their ex- periments stating that cod liver oil in its crude state was found to be 250 times as potent as butter in furnishing this constitu- ent. Our investigations were not outlined to bring out such extreme differences as we were primarily intent on a correlation of the fat-soluble vitamine with pigment content in comparison with the intense pigmentation of butter, and selected cod liver oil because we had at hand an excellent sample which in comparison with butter was practically devoid of yellow pigments—it had only a faint yellowish green color. If the vitamine were a yellow pigment it should therefore have shown only limited activity. The sample of oil was prepared from fresh cod livers with min- imum heat exposure. The livers were cut into small pieces, put into a steam-jacketed open cooker, and heated not to ex- ceed 180° F. The oil collecting on the surface was skimmed off as rapidly as it formed in the first 35 minutes of heating, filtered through paper, and bottled. As seen in Chart 1, this oil when fed at a level corresponding to 2 and 5 gm. of material in a kilo of ration was exceedingly active; it was far more efficient than any sample of butter fat that we have ever studied. This alone was one of the things which made us very skeptical in the early course of our studies of the assumption that the fat-soluble vitamine was neces- sarily a yellow pigment. Q4 Fat-Soluble Vitamine. VII Seasonal Variation in the Fat-Soluble Vitamine Content of Butter. Much has been written and said about the fat-soluble vitamine content of butter fat primarily because of its extensive use as an article of food and therefore its probable importance as a source of the fat-soluble vitamine in the human dietary. Due to the comparatively low intake of butter fat in the diet of the adult, the destruction of its vitamine in some cooking processes, the general occurrence of the vitamine in many foods such as green plant tissue, certain seeds and even roots, and the unde- termined requirements of man, it is questionable whether its vitamine content would now attract the attention that it does if it were not for the fact that butter fat was the material in which the vitamine was first discovered. Early in our studies we were impressed with the fact that butters varied decidedly in their vitamine content; most of them being very rich in this dietary constituent, but some being as poor as the average oleomarg- arine (5). Certain observations which we made also impressed us with the lability of the vitamine as we found it destroyed in heated butters and in butters kept under poor storage conditions (5), so that we did not feel at liberty to draw conclusions with respect to the primary causal factors involved. As our studies on the distribution of the fat-soluble vitamine progressed and indications of the occurrence of the vitamine with vellow pigments were obtained it appeared profitable to attempt to correlate these relations in butter fat especially in view of the fact, as is well known in dairy practice, that butter churned in late winter or early spring under Wisconsin condi- tions without artificial coloring is practically void of all color. If the vitamine content should be demonstrated to be of the same order of magnitude as the pigmentation then further presump- tive evidence of this relation would have been obtained. To this end there were prepared samples of butter fat in the latter part of the months of March, April, May, and June from cream obtained at the University creamery which was repre- sentative of the composite collection from a large number of dairy farms in the vicinity. The butter was churned in the laboratory, melted, and filtered at a low temperature, and then stored in a refrigerator till used in the experiments. .The butter H. Steenbock, M. T. Sell, and M. V. Buell 95 fat was fed in basal rations which have been repeatedly demon- strated to be very low in the fat-soluble vitamine content, though controls were never omitted. Originally, through the March series of experiments, the basal ration was one which we have used before, consisting of: casein, 18; agar, 2; Salts 32, 4; ether- extracted wheat embryo, 6; and dextrin, 70. In the April, May, and June series a white corn ration, consisting of: white corn, 40; casein, 14; Salts 32, 3; Salts 35, 1; and dextrin, 42; was used. This latter is an excellent ration and guarantees a sufficiency of the water-soluble vitamine being introduced with the white corn which is not always the case when a_ variable commercial product such as wheat embryo is used as its source. The butter fats of unknown value were introduced in these rations at the expense of so much dextrin. As illustrated in Charts 2, 3, and 4 there occurs a decided variation in the vitamine content of the different butters; but even gross inspection of the monthly collections made it evident to us that the variations in vitamine were not quantitatively reconcilable with the variations in pigment. To enable more accurate comparisons to be made we availed ourselves of the use of a standard color solution in a Duboseq colorimeter, which was prepared by dissolving 7 gm. of KeCrO, and 0.074 gm. of KxCreO07 in water and making it up to 100 cc. volume. Such a solution compares favorably with the color of June butter fat but as the intensity of pigmentation is reduced, as is the result in the winter butter fat, the effect of a residual yellowish green pigmentation becomes disturbing and comparisons are not so easily made. Nevertheless, the accuracy of the determinations exceeded by far the requirements of our work as the results of the feeding trials themselves cannot be evaluated with any great degree of accuracy. With June butter fat accepted as having a value of 100, May butter fat was found to have a value of 86, and March and April butter fats, a value of 2.8. The latter were therefore practically colorless. With these factors in mind, upon inspection of the growth curves, it becomes increasingly evident that the fat-soluble vitamine content of the butter fats does not run parallel to the intensity of pigmentation; otherwise in the first place May and June butter fats fed at the 0.5 per cent levels should have been far more potent than they actually were 96 Fat-Soluble Vitamine. VII in comparison with the March or April butter fats as they carried from 30 to 35 times as much pigment. In the second place when fed at different levels, 2 per cent for the March and April butter fats as compared with 0.5 per cent for the May and June butter fats, the former should not have exceeded the latter in efficiency as even then only from one-eighth to one-ninth as much total pigment had been introduced into the ration. These findings harmonize with those of Drummond and Coward (16) who arrived at similar conclusions. . Before the facts of these relations were obtained many attempts were made in the summer of 1919 to ascertain if any parallelism between vitamine and pigment content obtained by taking advantage of the fact that the carotin in butter fat is easily destroyed by heating. We heated butter fat in deep and in shal- low dishes in the presence and absence of oleic acid—as acids accelerate pigment destruction in butter very markedly—with and without aeration with hydrogen, carbon dioxide, and air. We expected that if vitamine and pigment were not identical under some of these conditions destruction of the one without destruc- tion of the other might be found to occur. Our results were entirely unsatisfactory as consistent duplication of results on different samples could not be obtained. As the selection of data bearing out a particular point at issue is not justifiable when unexplainable contradictory evidence is also obtained, the results of this work were not published. They served to .con- vince us, however, that the success of such experimental attempts depended largely upon good fortune as butter fat is too vari- able in fat-soluble vitamine content to be taken as a good source of vitamine for studies of this character. Since these experiments were carried out, Stephenson (17) has submitted data which tend to show that charcoal can be successfully employed in the removal or destruction of the pig- ment without causing complete destruction of the vitamine. Un- fortunately her experimental period is shorter than desirable, especially as she worked with animals of considerable size in which we have found under normal conditions the vitamine reserve to be high and continued normal growth for 8 weeks to be common. The sudden death of one individual is not re- assuring as pulmonary infections carry off some individuals on H. Steenbock, M. T. Sell, and M. V. Buell 97 a fat-soluble vitamine-poor diet without premonitory symptoms in the course of a few days; nevertheless her data are very sug- gestive especially in view of Palmer’s (3) observations on the feeding of pork liver. Fat-Soluble Vitamine in Beet Fats. Osborne and Mendel (18) and later Halliburton and Drummond (19) showed that beef fats might contain considerable amounts of the fat-soluble vitamine though in general their efficiency in furnishing this dietary constituent was not to be compared with butter fat. By fractionally crystallizing the beef fats from alco- hol, Osborne and Mendel obtained a very active fraction. The beef oils were found to be exceedingly active while the solid residue was inactive. In our work a somewhat similar product, the oleo oils from beef fats, prepared in commerce for the manu- facture of oleomargarine, were in some instances found richer in the fat-soluble vitamine than many butters (5). Subsequent to the publication of these results we became aware of the fact that the vitamine content as determined in our feeding experi- ments with these samples seemed to vary directly with the in- tensity of pigmentation. This led to the collection of additional data to determine if this was a mere coincidence or if it was com- monly true. During 1919 the experiments were confined to the investi- gation of the perinephric fat of animals of the Jersey, Durham, and Holstein breeds. The fatty tissue was ground in a meat hasher and extracted by heating slightly above the melting point in a steam oven and then straining and decanting the melted fats. ‘They were preserved in Mason jars in a refrigerator until utilized in the experiments. As seen in Chart 5, the Jersey fat was very active while the Durham fat gave no evidence of containing this vitamine. The same inactivity was shown by the Holstein fat. Both the Hol- stein and Durham fats were practically colorless; the Jersey fat, on the other hand was fully as pigmented as a sample of June butter. In 1920 we duplicated these experiments except for the fact that the samples were not taken from any particular breeds 98 Fat-Soluble Vitamine. VII but were selected promiscuously from slaughtered animals for color intensity. The dark beef fat was fully equal in color to June butter, the medium beef fat was two-thirds as colored and the light beef fat only one-tenth as colored. These values were obtained by measurement in a Duboseq colorimeter. The re- sults shown in Charts 6 and 7 are essentially of the same char- acter as those obtained the year before—the fat-soluble vitamine content roughly parallels the pigmentation. In view of the re- sults that we have obtained with butter fat, it is not to be con- cluded that this is necessarily always the case. The rapidity of fat deposition, its mobilization, and the variation in the assim- ilation of pigment with different breeds and individuals, no doubt all operate to modify the primary determinative effect of the composition of the ration. Just how the latter may influence the relations we have again had occasion to observe with the fat- soluble vitamine content of egg yolks.!. Normally, on ordinary rations light-colored yolks are low in the fat-soluble vitamine; yet by the selection of a special and unusual ration we have succeeded in producing light-colored yolks of normal vitamine content. Stability of Fat-Soluble Vitamine to Saponification. The study of the characteristics of the fat-soluble vitamine has presented considerable difficulties particularly due to mis- taken notions of its stability and solubility properties which were fostered by suggestions rather than conclusive evidence as presented by various investigators. McCollum and Davis (20) reported the transference of the fat-soluble vitamine from butter fat into olive oil after the butter fat had been submitted to a mild saponification at room temperature. This was sub- mitted as a preliminary paper in 1914 as it was stated that other experiments were under way and would be reported as soon as advisable. In the experiments detailed by them a number of difficulties can be appreciated which have made duplication very difficult as no confirmation of these attempts has been pub- lished. In the first place the drying of the soaps and the dis- sipation of the ether vapors from the ether-olive oil extract are processes not easily carried out under laboratory conditions with- out causing the destruction of considerable amounts of the vit- H. Steenbock, M. T. Sell, and M. V. Buell 99 amine. In the second place the olive oil extract as fed at a 3 per cent level and, therefore, equivalent to 6 per cent butter fat, did not leave a sufficient margin of fat-soluble vitamine to guar- antee its presence at the close of operations as many samples of butter fat without having been subjected to any treatment are ineffective when fed at this'level. In the experiments men- tioned, however, it is possible that the 20 per cent lactose (21) carried considerable vitamine so that but a small increment was needed to elicit a growth response. Nevertheless, all these facts made it appear very unprofitable to attempt to repeat these experiments especially as the vitamine was ultimately brought into the solution of a fat with no determination of the completeness of the saponification beyond an inspection of the solubility of the reaction mixture which in the presence of so much soap is far from satisfactory. These experiments are there- fore to be considered merely as a demonstration of the resistance of the fat-soluble vitamine to the mild saponification employed. In the light of this it was not surprising that Drummond (22) failed in demonstrating the resistance of the vitamine to the mild saponification of Henriques used by McCollum. He varied his procedure in that he attempted an ether extraction of the soaps but failed to show any activity of either the extract or an ether extract of the saponified residue when he fed the equivalent of 15 and 20 per cent of butter fat and whale oil, respectively. From our work on the extraction of the vitamine from plant materials, where we adopted the method of separation in use for the carotinoids, we have demonstrated repeatedly the re- sistance of the vitamine to saponification and its subsequent extractibility by ether (10). From our present work (Chart 8), it is evident that the fat-soluble vitamine as found in animal fats has similar properties. In two instances, Lots 741 and 969, the saponification was conducted at 37°C. for 4 hours; 300 gm. of the fat being treated with 600 cc. of 20 per cent alco- holic potash, which are the proportions of fat and alkali used in the methods of analysis of The Association of Official Agri- cultural Chemists (23). At the end of the 4 hour period 2,400 ec. of water were added and the aqueous alcoholic solution of soaps extracted three times with ether. The ether extracts were washed with a small volume of water and then evaporated 100 Fat-Soluble Vitamine. VII directly at room temperature in an air current on the ration. For control purposes a saponification was carried out with butter fat parallel to one run by the Official Methods using the same con- centration and excess of alkali, acting for the same period of time at the same temperature as the preparation. They gave the same saponification value indicating that the saponification in our butter fat preparation was complete. Nevertheless, we made another preparation in which the fat was boiled with 20 per cent alcoholic potash for one-half hour as required by the Official Methods but with no reduction in alkali concentration. The growth curves of Lot 972 bear testimony to the fact that even under these drastic conditions the fat-soluble vitamine was not destroyed to any appreciable extent. From this it can be con- cluded that it is not a fat or an ester and that it is not labile to heat in the presence of a high concentration of alkali. We desire to express our appreciation to Lord Brothers who furnished the cod liver oil and Armour and Company who fur- nished us with the beef fats. SUMMARY. In cod liver oil there is present a very high concentration of the fat-soluble vitamine with but small amounts of yellow pig-. ments. Butter fat shows a seasonal variation in the fat-soluble vita- mine content when obtained from stall fed cows during the win- ter and pastured in the summer as is the practice under Wis- consin conditions. The fat-soluble vitamine content of butter fat does not run closely parallel to the yellow pigment; yet in general, due to de- termination by their content in the feed, butters highly pig- mented are rich in the vitamine; butters low in pigment should be looked upon with suspicion. In beef fats the relations are somewhat similar; those most pigmented are also generally richest in their fat-soluble vitamine content. . The fat-soluble vitamine withstands severe methods of saponi- fication. This indicates that it is not a fat and probably H. Steenbock, M. T. Sell, and M. V. Buell 101 not an ester and makes possible the compounding of satisfac- tory fat-free synthetic rations for investigative purposes. ry Ot He CO LO BIBLIOGRAPHY. . Steenbock, H., Boutwell, P. W., and Kent, H. E., J. Biol. Chem., 1920, ii. p. Xl: . Steenkock, H., Science, 1919, 1, 353. . Palmer, L. 8., and Kempster, H. L., J. Biol. Chem., 1919, xxxix, 299. . Rosenheim, O., and Drummond, J. C., Lancet, 1920, i, 862. . Steenbock, H., Boutwell, P. W., and Kent, H. E., J. Biol. Chem., 1918, xxxv, 517. . Steenbock, H., and Gross, E. G., J. Biol. Chem., 1919, xl, 501. . Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xli, 81. . Steenbock, H., and Gross, E. G., J. Biol. Chem., 1920, xli, 149. . Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xli, 163. . Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xlii, 131. . Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1913-14, xvi, 481. . Stephenson, M., and Clark, A. B., Biochem. J., 1920, xiv, 502. - McCollum, E. V., J. Am. Med. Assn., 1917, 1xviii, 1379. . Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1914, xvii, 401. . Zilva, S. S., and Miura, M., Lancet, 1921, i, 323. . Drummond, J. C., and Coward, K. H., Biochem. J., 1920, xiv, 668. . Stephenson, M., Biochem. J., 1920, xiv, 715. . Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1915, xx, 379. . Halliburton, W. D., and Drummond, J. C., J. Physiol., 1917, li, 235. . McCollum, E. V., and Davis, M., J. Biol. Chem., 1914, xix, 245. . McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xxiii, 231. . Drummond, J. C., Biochem. J., 1919, xiii, 81. . Bull. 107, Bureau of Chemistry, United States Dept. Agric. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 102 Fat-Soluble Vitamine. VII THE FAT-SOLUBLE VITAMINE IN COD LIVER OIL ‘4 Gn. Et NVEE eC AR ACCRA Peet Sh N = Hi mre ~ ie a a Bad. Vain A ae tft | a alee wee Av R. aah e i e ae a Lz See ie BN NI ae Hh ae @ w oP oO ct el ty | 1S Lot 4072 Cod liver oi WE SHE REM HF Cuarr 1, Lots 1072 and 1073 illustrate the remarkable growth-promot- ing property of small amounts of cod liver oil as a source of the fat-soluble vitamine. Rats 4285 and 4281 both continued their phenomenal rate of growth beyond the curves of growth shown here, the former weighing 395 . gm. and the latter, 422 gm. 6 weeks later. Rat 4282 raised three young out of a litter of twelve in 5 weeks to an average weight of 40 gm. None of the other litters was raised. White Indian corn at a 40 per cent intake level was used as the source of water-soluble vitamine as it has been shown in numerous experiments to lead to nutritive failure as a source of the fat- soluble vitamine and yet it furnishes plenty of the water-soluble vitamine iy for normal growth. AT—SOLUBLE VITAMINE IN 0,5 PER CENT BUTTER FAT 80 40 120 Cuart 2. This chart shows the variable effects in growth responses when an attempt is made to introduce the fat-soluble vitamine into the ration by the substitution of 0.5 per cent of butter fat collected in succes- sive months for 0.5 per cent of dextrin. On the March collection in Lot 982 by the pronounced failure of growth and even maintenance in all the animals, by the dermal infections of Rats 3921, 3922, and 3923 and by the ophthalmia in Rats 3922, 3923, and 3924, there is left no doubt that a de- ficiency of the fat-soluble vitamine obtained. On the April collection, Lot 983, growth was considerably better but all the animals were afflicted with keratoconjunctivitis. On the May butter fat, Lot 985, growth was continuous but dermal nutrition was poor;no eye symptoms were observed. On June butter fat, Lot 1032, the experimental results were practically the same except that dermal malnutrition was not evident, and Rat 4122 con- tracted a prolonged bronchial infection. Young were not reared. 103 ie es i CIACN igi Sanu bil a wat Bh miele: | | Vy, Cuart 3. When the butter fat was increased to 1 per cent of the ration, growth was in all cases much improved with marked evidence of inferiority of the March product as fed in Lot 890. All of the rats contracted kerato- conjunctivitis before their death and Rat 3553 by its sneezing and coughing indicated the presence of a respiratory infection. The April sample, although not up to par, was evidently richer in fat-soluble vitamine con- tent, as the growth performance of the animals was much better and no indisputable symptoms of conjunctivitis were observed. There were, however, some indications of cutaneous malnutrition as the tails of the animals bore some infections towards the close of the experimental period. For May and June, Lots 986 and 1033, no special comments as to the nor- mality of growth appear to be called for. On the May product Rat 3940 even raised a litter of four without apparent difficulty. 104 FAT-SOLUBLE VITAMINE IN 2 PER CENT BUTTER FAT CS Saar say Saya Naa ea Lt tise fet et tae Dena Seca Ae Ll) | Tbh Co ag na eeec am Seal 9aa eee TOE AE ee ek Cuart 4. With the butter fat increased to 2 per cent of the ration deficiencies in the nutritive value of the collections, as far as evident were entirely eliminated, if we except the fact that Rat 3559 failed due to a localized caseous pulmonary infection. All other individuals remained normal, 105 Pete eked {fT Ration of|dext¥in djsplaged b Se se at mi dl we fe He Pa par djeplad ‘a LA Me i 2 fo} te ° md et f+] Ty 2 B38 x tas rm) | Ww vo ae’ ee El ae, POS Yee a sevagraccenne Cuart 5. This chart illustrates the growth performance of rats on the perinephric fats of cattle as a source of the fat-soluble vitamine. In Lot 902, Rats 3602 and 3604 were afflicted with keratoconjunctivitis, the eyes of the others being normal when the change to Ration B carrying the Jersey fat wasmade. Except for Rat 3603 the condition of the animals was poor so that the response to the improvement of the diet was probably not of the order of magnitude to be expected. This is suggested by the results shown by Lot 947. Lot 905. When the rats of this lot were changed to the fat-containing ration, all of the rats were affected with ophthalmia. The eyes of only Rat 3617 showed improvement subsequent to the change before death ultimately supervened. This was the only suggestion of the possible pres- ence of the fat-soluble vitamine; possible because improvement of eyes is sometimes though not generally observed without any change having been made in the diet, yet growth in such cases is not restored and death slowly results. Lot 947 when started out on a ration containing ten parts of Durham fat, such as was substituted in the ration of Lot 905, did not enable the rats to grow any longer or better than when the fat was not included. All of the rats showed inflamed eye conditions which promptly subsided shortly after the change to Jersey fat was made. This was true even in the case of Rat 3784 which however, due to its impoverished condition, died shortly thereafter. 106 YELLOW PIGMENTS AND THE FAT-SOLUBLE VITAMINE IN BEEF FATS Cuart 6. This chart shows the growth observed on the 1920 beef fat samples when the animals were given the fat addition from the beginning of the experiment. In Lot 1052, on the light beef fat samples, Rat 4201 devel- oped pulmonary infections and Rats 4203 and 4204, keratoconjunctivitis. The appearance of Rat 4202 alone remained fairly normal but rapid failure ensued after parturition which is very often observed on a fat-soluble vitamine-poor ration. In Lot 1053 on the medium beef fat Rat 4205 con- tracted conjunctivitis after 16 weeks on the ration, later by the 20th week its eyes turned purulent with complete recovery by the 26th week, but by that time indications of dermal malnutrition were very distinct; it showed loss of hair, localized infections on body, and a horny epithelial growth on itsnose. Rats 4208 and 4207 gave indications of dermal malnutrition only. These conditions in general suggest a deficiency in the fat-soluble vitamine content even though growth was fairly good. In Lot 1054 on the dark beef fat all the animals maintained themselves in good condition to the end of the experimental period. 107 Ganon om i. ers | | | || meen tested as, | part Whit@ cor 40 d Bpla ase 14 c/s a Salt wae rie | Fate aapam@ebe cbse: / / V nach 4 4068 2406 eeks Cuart 7. Chart 7 illustrates the relative efficiency of the same beef fats illustrated in Chart 6 as determined by their ability to induce recovery in rats which had given indisputable evidence of a fat-soluble vitamine deficiency in the ration. In Lot 992, Rats 3961 and 3963 had edematous eyes, respectively, at the end of the 6th and 7th weeks on the white corn ration. In the ease of the former, temporary improvement was noted from time to time; in the latter, permanent improvement extended over a period of 6 weeks, but in neither case was growth resumed. This illustrates, what we have often observed, that in the vast majority of cases less of the vitamine is required to main- tain normal eye conditions than to maintain growth. In Rats 3962 and 3964 the eyes were inflamed severely at the time of discontinuation of the trial. In Lot 993, on the medium beef fat, Rat 3967 indicated incipient inflam- mation with slight edema of the conjunctiva which was promptly cured upon change of ration. Rats 3966 and 3968 also showed some indications of an edema; in the former it persisted in spite of change of ration. On the dark beef fat in Lot 1017 previous to the change all rats except tat 4064 showed a severe ophthalmitis. Rat 4064 showed an enophthalmia. In all cases where the change of ration was made improvement was prompt and recovery complete. 108 H. Steenbock, M. T. Sell, and M. V. Buell 109 STABILITY OF THE FAT-SOLUBLE VITAMINE TO SAPONIFICATION eS a -sappnifipble resid oil j Case emuryo “put er fat = 7 Lot 972 Ratio Non-sappnifib Bie esidh OU itle conn SE et eee te Salts 35 Re, Dextirin LE Cuart8. This chart shows the resistance of the fat-soluble vitamine to destruction by saponification of the fats in cod liver oil and butter fat as indicated by the prompt recovery of growth in the rats when an ether ex- tract of a solution of the soaps was added to the basal rations. This consisted, in Lot 741, of a ration which had served in an experimental series to determine the fat-soluble vitamine content of butter fat. It is note- worthy that 0.7 per cent of this sample of July butter fat furnished no appreciable amounts of the vitamine. In Lots 969 and 972 our usual white corn ration was used. Inno case, previous or subsequent to the change of ration, was any abnormality of eye conditions observed. SUPPLEMENTARY PROTEIN VALUES IN FOODS. I. THE NUTRITIVE PROPERTIES OF ANIMAL TISSUES. By E. V. McCOLLUM, NINA SIMMONDS, ann H. T. PARSONS. (From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.) (Received for publication, March 21, 1921.) In 1915 McCollum and Davis! described a systematic*procedure for evaluating each of the several essential factors in foodstuffs. This procedure involves the feeding of the food under consideration as the sole source of nutriment to one group of animals, and to an- other the same food supplemented with single and multiple addi- tions of purified foodstuffs in every possible combination. Such additions include protein, inorganic salts, a source of fat-soluble A, water-soluble B, and as was later pointed out by Chick and her coworkers,” Cohen and Mendel,’ and others, water-soluble C. The latter factor is not essential in the diet of the rat. This procedure constitutes a biological method for the analysis of a foodstuff, and has been adopted by several students of nutrition. It has yielded results which have profoundly changed our basis of judgment as to the quality of a diet. Studies from several laboratories have established the general landmarks which enable us to appreciate the lines of procedure which must be followed if satisfactory diets are to be made up by combining the various types of animal and vegetable foods. In order to make such combinations of foodstuffs it is necessary that we should understand in detail the special qualities of each of the important natural foods. Such an understanding can be secured only through carefully planned experiments on animals in which 1 McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xxiii, 181. 2 Chick, H., and Hume, E. M., Tr. Soc. Trop. Med. and Hyg., 1917, x, 141. Chick, H., Hume, E. M., and Skelton, R. F., Biochem. J., 1918, xii, 131. 3 Cohen, B., and Mendel, L. B., J. Biol. Chem., 1918, xxxv, 425. 111 112 Protein Values in Foods. I each food is studied as the sole source of nutriment, then studied in combination with each of the other foods with which it may be used in practice. It is necessary to proceed from the simple to the complex mixtures in these studies. Ultimately it is hoped that diets can be planned which will promote the optimum of physiological well being, and therefore lead to the optimum in physical development, length of life, and the preservation of youth- ful characteristics. In publishing this series of papers dealing with the studies of the dietary properties of several types of food mixtures, several new observations will be pointed out. The interpretation of the results is based upon more careful and thorough observations than have hitherto been described in any similar studies. They include not only the rate and extent of growth, the fertility, and success in rearing of young, but also the period of life up to and including the onset of old age with its characteristic changes. Since animal tissues have a very prominent place in the diet of man in most parts of the world, it is of great moment to under- stand the value of these with respect to each of the essential dietary factors. Our knowledge of the nutritive qualities of animal tissues is still very incomplete. Watson and Hunter? showed that rats fed exclusively on muscle meats suffered severe malnutrition. Liver has, however, found great favor as a food for young fish in hatcheries. It has been shown? that lard does not contain appre- ciable amounts of fat-soluble A, whereas fats extracted from a glandular organ (pig kidney or cod testicle) are a good source of it. Liver and kidney have been shown’ to be a good source of both fat-soluble A and water-soluble B, whereas muscle tissue is very poor in both. Heart, a variety of muscle, was found on the other hand to con- tain sufficient of both fat-soluble A and water-soluble B to support growth for a time at least in young rats.7 ‘ Watson, C., and Hunter, A., J. Physiol., 1906, xxxiv, 111. ® McCollum, E. V., and Davis, M., J. Biol. Chem., 1913, xv, 167. Os- borne, T. B., and Mendel, L. B., J. Biol. Chem., 1913-14, xvi, 423. * McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xx, 641. 7 McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xxi, 179. Os- borne, T. B., and Mendel, L. B., J. Biol. Chem., 1917, xxxii, 309; 1918, Xxxiv, 17. McCollum, Simmonds, and Parsons 113 McCollum® pointed out that the mineral content of animal tissues such as muscle and glandular organs resembles that of seeds of plants sufficiently to indicate that it would not prove satisfactory as a source of inorganic elements in animal nutrition. No data exist showing the comparative values of the proteins of the several kinds of animal tissues. Osborne and Mendel’ have shown that growth takes place in young rats restricted to 18 per cent of protein derived solely from liver, kidney, muscle, or brain. This is a liberal amount, and is far above the plane of intake neces- sary for the support of normal growth in the rat when the quality of the protein is good. There is also reason to inquire into the possibility of the presence of toxic substances in the glandular organs. The presence of such powerful physiological stimulants as thyroxin and adrenalin in the thyroid and suprarenal glands, respectively, makes them unfit for human or animal food. We have been informed that the Eskimos do not eat the liver of the reindeer. This fact may per- haps be accounted for by the absence of a gall bladder in this spe- cies, and consequent high content of bile, which would give it a bitter flavor. The Eskimos are said not to eat the liver of the polar bear, to avoid that of a certain species of seal, and to believe the liver of the dog poisonous. The liver is concerned with so many types of transformations of organic substances which have their origin in metabolism that it seemed possible that certain of these may be present in sufficient amounts to be detrimental to one who eats freely of it. Similar consideration might lead one to inquire whether the kidney of an animal may contain sufficient amounts of certain metabolic products as to render it undesirable as a food. The glandular organs are rich in cell nuclei and consequently yield considerable amounts of purines when metabolized. These ultimately are converted in great measure into uric acid. There are conditions of perverted metabolism in man in which the excre- tion of uric acid and urates is interfered with and doubtless under such circumstances liver or kidney should not be eaten. There would seem little reason, however, why these organs should not be eaten by healthy persons as adjuvants to the diet. They possess 8 McCollum, E. V., The newer knowledge of nutrition, New York, 1918. 114 Protein Values in Foods. I dietary properties as distinct from those of muscle tissue as the leaves of plants do in contrast to the seeds. ; In regions such as Labrador and Newfoundland where the diet of the habitants consists essentially of wheat flour, molasses, fish, meats, tea, and raisins, beri-beri and scurvy are common. A condition popularly called night-blindness is also of frequent occur- rence among these people. On such a diet one would suspect the danger of developing xerophthalmia and apparently this is the ease. The successful treatment of night-blindness has recently been reported by the administration of cod liver oil. This oil is a good source of fat-soluble A and is very effective in the cure of xerophthalmia. Fresh liver is rich in fat soluble-A, water-soluble B, and water- soluble C, the protective dietary factors for ophthalmai, beri-beri, and scurvy, respectively. It should be easily possible to eradicate these dietary diseases in such regions as Labrador and Newfound- land by the use of fish livers as food. It is strange indeed that the natives of these regions have failed to discover the value of a . by-product of their fishing industry which would serve in a great measure to correct the conspicuous faults in their diet. For the purpose of comparing the biological values of the pro- teins of kidney, liver, and muscle, we have followed the procedure described by McCollum, Simmonds, and Parsons? of feeding the tissues singly as the sole sources of protein at planes so as to intro- duce 9 per cent of protein into the food mixture. They were sup- plemented with respect to all other factors so as to make a satis- factory diet, with the possible exception of the protein moiety. Lots 2475, 2476, and 2474, Chart 1, represent experiments of this type. Such experiments make it possible to compare the proteins of these animal tissues with those of a number of combinations of cereal and other seed proteins with milk which we have previ- ously studied.!° It has been shown that normal growth is secured when the diet contains 9 per cent of protein of high biological value. If much less than this content of protein is furnished by the food mixture, even when the protein is excellent, the growth falls distinctly below the curve of normal expectation. * McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxvii, 155. 10 McCollum, E. V., and Davis, M., J. Biol. Chem., 1915, xx, 415. McCollum, Simmonds, and Parsons 115 When proteins of several types such as those of cereals, legume seeds, and milk were compared in this manner it was found that the proteins of milk were superior to those of most seeds of plants yet examined. The seed which furnishes proteins nearest to milk in value for conversion into body proteins during growth is wheat, which therefore stands first among the cereals in value. From the records of Chart 1, which show the effects of feeding kidney, liver, and muscle proteins respectively at 9 per cent of the food mixture, it is apparent that the proteins of these substances are scarcely superior to those of certain cereals, especially wheat. We base this conclusion on the growth of young rats restricted to one of these sources of protein at the critical 9 per cent level. Normal growth is not secured with wheat proteins fed below this plane of intake. This result is very surprising indeed. It seems best explainable- on the assumption that the patterns on which the proteins of the muscle are constructed differ very decidedly from that of the liver or kidney, and presumably from the glandular organs as a class. The nutritive needs of the body involve the replacement of tissues of both organ and muscle types. Apparently glandular organs or muscle as the sole source of nitrogen in the food fails to serve for efficient transformation into new body proteins during the sym- metrical growth of the body tissues. They are complete but their transformation into body proteins cannot be very effectively accomplished (Chart 1). We have shown in a previous communication that the cereal grain proteins do not in general make good each other’s deficiencies or enhance to any great extent each other’s biological values when two of them are combined.’ Rye and flaxseed meal proteins form ‘a notable exception. These, when combined in certain proportions, form a mixture which is distinctly superior to the proteins of either component alone. The supplementary value of one protein for another depends on the yields by each of those indispensable amino- acids which are present in each of the sources in smallest amounts. We shall show in one of the following papers that kidney, liver, and muscle proteins have much greater values as supplements to the cereal proteins than the cereal or legume proteins have, with few exceptions, among themselves. The data in the succeeding paper illustrate the importance of animal tissues in the food supply 116 Protein Values in Foods. I when the diet consists mainly of such vegetable products as do not yield a mixture of proteins having a high biological value. For the special purpose of enhancing the quality of the protein in the diet they have the highest value. It must be kept in mind, however, that their use does not correct the mineral deficiencies of a cereal, tuber, and legume seed diet, and that when muscle meats are used as food they have little effect in raising the content of fat-soluble A in the resulting mixtures. In no instance, therefore, will one of these types of animal tissue supplement a cereal, tuber, and fleshy root type of diet so as to make it highly satisfactory. Our observations on the rats described in these experiments do not show any definite evidence of injury to the animals as the result of being fed excessively high protein diets. These diets were, however, essentially complete and fairly well proportioned ‘as regards all factors other than protein. It is not justifiable to seneralize from these results that such a high protein intake is safe forman. Our animals were not kept to determine the possible span of life or the time of appearance of the signs of senility, owing to the necessity of temporarily vacating the room in which the animals were kept. It is the custom for people in the United States to derive a high protein diet, when such is taken, in great measure from mus- cle meats, fish, poultry, eggs, and legume seeds. Menus containing such high protein foods will only in exceptional cases be completely supplemented by other constituents of the diet. The evil effects often attributed to excessive protein consumption may now with some confidence be attributed in many instances to faults in the composition of the diet in factors other than protein. Further studies are required to demonstrate the relative merits of diets of high and low protein contents when other factors are of compara- ble value in the two cases. We shall discuss this phase of nutrition on the basis of carefully planned experiments on animals in a later communication. There are some very interesting and important instances of successful nutrition among people who have lived almost exclu- sively upon a diet very rich in protein and derived from foods of animal origin. Weare indebted to Mr. Vilhjalmur Stefansson for the information that previous to about 1850 dental caries were very rare or absent from Iceland. During the last half of the 19th century cases have gradually become more and more common McCollum, Simmonds, and Parsons aly until today infected teeth are perhaps as common there as in most parts of the United States. No carious teeth were found among 96 skulls disinterred by Stefansson from a cemetery in Iceland dating from the 9th to the 13th centuries. These skulls are now in the Peabody museum at Harvard University and have been de- scribed by Hooton.!! The diet of the Icelanders previous to about 1850 consisted essentially of milk, mutton, fish, and fowl, but in some parts of the island they ate the eggs of wild birds. The only vegetable food eaten regularly was carrageen moss, but potatoes and turnips were eaten to some extent. The teeth of the natives and their general health were excellent as long as this diet was taken. The deterioration of the teeth apparently began about the time when cereals and sugar were regularly imported into Iceland as sources of food. The teeth of the primitive Eskimo were excellent. The younger generation in northern Alaska, whose diet is derived in a large measure from cereal grain products, canned foods, and muscle meats, similar to what would be purchased in a grocery store in the United States, has poorly calcified teeth which are often carious. We have collected numerous observations on the effect of dietary faults on the quality of the skeleton in the rat. These data make it clear that most profound differences in the extent of calcification and density of the deposited calcium phosphate can be effected by such faults as are found in the cereal, tuber, and muscle meat type of diet. The diet of the primitive Eskimo was very rich in protein but it was at least fairly satisfactory with respect to other factors. It consisted of muscle tissue and fat as the principal components, but all blood was carefully saved and eaten, and the glandular structures were regarded as dainties of especial delicacy. In addition, they regularly ate bone marrow and chewed the softer parts of bones, such as ribs and the epiphyses of the long bones. Such a selection of tissues suffices for the satisfactory nutrition of the rat and produces the fine physical development seen in the Carnivora such as the lion, tiger, jaguar, ete. The deterioration of the teeth of the Eskimo which occurred simultaneously with the modification of the diet due to contact with the white man is in 1 Hooton, E. A., Am. J. Physical Anthropol., 1918, 1, 53. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 118 Protein Values in Foods. I harmony with what we have been led to expect as the result of experimental studies of the types of combinations of ordinary foods which enter into the diet of man and animals in different parts of the world. The cereal and muscle meat diet or its equiva- lent, the bread, meat, and potato type of diet, is in all probability the cause of the deterioration of the teeth of the present genera- tion of “civilized”? Eskimos as it is among the people of the United States and Europe. CONCLUSIONS. The kidney, liver, and muscle of the ox contain proteins which, when they serve as the sole source of nitrogen, and are fed singly as the sole source of protein, but completely supplemented with respect to all necessary factors other than protein, are shown to possess about the same biological value as those of the wheat ker- nel. There is no distinct evidence of toxicity in either muscle, kidney, or liver tissue when fed at planes of intake sufficiently high to introduce from 35 to 70 per cent of protein into the diet. The first limiting factor in the kidney, liver, and muscle tissue is a lack of calcium. It is also necessary to add sodium chloride in order to insure prolonged well being. Carnivorous man and animals secure their sodium chloride by eating blood, and caletum by eating bone. Liver and kidney contain an abundance of fat- soluble A and of water-soluble B, and when fresh and raw, of water-soluble C. Muscle tissue is very deficient in these factors but does not entirely lack any one of them. Kidney proteins appear to have higher biological value than those of the other animal tissues yet studied. It has been our custom for years in preparing experimental diets to thor- oughly grind the several components of the food and to make a uniform mixture from which the constituent parts cannot be picked out by the ani- mals. In all cases iodine was given once a week in the form of potas- sium iodide-iodine in the drinking water, which was distilled. The liver, kidney, and steak, except when the contrary is stated, were steamed in a sterilizer until thoroughly cooked, subsequently dried, and ground. Prac- tically all visible fat was removed from both organs and muscle. The curves presented in the charts are typical representatives of a group of four to six animals which composed each experimental group. McCollum, Simmonds, and Parsons 119 Chart 1.—The curves in this chart illustrate the growth of young rats fed diets containing 9 per cent of protein derived solely from beef kidney, liver, and muscle, respectively. The inorganic addi- tions were of a character which completed, at least in a fairly satis- factory manner, the mineral content of these animal tissues. There can be no doubt that kidney proteins have a somewhat higher biological value than those of liver. Liver proteins, sur- prising as it may seem, have not been found in our experiments to be superior to those of muscle (steak). In Lot 2475 there were two females. One of these died in par- turition. The other at the age of 4 months had one litter of four young and successfully reared them. She died from unknown cause about 40 days after weaning her young. Two of her daugh- ters grew up and produced one and three litters of young (a total of seventeen), respectively. Of these ten were successfully weaned. Two granddaughters of the female described in the original experimental group were kept 63 months on the family ration, and although they appeared to be in good condition, neither proved fertile. The rats fed the kidney ration did not exhibit early signs of aging. In this respect they were superior to the groups fed diets containing comparable amounts of liver and of muscle. The records of these experimental groups fed 9 per cent of pro- tein from kidney, liver, and muscle, respectively, all other factors being more or less satisfactorily adjusted, show them to be typical examples of nutritional instability. - In Lot 2475 on the kidney diet, each succeeding generation was inferior to its parents. Lack of uniformity of vitality among individuals of the same group or family is observed with striking frequency in animals whose diets fall but little below the quality necessary to maintain the vigor of the species unimpaired throughout successive generations. It might be suggested that the failure of the animals fed liver to develop more satisfactorily, was due to the presence of toxic substances in this organ, which performs the function of degrading numerous foreign and poisonous substances derived from metab- olism and absorption from the alimentary tract and elsewhere. The records of Chart 2 show clearly that this is not the case. sryud eofaque yeorm Jo pues OTT TO—> 0 Eu e-D * 3 z 0 Vea xs nay ere Mi D D d BO USAIXep BY JO! WTB uya4xep 644 JO | 06 D B c é 9°92 Upe4xXeq | o°2 Tese+-issy RITES : a ¥ uve | Ser) pates | ees ee ce . L°et (zeeq) APUpPTy (yeq) eTosny , : Osby HO 79 BY : 7% “|_| SAPe BOL SRYED | MecCollum,-Simmonds, and Parsons 121 Neither of the two females fed the steak diet had any young, although they appeared to be well nourished and were kept under observation during more than 12 months. Three females on the liver diet were somewhat undersized, aged early, and never had any young, although they were kept under observation for more than a year. In the case of both the liver and muscle diets this was apparently due to the proteins from these sources not being of sufficiently good quality to make 9 per cent of protein from these sources sufficient to promote well being at the optimum, since in these diets all other factors were corrected. Composition of Salt Mixture 185. per cent INEM sige oaibebA td Sin GRIEG. 018 3.0:01055 1s ER CRE naa ar re 0.173 Pie sO. (aniny arouse sts oe cae. Se ss SF SNe Os Pe ans es 0.266 NaH2PO, + H.O olchel kvekoichcialaneohapcvel cls) si els/sleso/ spiel sale) el eheie\c) cis elsls 0.347 IRD BI O)a Ae Bek hye 600506 00 be Tee Son eee 0.954 CaH4(PO4)2H. aie cuokclaneiahetetametelohetetareteieie) evsvel< ve. cuchetete ¢ wvciisietaceve sss aue sire 0.540 12@ CHURN panss daca éb oct SOS) Cae ee 0.118 (CamlAChaveser ete Noe ee reise cick oa dole aioe aida 1.300 Chart 2.—The rats in these experiments were fed food mixtures which were satisfactorily constituted except for possible shortage of water-soluble B and fat-soluble A, which in each case were derived entirely from 25 per cent of kidney, liver, or muscle, respectively. The growth curves and fertility of the animals on the kidney and liver diets show that these amounts were satisfac- tory as sources of fat-soluble A and water-soluble B. In these diets the protein of the animal tissues was supplemented with 9 per cent of casein. Chart 4 shows, however, that 20 per cent of either kidney or liver suffices as the sole source of protein for nor- mal growth, reproduction, and rearing of young, for the fourth generation on the kidney diet appeared to have normal vitality. The rats of the fourth generation on the liver diet were inferior, although they were successfully weaned. Two females on the kidney diet, Lot 2163, had collectively four litters (fourteen young), all of which were reared. Two of the daughters were maintained during 10 months on the diet on which their mothers had lived. One remained sterile, the other had a single litter(five young) at about 4 months of age and never became pregnant afterwards. The young were in good condition when weaned. 1 O9TS OT OXY OST Ae 2 pues ts | Galea LOT “dyaaxey Jo 4 eet i= aed peqetdes __4ezy _1999nd JF 3 ike) i ha: = ome LOT] aa aa uta4 xeq 9°29 TO oe a Tox | O°T TOSN fp Lobe = ch 1) ae Belk “Ea oup ty 0°S2¢ a Te 122 McCollum, Simmonds, and Parsons 124 There were three females raised on the liver diet (Lot 2162). They had collectively fifty-six young (nine litters), of which thir- ty-three were weaned. The young which died became lethargic just before weaning time and died in this condition. We have at present no explanation for this peculiar behavior. Lots 2160 and 2161 were fed diets comparable to Lots 2162 and 2163, except that the factors, fat-soluble A and water-soluble B, were entirely derived from 25 per cent of muscle tissue (cooked and raw beefsteak, respectively). In both cases there was failure of growth after about 4 weeks. After a period of suspended growth during which one female in Lot 2161 had a litter of four young which were eaten by the mother shortly after birth, 5 per cent of butter fat was added to the diet of each group. In both cases there was a marked response to growth following this addition. This demonstrates that 25 per cent of round steak does not contain sufficient fat-soluble A to meet the needs of the young rat. Lot 2142, Chart 3, shows clearly that 20 per cent of steak does not furnish sufficient water-soluble B for the normal nutrition of young rats during growth. The records in this chart show clearly the remarkable difference between the glandular organs as compared with muscle tissue in respect to their content of both the factors, fat-soluble A and water- soluble B. This supports the view which we have _ repeatedly stated, that the dietary properties of a substance can be fairly accu- rately predicted from a knowledge of their biological function. Chart 3.—The three groups of animals whose curves are shown in this chart were fed diets comparable in all respects, except that the sole source of water-soluble B and protein was 20 per cent of the dry matter in the diet in the form of kidney, liver, and muscle, respectively. A comparison of these curves with those of Chart 1 shows that the protein furnished by this proportion of liver, kid- ney, and steak, respectively, suffices to support normal growth when other factors in the diet are properly adjusted. The animals fed the kidney ration, Lot 2144, were markedly superior to the other two groups. Representatives of four suc- cessive generations were grown upon this diet as their sole source of water-soluble B, and with no evidence of deterioration. In the original group there were three females. These produced collectively ten litters (forty young) but only six individuals were Bt i ic ie sali a li cs Pi: Lae dal tis e34nq) 14 xo Lord e2:| | 98s Heqgng| Cu | | wp t3xeC *l | £008, 1 ae | tox T | ToeN] 0 | | feup ty 7 a 10 79 BY | | pple LOI ‘S| LYyHO o9T 00¢ 124 McCollum, Simmonds, and Parsons 125 successfully weaned. The mothers destroyed their young soon after birth. Two daughters of the females just described were kept on the family diet. They had together six litters (forty young) and weaned only four individuals altogether. Here again the mortal- ity was the result of cannibalistic tendencies in the mothers. But one granddaughter of the original group was kept on the family diet. She grew normally and had six young (two litters) and successfully weaned all of them. One great granddaughter was brought up on the diet. She had three young (one litter) and weaned them successfully. With optimum nutrition these litters would have been two or three times as large as those produced. It might be suggested that perhaps the content of water-soluble B in this diet was below the optimum, and that for this reason the nutrition of the nervous systems of the rats restricted to this diet was faulty, and that the tendency to destroy the young was an expression of an abnormal psychology analogous to the psychosis observed in beri-beri or pellagra. The fact that the third and fourth generation females in this family were less cannibalistic than their mothers and grandmothers militates somewhat against this view. Lot 2143 derived all its antineuritic factor and protein from 20 per cent of liverin the diet. This was equivalent to 14.4 per cent of protein. The growth curves were normal. In the first group fed this diet there were two females. These had forty-six young (eight litters) and of these but twenty were weaned. Two second generation females had eighteen young (four litters) and weaned ten of them. Two third generation females had thirteen young (two litters) of which they weaned six. One fourth generation female was kept 53 months, when she died, never having had any young. None of these animals in any generation was kept to an advanced age. The young of the rats fed the liver diet were somewhat undersized but appeared to be vigorous. Lot 2142 was fed a diet comparable to the others described in this chart, but with all protein and water-soluble B derived from 20 per cent of muscle (round steak). These animals were mark- edly inferior both as regards growth and fertility to those fed kid- ney or liver at the same plane of intake. Two females were kept to the age of 15 months, an age which usually marks the end of 126 Protein Values in Foods. I fertility in the rat. One had a single litter of eight young but they died when about 2 weeks old. The other remained sterile. The rats in this group were still in good nutritive condition after a year on the diet. This chart, like the other records in this paper, shows clearly the superiority of glandular organs over muscle tissue as sources of water-soluble B. Chart 4.—These records show the behavior of young rats fed cooked dry beef kidney as the sole food, Lot 1253. Lot 1254 shows the growth curves of rats fed 97.5 per cent of kidney supplemented with sodium chloride and calcium carbonate. Lot 1255 was fed kidney, 94.5 per cent, supplemented with sodium chloride, calcium carbonate, and butter fat. Lot 1256 was fed a diet like that of Lot 1255 except that 14 per cent of the kidney was replaced by lactose. This last ration was designed to show whether lactose would tend to modify the bacterial flora of the alimentary tract. No beneficial effects of the modification were apparent. The rats restricted to cooked dried kidney as their sole food grew in a fairly normal manner. Two females had collectively fourteen young (a litter each), five of which were weaned. One of these young developed abnormal ribs suggestive of rickets. None of these young grew up. It is very remarkable that young rats confined to a diet of lithe could develop so successfully and reproduce and rear young. This tissue is very poor in calcium, and yields a great excess of acid on being metabolized. The protein content of this diet was not far from 71 per cent, yet because all essential food factors except calcium were so abundant, the animals were able to tolerate this deficiency and the abnormal protein content remarkably well. Lot 1254 contained two females, one of which became pregnant and died in parturition. The other had a tumor which became so large that she was chloroformed. There was but one female in Lot 1255. She had sixteen young (two litters) of which nine were weaned. Two second generation females had collectively seventeen young (a litter each) of which ten were weaned. Two third generation females had each a litter of young (seventeen) of which twelve individuals were weaned. The young appeared to be well developed, but were always greasy from their food. They drank much water. The cage had a strong odor. The protein content of the ration of Lot 1254 was 69 per Sset vr} lof ee 127 Re £0 Be TORN ee 128 Protein Values in Foods. I cent; that of Lot 1255, 67 per cent; of Lot 1256, 57 per cent. The results show that the rat is capable of growing and remaining in a state of health on a diet comparable to that of the strictly. car- nivorous animals. At least among the carnivorous animals of the Aretie regions the proportion of protein in the diet is not neces- sarily excessive at all times. Mr. Stefansson has informed us that it is not unusual for travelers to come upon a seal that has been killed and skinned, the subcutaneous layer of fat eaten, and the remainder of the carcass left practically untouched. The polar bear evidently prefers fat to protein as a source of energy. Lot 1256 contained two females. They had collectively twenty- two young (two litters each) of which fifteen.were weaned; two second generation females had together nineteen young, and weaned fourteen of them. One third generation female had one litter of six young and weaned them all. Chart 5.—The diet of the four groups of rats whose curves are shown in this chart was in all instances comparable to those in Chart 4, the only difference being the substitution of liver for kid- ney in the diets of Lots 1277, 1278, 1279, and 1280. Lot 1277, which was confined to liver as its sole food, grew very little and died from 4 to 6 months after being confined to the diet. Lot 1278 was fed liver supplemented with sodium chloride and calcium carbonate. On this diet growth was approximately nor- mal. The group contained three females. One of these had twelve young (two litters) and weaned eleven of them. The other two remained sterile. One second generation female remained sterile. Lot 1279 was fed liver supplemented with sodium chloride, cal- cium carbonate, and butter fat. Lot 2162, Chart 2, shows that even 25 per cent of liver as the sole source of fat-soluble A furnishes a sufficient amount of. this factor. There were three females in this group. They had thirty-four young (six litters) and weaned thirty-one of them. One second generation female grew up on the diet, and had six young in one litter. She weaned four of these. One third generation female in this family had a litter of six and weaned them all. The repro- duction records of this group were distinctly better than those of Lot 1278, whose diet was identical except for the 3 per cent of but- 130 Protein Values in Foods. I ter fat. We cannot explain the reason for the superiority of Lot 1279. . Lot 1280 was like Lot 1279 except that 14 per cent of lactose replaced a like amount of liver. This did not exert any notice- ably favorable effect on the well being of the animals. Two females each had a litter (collectively fourteen young) and weaned thirteen individuals. One second generation female had one litter of eight young and weaned seven of these. The young in Lots 1279 and 1280 appeared to be strong and in good con- dition but showed in all cases a peculiar condition which we have observed occasionally in abnormal animals; v7z., a wet and stained area around the urethral orifice. This not infrequently occurs in poorly nourished animals. Chart 6—The animals whose records are shown in this chart were fed a diet made adequate in all respects as far as could be judged, and with the protein derived solely from 50 per cent of dry beef liver. The protein content of the diet was about 35per cent. Lot 1281 contained three females. These had collectively thir- ty-five young (five litters) and weaned twenty-two. Two second generation females had one litter each, collectively nineteen young, of which number they weaned seventeen. These young appeared to be very well nourished. Lot 1282 contained two females. These collectively had five litters (thirty-four young). They weaned thirty-one of these young. Two second generation females were restricted to this diet. One had a litter of six, which she successfully weaned. The other female remained sterile. The young were apparently well nour- ished but badly urine-stained. There was no evidence that the inclusion of lactose benefited the animals. Chart 7.—The animals whose records are shown in this chart were fed either muscle tissue or blood, with and without certain purified food additions. Lot 1232 was restricted to a diet of cooked, dried, beef muscle. They were able to grow very slowly, but remained very much undersized, and died early. None of this group had any young. Two of the rats showed, toward the end of their lives, distinct signs of xerophthalmia, due to the lack of fat-soluble A in muscle tissue. | 7 Lp oe ae eae be + label ues is be & Hl e ¢ FAI £09 Ing ote xeqg U u ore 0603 08T $*08 qT OST o*os | etosny | ore Na Ded PE ME) i a S*t 08 [st | OOP | reporter | he oat |g | O°T | toe | oot | | TORN O°00T | — POdTQ xO) | fe e | . =| aa {36 et _|TPOeT | _jo*oot | 4900) ¢coerw 340 73 UOT 9 14079 Ry | {00 73 BY | peel LOT eeat LOT | Lzdz@ LOT | ee2t JOT *A | GUYHO wee = SI <1 i a 1 Loe | | | -—+- — a Se tagile | hemes 4 McCollum, Simmonds, and Parsons 133 Lot 2027 was fed dried ox blood as their sole food in Period 1. They declined rapidly on this and after 2 weeks were changed to a diet of muscle meat 50 per cent and dried blood 50 per cent. On this diet they were able to grow at arate approximately half the normal. A female in this group had a single litter of four young, which she destroyed within a few days. All these animals showed early signs of old age. Lot 1233 was fed cooked dried muscle (beefsteak) supplemented with sodium chloride and calcium carbonate. They grew but lit- tle better on this diet than on muscle alone, but one female had a litter of three young, which she destroyed soon after birth. Two other females remained sterile on this diet. That fat-soluble A was the limiting factor in this diet is shown by the records of Lots 1234 and 1235. Lot 1234 was fed muscle, sodium chloride, calcium carbonate, and butter fat. They not only grew in an approximately normal manner, but were fairly fertile and had moderate success in the rearing of their young. Two females had together twenty young (three litters) of which sixteen were weaned. One second gene- ration female was kept on the diet. She had one litter of five young and weaned them all. The young were somewhat inferior. Further studies are necessary to determine what modifications of this diet are necessary to secure greater fertility and higher vital- ity in the young. Lot 1235 was fed a diet of beefsteak supplemented with sodium chloride, calcium carbonate, butter fat, and 14 per cent of lactose. They were not superior in vigor, fertility, or success in rearing young to Lot 1234 which had more beefsteak in place of the lac- tose. Two females had thirty-two young collectively (five litters) of which twelve were weaned. Since these were all males no fur- ther reproduction records were secured. Chart 8—The animals whose records are shown in Chart 8 derived their protein and water-soluble B from 50 per cent of cooked, dried, muscle tissue (beefsteak). The protein content of the diet of Lot 1236 was about 35 per cent. The growth curves approximated the average and the animals appeared to be in a satisfactory state of nutrition. . There were two females in Lot 1236. Together they had thirty- four young (five litters) and weaned twenty-four of them. One 134 McCollum, Simmonds, and Parsons 135 daughter of one of these mothers was kept on the diet. She had one litter of eight young and weaned them all. Two of her daugh- ters grew up on the diet and each had a litter, together thirteen young, and weaned but three of them. The young in most cases appeared normal. ‘They were not urine-stained as many were on the liver diets. Lot 1237 differed from Lot 1236 in that 15 per cent of lactose replaced an equivalent amount of dextrin. There was no evidence that this carbohydrate exerted any beneficial effect over dextrin. Two females in this group had together thirty young (six litters) and successfully weaned them all. Two of the second generation females had each a litter (collectively fifteen young), of which eleven young were weaned. One third generation female had a litter of seven young and weaned them all. Chart 9—These records show the histories of three groups of young rats fed diets in which all protein and antineuritic factors were derived from raw muscle tissue (round steak). Lot 2056 was restricted in the first period to raw round steak as the sole source of nutriment. They grew no better on this than on cooked steak (Chart 7, Lot 1232). In a second period sodium chloride and calcium carbonate were added and these caused a slight response with growth for a time. The diet was too poor in fat- soluble A to admit of much growth. This is seen in the record of Lot 2058. Lot 2058 was fed a diet containing 50 per cent of raw, dried muscle (beefsteak) supplemented with sodium, potassium, calcium, chlorine, and fat-soluble A in butter fat. The diet contained 43.5 per cent of dextrin. On this ration growth was normal and the animals were apparently in good nutritive condition. Three females each had a litter of young (collectively twenty-two) of which twenty were reared. Two second generation females each had a litter (fourteen young) and reared them all. There was a period between the ages of 18 and 28 days when the young of the second generation appeared lethargic. They recov- ered later and appeared to be as alert as rats on the cooked steak. This was probably due to the effects of eating so much raw meat while still in a very immature condition. Lot 2057 had a diet like that of Lot 2058 except that 15 per cent of dextrin was replaced by a like amount of lactose. There was : ion SOOM 8S0¢ 8s0e v pS pug OT 38s £99 4Ng ut14xeC reales 107 ea o°¢ 4BJ 7044nNq : "i 13x0eq! Lo0k_ o°T Tox TOBN g*Tt fq089 5 0 a TORN] | eTosnu amy | 0°OS eTOShU ABYy ___ POT BY oT By ~ -ES0e LOTT | Tic i. 6962 LOT! 136 McCollum, Simmonds, and Parsons 137 no noticeable benefit from this modification of the diet. There were two females in this group. They had collectively twenty- three young (four litters) of which twenty-two were weaned. No young have as yet been secured from any of the daughters. These records when compared with those of Chart 8 indicate that no noticeable difference exists in the nutritive value of raw and cooked steak. Although steak is very poor in water-soluble B, there was a sufficient amount of it in 50 per cent of cooked steak to permit young rats to grow up and rear young. : Re tm ath a. = ; | RA. ~0ei' Nw SUPPLEMENTARY PROTEIN VALUES IN FOODS. II. SUPPLEMENTARY DIETARY RELATIONS BETWEEN ANIMAL TISSUES AND CEREAL AND LEGUME SEEDS. By E. V. McCOLLUM, NINA SIMMONDS, anp H. T. PARSONS. (From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.) (Received for publication, March 21, 1921.) In a former publication! we have shown that the proteins of the navy bean do not markedly supplement those of the maize kernel so as to improve their quality for the support of growth. This means that the proteins of this cereal and legume seed are both deficient in some of the same indispensable amino-acids. Our earlier studies also indicated that the cereal proteins do not have much of a supplementary value among themselves.? In a later paper of this series we shall present more records bringing out the extent to which the proteins of the cereal grains and the legume seeds enhance each other’s values for the nutrition of the rat. The data presented in the charts show the nutritive values of the protein mixtures resulting from combinations of the cereal grains and legume seeds with several representative animal tissues. We recently pointed out the surprising fact that when muscle tissue, liver, or kidney, serves as the sole source of protein in the diet, all other factors in which have been made satisfactory by suitable additions, the quality of the amino-acid mixture for transformation into body proteins during growth is no better than that of proteins derived from grains.’ It has always been 1 McCollum, E. V., Simmonds, N., and Pitz, W., J. Biol. Chem., 1917, 780bg, UPA 2 McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxvli, 155. 3 McCollum, E. V., Simmonds, N. and Parsons, H. T., J. Biol. Chem., 1921, xlvii, 111. 139 140 Protein Values in Foods. II accepted as a fact, hardly in need of experimental demonstration, that these animal proteins have exceptionally high biological values. Thomas came to this conclusion as the result of experi- mental data obtained on a full grown man as a subject. The periods of observation were very short, and a milk diet was used in the foreperiods which vitiated the results because of the sup- plementary effect on the foods being studied. Thomas’ experi- mental data represent digestibility and absorbability of proteins rather than biological value for transformation into body tissue.‘ TABLE I. Chart.| Lot. Source of protein. Growth. Fertility. | Mortality. 2 2193 | 3 wheat, 3} kidney. Excellent. | High. Medium. 2192 | 2 soy beans, } kidney. Fair. Very low.| High. 3 2479 | 2 barley, 4 kidney. Good. High. Medium. 4 2195 | 3 rolled oats, 3 kidney. | Fair. Medium. ey 5 2194 | 3 maize, } kidney. ss - High. 2191 | } navy beans, } kidney. sé Low. Low. 6 | 2190 | % peas, } kidney. re ds High. 2196 | Zrye,3 “ Good. Medium.| “ Of all the combinations of proteins from the two sources which we have studied, wheat and kidney proved to have the highest biological value. We have tested only one mixture of proteins from these sources; viz., that in which a seed furnished two-thirds and kidney one-third of the total. Since in many cases growth was essentially normal, the basis of judg- ment in the comparison of the values of protein mixtures was the variation in fertility and infant mortality. The combination of navy bean and kidney proteins forms a mixture having a higher biological value than any other animal tissue and legume seed which we have studied. Soy bean and kidney form a better protein mixture than soy bean with liver or soy bean and muscle. The results of our studies show that the proteins of kidney, liver, and muscle have remarkable values for the enhancement of the proteins of some of the cereal grains, and improve the legume seed proteins to an appreciable degree. Kidney, liver, and mus- cle proteins have without exception supplemented those of the cereal grains more satisfactorily than they do those of the legume seeds. There are marked differences in the efficiency of the sup- ‘ Thomas, K., Arch. Physiol., 1909, 219. McCollum, Simmonds, and Parsons 141 plementary relations between kidney proteins and those of several cereal grains which make it possible for us to differentiate be- tween their values in the case of the different combinations. The relative values of the several combinations of vegetable proteins with those of kidney with which we have experimented in this series may be illustrated by tabulating the seeds in the order of their efficiency for growth, when two-thirds of the protein is derived from the seed and one-third from kidney. Wheat—barley rye—oat—maize—soy hean—pea—navy bean. There is no marked differentiation between the kidney and oat as contrasted with the kidney and maize combinations. These are distinctly inferior to the combinations of kidney with barley, wheat, or rye. There is little difference in the biological values of the mixtures of proteins derived from peas or the two kinds of beans studied. These legume seeds combined with animal tissues are inferior to similar combinations of animal tissues with cereals. Table I shows in condensed form the effects of feeding kidney proteins as supplements to certain vegetable proteins on growth, fertility, and infant mortality. Among the several combinations of cereal and legume seed proteins with muscle tissue which we have studied, wheat is decid- edly the best. Growth and fertility were remarkable, but the infant mortality was high. Muscle tissue is distinctly less effec- tive than kidney as a supplement to the proteins of the seeds used, except in the case of wheat. The following order represents the biological values of combinations of seed with muscle in which the seed furnished two-thirds of the total protein and the muscle one-third. The values are arranged in a descending scale. Wheat—oat—barley—maize—pea—navy bean—soy bean. It is not possible to differentiate from our data between the values of navy bean and soy bean proteins combined with muscle. Table II shows in condensed form the effects on growth, fertility, and infant mortality of feeding muscle proteins as supplements to the proteins of several seeds. Liver proteins supplement those of the cereal grains and legume seeds in degrees which differ sufficiently to make possible rather decided contrasts in several cases. In general, liver proteins js 142 Protein Values in Foods. II enhance those of the seeds somewhat better than do those of muscle, with the single exception of the combination, wheat and muscle. The seeds which we have studied may be arranged in the following order to illustrate the supplementary efficiency of liver proteins for each, the best combination being on the left and decreasing from left to right. Barley—rye—wheat—oat—maize. ~ TABLE II. Chart. Lot. Source of protein. Growth. Fertility. Mortality. 7 2186 | ? wheat, | muscle. Excellent. | High. High. s 2189 | 2 rye, 4 iS Very good. RB a 2184 | ? navy beans, 3 muscle. | Poor. Low. of i) 2188 | 2? rolled oats,% “ Good. High. re 2477 | 2 barley, 4 muscle. a Low. is 10 2187 | 3 maize,4 “ Fair. ft es 2183 | 2 peas, 3 #2 i ef “ 2185 | 2? soy beans, 4 muscle. “4 se | fe Wheat and muscle gave an excellent growth curve and high fertility, but apparently because of a slight inferiority of this protein mixture as compared with certain others, the infant mortality was high. Wheat and muscle appear to form a protein mixture superior to rye and muscle. The growth curves of rats confined to a protein supply derived from rolled oats and muscle were better than the curves of those fed rolled oats and kidney. The latter, however, were more successful in fertility and in rearing young than the former. It is not possible from our data to decide between the values of the combinations of liver proteins with those of the legume seeds, peas, navy beans, and soy beans. A study of the records presented here, showing the effects of seed and meat diets, supplemented with respect to all factors other than protein, on the life histories of individual animals and on the family histories of successive generations confined to monotonous food mixtures, cannot fail to impress one with the fact that observations on the growth curves of a group of animals to maturity do not afford sufficient evidence to enable one to arrive at safe deductions concerning the quality of an experimental diet. Table III-shows the effect on growth, fertility, and infant mortality, of feeding liver proteins as supplements to the proteins of several seeds. McCollum, Simmonds, and Parsons 143 There are many who believe that if the diet affords sufficient calories and considerable variety, satisfactory nutrition will be assured. This may be far from true. So long as the selection does not include a variety of types of foods of unlike dietary values variety does not insure safety in any great degree. No matter how many cereals, tubers, and muscle meats such as steak, ham, chops, roasts, etc., may be taken, the diet will prove to be inade- quate. The inclusion of milk or leafy vegetables goes far toward TABLE III. Chart.| Lot. Source of protein. Growth. Fertility. Mortality. 11 2478 | 2 barley, 3 liver. Good. High. Medium. 2178 | 2 soy beans, § liver. Poor. Low. High. 12 2180 | 2 maize, 4 liver. Fair. Medium.| “ 13 2181 | 2 rolled oats, 3 liver. Good. High. e 14 2182 | 2 rye, 4 liver. Very good.| “ “ 2176 | 3 peas, 4 “ Fair. Low. a 15 2179 | 2 wheat, 3 “ Good. High. 8 2177 | 2 navy beans, 3 liver. Fair. Low. cs Rolled oats and liver formed a better protein mixture than rolled oats with kidney or muscle. This could not be discerned from an inspection of the growth curves or the records of fertility, but came to light through the comparison of the infant mortality in the three groups. Barley and liver made a better protein mixture than barley and muscle, but not quite so good as barley and kidney. Rye and liver also proved superior as a source of protein to mixtures of this cereal with either muscle or kidney. Navy beans with liver form a better protein mixture than combinations of this seed with muscle, but not quite so good as with kidney. making good deficiencies of such a diet in inorganic constituents and fat-soluble A. Fresh fruits and uncooked salad vegetables have a unique place in the diet as sources of the antiscorbutic factor water-soluble C. The substitution at frequent intervals of ham for chops will not tend to insure safety in nutrition. So large a part of the food sup- ply of man in the temperate and warmer regions of the world is derived from cereals and other seed grains, tubers, and edible roots, that in seeking variety in food attention should be fixed upon the importance of milk and the leafy vegetables suitable for’ 144 Protein Values in Foods. II greens, salads, ete., since these are so constituted as to correct the deficiencies of cereals, tubers, and roots.’ In examining the records of the experimental animals described in this paper it should be kept in mind that in studying the com- binations of proteins from animal and vegetable sources, we have in all cases added butter fat to furnish fat-soluble A and the inor- ganic salts necessary to correct the mineral deficiencies. The results do not, therefore, represent the supplementary effect of one of these foodstuffs for another except in respect to the protein factor. The results of these studies form a demonstration of the value of animal tissues as constituents of the diet, when the latter con- tains as much cereal products as is usually the case in America and Europe at the present time. At the same time it must be remembered that the animal tissues are by no means complete supplements for a cereal and legume seed diet. SUMMARY. 1. The proteins of kidney, liver, and muscle are remarkably effective as supplements for the proteins of cereals. There are demonstrable differences in the extent to which these animal tis- sues enhance the values of certain of the cereals. Thus, kidney, liver, and muscle are about equally effective as supplements to wheat. Maize proteins are less effectively supplemented by kid- ney, liver, or muscle than wheat, barley, or rye. 2. The three animal tissue proteins studied were found to have a supplementary relation to pea, navy bean, or soy bean proteins, but these combinations are very inferior to similar combinations of animal tissues with cereal grains as a source of protein. 3. Our results demonstrate that the proteins of kidney, liver, or muscle are more valuable for transformation into body tissues when combined with cereal proteins, than when each is fed as the sole source of amino-acids in the diet.* 4. Either muscle or glandular substance fails to supplement effectively the mineral deficiencies of cereals or legume seeds. The glandular organs are good sources of fat-soluble A. 5 McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxviii, 113. McCollum, Simmonds, and Parsons 145 Chart 1.—These curves show typical records of growth and repro- duction of rats fed from an early age on diets in which the sole source of protein was a cereal or legume seed. The amount of seeds was adjusted in each case so as to give the diets a protein content of 9 per cent. Such additions of butter fat and inorganic salts were made in each case as to correct dietary deficiencies other than protein of these seeds. The list of cereals here discussed includes wheat, maize, rolled oats, barley, and the legumes, peas, navy beans, and soy beans. Among these seeds wheat stands first in quality of protein when fed as the sole source of this factor. Oat and maize proteins have. approximately the same value, but we have gained the impression that oat proteins are slightly superior. None of the legume seeds is at all satisfactory as the sole source of protein in the diet at the plane of intake of 9 per cent of the food mixture. This fact is brought out by the curves of Lots 2376, 2510, and 2368. On adding casein in a second period growth was accelerated in each case. No young have been secured on diets of this type in which pro- teins were derived entirely from a legume seed or from maize. The fertility of rats fed oats to furnish 9 per cent of protein was so far below that of animals fed wheat protein in similar amounts as to indicate that the latter were in a distinctly better state of well being. Even in the case of the wheat diet the protein was not sufficiently good to permit of much success in rearing young. A few young could be reared by mothers which had grown up and were maintained on the oat diet containing 9 per cent of protein. These curves are presented as a basis of comparison for the records in the remaining charts described in this paper. These derive their content of protein (9 per cent) in each case from two sources. The object in each case is to compare the biological value of mixtures of proteins in stated proportions as compared with the values of each source when fed alone. Chart 2.—Lot 2193 was fed a diet from soon after weaning, which derived its protein content of 9 per cent from wheat and cooked dry kidney. All other factors in the diet were adequately sup- plemented. The wheat furnished 6 per cent and the kidney 3 per cent of the protein of the diet.. Growth was normal in this group. The state of nutrition of these animals was much superior PAM ia aval’ ce y a of 0°c yer £63 4ng oe | Fes BHZIUg | G°h UPt9xer g*6T UT xed bts in, Be le pee ae cael £0080 gers | 0089 *ujaqzxep O°T TORN; OW |. = roe jo 440d poget der *utayxep 0°06 eZzT ay O°SL fet reg uT@svo Jo qued|sed g go ytud pedetdes | 200 TRY 2ubT4 BIT =e Spotaod | és hl 0) S92 a Mae I 9 a od an a To o°e 38s f04gnd Z poysed 6°S9 Seal ca utzm4xeq {o°s yur e3gng vs | iz } | } g*t F008 L°ss uyaqgxeq; —“(ié‘*OS CCT FBT Fegang) OTE | FUT ARGH o°T TORN g*T £ooep G* Pe Uyt3 xed) | S*> UT M3 xe asc | _—sii{gst)|sates| ‘suqd Suygerdox o*t TORN ike £o08p [o°t | Eoovo | 6°? Sue q fos suegq Xaeu 4z TM ~ 18°0p es) eC a ol a TORN] rot | I Toen | po Fao 9422 OT SXTT poy+ed 0°09 | s3¥0 peTToy | 0°06 h voy | yo tae | 14079 CH | #40 FaeH tHOTIEY | thota wy | ome tor] 9L¢2 LOT pepe LOT! Cove OT “Tt Duvio | Ot$z LOT | ps 146 Bo. sezet ental! Ber tae | VN ea el ae mi CECA CARRE SE A PE a ae SA eRe SRP 148 Protein Values in Foods. II to those fed wheat as the sole source of protein when the content of the latter was limited to 9 per cent. Those rats which grew up on 9 per cent of wheat protein (Lot 2481, Chart 1) were as old looking at 14 months as were those in Lot 2193 at 18 to 20 months. Indeed, the animals of Lot 2193 were in such good condition that they compared favorably in appearance and in fertility with other experimental groups fed much higher protein intakes. There were two females in this group. They had collectively seventy-nine young (eleven litters) and weaned seventy of them. On a diet comparable in all respects but with all its 9 per cent of protein derived from wheat we observed 50 per cent mortality of the young. A somewhat higher protein content in the diet of Lot 2193 would apparently have shortened the nursing periods, which were longer than normal, and would have lowered the infant mortality. Four females in the second generation had seventy-five young (twelve litters) and weaned forty-nine of them. Two third generation females had forty-nine young (eight litters) and weaned thirty-six. The mortality of the young in this group was due entirely to destruction by the mothers. Frequently the young were bitten and injured or killed when approaching the end of the nursing period. One cannot say that animals, which had a diet containing enough protein to enable them to grow at the normal rate to maturity and to be as fertile as were these, were suffering from protein starva- tion. Yet it was due to the low protein intake (of the quality here used) which exerted a peculiar influence on the psychological reactions of the mothers and made them attack their young even after they were 2 or 3 weeks old. In rats in our colony which have highly satisfactory diets it is extremely rare that a mother attacks her offspring or indeed those of another. We not infrequently isolate among our stock animals two or three pregnant females in the same cage. They then care for their young in a common nest and nurse their own young and those of the other mothers promiscuously and without perversion of the maternal instinct. Lot 2192 was fed a diet in which the protein was furnished by soy beans and beef kidney. The diet contained 9 per cent of total protein, two-thirds of which came from soy beans and one-third from kidney. All the other factors were satisfactorily supple- mented. Growth was slow and the animals never reached full MeCollum, Simmonds, and Parsons 149 adult size. Their growth was much better, however, than that of rats restricted to the same amount of protein entirely from soy beans (Chart 1, Lot 2510). Two females grew up on this diet. One remained sterile. The other had nine young (two litters) and weaned three. Two second generation females remained sterile. The animals in this group were very old looking at 15 months of age. It is evident that 9 per cent of protein from these sources is not satisfactory for normal milk production. Chart 3.—Lot 2479 was fed a diet in which the protein was derived from barley and kidney. The barley furnished two- thirds and the kidney one-third of the total protein. The growth was more satisfactory than any we have observed in rats fed 9 per cent of barley protein only (see Chart 1, Lot 2483). Two females which grew up on this diet had collectively forty- five young (seven litters) and weaned thirty-one. One female in the second generation had nineteen young (three litters) and weaned eighteen. Two third generation females had forty-three young (seven litters) and weaned nineteen. One fourth genera- tion female was restricted to the diet but she failed early and died at the age of 110 days. These animals were in good condition at the age of 15 months, when they were discarded. Chart 4.—Lot 2195 was fed a diet in which the protein was furnished entirely by rolled oats and kidney The oats fur- nished two-thirds and the kidney one-third of the total protein. Their records were very much better than those of Lot 2484 (Chart 1). This is shown especially in the fertility and infant mortality of the two groups. Lot 2195 was somewhat undersized but of good appearance. They were not in such good condition as those which grew up on the wheat and kidney diet (Lot 2193, Chart 2). Two females grew up on this diet. One of these had twenty young (four litters) and weaned ten. The other died soon after giving birth to her first litter of six young. Two second generation females had thirty-seven young (six litters) and weaned twenty- three. One third generation female had one litter of five and weaned them all. These young were in good condition, but the nursing periods were long. The animals in this group were old looking at about 16 months. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 SPACED CO ie a Bh He POSEN COCA Sea SS08 93:16 CC SST Te CCCORECIE ES CCN Poe Teo SSR URNS SET ‘Fae 4 q 152 Protein Values in Foods. II Chart 5.--Lot 2194 was fed a diet, the protein of which was derived entirely from maize and kidney. The maize furnished two-thirds of the total protein and the kidney one-third. All other factors were satisfactorily supplemented. The growth curves were much better than were those of Lot 619 (Chart 1), which had a diet containing 9 per cent of maize protein alone. The animals of Lot 2194 on the maize and kidney proteins were never in good condition, but did not look very aged until about 18 months. Three females which grew up on this diet had thirty- one young (six litters) and weaned fifteen. They were only in fair condition and were small for their ages. One second genera- tion female grew up on the diet and had fifteen young (three lit- ters). She weaned four of her third litter. The other young she destroyed soon after they were born. Lot 2191 was fed a diet containing 9 per cent of protein, two- thirds of which was furnished by navy beans and one-third by kidney. All factors other than protein were satisfactorily sup- plemented. A diet exactly similar but with 9 per cent of protein derived solely from navy beans supports almost no growth (Lot 2368, Chart 1). The combination of navy beans and kidney is therefore vastly superior to bean protein alone. These animals looked very old at about 12 months. One female in this group had one litter of five young and weaned four, but they were very small. At 42 days of age the four weighed but 147 gm. A litter of four nursing a normally fed mother should reach a weight of about 350 gm. at this age. One other female in this group remained sterile. Two second generation females were restricted to the diet but neither one had any young although their growth curves were much better than those of rats confined to 9 per cent of navy bean protein alone (Lot 2368, Chart 1). Chart 6.—Lot 2190 was fed a diet containing 9 per cent of pro- tein, two-thirds of which was furnished by peas and one-third by kidney. All other factors were satisfactorily supplemented. The animals grew much better than a group confined to a similar diet with 9 per cent of pea protein as its sole source of nitrogen (Lot 2376, Chart 1). This shows that there is a greatly heightened value in the combination of proteins used in this diet as compared with pea protein alone. The rats fed this ration were somewhat SRRT GH Se SRS a 154 Protein Values in Foods. II | ae {4 CPR eta Ltt aN It Pia ce fe ON Sa BECHACtke McCollum, Simmonds, and Parsons Pap undersized, but appeared fairly well nourished in early life. They appeared senile when about 12 months of age. One female in this group had twenty-four young (four litters) and weaned four of her fourth litter. The other young were destroyed by the mother while very young. Another female remained sterile. Three second generation females were confined to the diet until they were 12 months old, but none of them had any young. Lot 2196 derived its sole protein from rye and kidney. The rye furnished two-thirds and the kidney one-third of the total. Except for the amount and quality of the protein the diet was well constituted. Although the growth of these animals was excellent and the fertility fairly high, the mortality of the young was very high. These rats were in good condition in early life but looked very old at about 16 to 18 months. Five females on this diet had collectively seventy-three young (fourteen litters) and weaned thirty-one. Many of these were destroyed by the mothers soon after birth. One female died after the first litter, and a second died after her second litter from undetermined causes. Two second generation females had thirty- one young (seven litters) and weaned nine. One third generation female had a litter of six. Our investigations show that cereal grain proteins are distinctly better supplemented by kidney proteins than are those of the legume seeds. The order of effectiveness in this supplementary relation is as follows: Wheat—barley: rye—oat—maize. There is no marked difference in the value of peas, soy beans, and navy beans in their supplementary relation to kidney proteins. Chart 7.—Lot 2186 was fed a diet comparable to those discussed in Charts 2 to 6 inclusive, except that muscle tissue (beefsteak) replaced kidney as a supplemental source of protein for a seed. This diet contained 9 per cent of protein, two-thirds of which was furnished by wheat and one-third by beef muscle. Deficiencies in inorganic salts and fat-soluble A were made good by suitable additions. The growth curves of this group, considering that the food mix- ture contained but 9 per cent of protein, are remarkable. All the Peo pt eh fete me CLL Es On Wc ik COONS SS oman Bee Ee: COCR TTS bia a < A ad T¥ latte ae saz hy boi ale ial | Ae ae oe. Kon’. a ico 2 eu EEVAS Z| a WAS ee ace ee Ly fl cls FA 4 = al 2 . oO ° °o So ° “2 fs = 8. 8 3S oa McCollum, Simmonds, and Parsons , Le animals grew at a rapid rate to a large size. This can be inter- preted only to mean that there is an excellent supplementary relation between the proteins of the wheat kernel and those of muscle tissue. This was likewise true of wheat supplemented with kidney (Lot 2193, Chart 2). The span of life was, however, somewhat short. The animals looked old at 16 to 17 months, whereas they remain vigorous up to 2 years at least when the nutrition is highly satisfactory. That they were not in this optimal condition, notwithstanding their large size and splendid fertility, is shown by the high mortality among their young. Al- though the biological value of the protein was high and the plane of intake sufficient to support growth and reproduction, the amount of protein of this quality was not good enough to maintain physi- ological well being to an advanced age or to suffice for the nutri- tion of the nursing young. Two females grew up on this diet. They had collectively sixty young (eight litters) of which only twenty-nine were weaned. Two females of the second generation had sixty-five young (eleven litters) and weaned twenty-three. One third generation female had nineteen young (three litters) and weaned only three. Many of the young were destroyed shortly after birth, but those which were weaned were in good condition. In several instances the mothers reared large litters (nine young; seven young). This shows that even 9 per cent of protein of the excellent quality formed by wheat and muscle in the proportions here used sufficed for milk production even when the litters were large. We differen- tiate between high infant mortality due to infanticide and that due to undernutrition of the nursing young. It is most remarkable that rats on certain types of faulty diets should fail to exhibit the normal maternal instinct, which is very strong in this species. Chart 8.—Lot 2189 was fed 9 per cent of protein in the form of rye and muscle. The cereal furnished two-thirds and the muscle one-third. The condition of these animals was not so good as that of Lot 2186 (Chart 7) fed a similar diet with the protein derived from wheat and muscle. They grew senile at about the same age as did Lot 2186. Two females had collectively thirty-nine young (seven litters) and weaned fifteen. Two females of the second generation had forty-four young (eight litters) and weaned fifteen. Two third Pie 2! HL? : | SAT Ee Fa rite ASS Cais Ce SRR R GRAVE NEY SSN ce Betacce cs ASSN ate : Oo (o) oO a ” eee ey oa? eee oe en McCollum, Simmonds, and Parsons 159 generation females, had each a litter (eleven young) and weaned six. One third generation female remained sterile. The young of all generations always presented an inferior appearance. The nursing periods were very long and in some cases the young remained undersized. In one instance two young which survived to the 41st day were still nursing and weighed but 60 gm. together. Lot 2184 was fed 9 per cent of protein in the form of navy beans (6 per cent) and muscle tissue (3 per cent). Other factors than protein were satisfactory. These rats grew poorly and their fertility waslow. The growth on this diet was better than we have seen on 9 per cent of navy bean protein alone. They were very old looking at 15 months. Two females had collectively ten young (three litters) and destroyed them all shortly after birth. Chart 9—Lot 2188 was fed a diet in which the total protein con- tent was 9 per cent, two-thirds of which was furnished by rolled oats and one-third by muscle tissue. The growth curves were much better than on 9 per cent of oat protein alone (Chart 1, Lot 2484), and the animals remained longer in a good condition. They were old looking at 15 to 16 months of age. Oat and muscle proteins are not of so good quality as a similar mixture derived from wheat and muscle, but of about the same value as a mixture derived from maize and muscle. This is shown by a comparison of Lot 2188, Chart 9, with Lot 2186, Chart 7, and Lot 2187, Chart 10, both with respect to the growth curves and reproduction records. There were two females in this group. They had seven litters (thirty-seven young) and killed them all. The young were de- stroyed by the mothers soon after birth... Another litter was born but destroyed before the number was ascertained. Lot 2477 was restricted to a diet containing 9 per cent of pro- tein. Barley furnished 6 per cent and muscle tissue 3 per cent. All other factors were made satisfactory by suitable additions. Their growth and ability to rear young were inferior to Lot 2186 (Chart 7) which was fed wheat and muscle tissue. They were very old looking at 14 months of age. Three females in this group had collectively seventy-five young (seven litters) and weaned only two. After these two were weaned ea eeTte 160 McCollum, Simmonds, and Parsons 161 and returned to the family cage they were killed by the older animals. Chart 10.—Lot 2187 was fed 6 per cent of maize protein and 3 per cent of muscle protein. Inorganic and fat-soluble A additions were made to correct the deficiencies in the maize and muscle mix- ture. The growth curves of this lot were distinctly below the optimum and inferior to the group fed wheat and muscle proteins. At 16 to 17 months they were very old looking and their offspring all presented an inferior appearance. Two females had collectively thirty-three young (six litters) and weaned thirteen. Two more litters were born but were killed by the mothers immediately after birth and the number was not ascertained. The nursing period in these animals was greatly prolonged because of the stunted condition of the young. Two females of the second generation were confined to this diet. They grew very poorly and never had any young. Lot 2183 was fed 9 per cent of protein derived from peas (6 per cent) and muscle (3 per cent). The growth of the rats restricted to this diet was not uniformily good, some reaching the full adult size in the usual period for well fed rats, others remaining perma- nently undersized. The curves of growth were far superior to any we have ever seen on 9 per cent of pea protein alone (Chart 1, Lot 2376). They were rough coated, old looking, and irritable at 12 months of age. Two females had each a litter of young (seven young collec- tively) and destroyed them soon after birth. Lot 2185 was fed 9 per cent of protein derived from soy beans (6 per cent) and muscle (3 per cent). The growth records were distinctly superior to those we have secured on 9 per cent of soy beans alone (Chart 1, Lot 2510). Their span of life was short. They were very old looking at 14 months. There was but one female in this group. She had two litters (eight young) but destroyed them soon after they were born. Chart 11.—Lot 2478 was fed a diet, the protein of which was furnished by barley and liver. It was comparable to those pre- viously discussed in which the protein was derived from cereal or legumes, supplemented with kidney or steak, respectively. The growth and reproduction records of these animals show clearly that liver is superior to muscle as a supplement to barley (compare Fe elds ee Paes A 162 fo ObN es Pee Jae 2s BESS Be OSS). Pe ae fay] ft ed FR | RET SRN eer eee NAL seg AE | eee | | NA IN SES SRA £ AP ERR ANE aids | | | | | NE = 163 164 Protein Values in Foods. II Lot 2477, Chart 9 with Lot 2478, Chart 11). These animals were in good condition at 16 months of age, although their coats were beginning to be rough. Three females were restricted to the diet. One whose growth was poor was sterile. The other two had collectively fifty-four young (ten litters) and weaned twenty-two. Four second genera- tion females were kept on the diet. Two of these failed to grow in a normal manner and remained sterile. The other two had twenty- six young (four litters) and weaned twenty-five. One third gener- ation female had nine young (two litters) and weaned them all. Lot 2178 was fed 9 per cent of protein, two-thirds of which was from soy beans and one-third from liver. The growth curves were distinctly below normal, but very much better than could be secured with 9 per cent of soy bean protein alone (Lot 2510, Chart 1). These animals were undersized and old looking at 14 months, which was somewhat earlier than in the case of Lot 2478 fed barley and liver, and distinctly earlier than the time of appearance of senile characteristics in rats fed wheat and liver (Lot 2179, Chart 15). Two females had collectively eighteen young (three litters) and destroyed them soon after birth. Chart 12.—Lot 2180 was fed maize and liver proteins as 9 per cent of the food mixture. As in the other diets described in this paper the cereal furnished 6 per cent and the glandular tissue 3 per cent. These growth curves were superior to what could be secured on 9 per cent of maize protein alone, but not so good as were ob- served in rats fed oats and liver (Lot 2181, Chart 13) or wheat and liver (Lot 2179, Chart 15). These animals were very old looking at 16 months and the young were always undersized and inferior in appearance. Two females had collectively fifty young (seven litters) and weaned but eight. The cause of the infant mortality in this family was mainly due to inadequate nutrition during the nursing period. This was greatly prolonged because of the stunted condition of the young. Those restricted to the diet after weaning did not thrive. Even in litters which were steadily reduced by the death of individuals at intervals of several days, the survivors did not grow better because of the lessened competition for the mother’s milk supply. — a 0 Sd Ns merry ee Bi 2 | MW i al oo | 2 a RY A Saas Sb Sele: a 6 RE SOGe SSA ae 09 Ae 4080 Gee COON RS ies. Eo eee CCC RI ERNE CCRC Soo me et eS Ne jdaaiege | | | | | SAa 166 Protein Values in Foods. II Two second generation females grew up on the diet and had collectively twenty-seven young (four litters) and weaned four- teen of them. One third generation female was restricted to the diet but never grew beyond about 75 gm. A record of infant mortality such as that of this group we know to be the result of poor quality or lack of sufficient quantity of milk secreted by the mother. It seems justifiable to conclude that it was quality rather than the amount of milk which was the determining factor, since the nursing periods were in most cases so prolonged beyond the normal period of 25 days. If the milk flow had been low it would hardly have persisted over so long a period and have enabled a few young to at last reach a state of independence. The young in the second generation were dis- tinctly better once they became able to eat the diet on which the mother was producing milk, than they were while still so imma- ture as to be confined to their mothers’ milk as their sole food. Chart 13.—Lot 2181 was restricted to a diet of rolled oats and liver, the plane of protein intake being 9 per cent of the food mix- ture. The oats supplied 6 per cent and the liver 3 per cent of the total protein. The inorganic and fat-soluble A deficiencies were made good by suitable additions of salts and butter fat. The growth curves of the group were normal and fertility essentially so, but the mortality of the young was high. The rats in this group began to look old at 14 months and deteriorated rapidly thereafter. The growth curves of this group were far superior to those of Lot 2188 (Chart 9) which grew up on a similar diet with beef muscle in place of liver. Two females in this group had collectively sixty-eight young (ten litters) and weaned forty. ‘Two second generation females had forty-three young (ten litters) and weaned fourteen. One third generation female had a litter of seven when she had been 6 months on the diet after weaning. . She weaned six of these. All the animals of this group were killed at about 18 months of age, and while they presented shabby exteriors their organs were found at autopsy to be in good condition and there were no lung infections visible. Chart 14.—Lot 2182 was fed 9 per cent of protein, 6 per cent being furnished by rye and 3 per cent by liver. The growth curves were excellent and the fertility was high. This shows that this fee PCNA SSCs COACENCAWECIE CCOCCITRS ALE AV ae SNS BU Peer aN CONTI RSET Ne Ets ° aie ek 2e'< sae Sees Se NWee! EOCACRERESE CLLRS, ae NOES By COS ERT Ce LLNS ao ASS ra ae ot te LIV V | SOREPo Or eer cae cAm ee TS BANK LY 5 Le: ae he oct ANES Le a a me —~_- any ete o*e lee Ip} yng | O9¢ 2° £9 ae ° OP UtH9 xo ai qT fo08y g°T Foov | & TOEN Ort (TORN | T°? (teatT Tp SATT o°Le | sBeg 0°0S ie ey xO 7] BY 200 73 BY 9LTZ LOT zete Lor “bl LYVHO 168 McCollum, Simmonds, and Parsons 169 combination of rye and liver proteins is of excellent quality, other- wise 9 per cent of protein would not have sufficed to promote growth so satisfactorily. The infant mortality of this group was, however, very high. This was due to the inadequacy of this amount of protein, even of excellent quality for growth, for the elaboration of milk of suitable quality for the nutrition of the young. Two females had collectively sixty-one young (nine litters) and weaned thirty-four of them. Three second generation females had fifty young (ten litters) and weaned only eight of them. One of these three had a litter of three and thereafter went 123 months before having a second litter. One rat (a male) of the third generation was restricted to the diet and grew to a size nearly normal to the adult. The rats in this group were first observed to show the coat changes characteristic of incipient retrogression toward senility at about 15 to 16 months. Lot 2176 was fed a9 per cent diet derived from peas (6 per cent) and liver (3 per cent). The growth curves were poor as compared with any secured with combinations of cereals and liver in compar- able amounts. They were undersized and their fertility was low. They assumed the characteristic coat which we have taken as an index to the transition from the youthful to the senile condition at about 13 months. Three females were restricted to this diet. One had a litter of five young but destroyed them soon after birth. The other two remained sterile. Chart 15.—Lot 2179 was fed 9 per cent of protein, 6 per cent being derived from wheat and 3 per cent from liver. Their growth curves were normal but they were never in so good a condition as those fed either oats or rye with liver (Lot 2181, Chart 13 and Lot 2182, Chart 14). Their fertility was very high but the mortality of the young was great. They began to look old at 18 months of age. Two females had collectively 127 young (eighteen litters) and weaned forty-two. Most of these young were destroyed by the mothers soon after birth. The mothers were apparently in good condition and the destruction of the young can be accounted for only on the basis of a peculiar psychological reaction of the mother owing to the nature of the faulty diet. boy } Ar ey, / 170 McCollum, Simmonds, and Parsons 174 Three second generation females had collectively twenty-three young (four litters) and weaned only one. This sole survivor was kept on the diet for 34 months but was inferior in development -and was discarded. Diets of this type in which the protein intake was about 18 per cent, and in which 11 per cent was furnished by cereals and legume seeds and 7 per cent by liver, kidney, or muscle, have proved excellent for promoting fertility and rearing of young with very low mortality. This proves conclusively that in the series of experi- ments reported in this paper the limiting factor in growth, fertility, and success in rearing young was protein. Lot 2177 was fed navy beans to furnish 6 per cent of protein and liver to furnish 3 per cent. The growth curves were inferior to any observed on combinations of a cereal and liver, but compar- able to those of rats fed peas and liver in comparable amounts (Lot 2176, Chart 14). Growth was, however, far superior to any secured with 9 per cent of navy bean protein alone (Lot 2368, Chart 1). ; These animals appeared very old at 15 months. There were three females in the group. One had a litter of two which she destroyed soon after birth. The other two remained sterile. Chart 16.—Lot 2392 was fed a diet containing but 4 per cent of protein derived from potato (1 per cent) and liver (3 per cent). A very small amount of nitrogen in this and the following rations was added in the alcoholic extract of wheat germ. The rats were unable to grow on this diet, all factors in which, other than protein, were of fairly satisfactory quality. In Period 2 purified protein (casein) was added. This modification of the diet was followed by a sharp response to growth. Lot 2394 was fed a diet identical with that of Lot 2392 except that muscle tissue (round steak) replaced the liver. The animals grew very slowly on this food mixture containing but 4 per cent of protein. In Period 2 the protein moiety of the diet was enhanced by the addition of casein. This led to growth at a rapid rate. Lot 2393 was fed a diet identical with those of Lots 2392 and 2394 except that kidney replaced the liver and muscle, respectively. This diet also contained 4 per cent of protein. Growth at a slow rate was possible on this diet. In Period 2 casein was added and this led to a stimulation of growth. It will be seen from the curves IT Protein Values in Foods. 172 OgT ase 60 te Of Upeseg | 002 McCollum, Simmonds, and Parsons 173 presented in the chart, each of which is representative of a group of four animals, that kidney protein supplements the nitrogen of the potato better than either liver or muscle proteins. These curves are included in this series to illustrate the fact that certain combinations of proteins are of sufficiently good quality to enable young rats to grow nearly as well on diets containing but 4 per cent of protein (potato and kidney) as they do on rations of similar value with respect to non-protein factors in which the sole source of nitrogen is 9 per cent of legume proteins (pea, navy bean, and soy bean). Compare Chart 1, Lots 2376, 2368, and 2510 with Chart 16. od y : P i 7 as 4 4 it 7 , 7 ony. iy UW? eae 9 [hed } > 5 ha vie ay arenas uh [oe ak th pgp Fe eR PN : ‘ d i i ) or fuse eed fe ty i upeile al hl is Ovi 4 7 > « st Pi ii fa on he) atta ’ a Fw F te Bee i : ’ . ; ie aj wid yh hy Pett 7 ' ~ ; . ’ i =f 4 . a ae SUPPLEMENTARY PROTEIN VALUES IN FOODS. III. THE SUPPLEMENTARY DIETARY RELATIONS BETWEEN THE PROTEINS OF THE CEREAL GRAINS AND THE POTATO. By E. V. McCOLLUM, NINA SIMMONDS, anv H. T. PARSONS. (From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.) (Received for publication, March 21, 1921.) It has become well established that the diet of man or animals may, when defective in sufficient degree, produce at least two dis- tinct syndromes, scurvy and beri-beri. Experimental studies on animals leave no room for doubt that a third specific deficiency disease, xerophthalmia or keratomalacia, is produced by lack of a definite organic factor essential for normal nutrition. There is much reason to suspect that certain eye conditions popularly refer- red to as night-blindness, and in the literature as hemeralopia may be, in part at least, due to lack of sufficient fat-soluble A. The widespread infections of the eyes seen in Egypt and elsewhere, may have their origin in lowered vitality of the eye structures due to lack of the dietary factor, fat-soluble A, which favors the inva- sion of the eyes by microorganisms. The evidence seems sufficient to warrant the conclusion that there are three deficiency diseases which occur in man as well as in animals. It is natural that the first step toward an appreciation of the relation of the diet to health in man should have been the result of casual observation on the extreme conditions, scurvy and beri-beri. The rarity of reference to the relation of faulty diet to the general health of peoples in the writings of those who are now discussing health problems, shows that it is not at present accepted by many that there is any important relation of the food supply. to well being, except in extreme cases of one-sided diet in which the defects are sufficiently grave to induce the development of a “deficiency”’ disease. The reason for this is easy to understand. Our stand- ards of what constitutes wellness and illness, as well as of what con- 175 176 Protein Values in Foods. III stitutes normal physical development, are relatively low. People are generally considered well until they admit that they are ill, or until they look so unwell as to attract attention. The difference between the average physical development of men and women, as contrasted with the best specimens in the community, is very great, yet we are accustomed to regard as normal many persons who are but poorly constituted physically. Malnutrition is painless, and leads to susceptibility to other agen- cies which may induce discomfort. The sequence of events is not ordinarily appreciated, and the cause, therefore, escapes notice. Low vitality, low resistance, and inefficiency, and a tendency to cumulative fatigue, are what we should expect in man to result from adherence to a faulty diet when the faults are of a lower order than would be necessary to bring about an attack of a “deficiency” disease. Although a few leaders in the new movement for the betterment of the health condition of children are urging the impor- tance of properly selected food, the idea is new and not generally - appreciated. There is much reason to believe that one of the most important contributing factors in industrial fatigue is the low re- cuperative power that results from an unsatisfactorily selected food supply. The data regarding the animals described in this paper, all of which were subjected to diets which were faulty to a certain extent, afford striking evidence that such types of diets in- terfere with the completion of the life history in a normal manner. This is true, notwithstanding the fact that in some instances the faults were by nomeansmarked. The extent to which the animals were injured, and the manner in which they were affected, afford food for serious reflection concerning the similar experience of man and its probable causes. The results of our experimental studies on the rat have led us to an appreciation of the short-sightedness of the view that a diet is satisfactory if it is not sufficiently poor to cause the development of a “deficiency” disease. Although the importance of a diet containing an abundance of milk and eggs has become generally accepted to have a distinct therapeutic value in the treatment of tuberculosis, it has not been widely appreciated that a similar diet should be very effective as a preventive of infection or of the flaring up of the disease. McCollum, Simmonds, and Parsons 177 We have given a great deal of attention to the problem of demon- strating the effects of relatively slight defects in the diet on the gen- eral health of the rat, and on its capacity to reproduce and rear young, and to remain vigorous to an advanced age. We have united the study of these problems with the study of the extent to which the proteins of certain foods supplement those of others. It has been easily possible to develop these lines together, and indeed it was not possible to pursue the one phase of the work independ- ently of the other, for in such systematic studies one meets with the nice gradations in quality of proteins which afford the oppor- tunity to make refined observations on the physiological well being of the animals as affected by the variations in the diet. We deem it a matter of great importance to discover in its incipi- ency when an animal is brought into a state of nutritive insta- bility. Under such conditions it is unable to support a burden of any appreciable magnitude, and it matters little whether the bur- den be that of producing milk, or bearing young, or of withstanding fatigue. It is such shades of difference in quality which occur in human experience in numerous cases. The type of experiment which we have come to employ, in which the performance of repro- duction and nursing of young comes under observation, brings to light this ‘twilight zone” of nutrition in which the vital powers of the body are below the optimum. It is desirable at this time to discuss the changes in appearance of the rat, and in its behavior as it grows old, in order to make possible an appreciation of the manner in which we have managed our experiments. The well fed rat exhibits up to about 18 months of age the ap- pearance of full vigor as shown by plumpness of the muscles; bright, - prominent eyes; clean, smooth, pink skin, easily observable on the feet and ears. The tail appears clean and free from scales. The coat appears to consist of hairs of approximately uniform length, and is glossy and well kept. Such animals are not apprehensive and permit themselves to be handled without becoming annoyed. They do not attempt to escape when handled with care. At a certain point in their life history there is a characteristic change in the appearance of the coat. This consists of the promi- nence of certain hairs which are scattered over the entire body, and which look coarse and are distinctly longer than the under coat. 178 Protein Values in Foods. III Physiologically youthful rats, irrespective of age, give attention to their coats. When the latter differentiates into hairs of two sorts the animals become neglectful of their toilets and look less clean and attractive than formerly. The skin on the ears, feet, and tail generally loses its waxy and healthy surface and appears dry, harsh, and often scaly. ‘ With this change in appearance there is usually seen a change in the habits of the animals. They tend to sit and sleep with the head tucked under the body or with the face downward. Healthy youthful rats sleep lying stretched out on their ventral sur- face or partly turned on one side, with the front legs extended and with the head resting on the paws. When the cage of youthful rats is opened they are awakened if asleep, and show an interest in their observer, but do not show signs of fear. Animals which are developing senile characters are generally dozing and their atten- tion is not usually attracted by careful opening of the cage. When touched they spring up in surprise and show irritation at being -molested. As time passes this behavior becomes more marked. The irritability, somnolence, dirtiness, roughness of the coat, and scantiness of hair become progressively more noticeable. To some extent, according to the nature of the fault in the diet, specific changes appear, such as attenuation of form and baldness over limited areas, but these latter are more characteristic of animals fed certain foods than they are of diets from different sources haying like specific dietary deficiencies. A further example of this peculiar effect of diet due to some obscure causes is seen in the frequent occurrence of very fine, short, glossy hair, sugges- ’ tive of a mole in rats fed largely on maize or kafir corn. Another type of observation which has proved to be of value as an index to the quality of the diet which enables one to - make fine shades of distinction in the value of the protein factor in a series of experiments which are properly planned, is the success of the females in rearing their young. This is a more sensitive index than is fertility, although in general low fertility and high in- fant mortality tend to run parellel. The fertility may, however, be high, and but a small number of young be reared to the weaning age. This mortality is, we believe, generally due to poor quality | ‘McCollum, E. V., and Simmonds, N., Unpublished data. McCollum, Simmonds, and Parsons 179 of the milk secreted by the mothers rather than lack of milk produc- tion. Itis not always possible to get satisfactory information on this point, but since the stomach when filled with milk can be readi- ly seen in a young rat during the first few days when the animal is properly illuminated, we have been able to observe the amount of milk produced in a sufficient number of cases to lead us to believe that in general the failure of the young to grow is due to deficiencies in the composition of the milk rather than to lack of sufficient amount. There is a marked tendency among female rats fed such diets as are described in this paper to destroy their new-born young. If © for any reason they become scattered about the cage the mothers will not be solicitous about returning them. This abnormal psy- chological reaction of the mothers toward their young represents a new and hitherto unstudied effect of faulty nutrition. It serves to emphasize the deep seated relation of the character of the diet to the mental reactions generally, for if the maternal instinct is so perverted, those attributes of the nervous system which are less deeply ingrained should be influenced in even greater degree. In contrast to the behavior of the mothers on experimental diets, details of which are given in the legends to the charts, it is of in- terest to contrast the behavior of the mothers in our breeding stock. It is our custom to isolate the females when it is easily observable that they are pregnant. This cannot be determined with cer- tainty until about the end of the second week after fertilization. For some years it has been our custom to isolate two or three fe- males in our breeding stock in a single cage. They each prepare a nest for the reception of their young, but when the cage is cleaned within a few days, and fresh bedding replaces the old, the young are mixed and returned to the cage together. From this time on the mothers do not separate their own young, but allow them to remain togetherin one nest, which may contain as many as twenty to thirty individuals. They nurse each other’s young in- discriminately. A mother will spread herself over the nurslings, and as many as possible attach themselves to her. After a time she will free herself from them and another mother will take her place. They seldom kill a young one and the infant mortality is almost zero. Only very infrequently have we seen a young rat under such circumstances unable to compete successfully with the others for his share of the milk supply. 180 Protein Values in Foods. } Ill In experimental animals on diets which are faulty, it appears, as stated above, that the milk flow is ordinarily sufficient to meet the needs of the developing young, but the quality may be poor with respect to some one or more factors, and cause failure of the animals to grow satisfactorily. It appears, however, that some mothers do not produce enough milk, at least after a time. We have occa- sionally seen a litter grow fairly well for 4 to 10 days, then all die within a few days. The cause of such behavior has in general not been ascertained. We have given some attention to determining how long a female rat may continue to give milk provided she is kept with young of a suitable size. In one case in which the young were removed and replaced by smaller ones at intervals of a few days, a female rat continued to secrete milk up to 141 days in quantity sufficient to induce some growth in four young about 8 to 10 days old. It is known that these young did not eat of the mother’s food supply.t When the fault or faults in a diet are sufficiently marked, growth is interfered with in the young, and the effects soon become appar- ent in the external appearance of the animals, even in the adult. We have made an effort to determine how sensitive the rat is to slight degrees of faulty composition in its diet. This can be done by prolonging the period of observation far beyond the growth period, and including a record of the fertility and success in rearing young and the time of the appearance of the first signs of old age. Since the character of the diet is but one of the factors which influence the well being of an animal, we have maintained the hygienic condi- tions such as illumination, temperature, ventilation, and oppor- tunity for exercise uniform throughout our entire rat colony. Fur- thermore, the differences in the condition and vitality of the animals as measured by the tests described are due in all cases to variation in the quality of the diet, which falls within the limits of what may occur in combinations of ordinary natural foods of animal and vege- table origin. As an example of the objective which we have had in mind in conducting these studies, the comparison of the relative values of the proteins in the several rations may be cited. The value of the proteins in a food or mixture, or the value of a purified protein, has not been studied by methods more sensitive than that of com- paring their efficiency for the support of growth. We have learned McCollum, Simmonds, and Parsons 181 that much more accurate comparison of quality in proteins can be made by maintaining a series of animals on a diet otherwise com- parable, but containing various levels of the same source of protein throughout the growing period and into adult life, so as to dis- cover the plane which just suffices for this result without inducing inferiority in appearance or size. Under these conditions the mother rat should she produce and attempt to suckle alitter would be confronted withan added demand for nutriment by the young. The young at the nursing age are more sensitive to faulty diets than are older animals which have been satisfactorily nourished during the first weeks of life. We have thus set up experimental conditions in which an unusual demand is made upon the mother, and extraordinary sensitiveness is characteristic of the young be- cause of their early age and lack of reserve power. It is of more than ordinary interest to find, as we have, that a diet may be sufficiently good to enable a group of animals to grow to apparently the normal adult size, and even to support a fair amount of reproduction and moderate success in the nursing of young to a state of independence, and yet be inadequate for the nourishment of a family through several generations. This fact is brought out well in many of our studies in which families during several generations have been restricted to a monotonous diet. If the diet is properly constituted with respect to all its parts the animals tolerate, at least to the fifth generation,such a monotonous food supply without any signs of injury. If, however, there are factors which are even slightly below the optimum, the vitality of the animals falls off in each succeeding generation, and the strain dies out. Sometimes this happens in the second, third, or fourth generation. An interesting difference in the effect of stunting of young ani- mals is seen, depending on the cause of the interference with growth. When the protein content of the ration is too low, young rats remain approximately normal in form, or at least they remain long and lithe. When the inorganic content of the ration is faulty on the other hand, they develop a short and stocky form. In the former case they respond to a better protein supply and look approximately normal, but when deformed by lack of sufficient calcium they never assume the normal form as the result of cor- rection of the diet.! THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 1 182 Protein Values in Foods. III The discussion of the growth, fertility, infant mortality, and appearance of the first recognizable signs of senility which is pre- sented in the legends to the charts, will make clear the fact that even very slight differences in the quality of the food mixture with respect to protein and fat-soluble A at least, can exercise a marked influence on the vitality of the rat. Animals may be brought to a state by feeding ordinary cereals and potato, supplemented in a fairly satisfactory manner except for the protein factor, in which their nutritive condition is unstable, and where very slight shifts in the character of the food for better or worse, make easily observ- able differences in their capacity to perform satisfactorily the func- tions of reproduction, or to retain full vigor to an advanced age. The experimental work reported in this paper is limited to a study of diets in which the protein was derived from a mixture of a cereal grain and potato. The quality of protein was estimated as accurately as possible by existing methods. In some cases casein was added to the diet in order to bring out the fact that the protein in a certain mixture was actually the limiting factor. The results show that the potato nitrogen serves to some extent to supplement that of the cereal grains, but that it is not so valuable for this purpose as are certain Senn tissues such as kidney, liver, or muscle.” The dita furnish definite evidence that there is no marked differ- ence in the capacity of the nitrogen compounds of the potato to enhance those of the different cereal grains, as was found to be the case in similar combinations of cereals with animal tissues. The latter are individually more effective with certain grains than with others, whereas the potato is less highly valuable in any combina- tion, and more similar in its relations to each. These studies lend support to the general proposition that it is not wise to attempt to combine foods which have the same func- tion, such as have the seeds and tubers. Both are storage tissues of plants and have in great measure the same dietary properties. They do not, therefore, serve effectively to make good each other’s shortcomings. * McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem.,. 1921, xlvii, 139. McCollum, Simmonds, and Parsons —-183 SUMMARY. We have described methods of experimenting which are more sensitive than rate of growth for yielding information concerning slight gradations of quality in foods. These involve fertility, success with the rearing of young, longevity, the preservation of youthful characters, and the stability of the nervous system. By establishing standards for comparison these indexes to well being reveal with great refinement the physiological condition of the animal. The supplementary values of the nitrogen compounds of the potato for those of the cereal grains and legume seeds have been studied witha view to making refined observations as to the biolog- ical values of several combinations. These experiments have brought to light the following facts: The nitrogenous compounds of the potato tend in some degree to enhance the biological value of proteins of cereals and legume seeds, but not to as great an extent as do the proteins of such ani- mal products as kidney, liver, muscle,? or milk. Marked differences are observed in the extent to which animal tissues enhance the quality of the proteins of individual seeds. No such selective efficiency has been observed in the relation of the amino-acids of the potato to those of cereal or legume seeds. The potato seems about equally effective as a supplement for all which have been studied. Chart 1.—These records are typical growth curves of rats fed diets in which wheat, rolled oats, maize, peas, and millet seed, respectively, furnish the only source of protein. The amount of each seed was adjusted so as to make the protein content of the diets 9 per cent of the food mixture. In each case the necessary inorganic salts and butter fat were added to make good the well established deficiencies of these seeds. Wheat proteins are seen to produce the best growth curves and the highest fertility. No young were secured from the animals fed maize, peas, or millet seed. A few young were reared on the wheat and oat diets. Charts 5 and 8 show that the infant mor- tality is in a great measure due to inadequate protein moiety of these diets. These records serve as the basis of comparison with those contained in the other charts. Bik: 7 i = = a4 - McCollum, Simmonds, and Parsons 185 In all cases the food mixture was ground together so that all its ingredients were consumed in the proportions shown in the formula, as is our invariable practice. Chart 2.—These curves illustrate the growth and reproduction of a group of rats fed from weaning time on diets, the protein of which was derived from millet seed and potato. This seed and tuber served as the only source of fat-soluble A. Lot 1398 contained about 11 per cent of protein (N X 6.25), approximately 9 per cent being derived from millet seed and 2 per cent from potato. The growth records were poor, but distinctly better than was observed in Lot 1383 (Chart 1), in which 9 per cent of protein derived entirely from millet seed was the sole source of nitrogen. No young were secured in the latter group, whereas afew were secured from Lot 1398. This group contained two females. These had together eighteen young (three litters) of which nine were weaned. ‘Two of their daughters were reared on the diet but remained sterile. The young of this group were all undersized for their age and the nursing period had to be prolonged to approx- imately twice the normal period of 25 days in order to enable the young to survive on the family diet. There is every reason to believe that the high mortality of the young in this group was the result of qualitative inadequacy of the milk secreted by the mothers. All these rats aged early. They were very senile at 8 to 10 months. Lot 1452 had a diet like that of Lot 1398 except that it contained 10 per cent of casein to supplement the protein. The good effects of this addition are easily seen in the character of the growth curves. Millet seed does not appear to be a very wholesome food grain. The fertility of the females in this group was far below normal and the mortality of the young high. These young were not destroyed soon after birth but only after they had time to show from retarded growth that the quality of the milk of the mother was inferior. Two females had together twenty-six young (three litters) of which twenty were weaned. Two daughters were confined to this diet. One had one litter of four young which she destroyed. The other had one litter which she destroyed, and died in the delivery of a second litter. Fae ets] | i | IN IN Je Hale CECE Ne oo et tl mE McCollum, Simmonds, and Parsons 187 Chart 3.—The animals in Lots 1407 and 1461 were fed diets similar to those of Lots 1452 and 1398, respectively (Chart 2), except that the former had an added source of fat-soluble A. Since millet seed contains distinctly more of this factor than wheat or oats, or than peas or beans, the effects of adding butter fat to this ration was scarcely noticeable. Lot 1407 contained three females. They had altogether thirty- four young (five litters) and weaned fourteen of them. Two females of the second generation were confined to this diet. One had a litter of six but they were very puny. Some of these were destroyed when very young. A litter which was nursed 11 days was then only about 60 per cent as heavy as it should have been if the mother’s diet had been satisfactory. Lot 1461 was fed millet seed, potatoes, butter fat, and casein in Period 1. Little growth was possible on this food. In Period 2 sodium chloride and calcium carbonate were added. Growth took place at a rapid rate after this addition was made. There were three females in this group. They had forty-six young (seven litters) of which thirty-one were weaned. One female of the second generation was restricted to the diet and had a litter of eleven young but killed them all, and died herself soon afterward. Other young from the original group on this diet were very inferior. Where these young were not destroyed when very small they remained stunted while nursing. This we interpret as being due to faulty composition of the milk secreted by the mother. It is probable this was the result of some toxic substance passing into the milk from the millet seed. These records all show that millet seed when fed in liberal amounts causes injury. This we provisionally interpret as due to ‘the presence of some toxic substance in milletseed. That ashort- age of fat-soluble A was not responsible for the results observed is shown in Chart 2. Among the farmers of the middle west there is a common belief that liberal feeding of millet causes injury to farm animals. The toxicity of millet seed is still under investigation. Chart 4.—The records in this chart are those of rats which were fed diets in which wheat and potatoes furnished the sole source of fat-soluble A. The protein content in the diet of Lot 1399 was approximately 9 per cent. That this amount of protein from these > 3 rt) ° aa am So Fisinseece EET) a Pa Tie NI 8 BAS » © RX ci 188 as Gap eaee ; .Anéneeue Be et CEES JER0 Ut Rae Cast Ts ae nN nu ei a] 189 190 Protein Values in Foods. III] sources is capable of inducing good growth and moderately high fertility is shown by a comparison with Lot 1408, Chart 5. The limiting factor in Lot 1399 is fat-soluble A. There were two females in this group. One had two litters (eleven young) but destroyed them soon after birth. The other remained sterile. Lot 1453 had a diet similar to that of Lot 1399 except that it contained 10 per cent of casein to supplement its protein content. This exerted marked improvement in their growth, fertility, and longevity. It is of special importance to compare these curves with those of Lots 1399 and 1408. The diet of Lot 1399 is very much improved either by the addition of protein or fat-soluble A. This illustrates a principle which many investigators have failed to grasp; v2z., that all the other factors in an experimental diet must accurately be evaluated before judgment can safely be formed con- cerning the quality of a food or preparation in respect to a factor under investigation. For example, the diet of Lot 1453 appears to contain sufficient fat-soluble A. Actually it is distinctly below the optimum in this substance. It contains the same amount of fat- soluble A as does the diet of Lot 1399, but this deficiency is not apparent in Lot 1453 for some months because of the excellent quality in all other factors. Deficiency in fat-soluble A, however, became manifest in the lowered nutritive value of the milk and caused injury tothe young, especially in the third generation. Two females in this group had collectively fifty-two young (seven litters) and weaned forty-two of them. One second genera- tion female had twenty young (three litters) and weaned eleven. One third generation female had eleven young (two litters) and weaned nine of them. The young were strong and vigorous in most cases. A few of the second litter obtained from a third gener- ation female were, however, quite undersized and puny. Chart 5.—The curve of Lot 1462, Period 1, illustrates the behav- ior of young rats fed a diet in which all the inorganic salts were furnished by a mixture of wheat, casein, and potatoes. No growth could take place on this diet even though all factors other than the inorganic elements were properly constituted. In Period 2 when calcium carbonate and sodium chloride were added growth took place at once and at the maximum rate possible. There were three females in this group. They had together forty-five young (six litters) and successfully weaned them all. a im K PN ee pees, SSE FERPA a WORE: Re ae 8 (ef ys 191 Ped Be) 3/3: Clie: ty » i) ® “8 7 =) 120 192 Protein Values in Foods. III One second generation female was restricted to the family diet. She had two litters (sixteen young) and weaned them all. One third generation female had a litter of nine and reared them all. The young were always in excellent condition. This is an example of the suecess which we regularly see in successful growth and rearing of young when the mother’s diet is satisfactory. Lot 1408 derived all its protein from wheat and potato. The protein content of the diet was about 9 per cent, two-thirds of which was derived from wheat and one-third from potato. This amount of protein from these sources did not support growth at the maximum rate possible but the results indicate that there is some improvement in quality by combining proteins from these sources. Two females in this group had twenty-one young collectively (four litters) and weaned eleven. One female in the second gener- ation had eighteen young (three litters) and weaned eight of them. Two third generation females had fifteen young (two litters) ~ of which twelve were weaned. The young in all these litters were small for their ages, and the nursing period was considerably pro- longed in all cases in order to bring the young to a state of inde- pendence. Since these litters were not large in the first or second generation, it would seem probable that the failure of the young to thrive in a normal manner was due to faults in the quality rather than to lack of sufficient milk. A comparison of the reproduction records of these groups (Lots 1462 and 1408) shows clearly that the infant mortality in the lat- ter was the result of an inadequacy of the protein of the diet. On - low protein diets, or diets containing protein of poor quality, we have regularly observed the necessity of a prolonged nursing period in order to fit the young to subsist on rations suitable for the adult. Chart 6.—Lot 2150 illustrates a typical record of a group of rats fed from weaning time on a diet in which the protein was derived from peas and potatoes. Two-thirds of the protein was supplied by peas and the remainder by potato. The diet contained 9 per cent of protein (N X 6.25). Growth on this diet was approxi- mately normal, showing that the mixed proteins of peas and potato at this plane of intake are far superior to pea proteins alone fed at this level (compare Lot 2376, Chart 1). Since dried potato is so fo) At » a o 8 BE SEE RACE peda Naar NT NIN, Ba | ip ga 2S i(Sals) los dgjddae Ai mee : eZ Lat / 7 2150 R172 2172 Pnd gen. 2172 rth o ng 194 Protein Values in Foods. III low in protein it is not possible to make a direct comparison of these mixed proteins with potato protein alone. That this content of protein from these sources is below the optimum is shown by a comparison of Lots 2150 and 2172. There were three females in this group. They had collectively six litters (eighteen young) but none was weaned. Two other litters were born but were destroyed by the mother before the number was ascertained. The rats in this group looked very old at about the age of 1 year. Lot 2172 had a diet similar to that of Lot 2150 except that it contained casein but no butter fat. The growth records were remarkably good, notwithstanding the fact that all the fat-soluble A was derived from peas (31.8 per cent) and potato (25 per cent). One male reached a weight of 360 gm., and a female, a non-preg- nant weight of 245 gm. There were two females in the group. They had together twen- - ty-three young (four litters) and weaned thirteen of them. The young were inferior in appearance, but did not develop xeroph- thalmia. One mother, however, developed inflamed eyes. Four second generation females were restricted to the food mixture but all died after being subjected to this diet for 4 to 5 months without having any young. Lot 1459 was fed a diet similar to that of Lot 2172, except that it contained about double the amount of peas. The first genera- tion fed this diet grew well, but the fertility was low and they aged very early. They were ready to die at about a year. There were two females in this group. They had collectively nineteen young (three litters) 4nd weaned nine of them. Two females in the second generation were restricted to the diet. One became pregnant, but died while giving birth to young. The other died at the age of 6 months. This ration contained a high protein content (about 27.5 per cent), more than half of which was from peas. Pea protein when fed at high levels apparently causes damage to animals. This diet (Lot 1459) contained con- siderably more fat-soluble A and protein in its content of peas than did that of Lot 2172. All other factors were equally satis- * McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxvli, 287. McCollum, Simmonds, and Parsons 195 factory or to the advantage (water-soluble B) in the case of Lot 1459, yet the nutrition of the latter group was distinctly inferior to Lot 2172. These results harmonize with the observation of Osborne and Mendel! that young rats fed purified pea protein as the sole source of this factor steadily declined. The records of Lot 1459 should be compared with those of Lot 1468 (Chart 7). The diets of these groups were similar but the latter contained more fat-soluble A, and this resulted in marked improvement in their condition. Chart 7.—The rats of Lots 1414 and 1405 derived their protein entirely from peas and potatoes. The pea protein constituted about 88 per cent of the total. The protein content of these food mixtures was 17 to 18 per cent. Even at this high level of intake these proteins are inferior to a mixture derived from wheat and potato, as is shown by a comparison of these groups with Lot 1408 (Chart 5). Lot 1414 contained two females. They had collectively three litters (nineteen young) and weaned all of them. One female of the second generation had one litter of three and weaned them. Some of the original group were kept on the diet more than 10 months but by 8 months they appeared old looking. Lot 1405 was fed a diet closely similar to that of Lot 1414 except that it contained no butter fat. The inferiority of these animals as compared with Lot 1414 was due entirely to the lower intake of fat-soluble A. A striking illustration of the danger of misinterpreting data obtained in nutrition experiments of this character is afforded by a comparison of four sets of records in these charts. Lot 2172 (Chart 6) derived all its fat soluble-A from 31.8 per cent of peas and 25 per cent of potato. The protein was enhanced by the addition of casein and the necessary inorganic salts were added to complete the mineral content. During the first year of life these animals appeared normal except for the high infant mortality. Lot 1459 (Chart 6) had more peas, the same amount of potatoes and casein, and the same salts. It, therefore, secured more pro- tein and more fat-soluble A than Lot 2172, yet the development of Lot 2172 was much more satisfactory. If we did not have the records of Lot 2172 we should have made the statement from the results of Lot 1405 that peas are very low in fat-soluble A. How- 4 Osborne, T. B., and Mendel, L. B., Z. physiol. Chem., 1912, lxxx, 307. ee eee eA eck) Vaio eA NCEE mH INS to ‘ag ENR Ce i SE COCA aS aT CCCI McCollum, Simmonds, and Parsons 197 ever, when the protein content was improved with casein, as in Lot 2172, the fat-soluble A derived from 31.8 per cent of peas and 25 per cent of potato had more beneficial effect than more than twice this amount taken with the same food mixture containing protein of lower biological value. The addition of butter fat (fat- soluble A) to Lot 1405, however, also made them develop distinctly better, as is shown in Lot 1414. Lot 1405 (Chart 7) was fed 72.5 per cent of peas and therefore more fat-soluble A than either Lot 2172 or Lot 1459 (Chart 6), yet because the casein was omitted in Lot 1405 the animals were very inferior. The protein and phosphorus content of Lot 2172 is lower than that of Lot 1405, yet the growth was much better in the former than in the latter. This was due to superiority in the quality of the protein in the diet of Lot 2172, and this factor was of sufficient biological significance to make a decided difference in the well being of the two groups. These illustrate border-line cases of malnutrition. They represent a type of diet in which a small variation of quality upward in one or another factor, as protein or salts, may so modify the early history of the experimental animals as to make a particular factor, e.g. fat-soluble A content, appear in one set of results to be adequate for the physiological needs of the animals, while a variation in the quality downward either in protein or salts, such variation remaining at the same time well within physiological limits, may entirely change the deductions with respect to the value of the constant factor fat-soluble A. This same idea is also brought out in Chart 8, Lot 1454, and also in Chart 11, Lot 1451 (see discussion under these charts). Lot 1468 in Period 1 was fed a diet of peas, potatoes, casein, and butter fat. The growth of the animals on this food was in no case more than half normal (Period 1). In Period 2 sodium chloride and calcium carbonate were added. Thisled toa prompt response with growth and reproduction. It appears probable from these results that there is less injurious effect of high pea protein inges- tion when its quality is enhanced by a casein addition than when the same pea protein content is taken without such improvement. This group contained two females. They had collectively four- teen young (two litters) and weaned them all. Two second gene- ration females had collectively twelve young (a litter each) and weaned eleven. These mothers were discarded after weaning 198 Protein Values in Foods. III their young. These animals presented a dirty appearance and were badly stained with their own urine. Chart 8.—Lot 2149 was fed a diet, the protein of which was derived entirely from rolled oats and potato. It contained 9 per cent of total protein (N X 6.25), two-thirds of which was furnished by oats and one-third by potato. Other factors than protein were satisfactorily supplemented. Growth on this diet was somewhat below the optimum, but the protein derived from this combination is of better quality than a similar amount of oat protein alone. This combination does not possess so high a biological value as does a protein mixture from wheat and potato in the same propor- tions. This is illustrated by Lot 1408 (Chart 5). Three females grew up on this diet. They had collectively thirty-one young (four litters) but none was weaned. The males were apparently in good condition at 7 months of age, but the females presented a poorer appearance. Those young which were “not killed by the mother grew so little while nursing that it is evident that the nutritive value of the milk was low. Lot 2173 derived all its fat-soluble A from rolled oats and po- tato. In other respects this diet was comparable with Lot 2149. The protein in the diet of Lot 2173 was enhanced by the addition of casein. Three females grew up on this diet. One female had two litters, another, one litter, collectively eighteen young, and weaned but four of them. A third female became pregnant but died while giving birth to her young. The young were in a very poor con- dition. Two daughters of these mothers were restricted to the diet but never had any young. All these animals aged early. Their span of life was only 5 to 7 months. ; Lot 1454 presents remarkable growth curves on a diet which all previous experience indicated to be very poor in fat-soluble A. All of this factor was derived from 62.5 per cent of rolled oats and 25 per cent of cooked dried potato. One male reached a weight of over 400 gm. Two females each had three litters, collectively forty- seven young, and weaned twenty-six. One daughter grew up on the diet and had two litters but lost them all during the first few days. These young were in poor condition and developed xeroph- thalmia in some cases. At the age of 11 months the animals appeared very old, but autopsy revealed nothing pathological. oa — “aA +» ee \ \ gl al. \ | —“™e. a » a 7 ’ te += 2 a ei, a > a 2 So =] +=] o fo) So STVY: oO So o ou ee Cee) ee cea. ee ee 199 \ \ Np ON fal 1454 ing Df yo D oD 200 Protein Values in Foods. III Our experience in studying the dietary properties of the oat ker- nel has demonstrated it to be the poorest of the cereal grains in fat-soluble A. Steenbock and Gross report the white potato to be an inadequate source of this factor. In interpreting the value of this diet with respect to fat-soluble A, Lot 1454 (Chart 8) and Lots 1409 and 1400 (Chart 9) should be compared. On the diet of Lot 1400 but little growth could take place, and xerophthalmia developed. Lot 1409, whose diet differed from that of Lot 1400 only in containing butter fat, was able to grow and reproduce. This was due specifically to the increased content of fat-soluble A. Lot 1454, whose diet differed from that of Lot 1400 only in that its protein content was improved by the addition of casein was also able to grow normally and reproduce and rear some young. The infant mortality was higher than that of Lot 1409. The content of fat-soluble A was the same in the diets of Lots 1400 and 1454. Such experimental results as these show the fallacy of deductions such as have been made by Hess and Unger® who fed infants during several months on a diet somewhat low in fat-soluble A but of excellent quality in other respects, and concluded that the factor fat-soluble A is not of great importance in practical human nutri- tion. Judgment on this matter must be based on a full appre- ciation of the difference between border-line malnutrition with its attendant grave dangers from infections or unfavorable reaction to any chance modification of the diet so as to reduce its quality in any way. Chart 9.—Lot 1409 was fed a diet in which the protein was all derived from rolled oats and potato. The content of protein in the food mixture was about 12.5 per cent, about 16 per cent of this being derived from potato, and 84 per cent from oats. All other factors were adequately supplemented. The rats which were confined to this diet never grew to the nor- mal adult size. There were two females in this group. They had collectively eighteen young (four litters) but only two were weaned. The mothers destroyed the young. One of these rats died during the birth of her third litter. One second generation female grew up on the family diet and had fifteen young (two litters) and * Steenbock, H., and Gross, E. G., J. Biol. Chem., 1919, xl, 501. ° Hess, A. F., and Unger, L. J., J. Am. Med. Assn., 1920, Ixxiv, 217. y E ; =, | e c=) aN CCCAYIN OR ok | ad Eze Potatoes 202 Protein Values in Foods. III weaned thirteen of them. One third generation female had one litter of six and successfully weaned them. Lot 1400 was fed a diet like that of Lot 1409 except that it did not have asupplemental addition of fat-soluble A. Growth on this diet was slow and incomplete. The rats all died in 4 to 5 months after being placed upon the ration. Xerophthalmia developed in three of the four rats confined to this food mixture. This diet with added fat-soluble A is capable of inducing nearly normal growth and supports reproduction and rearing of some young (Lot 1409). This ration with added protein has induced the maximum amount of growth in rats and supported reproduc- tion and rearing of some young, although the infant mortality was high (Lot 1454, Chart 8). This illustrates the fact that one is not justified in drawing conclusions concerning the content of fat-solu- ble A in a foodstuff without furnishing evidence that all factors in the diet are accurately evaluated. Lot 1463 was fed in Period 1 a diet consisting of rolled oats, potatoes, casein, and butter fat. The inorganic content of the diet was entirely derived from these sources. Practically no growth could take place on this food. In Period 2 sodium chloride and calcium carbonate were added. This led to marked response with growth to the full adult size. Three females in this group had collectively thirty-eight young (six litters) and weaned thirty-six. One second generation female was restricted to the diet. She had fifteen young (two litters) of which fourteen were weaned. One third generation female had a litter of nine and weaned them all. These young were in all cases in excellent nutritive condition. A comparison of the reproduction records and infant mortality of Lot 1463 with those of Lot 1454 (Chart 8) shows clearly the beneficial effects of a liberal supply of fat-soluble A. This is shown by a mortality among the young of 45 per cent in Lot 1454 in the first generation and of 100 per cent in the second generation. In Lot 1463, 95 per cent of the young were weaned. The ap- pearance, size, and condition of the young of these two groups presented as marked a contrast as did the infant mortality. Chart 10.—Lot 1406 derived all its protein from 69.5 per cent of maize and 25.0 per cent of potatoes. All other factors were Ratio: a oO S 3 gS aa r fat Case ih Butt & 260 204 Protein Values in Foods. III satisfactorily adjusted by suitable additions. Growth was some- what retarded ‘because of the quality in the protein factor. Three females grew on this diet. They had collectively thirty- five young (six litters) and weaned eleven of them. ‘Two second generation females were restricted to this diet. One had a litter of two young and after a long nursing period weaned them in poor condition. The other remained sterile. The animals in this group were always somewhat undersized and of poor appearance. Lot 1460 in Period 2 had a diet like that of Lot 1406 except that it contained 10 per cent of casein. In Period 1 no inorganic salts were added. Without these growth was suspended, but with them the animals grew rapidly. This shows that in Lot 1406 the quality and amount of protein was the limiting factor. Three females grew up on this diet. They had collectively fifty young (seven litters) and weaned forty-eight of them in good condition. One second generation female had seventeen young (two litters) and weaned fifteen of them. One third generation _ female had fifteen young (two litters) and weaned seven. The young were vigorous and active but their coats were not as glossy and well kept as those of other rats which we have observed. The contrast between these young and those of Lot 1406, on a lower protein plane, was very marked. Chart 11.—Lot 1397 derived both protein and fat-soluble A entirely from maize and potato. The maize used in these experi- ments was of the yellow variety. A comparison of these growth curves with those of Lot 1451 (Chart 11) shows that notwithstand- ing the high content of yellow maize in this food mixture, protein was the first limiting factor in preventing normal development. Lack of sufficient fat-soluble A was, however, a very important deficiency in this diet, as shown by comparing the curves of Lot 1397 with those of Lot 1406 (Chart 10). There was more fat-soluble A in the diet of Lot 1397 than in that of Lot 1451, yet the satisfactory content of protein in the latter diet made it appear, in the absence of a complete series of biologi- cal tests, to contain a satisfactory amount of this factor. Al- though certain yellow maizes doubtless contain more fat-soluble A than certain white varieties it seems to us that there has been some exaggeration of the value of yellow maize as a source of this 2 AR Bie s Be Af. AE RRS eer iz. Ce ee Loe ei ct NA Chia RNS ya CC te Po RT ae i ee SESE SANS CORES CERIN NE eq OB = g 205 206 Protein Values in Foods... III factor.?. If we were limited to experiments with diets of the type fed Lots 1451 (Chart 11) and 1454 (Chart 8) we could easily have fallen into the error of interpreting the results as indicating that diets containing 60 to 70 per cent of yellow maize or even rolled oats supplemented with potato to the extent of 25 per cent of the food mixture would prove satisfactory in their content of fat-sol- uble A. More carefully planned experiments eliminate the dan- ger of error in studies of this nature. Lot 1451 contained two females. They had collectively sixty- four young (eight litters) and weaned fifty-nine. Two second generation females had five litters (twenty-seven young) and weaned twenty-two. Two third generation females had collec- tively eleven young (two litters) and weaned ten. All the young were in good condition, but in a few cases the hair was short and. silky in appearance. 7 Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xli, 81.- SUPPLEMENTARY PROTEIN VALUES IN FOODS. IV. THE SUPPLEMENTARY RELATIONS OF CEREAL GRAIN WITH CEREAL GRAIN; LEGUME SEED WITH LEGUME SEED; AND CEREAL GRAIN WITH LEGUME SEED; WITH RESPECT TO IMPROVEMENT IN THE QUALITY OF THEIR PROTEINS. By E. V. McCOLLUM, NINA SIMMONDS, ann H. T. PARSONS. (From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.) (Received for publication, March 21, 1921.) Those who have discussed the world’s food supply during the past few years have talked in great measure about the cereal grains as the most important and economical source of human food. In- deed, for many years the cereals and legume seeds have been increasingly cultivated both in Europe and America to meet the needs of their rapidly increasing populations. Bread grains have actually become the staff of life of a large part of the human family. It is now recognized by students of nutrition that the cereal grains individually and collectively are incomplete foods.' The war has taught through tragic experience that children can- not be kept alive for a very long period on sucha food supply, although the requisite calories and energy may be supplied. The history of scurvy furnishes numerous examples of the dangers attending the restriction of the diet of adults to cereal products, legume seeds, and meats. Nevertheless, statisticians and persons in charge of feeding large groups of people in armies, prisons, asylums, hospitals, and labor camps, still in many instances show lack of appreciation of the dangers of limiting people to certain types of restricted diets. It was natural that in the early efforts to discover the fundamen- tal truths regarding the nutritive requirements of mammals, inves- 1McCollum, E. V., The newer knowledge of nutrition, New York, 1918. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1920, xli, 275. 207 208 Protein Values in Foods. IV tigators should have attached much importance to the rate of growth during limited periods by young animals fed diets of dif- ferent types. It was spectacular to observe complete failure in one case on a diet with which a chemist could find nothing amiss, and successful growth for weeks or months on another diet which according to older views should have been no more satisfactory than the first. With advancing knowledge, however, it became possible to interpret the cause of failure or success with any given diet, and new goals were set up by those who were able to make progress in nutrition studies. In our laboratory we have for several years sought to approach the solution of the problem of what constitutes the optimum diet for the purpose of promoting growth, of supporting highest fertility, greatest success in rearing young, and of preserving for as long a period as possible the char- acteristics of youth, and of extending to the extreme limit the span of life. With this objective we have come, as the result of much experience, to question whether the extension of the use of cereal grains in the diet of man has not already passed the limit of safety. At least it is more necessary now than formerly that the remain- ing components of the diet should be chosen with knowledge and care in order to correct the deficiencies of the cereal, tuber, and muscle meat mixture, or the bread, potato, and meat type of diet which has become so prominent a feature of the nutrition of many American and European families at the present time. It has been already pointed out that the cereal grains, legume seeds, and mus- cle tissue meats are too poor in calcium, sodium, chlorine, and fat- soluble A to meet the physiological requirements of a mammal. Mixtures of these in any variety are little, if any, better sources of the essential mineral elements or of fat-soluble A than are the individual foods themselves.’ Primitive man ate everything he could secure which was edible. His animal food included the flesh of such game as he could catch, and also fish, eggs, birds, shell-fish, insects, etc. Among the vege- table products which he doubtless ate were fruits, berries, fleshy roots, nuts, and a few other seeds of plants, among which were the seeds of those grasses which have since been developed into our * McCollum, E. V., Proceedings of the Institute of Medicine of Chicago, 1920, iii, 13. McCollum, Simmonds, and Parsons 209 cereal crops. There are relatively few regions where nuts are sufficiently abundant to furnish a regular article of diet for a sparse population during even a few months in the year, and the supply of cereal grains was even more inadequate. The cereal grains are the seeds of several grasses. In a country where no agriculture was practised, grasses would be cropped by grazing animals and the development of seed greatly interfered with. As Huntington points out, extensive agriculture was impossible until after animals were domesticated.* Such seed as was produced was born on isolated and scattered stems, and would be difficult to harvest in appreciable quantities. One exception was the wild rice plant which grew in the water and was therefore protected to some extent from grazing animals. It was more abundant in certain places than the seeds of any land grasses were likely to be in the unmolested fruiting condition. Rice was therefore harvested from very early times in Asia and in some of the northern states of America. Even in these favored regions of shallow lakes and rivers, however, rice never formed a principal constituent of the diet of the Indians, but only an adjuvant in the fall and early winter. Grass seeds of the type of the cereal grains are always eagerly sought for at ripening by birds, and the harvest time would naturally be short. Maize was never a prominent article of diet among the Indians, but only served to vary their otherwise carnivorous food supply. All the higher apes eat more or less of tender leaves which have mild flavors. After man reached a stage of develop- ment where food was regularly cooked he was able to eat coarse vegetables of the leafy type in greater variety and in larger amounts than when he had to eat them raw, because of the diffi- culty of digesting some of them. Pot-herbs early became a regular part of the diet of man as he advanced toward civiliza- tion. They are today the outstanding feature of the diet of the Chinese and Japanese. The great increase in the consumption of cereal grains in vari- ous forms as flours, corn-meal, corn grits, rolled oats, and in the form of the many breakfast foods found on the market as human food is an incident in connection with the development of modern industry, and the change from a rural to an urban life. This has forced a great part of the population to depend upon the 3 Huntington, E., Civilization and climate, New Haven, 1916. 210 Protein Values in Foods. IV remainder for food while they operate the machinery of industry. The land yields calories and protein in greatest abundance when farmed to cereals and leguminous plants, rather than when used for the production of milk or meat. This type of farming has therefore been encouraged, and wheat and maize production has been stimulated year by year to higher and higher levels until the world’s capacity has nearly been reached in this respect. This great consumption of grains has changed the character of the diet . profoundly from what it has ever been before in human history, and in a manner which tends to undermine the vitality. The national dietetic sin of America and many parts of Europe has grown to be close adherence to a meat, bread, and potato diet, or other foods which have similar dietary properties. When it is remembered that the cereal grains are now all but universally decorticated and degerminated for the purpose, of producing products which can be kept without commercial hazard, and that these are decidedly poorer in their dietary properties than are the seeds from which they were milled, the situation can easily be appreciated. The diet of cereals, muscle tissue meats, and tubers (the meat, bread, and potato type) is not satisfactory for the nutrition of man or animals, and is of distinctly poorer quality when highly milled products are used in abundance.‘ We wish to emphasize in connection with this study of the man- ner in which the cereal grains supplement, or rather fail to sup- plement, the proteins of other cereal grains and legume seeds, the fact that the widespread use of this class of foods represents an inno- vation in man’s diet, and one which is not for the best. The cereals may well be used as articles of human food, but it is wiser to uti- lize more of the land for the extension of the dairy industry in order to increase the supply of milk and other dairy products, than to seek to extend as far as possible the production of crops which yield the greatest returns in such food units as the chemist has long recognized, but which fall short of the requirements of mammals in respects which we have but recently been able to appreciate. When one studies the charts described in this paper it is difficult to avoid the conclusion that a diet composed too largely of cereal ‘McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxviii, 113. McCollum, Simmonds, and Parsons 211 products, and not satisfactorily supplemented by foods which cor- rect their deficiencies, will tend to lower the vitality and promote the early development of senile characters. We cannot agree with Daniels and Nichols that the consumption of legume seeds such as the soy bean should be increased.’ It is much better to use these seeds for feeding dairy cows, for their deficiencies can be made good by the latter through the consumption of forage plants, and used as a source of milk formation. Milk forms the most satisfactory corrective food to make good the deficiencies of the cereal, muscle meat, and tuber diet now in such widespread use. SUMMARY. The charts presented in this paper bring out the following facts: Earlier observations on the inferior character of the proteins of the legume seeds have been confirmed. It is further shown that protein mixtures derived from two such seeds, the selection being made from navy or soy beans or peas, are little, if any, better than the proteins of the individual seeds, which compose the mixture when fed as the sole source of nitrogen. Two cereal grains when combined fail to form a protein mixture which is markedly superior to the same amount of protein from a single grain for the nutrition of the rat. In certain instances the improvement in the quality of the pro- teins is decidedly great when a cereal grain is supplemented with a Jegume seed. Conspicuous examples of such enhancement of proteins are wheat and navy beans (Chart 12), and wheat and peas (Chart 13). Table 1 presents the records of the weights of several litters of young rats, the mothers of which were confined to diets which were essentially comparable in quality in all factors other than protein (Lots 2369, 2365, 2370, and 1236). The rate of growth of the nursing young served as an index to the extent to which the protein moiety of the food mixture met the needs of the mother for her own maintenance and for milk production. Lot 2369 should be contrasted with Lot 2069. Each litter con- tained four young. Lot 2369 derived its protein (9 per cent) from maize and peas. Growth was retarded, indicating that the quality of the milk was below the optimum. Lot 2069 derived its 5 Daniels, A. L., and Nichols, N. B., J. Biol. Chem., 1917, xxxii, 91. TABLE I. This table shows typical records of nursing mothers on diets contain- ing 9 per cent of protein from different sources, as contrasted with others on diets equally well constituted in respect to all other factors and containing higher protein contents of good quality. Lot No. Weight of 9 when young were born. Weight days later. Weight of 2 when through nursing. No. of young. Weight. Source and content of protein. m. 144 —) 2369 2069 2365 2370 |192 2153 |175 2370 1236 days |gm.| days |gm. 18 |150) 46 10 |232 120 22 |205 37 |170 28 |192 or Or days \gm.| days 18 | 71] 31 10 |100 12 | 74 15 |137 16 (133 212 22 gm.| days |gm. 107| 46 |123) Maize 6 per cent protein, peas 3 per cent pro- tein (see Chart 9). 197 Degerminated ce- reals, peas beans, steak, and cabbage (18.2 per cent protein). 102) 45 |210) Barley 6 per cent protein, navy beans 3 per cent protein (see Chart 8). 160| 37 |175| Wheat 6 per cent protein, peas 3 per cent protein. Degerminated ce- reals, peas, beans, steak (19 per cent pro- tein). 277 1167| 44 |250| Wheat 6 per cent protein, peas 3 per cent pro- tein (see Chart 13). 286| 40 |544| Steak 50 per cent, salts and butter fat, 35 per cent. protein. McCollum, Simmonds, and Parsons 213 4 Lot 2069. Lot 2153. Lot 1236. Bolted flour...... 30.0 Bolted flour...... 30.0 Round steak Corn-meal....... 15:5 Corn-meal....... 19.5 (cooked)....... 50.0 Rice seen. ca ShOPernIcets sos eee 9:5 SNaCier race. 1.0 Rolled oats...... 8.0. Rolled oats...... 9.5° KGa. eee 1.0 Re AS seis sive oe SMO MECAS hoes «ico oes 9:5 ‘Ca€O;. een 1.5 Navy beans...... 8.0 Navy beans...... 0.5) -) Dextrinnss--eeeee 43.5 Round steak Round steak Butter tateeeeeee 3.0 (cooked)....... 10.0 (cooked)....... 10.0 Cabbage (dry).:. 10:07 NaCl... ..2..-... a LENG] ee oe AOD CaCOs: 25. es ckiasic 125 protein from a variety of sources, and the amount was approxi- mately double that in the diet of Lot 2369. These young were more than 25 per cent heavier at 10 days of age than those of Lot 2369 were at 18 days. The nursing mother in Lot 2365 derived her protein (9 per cent) from barley and navy beans. The young nursing this mother fell far behind the growth of those which nursed a mother of Lot 2370, which derived its protein (9 per cent) from wheat and peas. The former litter of five young at 18 days weighed collectively 70gm. Thelatter at 12 days weighed 74 gm. This difference in the nutrition of these nursing young was solely due to the differ- ence in quality of protein in the two food mixtures. Other equally interesting comparisons can be seen in the table. Lots 2069 and 2153 received 18 and 19 per cent of protein, respectively, and from a variety of sources. Their diets were, however, decidedly below the optimum in their content of fat- soluble A, and for this reason would not serve to maintain the vitality of a family through successive generations. A further discussion of this type of diet and its effect on quality of milk secreted and on the span of life will be given in a later paper. Chart 1.—The curves presented in this chart are typical of groups of rats which were fed from an early age on diets which derived their protein contents from: (1) peas; (2) soy beans; (3) peas and navy beans; and (4) soy beans and navy beans. In each case the content of protein in the diets was 9 per cent. Lot 2367 derived two-thirds of its protein supply from peas and the remainder from navy beans. Lot 2366 derived two-thirds of its protein from soy beans and one-third from navy beans. Deficiencies in the THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, no. l Lot 26867 had a eal cas aS oe O° ire) on @ 9 n “ 2 a a ss 229 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIT, NO. 1 ~ 230 Protein Values in Foods. IV in all cases. A litter of eight weighed 65 gm. at 14 days. At 22 days the mother had reduced the number to six, which weighed 55 gm. At 48 days there were but five remaining. These weighed 111 gm. At 76 days but three remained. These weighed LO7 gm. Lot 2363 was fed 9 per cent of protein, 6 per cent being derived from rye and 3 per cent from navy beans. The growth records and fertility were decidedly below normal. The nursing periods were greatly prolonged and the infant mortality was high. After being restricted to the diet for 18 months the animals looked very old. Two females had collectively thirty-one young (five litters) and weaned twenty-two. A litter of nine young was reduced by the mother to eight by the 11th day. These weighed 59 gm. They were reduced to five by 34 days, and the litter weighed 80 gm. At 55 days the litter was reduced to three, weighing 85 gm. This is less than one-third the normal weight for this age. Two second generation females had twenty-three young (four litters). One third generation female had a single litter of two, but de- stroyed them soon after birth. Chart 11.—Lot 2361 was fed maize and navy beans to furnish 9 per cent of protein in the diet. Maize furnished 6 per cent and navy beans 3 per cent of the protein. The animals grew slowly and remained somewhat undersized, their fertility was low, and infant mortality high. In Lot 814 we’ have secured much better growth and reproduction on a diet containing 7.4 per cent of maize and 4.4 per cent of navy bean proteins, respectively. This is a remarkable illustration of the physiological effects which may result from a small variation in the amount of protein in the diet. There were two females in this group (Lot 2361). They had fourteen young (three litters) and weaned eight. The young were small for their ages. One litter of seven was reduced by the mother to six at 17 days. These weighed but 60 gm. At 42 days the six weighed 145 gm. One second generation female was kept on the diet 113 months but never had any young. Lot 2379 derived the 9 per cent of protein in its diet from rye (6 per cent) and soy beans (3 per cent). The animals remained somewhat undersized but appeared to be in good condition. Two females had forty-three young (five litters) and weaned fifteen. The young were never in good condition and the nursing 7McCollum, E. V., and Simmonds, N., J. Biol. Chem., 1917, xxxii, 54. qes_4eqang ae ee 232 Protein Values in Foods. IV period was long. ‘Three second generation females grew up on the diet. ‘Two of these remained sterile. The other had‘a litter of four and weaned one. One of the females of the original group had a litter of eleven. At 8 days these weighed collectively 85 gm. By 20 days they were reduced by the mother to ten. These weighed 110 gm. On the 35th day five of these died. On the 41st day but two were left. These weighed but 37 gm. This was less than one-third _ what they should have weighed. These two remaining ones were killed and eaten by the mother at 51 days. The history of this litter of young is typical of many we have seen in groups of animals restricted to diets which were satisfactory in every respect except in quality or amount of protein. Chart 12.—Lot 2362 took a diet containing 9 per cent of protein derived from wheat (6 per cent) and navy beans (3 per cent). The growth records and fertility were good but the infant mortal- ity was high. Two females had thirty-seven young (nine litters) and weaned eleven. Many young were destroyed by the mothers soon after birth. Two second generation females had thirty-five young (nine litters) and weaned seventeen. One second generation female remained sterile. The young were always inferior in size, appearance, and vigor. Chart 18.—Lot 2370 derived its 9 per cent of protein from wheat (6 per cent) and peas (3 per cent). The growth curves of this group were the best we have observed in rats fed but 9 per cent of protein derived from a combination of vegetable proteins from two sources. The animals reached full adult size, the fertility was high, and the success in rearing young was better than in any other group described in this series, or in any instance in our experience where rats were grown and maintained on a diet containing but 9 per cent of vegetable proteins. Two females had forty-nine young (nine litters) and weaned thirty-four. Two second generation females were grown on the family diet. One remained sterile. The other had twenty-six young (four litters) and weaned twenty-one. One third genera- tion female had ten young (two litters) and weaned eight. The young were always somewhat small for their ages and the nursing periods rather prolonged because of the limitation placed upon the mothers by the low protein content of the diet. kk AS ae Ge ee Lider ee NN A Te ME GSN ASR & Coo PAAP NE PL Nien Le Saab ae Cr ret tS CCAR PR See (aon Ger CONE SE Lee RN | Ce eS GEER Hg NS to © = D | = . Ame ine = Pl ee caeeee SUPPLEMENTARY PROTEIN VALUES IN FOODS. V. SUPPLEMENTARY RELATIONS OF THE PROTEINS OF MILK FOR THOSE OF CEREALS AND OF MILK FOR THOSE OF LEGUME SEEDS. By E. V. McCOLLUM, NINA SIMMONDS, ann H. T. PARSONS. (From the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore.) (Received for publication, March 21, 1921.) The nature of the dietary deficiencies of the cereal and legume seeds is now well understood.! It has also been shown that milk and the leafy vegetables occupy a unique place among our ordinary foodstuffs in that they are the only foods regularly con- sumed in moderate quantities which are of a nature to correct the mineral deficiencies of cereals, legume seeds, tubers, and fleshy roots, or to adequately supplement them with respect to fat-solu- ble A. It is well known from laboratory experiments on animals as well as from agricultural experience, that milk proteins tend to enhance the value of vegetable proteins generally.2. Specific infor- mation as to the extent to which milk proteins supplement those of individual vegetable foods is still wanting. The studies reported in this paper form a contribution to this phase of our knowledge of practical dietetics. In these experiments the proteins of the diets were derived from half skimmed milk powder (Merrell-Soule) and a single plant seed or tuber. In order to bring out more clearly the extent of the supplementary relations between the proteins employed we have in all cases limited the content of this factor to 9 per cent of the food mixture. We have already pointed out that in order to se- cure a normal growth curve in the rat on this low plane of protein 1 McCollum, E. V., The newer knowledge of nutrition, New York, 1918. McCollum, E. V., J. Biol. Chem., 1914, xix, 323. 235 236 Protein Values in Foods. V intake, the quality of protein for growth must be good. When the growth curves fall below normal the extent of retardation of development serves as a good index to the extent to which the quality of any mixture under investigation falls below that of the best combinations which we have been able to discover. When, in addition, we observe the animals throughout their reproductive period and secure records of fertility and infant mortality, and the time at which the first signs of old age appear, we have the most sensitive indexes to physiological well being which it seems proba- ble will ever be observable. Proteins of good quality will induce normal growth when fed at the plane of intake of 9 per cent of the food mixture, and may induce a fair degree of fertility. In order to secure high fertility and low infant mortality the proteins must be of excellent quality. In a former paper‘ we have described comparable studies with diets in which the proteins were derived from combinations of either liver, kidney, or muscle with cereal or legume seed proteins. The plane of protein intake was uniform (9 per cent) in all cases. These records, together with those presented in the present paper, form, therefore, a contrast between the value of milk proteins on the one hand and animal tissue proteins on the other, as supple- ments for a number of vegetable foods with respect to the protein moiety. The results bring out in a very striking way the unexpected superiority of animal tissue proteins for the svecial purpose of enhancing the value of various plant seed proteins. In our num- erous studies of this phase of nutrition we have demonstrated that with diets of the type containing 9 per cent of protein, and with all other factors satisfactorily adjusted, the proteins of kidney produce the best results we have yet observed. The biological value of the proteins of animal tissues for growth or for maintenance of health differs in an easily demonstrable degree. An inspection of the results of our experiments with diets of the type here employed warrants arranging the proteins of a number of animal and vegetable foods of great importance in a series show- ? McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1919, xxxvil, 155. . * McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1921, xlvii, 139. ——- McCollum, Simmonds, and Parsons 23% ing their relative nutritive values. In the following scheme we have arranged the several foods in order of the biological value of their proteins. The best is placed at the left and the series is a descending one. (Muscle Milk ~r.:,. (Soy beans ; .__} (round steak) {Maize} Beef kidney—Wheat— ae | Barley wrlOats — Navy beans (beef [Rye |Pea In our earlier studies of the cereal grains’ we became convinced that wheat proteins were of somewhat lower biological value than more recent experimental data would seem to indicate. This may perhaps be accounted for by differences in the proteins of various samples of wheat. It is well known that the dough-form- ing quality varies markedly in wheats. This property depends upon the peculiar nature of the proteins of this grain and may be due to lack of uniformity in the relative amounts of the individual proteins contained in the seed. It should be kept in mind that such a differentiation in biological value of proteins from these foods will not be apparent unless the experimental procedure is appropriate to bring them out. The several foods included in the scheme must be fed, with other diet- ary factors satisfactorily constituted, at such a plane of intake as to furnish the critical level of 9 per cent of protein in the diet. This is the only method we have been able to devise to show these differences. The observations must include not only the period of growth but also the fertility, the success with young, and the period following the completion of growth to the point where senile characters are apparent. We were surprised to find how consistently combinations of milk proteins and cereal or legume proteins fail to show as high biological values as can be demonstrated for kidney, liver, and muscle proteins combined with those of certain cereals. It should not be lost sight of that milk has an effective supplementary rela- tion to cereals both with respect to the inorganic and fat-soluble A deficiencies of the latter, whereas muscle meats supplement them § McCollum, E. V., Simmonds, N., and Pitz, W., J. Biol. Chem., 1916-17, xxvili,211. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1919, xxxvii, 557. 238 Protein Values in Foods. V only with respect to the protein factor, and glandular organs only with respect to protein and fat-soluble A. This fact is brought out in a striking way by the records in Chart 5. When pasteurized or boiled milk, or cooked glandular organs are contained in the diet they do not effectively supplement a cereal and legume seed mixture with respect to the antiscorbutic factor since this is a labile substance. This factor is essential in the nutrition of man, monkey, and guinea pig, but need not be furnished by the diet of the rat or prairie dog, since they are apparently able to synthe- size it.® SUMMARY. We have described in this and preceding papers experiments which were so planned as to compare the relative merits of animal tissue proteins and of milk proteins for enhancing the value of the proteins of each of the following vegetable foods: barley, peas, soy beans, rye, maize, navy beans, wheat, potato, and rolled oats. These indicate clearly that animal tissues such as liver, kidney, or muscle, are superior to milk for the specific purpose of making good the deficiencies of the proteins of the seeds and tuber mentioned above. Milk, however, is an effective supplement for these vegetable foods with respect to other factors as well as protein. This is especially true of calcium and fat-soluble A. In making deductions from these results it should be kept in mind that muscle tissue supplements seeds, tubers, etc., only with respect to the protein factor, and that other deficiencies of even greater importance for the well being of the body are always met with in that group of vegetable foods which are functionally storage tissues of plants; v7z., seeds, tubers, and roots. Chart 1.—Lot 2391 was fed a diet containing 9 per cent of pro- tein derived from barley (6 per cent) and milk (3 per cent). All other factors in the diet were made satisfactory by suitable addi- tions of inorganic elements and fat-soluble A (in butter fat). Growth took place at a subnormal rate and the full adult size was never attained. The animals in this group looked rough coated and old at 15 months. * McCollum, E. V., and Parsons, H. T., J. Biol. Chem., 1920, xliv, 603. ene yeeO® > a 3 Beet ba ee Pier eer wobehetedeoh Ledenpatet petrol Al SERRE ae 4244 ae Me Sate tae ane cnoehe ae eee Mewes... 0°89 AXE es - 14x0q g°T £0089 £0089 0°l TORN TOBL 6 mae yu pesropaodg £°6 yu AO rd sted 0°0S ROT Teg COs io Fe Paes ss OF 08 Oct 002 239 240 Protein Values in Foods. V Two females grew up on this diet. One remained sterile. The other had eleven young (three litters) but destroyed them soon after birth. Lot 2390 derived its diet, containing 9 per cent of protein, from peas (6 per cent) and powdered milk (3 per cent). The protein of the diet was the limiting factor. The animals all grew poorly, but better than they could have grown on 9 per cent of pea protein alone. These rats were very apprehensive and could be weighed only with difficulty because of constant efforts to escape. They were so excited when handled that they would not sit still in a a small covered box but would constantly spring up and strike the lid with their heads. The hair was very short and fine, and had a silky appearance, which we have never seen on rats fed highly satisfactory diets. The same type of coat has been frequently met with in rats restricted in a great measure to maize as a source of protein. Less frequently we have seen these “‘mole-skin” rats - in groups which derived their protein from kafir corn. Two females were restricted to this diet but never had any young. Lot 2389 was fed soy beans and powdered milk as a source of protein. The total protein content of the diet was 9 per cent. The soy beans furnished two-thirds and the milk one-third of the total. While the growth was much better than we have ever seen on 9 per cent of soy bean protein alone, the rate of growth was distinctly below normal. The animals remained undersized, and their fertility was very low. There were three females in this group. One had a litter of seven. At 14 days they weighed 57 gm. At 21 days one had died. The remaining six weighed 62 gm. At 27 days but four survived and these weighed collectively but 45 gm. They were very puny and incapable of growing on the mother’s milk. The other two females remained sterile. Chart 2.—The rats of Lot 2386 were fed 9 per cent of protein derived from rye (6 per cent) and milk powder (3 per cent). Growth was slow but the animals reached nearly the adult size after some delay. These rats aged decidedly early. They looked old at 14 months. The second generation were all more under- sized than the first. We have observed in many cases where the food mixture was faulty to a slight degree and a family was con- 242 Protein Values in Foods. V fined to the diet through several generations, each succeeding generation was smaller than the preceding one when growth was completed. There were three females in the group. ‘Two of these remained sterile. The other had twenty-eight young (three litters) and weaned eleven. The nursing period was, however, very long. A litter of eight was reduced by death to five by the 32nd day. These weighed 108 gm. At 39 days they weighed 121 gm., and were in a poorly developed condition. They were less than one- third the normal size for their age. One second generation female had a single litter but destroyed it soon after birth. Lot 2385 was fed 9 per cent of protein derived from maize (6 per cent) and milk powder (3 per cent). All other factors in the diet were made satisfactory by suitable additions. Growth was slow but the animals reached nearly the full adult size. There were three females in the group but none ever had any young. The hair of this group was short and silky and suggestive of a mole skin. Chart 3.—Lot 2388 derived the 9 per cent of protein in its diet from navy beans (6 per cent) and milk powder (3 per cent). The animals grew slowly and remained permanently undersized. They lived surprisingly long on this diet on which they grew so poorly. The history of the group on 9 per cent of protein from navy beans and milk is comparable to that of Lot 2390 (Chart 1), which was identical except that peas replaced the beans. The same diet with soy beans in place of peas or navy beans produced distinctly better growth (Lot 2389, Chart 1). Lot 2384 derived the 9 per cent protein in its diet from wheat (6 per cent) and milk powder (3 per cent). The combination of wheat and milk proteins is better than a similar amount of protein from wheat alone. With the exception of the diet of oats and milk (Lot 2387, Chart 4), this food mixture was superior to any other combination of seed with milk proteins which we have studied. The animals appeared old after about 19 months on this diet. There were two females in the group, one of which died after being 45 months on the diet. The other had forty-one young (seven litters) and weaned nineteen. The nursing periods were long in all cases. The young were not destroyed in the ruthless oe eee qng JU pedepaod qBou HO 3 By Fai LOT ose 243 244 Protein Values in Foods. V manner frequently observed, but died at intervals from unde- termined causes. A litter of five young weighed but 103 gm. at 23 days of age. At 58 days four weighed 141 gm. They appeared to be in good condition but were undersized. Two second generation females had but five young (one litter) and weaned one. Chart 4.—Lot 2403 derived the 9 per cent of protein which its diet contained from potatoes (6 per cent) and milk powder (3 per cent). Growth was somewhat below normal and they remained undersized. These rats aged very early. They looked as old at 1 year as many better nourished animals do at 18 months. Three females had twenty-seven young (five litters) and weaned only two. The nursing periods were long. A litter of five weighed 40 gm. at 15 days. At 26 days they were reduced by death to three, which weighed collectively 47 gm. At 60 days but two were left. These weighed together 70 gm. This is less than half what they should have weighed at this age. The nitrogen of the potato is in great measure in the form of simple substances of a non-protein nature. These substances are evidently not of a character which supplements the proteins of milk to any marked extent. We have pointed out elsewhere that the nitrogen of the potato when fed as the sole source of this factor is not of so high a value as some have reported.? Lot 2387 was fed 9 per cent of protein, two-thirds of which was derived from rolled oats and one-third from milk powder. This combination of proteins seems to have a higher value than any other cereal and milk mixture we have investigated. But little inferior to this is the wheat and milk combination. We have in some earlier experiments seen better curves of growth on about this amount (8 per cent) of protein from a mixture of oats and milk. After 16 months on the diet their coats (Lot 2387) were somewhat rough, but the animals were still vigorous. Three females had thirty-three young (five litters) of which six were weaned. Three other litters were destroyed before their numbers could be determined. The nursing periods were long. A litter of seven young weighed 66 gm. at 15 days of age. At 34 7McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1918, xxxvi, 197. 246 Protein Values in Foods. V days they weighed collectively 107 gm. At 60 days six of these weighed 202 gm., or less than half the normal for this age. Two second generation females grew up on the diet. One was sterile. The other had one litter of three after she had been 8 months on the diet. She weaned them all after a long nursing period. Chart 5.—Lots 2148 and 2147 show the relative merits of milk and of muscle tissue as supplements to a mixture of foodstuffs consisting of articles which are functionally storage organs of plants. The mixture, aside from the milk or muscle meat, con- sisted of two fleshy roots, red beet and yellow turnip; a tuber, the potato; two legume seeds, pea and navy bean; and two degermina- ted cereal products, wheat flour and corn-meal (maize). Notwithstanding the wide variety in such a list of foods, and an appropriate chemical composition as indicated by the ordinary food analysis, it does not promote growth in young animals nor support the vitality of adults as measured by fertility, success in rearing young, or in deferring the onset of old age. Muscle meat (round steak) supplements a mixture of cereals, legume seeds, fleshy roots, and tubers only with respect to the pro- tein factor. Milk on the other hand enhances not only the protein of these vegetable foods, but likewise makes good their mineral deficiencies and also the shortage of fat-soluble A which all such mixtures exhibit. Lot 2147 in which the vegetable diet of storage organs is sup- plemented only with muscle meat, failed to grow normally. The curve shown is typical of a group of six animals restricted to this diet. They never had any young and aged very early. They looked extremely rough coated after 6 to 8 months on the diet. Lot 2148, on the other hand, whose diet was similar in all respects but contained 10 per cent of whole milk powder, grew normally and remained in much better condition to an age of about 18 months. These animals showed fair fertility and success in rearing their young. The milk supplemented not only the proteins of vegetable origin but accomplished what was of greaterimportance; viz., the correction of the inorganic deficiencies and made good in great measure the lack of fat-soluble A. The bearing of such observations as these on practical human dietetics will be easily appreciated. 09324 0d OTs HO T9 By ig CUE eS tea A 00¢ nameencsiten? TEER ae” ea Tout OLos ny sueeq) Lae aes 247 a STUDIES IN THE VITAMINE CONTENT. II. THE YEAST TEST AS A MEASURE OF VITAMINE B. By WALTER H. EDDY, HATTIE L. HEFT, HELEN C. STEVENSON, AND RUTH JOHNSON. (From the Department of Physiological Chemistry, Teachers College, Columbia University, and the Department of Pathology, New York Hospital, New York.) (Received for publication, March 30, 1921.) Since Williams (1) first suggested the identity of Wildier’s (2) “bios” with water-soluble B vitamine and proposed as a test for the vitamine the measurement of yeast stimulation, the procedure has undergone considerable investigation and criticism. As a result we now have a choice of several methods of applying the test which may be classified as follows: Measurement of yeast stimulation (a) by counting the cells; (b) by weighing the cells produced (1, 3); (c) by determination of the CO, produced (4); and (d) by determination of the volume of cells produced (5, 6). Of the methods presented to date the authors have found that the method devised by Funk and Dubin (5) combines quantitative accuracy with the simplest technique. In the work reported here we have applied this method. In the field of criticism various types of questions and doubts have been advanced as to the specificity of the test and its adapta- bility as a quantitative instrument for vitamine measurement. The principal objections to its use may be reviewed briefly. Souza and McCollum (7) call attention to the sensitivity of yeast cells to many types of stimulation and advance data to illustrate this view as evidence against the accuracy of the test for evaluat- ing vitamine content. More recently MacDonald and McCol- lum (8) have shown by a series of transplants that yeast will grow in a culture medium practically devoid of all but a trace of “bios” and suggest that this result is explicable on one of two hypotheses; v7z., that either yeasts do not require “bios’”’ for 249 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 250 Vitamine Content. II growth or:that they can synthesize enough to meet their needs. Either explanation would make the test inaccurate for deter- mining total vitamine in an extract. Emmett and Luros (9) and Emmett and Stockholm (10) deny that the “‘bios’’ is vitamine B and present evidence to show that the stimulative factor possesses neither antineuritic power nor the power to induce growth in white rats. They grant the presence of a stimulative factor in extracts of vitamine-rich materials but imply that it is either a new vitamine or a fa¢tor of another sort. A series of articles has recently appeared reporting work by three authors, Fulmer, Nelson, and Sherwood (11). In these articles they point out that the stimulative effects of alfalfa and wheat embryo extracts upon yeast growth are not directly proportional to the weights of the materials extracted. Funk had already called attention to this fact in his article describing his centrifugation method. Second, they present evidence tend- ing to show that the stimulative effect of an extract or culture medium is greatly altered by varying the concentrations and kinds of mineral salts therein. In this connection they propose a formula for a culture medium (Medium F) which they believe will produce maximum stimulation to yeast growth and which will not be improved or altered by the addition of extracts of organic substances unless the latter disturb the optimum salt concentrations of the medium. In brief they imply that “bios’’ is a matter of salt concentration. Third, they advance the claim that the stimulative effect of alfalfa extracts upon growth of yeast cells cannot be changed by the treatment of the extract with alkali and this fact indicates that the stimulative factor cannot be vitamine B. Should all these criticisms prove valid it would seem idle to proceed further with the development of the test. In reviewing these papers and our previous work we are not yet convinced that the time has come to reject the test as worthless. In the present paper we wish to present our accumulated data of the past 6 months which have been devoted to somewhat detailed investigations of behavior of the test under varying conditions. Since the presentation of the Funk-Dubin method (6) at New Haven in May, 1920, we have adopted that technique. For ON whe Se Eddy, Heft, Stevenson, and Johnson 251 various reasons we have modified the reported method somewhat, but in the main it is essentially as described by the authors. Since, however, all the results of this article are based on the use of this test, it may be worth while to briefly describe our procedure. Method. To a basal diet of 9 cc. of sterile culture medium (Nageli solu- tion, Medium F, etc.) in a sterile test-tube is added 1 cc. of the sterilized extract of the material to be examined. A pure culture of Fleischmann yeast is maintained on an agar slant and 24 hours before the test is to be made, a transplant is made to a fresh agar slant. (Funk and Dubin hold that brewer’s yeast is better adapted to the purposes of the test since it is always bottom growing and packs more readily on centrifugating.) One stand- ardized platinum loopful of the 24 hour yeast is used to inoculate the contents of each tube, the tube is then stoppered with cotton and incubated. In the experiments that follow, unless other- wise stated, the culture medium used by us has been the Nageli solution. Our incubators are set for 31°C. and are so maintained that their maximum temperature variation lies between 30 and 35°C. For various reasons we have selected 72 hours as our period of incubation. At the end of the incubation period the yeasts are killed by plunging the tubes into water heated to 80°C. and the tubes are maintained at this temperature for 15 minutes. Unless the yeasts are killed they will not pack well in the subse- quent contrifugation. - The contents of the tube are next transferred to a Hopkins’ vaccine centrifuge tube which has a capillary tip graduated from 0.00 to 0.05 ec. with five markings. With a magnifier and a scale it is possible to read accurately to tenths of the divisions etched on the tip; 7. e., to thousandths of a ec. The tube and its contents are then centrifugated 20 minutes at a speed of about 2,500 revolutions per minute. In the preparation of our extracts various methods were em- ployed depending upon the nature of the source. We have found that boiling water will in 3 hours extract all the yeast stimulative substance from a small amount of dried material unless consider- able starch is present. In that case we have resorted to extraction 252 Vitamine Content. II with 95 per cent alcohol, filtered, evaporated to dryness on a steam bath, taken up the residue with water, filtered again, and diluted or concentrated the filtrate as desired. If much fat is present a preliminary extraction with alcohol-free ether will prepare the material for water or alcohol extraction. We have used each and all of these methods as the circumstances war- ranted. When extracting food materials it is customary to dry them to constant weight at 60°C. in an air bath. The filtered watery extracts used in the tests are determined colorimetrically for pH. In practice, if the hydrogen ion concentration falls between a pH of 6 and one of 7 no further treatment is applied to the extract. If the concentration is over 7 or under 6 we have neutralized with HC] to litmus and restored to the original volume by dilution or evaporation. Excess of alkali not only retards yeast growth but also tends to precipitate the calcium phosphates from the culture medium. Once prepared, the extracts are placed in cotton stoppered Erlenmeyer flasks and sterilized in an Arnold steam sterilizer. Two 30 minute treat- ments with a 24 hour interval are usually sufficient to secure sterility and once sterilized the flasks and contents can be kept indefinitely. Its Our first experiments aimed to apply the above technique to a series of materials whose relative vitamine B content had been already established by rat-feeding tests. We, therefore, selected for this purpose the materials described by Osborne and Mendel (12) in a previous article in this Journal. Our tests with these sources fall into three series. In our first series we obtained fresh materials of the kind described by Osborne and Mendel, dried them at 60°, and made our extracts by boiling the dried materials with water. These preparations are those listed on the chart as “‘Dr. Heft’s preparations.’”’ To make sure that our extraction was complete we reextracted the residues until tests showed that the stimulative factors were reduced to the limits of the controls. Since water was used in the experiments it does not follow that we obtained all the vitamine that a rat gets out in digestion, but with that reservation, we feel sure that our tested extracts contained all of the vitamine that¥was_ex- | | Eddy, Heft, Stevenson, and Johnson 253 tractable by water. The final extracts were combined and con- centrated to a volume such that 50 ee. contained the extractable TABLE I. A comparison of the vitamine values of a series of materials as deter- mined by Osborne and Mendel (12) in a rat-feeding experiment with the values given by the yeast test applied to water extracts of similar materials (Dr. Heft’s preparations). The Osborne and Mendel results. Gain of weight of rats Gain of weight Materials used. fed eum dani: = Average. Ae ar ie: =I Average. te. reBtanlh aca Naeional tees Mla cae ees Afallia'.. 5.0. 200} 179} 146 525] 175| 1 | 144] 121) 106] 371] 124] 1 Clover. 3 197 7a ASO 524, 174; 2 | 112/ 102) 99) 313) 99) 2 RomaOn.--6. 122] 120) 118) 115} 475) 119) 3 88} 70} | 158] 79} 3 Spimach...... 119} 101} 81) 75; 376} 94) 4 87| 61} 32) 180} 60) 4 Cabbage..... 99) 91) 58} 248) 82) 5 52| 49) 30) 131} 43) 5 aN ps. 96} 68) 66 aA AeA (8) 46} 30) 20) 96) 32) 7 @arrot..2.: .. 78| 78) 66) 46] 268) 67) 7 49} 38] 36] 123] 41] 6 timothy ..,... 42) 40) 10 OZ roles |) 22 5) —2| 2oleaSians ECU... 5 2 s.: ©) 8) Se =o) ae No data on 0.5 gm. The yeast test results. 1 cc. of water extract contained the extractable ‘““bios’’ of 0.02 gm. dried material. 1 cc. of extract and 9 cc.” of Nigeli solution were used in each determination. Materials Individual determinations in thousandths of a § é in Osborne and |* ee. of yeast cells. = a EY S Mendel order a q = N of value. Heel eat atta Peeler on lize dicgeeeagmecloagn| fovea aie : Alfailia:. «2.2% ait (0) |) 2855 1) B83] 2 |) || ley) sl |) SSE 1) a) AAP | 304| 35 5 @lover.... 58 | 48 | 46 | 45 | 43 | 40 | 40 | 40 | 38 | 38 | 436] 44| 3 Lomato..!.. .- 55 | 55 | 49 | 48 | 42 | 42 | 39 | 39 | 39 | 408} 45 |] 2 : Spinach. ..... 82 | 81 | 76 | 70 | 70 | 69 | 66 | 63 SHA) TA 1 Cabbage...... AGA Ale oonltOom i 2On | 20n Zon! 2 252) 31 7 | TET OS paeeae Bist || ate) |) Gi |) ain || aHh i) SES I SEs Il 24s) I 246) 305) 34 | 6 Carrot... .. 51 | 50 | 42 | 42 | 42 | 41 | 41 | 40 | 40 389} 438 | 4 mo phy.. ...: AQN AO 33 WSee Ae el 2oo| 250 2a 1-22 | 22> 2902958 JBYS20io 5 fae QO 264260 2ameot a eae 22220 8 214) 24 9 Controle)... . P| May ey | a) Ps 8 8 | 23 2 1 alta? el, material from 1 gm. of dried material. Table I shows the results obtained with the materials of the first series and Chart I is a graphic presentation of the same. re CHART Tf COMPARISON OF FEEDING TESTS ©THE YERST VITAMINE TEST [intitas Z. Soyer ResUUTS-1qm feddaly | T.O%,M. FesuuTs: D5 gn Fed duly =|2 oe : m Be: BTL AT mie aia 0°%Ms a TW-WT Vatuing Concas. ee rma Es site ee io f PE NY 2 led? 1 cum y cs Zl : fae mee : BL AR os eae ey SAH HM es al \ al (E03, Na et eS Ee Te] Teal cite 4 _ Igmi in 19D. 20 L i en eS —=- | ; Eddy, Heft, Stevenson, and Johnson —.255 While we may justifiably draw from these results an approxi- mate agreement with the Osborne-Mendel conclusions the agree- ments are not exact and in the case of spinach there is a very marked discrepancy. We felt at the time that our variations might quite possibly be due to variability in the vitamine con- tent of our materials as compared with the Osborne and Mendel TABLE II. Results of the yeast test applied to varying concentrations of water extracts of the actual materials used in the Osborne and Mendel experi- ments.* : A. 1 ce. of extract tested carried the extractable ‘‘bios’’ of 0.02 gm. dried material. 1 cc. extract plus 9 cc. Nageli solution used in each test. Materials in Readings in thousandths of a cc. Peete Order Osborne and Mendel Total. nee 0 order of value. 1 2 3 A 5 6 7 8 value. /:\Ibi3) Wie NO). |) G8} |) Za | Biz || Bie |) Bi70 |) Ska] Bie) |, Gay || 25 4 Cloverc ccc aaa: 62) | 59) | Poon 49) aoe 45) AQ Ae oil 2 ROTMMALOW A. a. so 2 55 | 49 | 45 | 44.) 43 | 42) 41 | 39 | 358 | 45 3 Spimachhe . ose... 68") 66) | GouleG2Zalnad Nod) 52) |.528|) 479 | 59 if Wabbage:........ 49 | 47 | 44 | 40 | 36 | 36 | 32 284 | 40 6 INCI) OR Rear 50: | 45 | 40 1405/39) | 38 | 38 | 37 | 327) 41 5 (COMO aes SAee a0) |) aie || SH! | 4! | Sh | a) | SO eee) Pay || Sail 8 mM OOM. a 3. 28) |p 2hl |) 20) | Si || Bi. ato: rei || SB ls SIU) ei7/ U RG, 6 Bee ae PAS || PRE | OK! || 8% || OBS OAL X0) 160 | 22 9 Wontrolss. 4-6: DA Si | 5 Des UG B. Concentration: 1 ec. carries ‘‘bios”’ C. Concentration: 1 ee. of 0.016 gm. dried extract. carries 0.01 gm. eyelet Bes; Materials. To ese paalesel eG eSel Gelito | a |. | Ses ane = eels Sle mle PARR... condos 38] 36] 34] 29] 137) 34 | 6 | 37] 38) 40) 45] 160) 40) 1 lover: 5.2... see 45| 46] 47| 48] 186] 46 | 2 | 35} 36) 37] 38] 146) 36 | 3 BROMINE TOs ss... dane 32) 33) 35] 42] 142) 35 | 5 | 28) 30} 35) 47/ 140) 35 | 4 Spinach. ...3. 204). 46| 49] 50} 52} 197/ 49 | 1 | 36} 38) 38) 39) 151) 37 | 2 Wabbace:...... cee 34| 34] 35) 40] 143) 36 | 4 | 21) 22) 24) 24, 91) 22 | 6 PRUETT Dee. ss See 24| 26] 27] 30) 107| 27 | 7 | 18} 18} 20) 20| 76) 19} 8 Warnotices c.< 2+ oe 20} 22) 30] 32] 104) 26 | 8S | 21) 22) 24) 24) 91) 22) 7 simothiy...:.-. 41) 42) 43] 46) 172) 43 3 | 23} 26} 30) 34) 113) 28 5 Weetemern oe +. <> oe ASS) 1926 yestiezOe 9) P13) TS to 62) ol5 9 Wonton si). tae 24) Be Au 2 Naa to, Al PP AY 25 ahd 256 Vitamine Content. II TABLE II1—Concluded. D. Concentration: 1 ec. carries ‘‘bios’’ E. Concentration: 1 ee. of 0.006 gm. dried material. carries 0.002 gm. Pail wee Materials. vy) oo eaelneed ae bes | 1. | 2 |-o (ae een ee = aes) Sivels eies PA alfa once siete 26} 28} 29) 31) 114) 28 2 | 26) 26) 28) 29) 109) 27 2 Clovers> 2 ses 36} 38) 40} 42) 156) 39 1 | 34) 35) 37] 38) 144) 36 1 SPoOmatOyaecie sisal 25) 25] 31) 32] 113) 28 3 | 22) 23) 24) 28) 97| 24 5} Spinach. ;..:..3.- 27| 27) 27| 30} 111) 27 5 |) 22) 2222125) Son 5 Cabbage... 5--cer 20 20} 20 | 6 | 17) 17| 18) 19) 71} 18 6 Ubbtu lh }oenenouspuc 15) 16} 17; 17} 65) 16 7 | 13} 13) 15} 16) 57| 14 7 Carrots. oso one 10) 11) 14) 17} 52) 13 8 5| 5) 19) 23) 52113 8 SPimothy.. sees 25| 26) 29) 32) 112) 28 4 | 18] 21) 25) 30) 94) 23 4 IBeet si... gueaene 10) 10} 11) 14) 45) 11 9 9} 10) 10) 10; 39) 9 9 @ontrolzs:.. oe 2-2 4) 2110) 22 4) 23a * For graphic presentation see Chart I. sources. To check this we sought a test upon the actual materials used in the Osborne and Mendel experiments and these materials were kindly furnished us for the purpose by Professor Mendel. With the actual materials at hand we repeated our tests and in this set of experiments we varied our procedure by testing various concentrations of each extract. Table II gives the actual results and these are also reproduced graphically in Chart I. The most significant features of these results are the extreme variability in individual determinations in the higher concentra- tions, lessened variability, and closer approximation to the feeding test results when diluted extracts are used. The behavior of the diluted concentrations is we think more than a coincidence. Funk (5) and others (11) have shown that the curve of stimu- lation under varying concentrations approximates the loga- rithmic curve and that to get sharp contrasts between two extracts it is necessary to test dilutions which will fall on the steep part of the curve. This point is emphasized in the next series of experiments. Il. The results with the varying concentrations suggested that we proceed next to the establishment of the curve of reaction of a single material. To this end we first selected dried alfalfa meal 1+. lia Sia meas AR * Eddy, Heft, Stevenson, and Johnson 257 as our material and, keeping all factors constant except concen- tration, proceeded to determine the stimulative activity in each concentration. To obtain our extract, 400 gm. of dried alfalfa meal were repeatedly extracted with boiling distilled water, the filtered extracts combined, refiltered, and concentrated to a degree that 1,000 cc. contained the extractable material from 400 gm. In working with this extract we were at first bothered by the tendency to sedimentation on standing or centrifugating. To avoid this complication we employed a procedure described by Osborne and Wakeman for removing protein from spinach ex- tracts. This consisted in diluting the water extract with 95 per cent alcohol to 40 per cent of the volume and filtering off the precipitated protein complex. This procedure is attended with variable results. Sometimes the precipitate carries down with it all the stimulative substance in the extract. At other times very little loss results. In the extract which we used tests showed that the loss was less than 0.002 cc. expressed in terms of yeast tests made before and after treatment. The alcohol treatment naturally diluted our extract and to remove the alcohol and restore the desired concentration the filtrate was concen- trated on the water bath to the original 1,000 cc. The tests reported below were made on this material and variations in concentration are expressed as fractions of the maximum con- centration. Table III and Chart II show six series of tests. In Series A, B, D, and E each determination was made by com- bining 1 ce. of the alfalfa extract with 9 cc. of Nageli solution. In Series C, Fulmer, Nelson, and Sherwood’s Medium F was substituted for the Nageli solution. Series F was obtained with a filtered autolysate of Fleischmann yeast cakes. One pound of the moist cakes was mixed with water and allowed to autolyze. The mixture was then filtered and concentrated to such a volume that the highest concentration used represents the extractable material from 3 gm. of Fleischmann yeast cake in each ce. of extract. The general shape of the curves of Series A and B is the same, the only difference being in the height of Series B and in the lower control value with this extract, possibly due to repeated heating in the 2 months that elapsed between the two tests. Both show a steep ascent to an optimum with little variability to the left ane | 258 Vitamine Content. II of the optimum. On the right of the optimum there is not only much wider variation in the test results but a marked decline. These results confirm those of Fulmer, Nelson, and Sherwood (11). These peculiarities show the futility of attempting to compare the value of two extracts unless the curves of the two are known and the impossibility of depending upon comparisons based on weights TABLE III. The yeast growth curve determined by varying the concentrations of a given extract. Time of incubation 72 hours in each determination. The results are given in thousandths of a ec. of yeast cells. Series A. Alfalfa extract plus Nigeli solution. Maximum concentration represents the extractable material from 0.4 gm. dry alfalfa meal in 1 ce. of water. Concentration ...... 1 |0.9)0.8| 0.7) 0.6) 0.5 |0.45| 0.4 |0.35) 0.3 0.25) 0.2 |0.15) 0.1 | 0.05 22| 23] 27| 30) 24] 24) 29) 34) 36) 35) 36] 29) 30] 21) 20 23} 24) 28) 30) 25) 28) 31) 35] 38] 37) 36) 30) 34) 24) 22 24| 28) 31/ 33) 28) 28) 33) 35) 39) 37/ 36) 32) 34) 27) 25 26} 29) 33) 35) 35) 29) 35) 37/ 39] 37 32 27| 30 29 o7 39 33 27 ol 38 40 34 ol bo 38 40 34 dl 36 41 39 32 AoONIDIAD | Control. Totals: 95/104 119 128 112|240) 128/254) 152/306|108/259| 98/220) 97) 37 Average....... | 24) 26 29) 32| 28] 30} 32) 36) 38] 38) 36) 32) 32) 27) 24) 6 Series B. Made 2 months later but with the same extract of alfalfa used in the Series A tests. 3 Concentration...) ss Reta iaese Winis? | cca les ela eT ey || Ryo] eve | ee = 3 a = = ~ 3 Y=) = > oO N Nn _ - So o es|o}o|o| Stelelolclale age sia 17| 24| 25| 23] 28] 28] 30] 29] 28) 29] 23] 25) 21\18]16/13/14] 2 20) 26) 25) 25) 29) 29) 33] 33) 29] 29) 27) 26) 21)18/16)15)15) 2 an a Lee > | | | | Wotal:. soca 103/133 129)189}162/162 164 164 153 150 142/133)110,93)86 74/81) 4 | Average.....| 20] 27| 26] 28) 32] 32] 33] 33) 31| 30| 28) 27) 22/19117|15/16| 2 Eddy, Heft, Stevenson, and Johnson 259 TABLE IlI—Continued. Series C. Made with the same extract of alfalfa as Series A and B but with Fulmer, Nelson, and Sherwood’s Medium F in place of Nagelisolution. 3 S| Sabet: ol] S| Sila essere 30] 37| 32] 34] 30) 32} 28] 28) 26) 27) 26} 23) 20)18)18]17/18}13 37| 38] 32) 35] 30} 33) 29] 29) 30) 28) 26] 25) 22/18)19/18)19]14 42) 42) 42) 35} 31) 34) 29] 32) 30) 28). 27) 25) 23/20)20)19|20)15 43) 55| 44! 46) 34) 34} 33) 33) 30] 30) 29) 26} 23/20/20)19/20]16 62| 55] 45] 47) 36) 35] 35} 34) 33} 30) 29) 26] 23/22/21/20/21|17 Total. ..... .|214/227|195/197|161/168)154/156 149)143/137|125/111/98/98/93/98)}75 I ed fact i i ed Average.....| 43) 45] 39 39) 32|.33] 31} 31) 29) 28) 27) 25) 22/19)19)18/19]15 Series D and E were made with alfalfa extract and Nigeli solution at the same time as Series B in order to determine the curvature of the steep part of the alfalfa curve. A concentration of 0.1, therefore, represents the extractable material from 0.04 gm. alfalfa in 1 ec. of water. Series D. z Concentration .\% 5 <..e0¢ a eo 250) 2a N24 122, 1°20) 19) |) 16.) 16) 14. tal Simone? 3... ee 125] 115) 114] 106) 95 | 87 | 79 | 76 | 69 | 51 | 4 PAWIETACC\ 2s ciel a ene Ds PB BB Sal sy aye ea ie Mae ae | ae) Series E. z Concentration... ... 0.0.25... 0.01 | 0.009} 0.008) 0.007) 0.006) 0.005) 0.004| 0.003) 0.002) 0.001 g is) Oo OuMRSvimp oan ad (ice cor |) “4 [8 Sie eae Onl, OF eleeouie fhe toh Gide oA Se eee Gaul senna ee heed Bele soollas: FOE) POS OM Reti. Pd) Gtrs "5 Sls AOR TOMS Caines ere Wed te) Gol Se). Skewes SOUS. Moto. coc eee 478 46) 43°40) 35 | 35 | 304-25) )-22)) 1594 PAV OTAIDE iste ticle sth ore OF Om liaeee Se eee lami a dag ae O 4) 3) 42 260 Vitamine Content. II TABLE I1I—Concluded. Series F. Made with yeast autolysate in place of alfalfa extract. In the maximum concentration 1 ec. of autolysate contains the extractable sub- stance from 3 gm. of Fleischmann yeast cake. In this series the autolysate was combined with Nigeli solution. s Concentra- 5 tion...... = rapa pd | | aerate) ee sie |S 5/3s/3s Ss Bis 5 ~lololol[olototolelto si colo sles sisi 68) 44) 62} 68} 35) 63) 60) 65) 52 eo) 69) 55| 27/82/47/45/41/28|23) 2 68} 70) 63) 70) 36) 65} 68) 80) 60) 69] 70) 69) 36) |48/47/42/389/30) 2 70 70) 38) 68) 68 67; 80 46 2 70 52| 70| 70} | 67) 80 46 : 2 70 70} 75) 70 69 50 70 70 | 56 Total. . |346/114)125 208/231 341 406 145 385|294)139 124/261 32 5l92 8367/53) 8 Aver- age..| 69) 57| 62) 69 46) 68] 67| 72| 64| 73) 69) 62) 43/382/47|46/41/33/26) 2 of extracted materials as already pointed out by Funk and Dubin (5) and Fulmer, Nelson, and Sherwood (11). The shape of the curve also established the following data concerning the behavior of alfalfa extract. First, that no matter how concentrated the extract, it was impossible to develop a stimulative effect greater — than the production of about 0.04 cc. of yeast cells in 72 hours for each ce. of extract used; second, that increasing the con- centrations beyond the optimum lowers the production of yeast. The question naturally arises as to whether these features are common to all extracts; whether the decline to the right of the optimum is due to hydrogen ion effect; and whether time of incubation is a factor? The yeast autolysate curve shows that while its shape is essentially the same as that of alfalfa in showing steep rise, optimum concentration, and decline in higher con- centration, its power to produce growth of yeast cells exceeds the optimum power of alfalfa extract. This fact alone would be sufficient to show that in making the yeast test we are concerned with other factors than the presence of vitamine B and its con- centration. In Table IV are given the results obtained by apply- ing the test to a variety of materials. CHAT. EFFECTOF VARIATION is CONCENTRATION on THE TOOT LALFALFA EXTRACT + NAGELI -Concentrations 0-1C -Seties ARB : IC= Extract of o4gm. Alfalfa in ee. eo TL houts pare is = SEIS bi) -_ Sess u ee mal ual: E » tT TAS al tet i LLL WUE ETE] VT A i Se a eS a | Oh ge te SER Phos a ‘2 er at —— top [te sqaTleischman\east| | Cake infected | 262 Vitamine Content. II TABLE IV. Hesults of applying the yeast test to varying concentrations of extracts of the materials named. Test results with a dilute water solution of Funk’s 1913 crystalline com- plex (vitamine). 0.0079 gm. was dissolved in 75 ce. of water. This solu- tion strength is marked 1 in the series below and further dilutions were made for test purposes as indicated. The results are given in thousandths of a ce | 3 Concentration...........04+: 1 |0.8|0.5 | 0.4 | 0.3 | 0.2 | 0.1 |0.08| 0.05 |0.03| = 3) Noe AG S| A) 4a a A eee TAN aa 1G) |)-26\) 6. Aa eee id 7 eal, 6) 65)? aa ee al ea aaa Ta eedenlied «|| 6: | G0) a | ee 8 8 7 6 6 5 5 4 5 4 Average: 225 & atin Ciel 6) 5 | 5 | Aaa 4 as eee A comparison of extracts made from gland material extracted first with neutral alcohol and the residue reextracted with acidified alcohol. 1. Neutral alcohol 2. Acid alcohol extracts Gland. extracts of the residues original glands. from 1. E 3 Concentration....... On Onesie! ololg re) alo m| alo Ooo los kh 20} 22) 23] 18 Sheep pancreas. 13] 13} 13] 11) 3 5 29| 23] 24| 20/6 | C equals 2 gm. | 13] 15] 14] 11] 2 Ee ed ee fresh gland in a |—|/—-|——|——— Average......-. 24| 22) 23) 19151] ee. 13) 14) 13) 11) 2 15} 13} 9} 7| 5 | Human pancreas. | 10} 9} 8 5) 3 15) 4) 3) 7G 10) 10) 8) 6) 2 Average....... 15] 13} 11) 7}5] 10} 9| 8] 5/2 9} 8| 6 4| 5 | Human liver. 8| 7 5) 43 10) 8| 7 4| 6 8| 7 7 42 Average....... 9) 8 6 4\ 5 | 8| 7) 6) 412 Eddy, Heft, Stevenson, and Johnson 263 The only new point brought out by these tests relates to the gland extracts. Swoboda (13) in a recent article in this Journal advanced the view that Williams’ results with pancreas were due to his use of acid in making his extract, since with the method employed he was unable to get stimulative effects on yeast growth with pancreatic extract. He formulated a hypoth- esis of vitamine existing as a vitaminogen. The tests in Table IV were designed primarily to test this view. The results fail to confirm either of his contentions. We get stimulation well above the control with a neutral alcohol extract of pancreas and the residues on reextraction with acidified alcohol yield extracts of lower potency. If the vitaminogen view were correct the latter should exceed the first in power. The concentrations used were not sufficiently great to add much data to the question of curve shape, or location of optima. A study, however, of the tests with the Osborne and Mendel materials enables us to con- clude that the optima vary with the material extracted. The decline of growth in our concentrations of alfalfa extract higher than the optimum could not be due to the pH, for its values (determined colorimetrically) in these inhibitory concen- trations differed too little from that of the optimum concentration to function as an inhibitory factor in this experiment. To answer the query as to the effect of time of incubation on our results we tested out the rate of growth of yeast cells in two concentrations of the alfalfa extract as shown in Table V and Chart III. The concentrations selected were the one found optimum in the Series B tests and the full concentration of the extract. The results show that after 24 hours and up to a period of 19 days the time factor does not change the result. The concentration found optimum in the earlier tests retains its superior stimulative potency regardless of the time of incubation. These results do not answer all the questions raised by the alfalfa curve. They do, however, make it clear that if vitamine B is one of the factors concerned in the combined ‘“‘bios” effect of an extract it is only one of several factors concerned and that the use of the yeast test for quantitative measurement of vitamine content must await the development of a medium which shall be optimum for all factors except vitamine, exactly the situation that had to be met in the development of basal diets for rat- feeding experiments. 264. Vitamine Content. II] Il. The assumption that it is possible to develop a basal diet for yeast cells, optimum in all except vitamine B implies that at least one of the stimulative factors is vitamine B. If Fulmer, TABLE V. The effect of the period of incubation on the rate of growth of yeast cells in the optimum concentration of alfalfa extract as contrasted with the higher concentration of the extract.* Concentration I. Concentration II (optimum). E 1 ce. contains the extract of 0.4 gm. 1 cc. contains the extract of 0.15 gm. Period of of alfalfa. of alfalfa. incubation. Test. Test. Average. Test. Test. Average. hrs. a 2 2 2 0 0 0 6 2 eS 2.0 if iL 1 8 2 3 2.5 2 2 2 12 3 3 3 2 2 2 24 6 6 16 16 40 13 17 15 22 23 22.0 48 15 20 17.5 27 29 28 64 17 22 19.5 29 29 29 days i 6 6 16 16 2 15 20 eae 27 29 28 3 18 20 19 30 33 31.5 4 29 33 31 38 38 38 5 26 30 28 32 35 30.0 Zl 27 27 27 30 36 33 8 28 36 32 40 41 40.5 9 27 26 26.5 38 40 39 11 24 28 26 34 36 35 18 26 26 25° 31 40 34.5 14 28 31 PAIRS, 35 37 36 15 29 33 31 34 37 35 16 30 32 il a4 36 36 36 19 30 31 30.5 36 37 36.5 * For graphic presentation see Chart III. Nelson, and Sherwood’s contentions (11) are true even this factor is eliminated and further attention to development of the test is useless. Their contentions rest upon two different pieces of evidence; viz., the claim that by proper selection of a medium Eddy, Heft, Stevenson, and Johnson 265 of known constituents it is possible to obtain a medium that will produce a growth of yeast cells which is not improved by the addition of organic extracts, and second that the stimulative power of extracts on yeast growth is not affected by alkali treat- ment of these extracts, vitamine B being known to be extremely sensitive to the destructive effect of alkali, especially when com- bined with heat. RATE TEAIT GROWTH DAT AYS h & VAd AS To settle these points we felt that a fair test would be to sub- stitute for the Nageli solution the Medium F of Fulmer, Nelson, and Sherwood and see if our tests would still yield increased growth phenomena when this medium was supplemented with alfalfa extract. The differences in the two media are shown below. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 266 Vitamine Content. II Nageli solution. Medium F., 100 ce. distilled water. 100 ec. distilled water. 1 gm. ammonium nitrate. 0.188 gm. ammonium chloride. 0.05 “‘ ealeium phosphate. 0.10 ‘* calcium chloride. 05 “ KSHPO; 0.10 ‘* KsHPO,. 0.25 “‘ magnesium sulfate. 0.04 “ precipitated calcium 10.0 ‘‘ dextrose. carbonate. 0.6 «dextrin. 10.0 ‘* sucrose. In using the F medium it is obvious that the calcium carbonate will sediment with the yeast cells unless removed before centri- fugating. We accomplished this by adding dilute HCl, drop by drop, to the tube contents just before centrifugating. The results of this test with varying concentrations of alfalfa extract added, are shown in Series C of Table II and in Chart II]. They show that while, as evidenced by the control figures, Medium F is superior to Nageli solution as a basal medium it is still capable of stimulation by every concentration of the alfalfa extract used and that while the optimum growth obtained is greater than that obtained with Nageli solution the shape of the curve is essentially the same as with Nageli solution. These results seem at least to make questionable Fulmer, Nelson, and Sherwood’s first contention. To test their contention regarding the effect of alkali we selected both alfalfa extract and yeast autolysate for experimental ma- terials. Our alkali-treated extracts were obtained in two ways. A portion of the extract was measured out and make up to 10 per cent strength with solid NaOH. This mixture was then autoclaved at 15 pounds above atmospheric pressure for 3 hours. After autoclave treatment the extract was neutralized with HCl to litmus and evaporated to its original concentration. Except for the action of the alkali on the components of the extract the sole change made in composition was an increase in NaCl, 1 cc. portions of the treated extract, diluted to provide varying concentrations, were then combined with 9 cc. of Nageli solution | in one experiment and with Medium F in another. Previous experiments had provided us with the control data (see Table II and Chart II). Table VI and Chart IV give the results. TABLE VI. The effect of alkali treatment upon the stimulative powers of alfalfa and yeast extracts.* Studies of alfalfa extracts. (C equals the extractable material from 0.4 gm. of dried alfalfa in 1 cc. of water.) CS Pe PSOE nea Nageli solution 0.95C 2026) 28 24 20 used. 0.75C Bd, PAE POAT 25 27 See Table III. 0.55C DYE SP WIR 27 28 0.475C 202 la 28 27 32 0.375C Bs) Pal BM 28 33 0.275C 2B} i DS 24 31 0.175C a) Da WE Us 28 0.075C AGG 17 16 22 0.035C Wee ee aly 15 17 0.015C 10 LOR wt 10 16 Control. 2D» 2 2 | 2 Medium F used. 0.95 UR) DRY Dey 43 0.75 2122 28 24 45 0.55 Se 220 26 23 39 0.475 20 24 26 23 32 See Table III. 0.375 ifs 240" Gal 19 31 0.275 16, 18 16 29 0.175 ib, iets} 12 27 0.075 1@) “py ae 11 22 0.035 ily lh, 2 11 19 0.015 OPO 10 10 19 Control. alt dl 4 15 ; Studies of yeast autolysate. (C equals 0.75 gm. in 1 cc.) @ % pauccntrations sea Se with egersare Alka ly treste@ with Areranes 0.95C we P15, S33 Dif DP) 93). DRS 22 ’ 0.05C - DA De OES 24 ii aS 17 : 0.475C 7A) BBY BE D2? 1G) 16S 1G 15 0.375C ZO e251 a 26 24 tse Toee16 15 0.275C 18 18 20 18 1 14 IB 12 0.175C ily ates Ue 17 bier lO) 11 0.075C 16) 1183, 118° 13 TOR LO te tt 10 0.035C Sa eGise 29 9 SOF 9 9 0.015C Dake) 52, 2 DO el. 5 Control. DA Re 2 Bo BS 3 * For comparison with untreated yeast autolysate see Chart IV. 267 (NIT HE pep OF ALKALI +t CAT ON THE YEAST TLOT Acts mr TT ee fre = ial nlTeatetg Yast mu 1 ma }hali Apel : 2p THE-RESULTS- OFAN: “ATTEMPT-TO-EVALUATE- EXTRACTS % ALKALI + HEAT The average of 7 eis. made on each of 2 Conce nitalions isTecotded O's = agucg cuit TCHR Mhal) extacis _O’=unneutrahised alkali exttacts | 1.0L Y — - l 268 Eddy, Heft, Stevenson, and Johnson 269 When these results are examined carefully the conclusions are not so definite as the figures might at first indicate. They seem to show destruction by the alkali of some of the power but the extent of the destruction is not nearly so great as would be expected if vitamine B formed a large part of the stimulative factors. It is also easy to see why Fulmer, Nelson, and Sher- wood reached their conclusion as to practically no destruction if they tested only the higher concentrations, for in the higher concentrations the effect is much less marked, especially in the experiments with Nageli solution as the basal medium. These facts show up more distinctly in the charted results. We there- fore resolved to make another test. If vitamine B is one of the factors and if it is destroyed by alkali the extracts of the Osborne and Mendel series ought to show a marked reduction in stimu- lative power after alkali treatment. To determine this we made a new set of extracts of those materials. These sets consisted of three series obtained as follows: Series A represents aqueous extracts of 1 gm. of dried material made up with water so that 1 cc. carried the “‘bios” of 0.01 gm. of dried material. Series B consists of a similar set obtained by extracting 1 gm. of the dried material with 0.1 n NaOH, filtering, neutralizing with HCl to litmus, and diluting until 1 ec. carried the “bios” of 0.01 gm. of dried material. In both these extractions the material was boiled continuously for 3 hours in the extractant. The third series was obtained by taking 50 cc. portions of Series B, adding 5 ce. of 0.1 n NaOH to each portion, heating at 100°C. for 30 minutes, and then testing without neutralizing. We assumed that the order of test effects would be in all cases highest with Series A, Series B coming next, and Series C third. The actual readings are given in Table VII and graphically in Chart IV. These results fail to confirm the conclusions from Table VI and seem to give excellent confirmation of Fulmer, Nelson, and Sherwood’s view that the stimulative factor is not appreciably affected by alkali treatment. These two sets of experiments, therefore, leave that question unsettled. From these results and from the previous ones it becomes increasingly evident that the growth of the yeast cells, as deter- mined by any one of the methods devised to date, is a resultant of so many different factors, mutually interacting, that to interpret 270 Vitamine Content. II these growths as a quantitative measure of vitamine content is unjustified. All the evidence except that based on alkali treat- ment seems to argue in favor of vitamine B as at least one of the TABLE VII. Effect of alkali treatment on the Osborne-Mendel preparations. Only the averages are given. est |l|| ‘eo : = 3 - 3 7 = o = = + = ° Materials tested ./.c.cc deve deeseecc = 2 S S 2 I 2 ° = 5S 3 5 & = fa) be a =I ct S = = ° a 3 3 a aed oO ° = S) a mM 1@) a é) oH fea) 6) Full concentration of aque- OuUBLextract: 220 02055,- ase Aalto 20.| 27 | 16") 14) 18 ye Ogi Neutral alkaline extract....| 19 | 19 | 20 | 19 | 21 | 14 | 24] 26; 7) 1 Alkaline extract with 5 ce. eke 2; AEA oh eS Rae A eS 26 | 14) LS S228 Half concentration of aque- OUStCXET ACh. 22m eee eck 16/48) 18 2)10 | 14 17 163) 13 Sale _ Neutral alkaline extract....| 14 | 18 | 13 | 15 | 16) 16| 18 | 16| 6] 2 Alkaline extract with 5 ce. SCAT: |. .: eee been es). ae 197 )19) | 20°) 18°) 1289 20 aS Sale Fourth concentration of aqueous extract.......... 13) | 14) 14) 9 | 10 | 16) 15s) 10 ome Neutral alkaline extract..../ 13 | 15} 11 | 11.) 13) 11 | 14) 13 | 642 Alkaline extract with 5 ce. Baler se atanecr. pee omens 165/14) 15") 14) 10) Stan Teh 2a ee Eighth concentration of aqueous extract.......... CeO) 10:) 6) 7 14 eae Shoe ae Neutral alkaline extract..... 5|12]} 7/10] 8/] 8/|12/]10|] 4] 2 Alkaline extract with 5 ee. A so) | ey st Le Ma rc F 971799) AS 9) Oi Sel AAAS eee ee Sixteenth concentration of aqueous extract.......... 91 8) 9.5) 9 | 9) 11 | aeeioes Neutral alkaline extract:..-| <2.) S0|| 5) 6 |. 4) 617 | SaaieeSaleez Alkaline extract with 5 ce. alkali)? yh 2s eee Beleon| °3 |) 5!) Saal wae 4 | ee ee ee growth stimulants while the alkali evidence is contradictory and incomplete. There seems, therefore, only one alternative; - namely, to attempt to separate and identify the factors concerned in the growth stimulation and if possible devise a medium that ee Es ee Eddy, Heft, Stevenson, and Johnson 271 can justify its use as a basal diet for yeast when used to test vitamine content. We have begun the development of such experiments and a few of our preliminary results follow. Ry Professor McKee of the Department of Chemical Engineering of Columbia University has recently prepared two sets of carbons, one specially adapted to the adsorption of acid-reacting sub- oa A STUDY OF THE BEHAVIOR OF Mc KEES CARBONS ICC | BASE ADIORBING C. | ACID ADSORBINGC.| LLOYD'S REAGENT | PHOSPHOTUNG STIC ACID | | BEDSRE! AFTER | BEFORE) AFTER | BCTORE LATTER pec ee (ARTER: |: Some |, + fA EXTINCT] | te — et — et TESTED en ee eS ee y BEFORE avo eee ates nae Ce Sere = ieee ge | . =a a oe Fe AFT ER i Teen ee ee a mn a ee oe ed a ae Singe d's.= 5 ned bb me] . (Errecronn= pce | CONTRO | rmecmer so CONTROL EITR- BEFORE, 090%? TREATMENT | | LT CURVEOF | do: | GU-ACETICEL. [eee EN i Se stances and the other to the adsorption of alkaline-reacting substances. Samples of these were furnished by him for our use. We first determined that the base-adsorbing carbon had a power of adsorption for the yeast erowth-stimulating factor that was quite as good as treatment with fullers’ earth (Lioyd’s reagent), or precipitation with phosphotungstic acid (see Chart V). With this fact established we proceeded to treat a known concentra- tion of alfalfa extract with this carbon. After a few hours shaking oe Vitamine Content. II and then allowing the mixture to stand for 24 hours the carbon was filtered off on a suction funnel. This carbon was then washed on the funnel and by shaking repeatedly with distilled water until all adherent material that was removable by this washing was eliminated. The washed carbon was then boiled with glacial acetic acid and the acid extract filtered off through an alundum filter. This acid extract was evaporated to dryness on the steam bath and the residue taken up with distilled water and neutralized. Its volume was made up with water to equal that of the alfalfa extract from which it was derived. This extract in full concen- tration and in several dilutions was tested with the yeast test and the results are shown in Chart V. When compared with the test strength of the solution from which it was obtained the recovery of the stimulation factor by this method seems to have been rather high. With a portion of this extract we repeated the alkali treatment and obtained an unmistakably destructive effect, though not complete destruction. This result leads us ~ to retain our’ faith that one of the factors concerned is either vitamine B or one that resembles it somewhat closely in properties (14). Analyses are now being conducted on the carbon recovered factor to obtain a possible clue as to its nature. If we picture the action of a factor on a yeast cell we may assume that the process might well consist of three steps; first diffusion, by which it comes in contact with the cell; second, possibly adsorp- tion or at any rate penetration into the cell; and finally, its specific stimulation to protoplasmic production. If this is true the fol- lowing conditions will exist and influence the growth of the cell; viz., number of factors present, concentration of these factors, and rate of diffusion of factor to cell. It occurred to us that the failure of high concentrations to permit optimum growth might be a matter of diffusion. The following tests were devised to test this view. The two concentrations of alfalfa extract used in the time of incubation test (Table V) were selected, and two sets of tubes prepared as usual. One set was kept perfectly quiet at room temperature, after inoculation with yeast suspension for 5 days. The second set was treated in the same way except | that at intervals in the 5 days they were transferred to the shaking machine and shaken a total of 15 hours in all in the 5 days. The Eddy, Heft, Stevenson, and Johnson PA gs: rate of growth in the two sets is shown in Table VIII. Having thus shown that diffusion is an important factor, a second test was devised as follows. Two sets of tubes were prepared as before but the 1 ce. of extract was in one set of tubes placed within a collodion sac suspended in the Nageli solution. The effect of this process was to reduce slightly the growth rate -but there is TABLE VIII. The effect of changing diffusion conditions upon the yeast test. Shaking experiment. Full concentration of alfalfa. ‘Optimum concentration of alfalfa. Test. ae Test. ee 5 days with no Shalcinge 5 os: LOPES ett 12 | 22° 25° 22 27° 22 24 5 days with a ‘total of 15 hrs. shaking....... 22 Non2 Ul GaZonls 20 29 35 39 34 34 Control (still)... De, 2 Control] shaken.. 3 4 4 4 Collodion bag experiment. Full concentration yeast Half concentration yeast autolysate extract. autolysate extract. Test. eine Test. pe 1 ec. extract mixed with Nageli solution in tube.. 84 80)| 82 Ue (au) 7B 1 ce. extract suspended in collodion sac.....:...| 67 62 60 82] 68 | 64 56 61 61] 60 ‘CLT AC) a eo a 2 2 no doubt that the growth factor passes through the membrane; 1.e., is highly diffusible through membrane. The results are shown in Table VIII. We are now trying to determine whether by a study of the content of the collodion sacs before and after diffusion, light may be thrown on the nature of the factors. These few experiments are cited merely as illustrating the com- plexity of factors taking part in a yeast test. 274 Vitamine Content. II SUMMARY. 1. When the yeast test is applied to materials already evaluated as to vitamine content by rat-feeding experiments, the results show only approximate agreement. The agreement is more marked, however, when the extracts are dilute. 2. A study of the curve formed by plotting all the determina- tions obtained with varying concentrations of an aqueous extract of alfalfa shows that the reaction does not give the appearance of a monomolecular reaction. From the control point to the optimum it approximates the shape of the logarithmic curve but to the right of the optimum point there is a distinct decline indicating inhibitory factors in the higher concentrations. 3. A comparison with curves obtained from other extracts shows that the optimum growth varies not only with the con- centration but with the nature of the extract tested. 4. A study of the effect of extracts upon growth of yeast cells, using Fulmer, Nelson,and Sherwood’s medium F in place of Nageli solution fails to support their contention that the growth stimulus is purely a matter of concentrations of known constituents. 5. Certain results, following the use of alkali, seem to indicate that if the solution treated is sufficiently dilute, the destructive effect of the alkali will appear; but attempts to verify this with extracts of the materials used in the rat-feeding tests gave most contradictory results. In any case it seems evident that at least some of the factors concerned in the stimulation are not affected by alkali treatment but the data is not sufficiently complete to justify the view that vitamine B is not present and therefore not one of the functioning stimulants. 6. The cumulative effect of the data obtained is to suggest that in its present state the test is distinctly unreliable as a quantitative measure of vitamine content. On the other hand it suggests interesting possibilities as a method for studying the kinds and behavior of growth stimuli. 7. Experiments are cited that illustrate some of the factors that enter into the growth results and a method of analyzing their nature is given. 8. Until a basal medium is worked out that provides an opti- mum of all the factors except vitamine B the test must be con- sidered of little value in the estimation of true vitamine content. Eddy, Heft, Stevenson, and Johnson 219 BIBLIOGRAPHY. . Williams, R. J., J. Biol. Chem., 1919, xxxviii, 465. . Wildiers, E., La Cellule, 1901, xviii, 313. Eddy, W. H., and Stevenson, H. C., J. Biol. Chem., 1920, xliii, 295. Bachmann, F. M., J. Biol. Chem., 1919, xxxix, 235. Funk, C., and Dubin, H. E., J. Biol. Chem., 1920, xliv, 487. . Funk, C., and Dubin, H. E., Proc. Soc. Exp. Biol. and Med., 1920, may NO: . Souza, G. de P., and McCollum, E. V., J. Biol. Chem., 1920, xliv, 113. . MacDonald, M. B., and McCollum, E. V., J. Biol. Chem., 1920-21, xlv, 307. . Emmett, A. D., and Luros, G. O., J. Biol. Chem., 1920, xliii, 265. . Emmett, A. D., and Stockholm, M., J. Biol. Chem., 1920, xliii, 287. . Fulmer, E. I., Nelson, V. E., and Sherwood, F. F., J. Am. Chem. Soc., 1921, xliii, 186, 191. . Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1920, xli, 451. . Swoboda, F. K., J. Biol. Chem., 1920, xliv, 531. . Osborne, T. B., and Wakeman, A. J., J. Biol. Chem., 1920, xlii, 1. . Williams, R. J., J. Biol. Chem., 1920, xlii, 259. NOTES ON APPARATUS USED IN DETERMINING THE RESPIRATORY EXCHANGE IN MAN. I. AN ADAPTATION OF THE FRENCH GAS MASK FOR USE IN RESPIRATORY WORK. By CAMERON V. BAILEY. (From the Laboratory of Pathological Chemistry (Respiratory Division), New York Post-Graduate Medical School and Hospital, New York.) (Received for publication, May 2, 1921.) Considerable difficulty has been experienced in finding a suita- ble breathing appliance to be used with respiration apparatus. To insure absolute rest and normal type of respiration it is essential that the subject suffers no discomfort. The appliance should per- mit him to breathe through the nose, mouth, or both as is his cus- tom; otherwise abnormal conditions are imposed, and his atten- tion being focused on the respiratory act, unnatural breathing results. Appliances necessitating the insertion of tubes into the mouth have the additional defect of causing salivation, with a resulting frequence of deglutition and, in the closed circuit apparatus, the possible swallowing of oxygen. Of the appliances at present in use, the half mask used by Boothby and Sandiford! presents none of these defects. In practice, however, difficulties arise in the use of the half masks; the engaging surfaces are small and the bony support of the tissues deficient; in the absence of molar teeth, the cheeks sag inwards during inspiration and leaks are apt to occur. In the attempt to avoid leaks the mask is frequently applied so firmly to the face that extreme discomfort results. The rubber gas mask used in the French army is admirably suited to this work.? It is made of thick rubber, covers the whole 1 Boothby, W. M., and Sandiford, I., Laboratory manual of the technic of basal metabolic rate determinations, Philadelphia and London, 1920, 35. ? The gas masks and valve attachments may be obtained from the C. M. Sorensen Co., 177 East 87th Street, New York. 277 278 Respiratory Exchange in Man. I face, and presents broad surfaces which closely engage the fore- head, sidesof theface,andjaw. Thetissues in these regions are well supported by the bony framework of the face and the mask readily adapts itself to these fixed surfaces. It is held in place by elastic straps passing around the head. In emaciated subjects, leaks may occur above or below the zygoma, in this area the pull of the straps is In the same plane as the surface of the face. In such rare in- stances the leaks are readily overcome by placing 6 inch rubber sponges over these areas of the mask and binding them firmly in place witha 3 inch bandage. In this mask, the incoming air is directed | cl fl pore See ] ~<—=70 GASOMETER Brea: upwards towards the windows, the opening of the expiratory tube being opposite the nose and mouth. In this way the space is perfectly ventilated and no discomfort results. Rubber flutter valves are eminently satisfactory for use with these masks. They are conveniently enclosed in flattened glass tubes introduced as near the mask as practicable. A satisfactory arrangement of mask and valves for the Tissot method is shown in Fig. 1. The subject reclines in a wheel chair in the rest room, the tubes pass through the wall into the laboratory where the valves are mounted, the tubes then lead directly to the air supply and to the gasometer. A window permits the operator in the laboratory to observe the C. V. Bailey 279 subject, while at the same time he can follow the respirations by watching the movement of the valves and start and stop the test by turning the 3-way valve on the gasometer. The incoming air enters the room through a 4 inch pipe, the end ‘of which is closed by a rubber bathing cap held in place by a rubber band. This takes up the pressure of gusts of wind and prevents it blowing through the valves. A 2 foot length of 24 mm. rubber tubing leads from the large pipe to the inspiratory valve, this is for the purpose of trapping any expired air which might backlash through the valve. In using the mask with closed circuit appara- tus double tubes lead to the oxygen reservoir, the expiratory tube, containing a flutter valve, leads through the CO, absorbed. With portable apparatus the valve case can be made of transparent celluloid which is not readily broken. In this type of apparatus the use of the mask with a single tube and without valves is unsat- isfactory as the dead space is increased to too great an extent. i —_—— . = ne : te) +a i ® ' poe? = ‘ k > y yi ve. - ae yal @ rn ie LS Moy .' ‘ ' Ts a we ae ee apa ij Ne WTR BID HE ge ous a me vag ~ 4 =i. r % UF y i = NOTES ON APPARATUS USED IN DETERMINING THE RESPIRATORY EXCHANGE IN MAN. II. A SAMPLING BOTTLE FOR GAS ANALYSIS. By CAMERON V. BAILEY. (From the Laboratory of Pathological Chemistry (Respiratory Division), New York Post-Graduate Medical School and Hospital, New York.) (Received for publication, May 2, 1921.) This appliance is designed for readily collecting and holding samples of gas and for transferring them to a gas-analyzing burette without danger of dilution or loss of mercury. The bottle shown in Fig. 1 is 19 em. high and has a gas capacity of 60 cc.; this is a convenient size for use in conjunction with a gasometer and the Henderson? modification of the Haldane gas analyzer. 1 This bottle may be obtained from the C. M. Sorensen Co., 177 East 87th Street, New York. * Henderson, Y., J. Biol. Chem., 1918, xxxiii, 31. 281 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 282 Respiratory Exchange in Fria. 4. Fra. 6. Fia. 7. Man. II Fie. 3: Fie. 8. SHY et ay or ay C. V. Bailey 283 The bottle as illustrated consists of parallel glass cylinders communicating at the bottom through a small opening. The cylinders are firmly cemented into a metal case which forms a base. The top of one cylinder has a funnel-shaped inversion; the other tapers off to a 2 mm. capillary tube to which a 2-way stop- cock is fused. The second opening in the stop-cock communicates with a capillary tube which bends over the inverted top of the first cylinder. The stop-cock terminates in a straight spout of capillary tubing 3 cm. long. The appliance is half filled with mercury, and before use is flushed out with 1 per cent sulfuric acid. The series of small drawings illustrate the manipulation of the bottle. Fig. 2. It will be seen that by tilting the bottle forward, the mercury displaces the air in the gas compartment and in the barrel of the stop-cock. Fig. 3. If the stop-cock be completely reversed while the bottle is in this position, these parts remain filled with mercury and the spout is in communication with the bent capillary tube through which the gas to be sampled is blown. Fig. 4. The stop-cock is returned to its original position and the sample of gas is drawn into the bottle by tilting it backward. Fig. 5. By turning the stop-cock while the bottle is in the last position the sample is trapped in the compartment under pressure and the spout again communicates with the curved capillary tube. Fig. 6. The sampling bottle is now placed on a shelf overthe analyzer framework; the spout connected to the top of the gas burette and the mercury in the latter forced through this commu- nication rendering it free from air. Fig. 7. The stop-cock is now turned to the first position, the mercury in the burette lowered, and a sample of the gas is drawn into the gas burette. Fig. 8. The stop-cock is again reversed and sufficient gas remains for five additional analyses. a 7 a | <> Wu i be o) a io. “re Se eth en : "5 Vi ales A 7 — a“ : | ‘g Os ait e ra ~ rf . rv iy * a ; ' 4 its fefuene 2 a » STM? % : aH 1 Ne ae allied wealth 6 . DuhiLad i Ale : + AO URE Be nm atl Bau ae . *, if Lee fu 7s paren yetih9 im fh qa’ : yep , | _ aA A yet ‘ ' ' ee. 4s . * ' i a i eh Mies STUDIES ON THE DIGESTIBILITY OF PROTEINS IN VITRO. II. THE RELATIVE DIGESTIBILITY OF VARIOUS PREPARATIONS OF THE PROTEINS FROM THE CHINESE AND GEORGIA VELVET BEANS. By HENRY C. WATERMAN anp D. BREESE JONES. (From the Protein Investigation Laboratory, Bureau of Chemistry, United States Department of Agriculture, Washington.) (Received for publication, May 19, 1921.) The ill effects (1, 2) which have sometimes followed the use of velvet bean meal as the principal source of protein in stock feeds have lead to an investigation by Miller (1) in which he isolated a toxic substance, 3, 4-dihydroxyphenylalanine, from the seed of the Georgia velvet bean, Stzzolobiwm deeringianum. This discov- ery seemed at the time of its publication to furnish a sufficient explanation of the objectionable properties exhibited by these beans. But feeding experiments recently made in this laboratory (3), while they tend to confirm the supposition that the velvet beans contain a toxic substance, have brought out also the inter- esting fact that the isolated proteins prepared from the Chinese (Stizolobium niveum) and Georgia velvet beans by dialyzing their saline extracts are no better tolerated than is the bean meal. There must, then, be some limiting factor other than the dihydrox- yphenylalanine alone. Amino-acid deficiency cannot account for the failure to promote growth, for analyses (4, 5) have shown these proteins to be adequate except possibly with respect to cystine; and. no improvement resulted from the addition of this amino-acid. Further, the proteins as obtained by coagulation gave normal growth. The most reasonable hypothesis to account for these observations seemed to be that the proteins in the raw state as prepared by dialysis are not sufficiently digestible to be available for animal nutrition, while the coagula were cooked, by the boiling incident to the process of preparation, enough to ren- der them sufficiently digestible. The primary purpose of the ex- periments described in this paper was to test the correctness of this 285 286 Digestion of Proteins in Vitro. II conclusion. ‘We wished also to secure further data on the extent to which our results derived from experiments 7n vitro run parallel with those of growth and utilization tests made with animals. The method employed was essentially that of Waterman and Johns (6), described in the first paper of this series. Inasmuch as proteins from different sources were to be compared in the pres- ent case, the results were calculated on the basis of the total amino nitrogen of the protein, minus the free amino nitrogen (3 the lysine nitrogen), as suggested in the first paper. The formula for the calculation of the percentage of digested nitrogen becomes, then, DN =WNga — No Na Na = mg. amino N found in the reaction mixture after digestion, cor- rected for the blank due to the reagents used in the Van Slyke amino N apparatus. Np, = mg. amino N produced by self-digestion from the enzymes in a blank digestion; corrected as above. Na = mg. total amino N, derivable by complete hydrolysis from the protein, minus one-half the lysine nitrogen. < 100 where The experiments confirm our theory based upon the results of the feeding experiments, that the failure of raw dialyzed Chinese velvet bean protein was due to partial indigestibility. The figures obtained for the digestibility of the coagulated proteins of the Chinese and Georgia velvet beans are nearly twice as great as those given by the uncooked dialyzed proteins in either case, and agree well with those yielded by cooked phaseolin (6) and by casein (Table IV). The figures found for the raw dialyzed pro- teins are about 8 per cent lower than those given by raw phaseolin. This corresponds well with the growth experiments in these two cases. It seemed almost certain that the difference between the raw dialyzed and the coagulated velvet bean proteins was due simply to the cooking involved in the preparation of the latter material. There remained, however, the possibility that the coagula contained protein not precipitable by dialysis and that their greater percentage of digestible nitrogen might have been due in part to these other proteins. In order to eliminate this ques- tion we made cooked preparations of the protein obtained by dial- ysis from each of the two species of velvet beans and have compared their digestibility with that of the corresponding raw dialyzed pro- Ye CO H. C. Waterman and D. B. Jones 287 teins. Both cooked preparations showed a digestibility agreeing closely with those of the corresponding coagula. In addition to the difference in the percentages of digestion nitrogen, certain purely qualitative indications were observed which also point to a marked superiority in digestibility of the coagulated and the cooked dialyzed preparations over those of the raw dialyzed proteins. In the first place, there appeared in the digests of the raw dialyzed samples, on neutralizing the acid after digestion with pepsin, a bulky, light gray, flocculent precipitate apparently consisting of incompletely peptonized protein. The coagulated and the cooked dialyzed protein formed only a small precipitate at this point. Also, when the digests are heated, to inactivate the enzymes at the end of the digestion period with trypsin, a small amount of coagulation always occurs, although the solution is distinctly alkaline. In the present case this precipitate was always considerably greater in the reaction product of the raw dialyzed, than it was in those of the cooked or the coagulated proteins. Again, in making the determinations of amino nitrogen on the filtrate from these reaction mixtures, we found that the digestion products of the raw dialyzed samples foamed persistently in the Van Slyke apparatus, filling both the deaminizing bulb and the gas burette with a thick froth which made it almost impossible to carry out the analysis accurately, unless diphenyl ether or some other foam inhibitor was used. This behavior of the digests of the uncooked preparations is very characteristic of solutions containing unhydrolyzed protein. The digests of the cooked and the coagulated samples, on the other hand, gave practically no trouble of this sort. Finally, if the fil- tered, slightly alkaline digestion products were made just acid with acetic acid a further precipitate was produced, and, here again, its amount was considerable in the case of the raw dialyzed preparations and very slight,a mere cloud hardly greater than that, yielded by the blank digestion, in the case of the cooked or the coagulated protein. This combined evidence leaves little doubt that the difference in digestibility between the raw dialyzed and the coagulated vel- vet bean proteins is due simply to cooking. Also, the experiments as a whole tend decidedly to confirm our supposition that incom- plete digestibility is one of the limiting factors in the nutritional 288 Digestion of Proteins in Vitro. II failure of raw velvet bean meal and the only limiting factor in the similar failure of the protein isolated by dialysis. EXPERIMENTAL. Preparation and Analysis! of the Proteins —The coagulated and the dialyzed proteins from the Chinese velvet bean were made? in accordance with the direction of Johns and Finks (4). The dried preparations were ground to pass a 100 mesh sieve, the pow- dered material was exposed to a filtered current of air to come to equilibrium with atmospheric moisture, and the total nitrogen, not corrected for ash or moisture, was determined. The total amino nitrogen of each preparation, minus its free amino nitrogen (2 the lysine nitrogen), was then calculated from the figures of the Van Slyke analyses given in the paper referred to above. The coagulated Georgia velvet bean protein was prepared like that from the Chinese variety. The dialyzed protein was preci- pitated directly from the saline extract without preliminary frac- tionation by ammonium sulfate. Both of these preparations, therefore, contained a mixture of the a- and f£-globulins of the Georgia bean, and in addition to these the coagulum contained the traces of albumin present in the extract. The dried mixtures were prepared for analysis and their total nitrogen determined as above described; but the presence in each of more than one protein made necessary a different procedure in the estimation of the amino ni- trogen. An average of the amino nitrogen figures, taken from the nitrogen distribution given by Johns and Waterman (5) for the a- and $-globulins, corrected for free amino nitrogen as above, and weighted according to the relative yields of the two proteins, was used as the basis of the calculation in the case of the dialysis prod- uct. The maximum error which could be introduced into the eal- culation of the digestion nitrogen by the use of the value thus derived would not be significant for the purposes of the present experiments. The coagulum, however, contained albumin, the 1 The elementary analysis of the preparations was made by S. Phillips of this laboratory. 2 This material was prepared by C. E. F. Gersdorff of this laboratory. Both varieties of the beans were furnished by the Bureau of Plant Indus- try, United States Department of Agriculture. H. C. Waterman and D. B. Jones 289 relative quantity of which could not be estimated with any approach to accuracy. A sample of about 3 gm. of this material was hydrolyzed, therefore, and freed from ammonia as in the determination of the distribution of nitrogen by Van Slyke’s method. After expelling the ammonia, the hydrolysate was acidi- fied with acetic acid, concentrated to a syrup, transferred to a 200 cc. graduated flask, and made up to the mark with distilled water. The amino nitrogen in this solution was then determined by means of the Van Slyke apparatus, and the N, calculated from the value thus obtained. Digestion with Pepsin—The procedure was the same as that previously described (6), except that 0.1 N hydrochloric acid was substituted for the 0.1 Nn sulfuric acid used in the first experiments. Two or three samples, approximately 0.500 gm., of each of the proteins to be compared were suspended each in 25 cc. of 0.1 N hydrochloric acid, 25 cc. of a 0.2 per cent solution of pepsin in the same reagent were added, and the mixtures incubated for 14 hours at 37°. The raw dialyzed proteins from both the velvet beans readily dissolved in the dilute acid, while the coagulated and the cooked dialyzed proteins were not completely dissolved until near the end of the digestion period with pepsin. Digestion with Trypsin.—After the peptic digestion the 50 ce. of 0.1 N acid were neutralized with 5 ce. of N sodium hydroxide and 5 cc. of a6 per cent solution of trypsin in 0.1 N sodium hydrox- ide were added. The reaction mixtures were then returned to the incubator and digested for 24 hours at 37°. The activity of the enzymes was then destroyed by heating to 80° on the steam bath, the solutions were cooled and filtered, and amino nitrogen was determined. With each set of experiments a blank digestion of the enzymes alone without any added protein was carried out in exactly the same way as were the digestions of the samples, in order that the amino nitrogen set free by the self-digestion of the enzymes might be determined and a correction made for it. The resulting data, together with the percentages of digested nitrogen calculated according to the formula given above, will be found in Tables I, II, and III. The average digested nitrogen values for casein, cooked and raw phaseolin, and for each of the six velvet bean preparations studied are presented for comparison in Table IV. TABLE TI. ‘Comparative Digestibility of Coagulated and Dialyzed Chinese Velvet Bean , Proteins. Digestion N, calculated on basis of Na Amount of | Combined Amino N |Amino N of Preparation. sample amino N in | after diges- | blank diges- (protein). |sample (Ng).| tion (Nqg). | tion (Np). mg. mg. mg. mg. per cent Coagulated....... 1 501.3 51.13 48.09 18.24 58.0 2 501.8 51.18 48.59 18.24 59.1 (ALVEL ADELE Shei. Rene Pee es eis aolal ts eleide fo A eT nee 58.6 Dishyzed... 2s acn .+ 1 502.1 54.23 30.51 18.24 22.4* 2 502.0 54.22 33.99 18.24 28.9 AV CT AT CL MLat nie cr ate tfos amie ronioe ticks) oil vis ba, 4 alajy SRE Or Rete ai 28.9 Coagulated....... 3 500.9 51.09 47.72 19.20 55.8 4 501.2 51,12 47.72 19.20 55.8 IA VCE AD CHE ters Etre are ats Sletei ss Seta hs a's Lavage eR Ee, SUPE 55.8 Dialyzed.... ce... 3 | 501.3 54.15 35.40 19.20 29.9 4 501.2 54.14 35.90 19.20 30.8 5 501.2 54.14 35.73 19.20 30.5 AAV CY SD Oca tractors eas Cede MeL Cibie ssi sje ba nace ss slat een oe iae cae ee 30.4 General average. per cent Peal med Seaee hess Pee ee kee ovis be ehig Lee ue Re A eee 29.7 Coapgitlateds 36.0.5). 206 ASSES Stl ACs ad OARS Ree eee 57.4 * Not included in the averages. TABLE II. Comparative Digestibility of Coagulated and Dialyzed Georgia Velvet Bean Proteins. Amount of | Combined | Amino N | Amino N of Digestion N, ; : : : : leulated Pre Yo ee | SaIo UNS, Sacer Gale | Clan as ea ee re mq. mg. mg. mg. per cent Coagulated....... 1 501.0 53.96 48.56 18.80 59.2 2 501.0 53.96 48.89 18.80 55.8 Averaper: cic See: ek ee CE idl to. . |. eee 55.5 Dialyzed.......... 1 501.5 56.02 36.50 18.80 31.6 2 501.6 56.03 36.66 18.80 31.9 AVOLARE ..: iy sic cde were a ee oe oes 3 bo ys 31.8 Et ©: TRELLerinian and D. B. Jones 291 TABLE III. The Effect of Cooking upon the Digestibility of Dialyzed Velvet Bean Proteins. Amount of | Combined | Amino N_ |} Amino N of Digestion N, Preparation. sample amino N in| after diges- | blank diges-| © Ha aise f (protein). |sample (Ng).| tion (Ng). | tion (Np). | ° oh S mg. mg. mg. mg. per cent Georgia. Dialyzed, cooked*...... 1 501.1 54.38 48 .53 18.86 54.5 2 501.3 54.40 48.65 18.86 54.8 EASTGATE pe RS =, ec a OA eRe oie Ce SALT Georgia. Dialyzed, raw. .3 | 502.3 56.10 37.11 18.86 32.5 4 502.0 56.07 37.34 18.86 32 TELE 20) MP Ot 69 5 2rd ce RA a S2ut Chinese. Dialyzed, cooked*... .... 1 501.2 52.18 48.02 17.65 58.2 2 500.6 92.12 47 .62 17.65 5X5) i 072 YS et al be A oo Soh ch i he 57.9 * An aqueous suspension was boiled 2 hours, evaporated to apparent dryness on a steam bath, and ground to 100 mesh powder. DISCUSSION. From the properties of the walks bean proteins brought out by these experiments we may conclude, in the absence of experi- mental evidence to the contrary, that our hypothesis regarding partial indigestibility as the limiting factor in the failure of the raw dialyzed Chinese velvet bean protein to promote growth is the correct one. The nutritional inadequacy of the uncooked protein, together with the fact that a normal growth rate can be secured withthe coagulated protein, is then to be referred to difference in digestibility alone. The raw ground seed would have the double disadvantage of toxicity and non-available protein content, while the cooked meal would probably still contain dihydroxyphenyl- alanine. Our experiments also justify us in predicting with some confidence that the raw dialyzed protein from the Georgia velvet 292 Digestion of Proteins in Vitro. II bean will behave in nutrition experiments like that from the Chinese variety; and that cooking under conditions similar to those described above will render either of these dialyzed prepa- rations capable of supplying the protein requirement of normal growth. TABLE IV. Comparative Summary of Digestibility Experiments in Vitro and Nutritional Experiments in Vivo, on the Velvet Bean and Other Proteins. Digestibility. Result of growth Protein. Heise experiments. per cent Chinese velvet bean coagulum......... 57.4 Normal growth (2). Georgia ‘“ od Le i ae 55e5 cs “ (2). Chinese “ “ , dialyzed protein. 30.4 Supports neither growth nor life (2). Georgia “ ti ‘ rs BPS Not tried. - r cooked dialyzed Prove yee os Se ead oo. 54.7 rc 4 Chinese velvet bean, cooked dialyzed DLOUCIN Me Ae eee ae ree care = OR oc 57.9 ~ a8 Phascolim era weet... cee crest etiam sos 39.5 Does not support growth, with or without cystine (7). Phaseoliny cookedsneecds cess see Soni Normal growth when supplemented with cystine (7). CABG Sree Sere tae Meee i adn ss 61.4 Normal growth. * Recalculated from Table II, first paper (6). + New determination, using hydrochloric in place of sulfuric acid in the pepsin digestion. : With regard to the relation between the values obtainable in determinations of the type under consideration, and the results of growth experiments with animals, the figures presented in Table IV give some indication. Six of the nine preparations listed have been fed as the sole source of protein in an otherwise adequate diet. Of this group, four produced normal growth in animals and gave relatively high digestibility values in vitro. Cooked phaseolin required the addition of cystine, it is true; but cystine did not improve the raw phaseolin diet, and even without ee H. C. Waterman and D. B. Jones 293 the addition of cystine the cooked phaseolin maintained the weight of the animals for some time. The other two proteins tested supported neither growth nor life, and they showed a relatively low digestibility 7n vitro. Finally, the greatest diges- tibility so far observed in our experiments is that of casein, the principal component of the natural food of young mammals. Thus all the evidence at present available supports the assump- tion tentatively put forward in our first paper on this subject, that an indigestibility sufficient to interfere seriously with the capacity of a protein to furnish the amino-acid requirement of normal growth may readily be demonstrated by this method. Concerning the relation existing between these estimations of digestibility 7n vitro and absolute digestibility as determined in the animal organism, however, we have as yet no reliable infor- mation beyond the fairly safe inference that figures for different proteins as found by the two methods may be expected to vary in the same direction and with a rough proportionality. Any direct arithmetical ratio is probably precluded by the subtraction of the considerable blank correction for the self-digestion of the enzymes in calculating the results of the estimations in vitro, since, of course, this self-digestion is considerably greater in the blank digestion where no substrate is present than it is in digests containing a protein sample. It is even possible that self-diges- tion is so far retarded by the substrate that a less error would be introduced by omitting this correction entirely than by including it in the calculation. Bayliss (8) reports an experiment in which trypsin, acting upon casein as a substrate, showed “‘no appreciable destruction up to eight hours.” It is of interest to note in this connection that if the data of our experiments be calculated without subtracting the blank they yield figures much like those obtained by the absolute method in similar cases. Various experiments with casein, for instance, when calculated on this basis gave values ranging from 99.5 to 100.2 per cent. The values for the cooked and the coagulated velvet bean proteins lie between 90 and 100 per cent on this basis, and those for the raw velvet bean proteins would be about 65 to 70 per cent. The figure 73.6 has been given as the digestion coefficient for the nitrogen of velvet bean meal (beans and pods) when fed to steers (2). Until we have further experimental data bearing upon this point, 294 Digestion of Proteins in Vitro. II however, we prefer to regard our results as purely comparative, and to include the blank in the calculation. The chemical nature of the indigestibility of certain proteins, which manifests itself in animals by a failure to promote growth and zn vitro by a relatively low yield of amino nitrogen set free by proteoclastic enzymes, also presents a problem of which little isknown. The most probable answer to the question seems to be suggested by the work of Fischer and Abderhalden on the hydrol- ysis of polypeptides by proteoclastic enzymes. These investi- gators demonstrated that certain polypeptides are hydrolyzable by trypsin while others, containing the same amino-acid residues united in a different order, are not attacked at all. The presence of one or more of the essential amino-acids in the form of such stable combinations would, of course, explain the failure of a protein to promote growth, and would lower the yield of amino nitrogen set free from the protein by enzymes in vitro. A study of the undigested residue precipitable by dilute acid from the solutions remaining after our digestion experiments may throw some light upon this point; and it is intended to make such a study the basis of a future report. SUMMARY. Estimations of the comparative digestibility in vitro by the method of Waterman and Johns of six preparations of the proteins of the Chinese and Georgia velvet beans indicate: (1) That partial indigestibility is the limiting factor in the failure of raw dialyzed Chinese velvet bean protein to promote growth, and that the normal growth secured with the protein prepared by coagulation from either bean is probably to be attributed to an increase in digestibility brought about by the boiling incident to the prepa- ration of the latter material; and’ (2) that cooking under the con- ditions described renders the dialyzed protein from either seed as digestible as the coagulated product, and that probably, there- fore, these cooked dialyzed proteins will support growth as well as do the coagulated proteins. The double disadvantage of toxicity and non-assimilable protein content amply explains the behavior of the raw ground bean, while the cooked meal probably still contained dihydroxyphenylalanine. H. C. Waterman and D. B. Jones 295 The experiments also support the contention that the results of such estimations run parallel with the results of growth experi- ments, in as far as differences in the digestibility of the proteins are concerned. BIBLIOGRAPHY. 1. Miller, E. R., J. Biol. Chem., 1920, xliv, 481. 2. U. S. Dept. Agric., Farmer’s Bull. 962, 1918, 38. 3. Finks, A. J., and Johns, C. O., Am. J. Physiol., 1921, in press. 4. Johns, C. O., and Finks, A. J., J. Biol. Chem., 1918, xxxiv, 429. Jones, D. B., and Johns, C. O., J. Biol. Chem., 1919, xl, 435. 5. Johns, C. O., and Waterman, H. C., J. Biol. Chem., 1920, xlii, 59. 6. Waterman, H. C., and Johns, C. O., J. Biol. Chem., 1921, xlvi, 9. 7. Johns, C. O., and Finks, A. J., J. Biol. Chem., 1920, xli, 379. 8. Bayliss, W. M., The nature of enzyme action, New York, 3rd edition, 1914, 76. THE METABOLISM OF NITROBENZALDEHYDES AND NITROPHENYLACETALDEHYDE. By CARL P. SHERWIN anp WALTER A. HYNES. (From the Chemical Research Laboratory, Fordham University, New York.) (Received for publication, March 26, 1921.) Several investigators have shown that the metabolism of the o-, m-, and p-nitrobenzaldehydes is entirely different in the body of the dog from that found in the organism of the rabbit, and moreover, that the fate of the o-nitrobenzaldehyde is entirely different from that of the m and p compounds in the same animal organism. We thought it of interest to determine the fate of these three compounds in the human body, and also to include in the work p-nitrophenylacetaldehyde, which, up to the present time, had not been employed in such experiments. 0-Nitrobenzaldehyde. The pure substance,! (melting point, 44°C.), in capsules, was ingested by a man in 2 gm. doses. Because of the toxicity of the compound, only two such doses could be administered. The urine was collected, evaporated to a small volume, acidified with sulfuric acid until the urine showed an acid reaction to Congo red, and repeatedly extracted with ether; the ether was evaporated to dryness and the oily residue taken up with water. After standing 2 days, crystals were recovered from the water solution and dried in a desiccator over sulfuric acid. The melting point of the crystals, 142-144°, showed the substance to be o-nitro- benzoic acid. After the first 2 gm. dose of the aldehyde, 1.7 gm. of the acid were isolated from the urine, while after the second dose, 1.8 gm. were found, thus giving a yield of, respectively, 77 and 81 per cent of the substance fed. . 1 The o-nitrobenzaldehyde used in this work was furnished by Dr. C. G. Derrick of the National Aniline Works. 297 298 Metabolism of Nitroaromatic Aldehydes Two doses of 2 gm. each of the o-nitrobenzaldehyde were fed to a dog of 10.5 kilos body weight. From the urine of the dog we isolated 75 and 80 per cent of the substance in the form of o-nitrobenzoic acid. It appears, therefore, that the fate of the o-nitrobenzaldehyde in the human body is much the same as in the organism of the dog. These results agree with those of Sieber and Smirnow (1), and Cohn (2), who used dogs for their experiments. Cohn, however, when using rabbits for the same work, was unable to recover more than 10 per cent of the substance fed in the form of o-nitrobenzoic acid. We repeated this work on rabbits and were able to confirm his results. o-Nitrobenzaldehyde was found to be much more toxic than the corresponding m and p compounds. A man of 70.5 kilos body weight, after receiving a 2 gm. dose showed every sign of intoxi- cation, followed by marked albuminuria. m-N itrobenzaldehyde. The same subject ingested three doses of 2 gm. each of this compound within a period of 96 hours. The urine was collected and treated as in the previous experiment. 4.21 gm. of a sub- stance melting at 139-141° and 2.55 gm. of a substance melting at 160-162° were recovered from the urinary extract. The former substance, m-nitrobenzoic acid, as shown by its melting point, represented 63.51 per cent of the m-nitrobenzaldehyde fed, while the latter substance, m-nitrohippuric acid, is equal to 24.83 per cent of the substance fed, a total of 88.34 per cent. After 6 gm. of the aldehyde were ingested by a second individual, the ratio of m-nitrobenzoic acid to m-nitrohippuric acid excreted was entirely different. In this case, about 75 per cent of the aldehyde was found in the urine as m-nitrobenzoic acid and only 5 per cent as m-nitrohippuric acid. The m-nitrobenzaldehyde proved entirely non-toxic, apparently causing no inconvenience to the subjects in doses as large as 5 and 6 gm. Previous work (1) on dogs has shown that after feeding this substance, m-nitrobenzoic acid and m-nitrohippuric acid could be recovered from the urine, while after feeding to rabbits (2), an entirely different substance, m-acetylaminobenzoic acid was found in the urine. — = ——— C. P. Sherwin and W. A. Hynes 299 p-Nitrobenzaldehyde. This substance, in capsules, was ingested by a man in 2 gm. doses. A mixture of the corresponding benzoic and hippuric acids was obtained. Out of a total of 4 gm. taken by the subject, 62 per cent was isolated from the urine as p-nitrobenzoic acid and 19 per cent as p-nitrohippuric acid. p-Nitrophenylacetaldehyde. This substance, prepared according to the method of Lipp (3), by the treatment of p-nitrophenyl-8-chlorolactic acid with sodium carbonate, melted at 84-86°. The p-nitrophenylacetaldehyde was fed to rabbits in 1gm. doses with apparently no ill effects. The urine was collected for 36 hours and treated as usual, except that extraction was first carried out with ether, followed later by two extractions of the evaporated urine with alcohol and then with ethyl acetate. From the ether extract we obtained a yellow oil on evaporating almost to dryness. This oil was difficultly soluble in hot water and after standing in the ice box for 48 hours crystallized out as yellow needles melting when dry at 151-153°. This substance was proven to be p-nitro- phenylacetic acid by its melting point as well as by its titration value. After feeding 4 gm. of p-nitrophenylacetaldehyde to rabbits, 3.86 gm. of p-nitrophenylacetic acid, corresponding to 88 per cent of the substance fed, were recovered from the urine. None of the substance fed, or any of its derivatives, could be found in either the ethyl acetate or alcohol extracts. 5 om. of this substance were again fed to rabbits, 4.23 gm. of p-nitrophenylacetic acid, corresponding to 77.1 per cent of the substance were recovered from the urine. 5 gm. of the substance in capsules were fed to a dog and the urine treated in the same manner as the rabbit urine. From the ether extract 3.92 gm. of p-nitrophenylacetic acid were recovered, or 71.35 per cent of the aldehyde fed. No p-nitrophenaceturic acid nor any derivative of p-nitrophenylacetic acid could be found in either the alcohol or ethyl acetate extracts. These extracts were evaporated to dryness, the residue was redissolved in water and fractions of this solution were strongly acidified with sulfuric acid 300 Metabolism of Nitroaromatic Aldehydes and boiled for 2 hours. These were then cooled and extracted with ether to determine the presence of any p-nitrophenylacetic acid which might previously have escaped extraction in the form of an insoluble conjugation product. The absence of the acid at this point shows that no conjugation product of the acid existed in the urine and that only an oxidation of the aldehyde to an acid had resulted from the passage through the body of the dog. A man ingested a total of 5 gm. of the p-nitrophenylacetal- dehyde with much the same results as were previously shown for the dog and rabbit. In this case 70.13 per cent of the aldehyde was excreted in the form of p-nitrophenylacetic acid. Thus, in each case where the p-nitrophenylacetaldehyde was fed, there was only an oxidation of the aldehyde to the carboxyl group, which is entirely comparable to the action of the three nitrobenzaldehydes; but in no case was there a reduction of the nitro group to an amino group as was found to be the case when m- and p-nitrobenzaldehydes were fed to the rabbit. There was also no conjugation of the carboxyl group with glycocoll as was seen when m- and p-nitrobenzaldehydes were fed to dogs and which was to be expected, as we (4) have previously shown that p-nitrophenylacetic acid when fed to dogs is to some extent combined with glycocoll and excreted as p-nitrophenaceturic acid. CONCLUSION. The fate of o-, m-, and p-nitrobenzaldehydes in the human body was much the same as that previously shown for the dog. In each case oxidation took place with the formation of the corre- sponding acid. Inthe case of the 0 compound about 90 per cent was excreted as the o-nitrobenzoic acid, while the m and p com- pounds were excreted to a large extent as the m- and p-nitro- benzoic acids, but also to a small extent combined with glycocoll and excreted as m- and p-nitrohippuric acids. In no case was there a reduction of the nitro group. p-Nitrophenylacetaldehyde was fed to rabbits, dogs, and a man. In each case there was only an oxidation to the corre- sponding acid, with no reduction of the nitro group or combination with either glycocoll or glutamine. C. P. Sherwin and W. A. Hynes 301 BIBLIOGRAPHY. 1. Sieber, N., and Smirnow, A., Monatsh. Chem., 1887, viii, 88. 2. Cohn, R., Z. physiol. Chem., 1892-93, xvii, 274; 1894, xviii, 183; Arch. exp. Path. u. Pharmakol., 1905, liii, 435. 3. Lipp, A., Ber. chem. Ges., 1886, xix, 2643. 4. Sherwin, C. P., and Helfand, M., J. Biol. Chem., 1919, xl, 17. FAT-SOLUBLE VITAMINE. VIII. THE FAT-SOLUBLE VITAMINE CONTENT OF PEAS IN RELATION TO THEIR PIGMENTATION.* By H. STEENBOCK, MARIANA T. SELL, anp P. W. BOUTWELL. (From the Department of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, May 17, 1921.) In the classification of naturally occurring foods which enter into the make-up of the human dietary the seeds were early given a position of rather uniform value. Thus in 1919 McCollum (1) makes the statement: “By the application of the biological method of analysis of a food-stuff to each of the more important seeds employed in the nutrition of man and animals, the fact was brought to light that they all resemble each other very closely in their dietary properties. The list of seeds examined in- cluded,—wheat, corn, rice, rolled oats, rye, barley, kaffir corn, millet seed, flaxseed, pea and both the navy and the soy bean. . . . All are, with the exception of millet seed, below the optimum in their content of the dietary factor, fat-soluble A.”’ From our present knowledge it is very evident that this state- ment merits considerable qualification, for while seeds as a class are apparently never as rich in the fat-soluble vitamine as the leafy parts of plants yet some contain considerable amounts of this vitamine, and among these millet seed, according to our investigations, does not occupy a place of special prominence. In fact, though such varieties of millet may be in existence we have never had as uniform success with these as a source of fat- soluble vitamine as we have had with yellow Indian corn. That there are other instances where exception must be taken to this statement was brought out in the further development of * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 303 304 ~ Fat-Soluble Vitamine. VIII our theory that the fat-soluble vitamine is biologically related to certain yellow plant pigments. We have already shown that in Indian corn (2) those varieties carrying yellow pigment are rich in the vitamine while those free from it contain very little of this constituent. Further investiga- tion has brought out the fact that in butter fat and in beef fat (3), while there is no absolute parallelism between vitamine and pig- ment content, yet probably due to their simultaneous occurrence in nature as well as resemblance in properties, where the pigmen- tation is high the tendency for high vitamine content obtains. In this paper it is desired to bring out the relation of vitamine and pigment content that has been found to hold among different varieties of edible peas. EXPERIMENTAL. Six samples of peas were used for the comparisons. ‘Two were purchased in the open market, the one as a sample of split peas sold for cooking purposes having been practically freed from all pericarp; the other was a small green pea of an unknown variety which is commonly used in pigeon feeding. The remaining samples were purchased from a local seed house as pure bred varieties. Pigment estimations were made by extraction with alcohol, fol- lowed by saponification and comparison in a Duboseq colorimeter. 30 gm. of peas, finely ground, were extracted with hot 95 per cent alcohol in a Soxhlet extractor for 6 hours. The extract was then saponified over night with 10 cc. of 10 per cent alcoholic potash which had been prepared with the usual precautions. After dilution with water the alcoholic soap solution was extracted by shaking repeatedly with ether. The ether was then volatilized, the yellow pigments taken up in 40 ec. of alcohol and quantita- tively compared with one another in the Duboseq colorimeter. The results of this comparison are shown in Table I. The analysis for relative vitamine concentration was made by the use of the rat as the experimental animal following the tech- nique described in previous publications. Four animals were fed in each group, one group being used for each sample. The results as presented in Charts I and II show the complete data obtained with the various groups when fed peas at a 15 per cent level as the sole source of the fat-soluble vitamine in their diet. Steenbock, Sell, and Boutwell 305 TABLE I. Relative Yellow Pigment Content of Green and Yellow Peas. Color. Designation. : Relative pigment values. Yellow. Commercial split. 39 & Marrowfat. 44 “e Canadian field. 55 Green. Small green. 76 Gt Alaska. 100 se Scotch beauty. 95 YELLOW PIGMENTS AND THE FAT=S =a ey | 1 11 1 1 PEPE SCECEEE Sauer SePSuEr se O70 psi } | ager 15 40 : 7 ‘pe ts of dex trin disp Paar iy SE om Sie ne lei eae et |e CuHart 1. Fat-Soluble Vitamine. VIII 306 Be eer epee ene | oe ea ee a ee D2 RSRE PE aPaaeee ies ees aie ese ee COSA CCC CRS Bock ELerN Tele Se COB CEC CATON TAKE feeersieioNal NT TS REN TOA RENO OW SoO. Sie soe a See aot ee Oh «0 SI CHART 2, Steenbock, Sell, and Boutwell 307 In Chart I are shown the growth curves resulting when yellow peas were used as the medium for the introduction of the fat- soluble vitamine in the diet. In view of the abundance of hemi- cellulose found in peas it was surmised that the peas would have to be cooked to avoid causing digestive disturbances. This was done with the commercial split peas fed to Lot 1077 but not with any of the other samples as in the course of other feeding trials with peas it was found to be unnecessary although tympanites was occasionally observed. All the animals in the three lots contracted eye infections and four developed respiratory trouble in addition. In Lot 1001 two of the rats; vzz., Rats 3997 and 4000 responded gradually when butter fat was added to their ration. With the others no improvement of ration was attempted, yet from the consistency of the data as a whole no doubt is left that failure of growth and maintenance was due to a fat-soluble vitamine deficiency. That yellow peas are not to be classed with seeds of very low fat-soluble vitamine concentration, such as wheat, oats, and barley, is shown by the very appreciable growth which occurred during the first 2 months on the experimental ration. On the basal ration without supplementation with peas young rats of the age used in these experiments rarely grow longer than 6 weeks. Further experimentation with the amount of peas increased to 50 per cent of the ration gave more conclusive evidence of an appre- ciable fat-soluble vitamine content. On the marrowfat peas one rat with an initial weight of 60 gm. rose to 285 gm. after 4 months subsistence on the ration; yet its intake of the fat-soluble vitamine was not sufficient, as 4 weeks later, it as well as two others of this group, died from respiratory infections at an approximate weight of 170 gm. The results of feeding green peas are shown in Chart II. Though fed at the same level as the yellow peas; v7z., 15 per cent, the curves of growth in themselves show their superiority as a source of the fat-soluble vitamine. In these three groups by way of contrast there was observed only one instance of suggested incipient ophthalmia and one instance of respiratory infections which occurred, respectively, in Rats 4267 and 4265. In the com- plete failure occurring with Rat 4268 the cause of death was ob- viously of a foreign nature as no symptoms indicating fat-soluble vitamine were observed. 308 Fat-Soluble Vitamine. V-IEE SUMMARY. In ripe peas, out of six samples investigated, those of a green color, also carrying considerable yellow pigment, were far richer in their fat-soluble vitamine content than yellow peas which con- tained much less yellow pigment. BIBLIOGRAPHY, 1. McCollum, E. V., The newer knowledge of nutrition, New York, 1919, 38. 2. Steenbock, H., and Boutwell, P. W., J. Biol. Chem., 1920, xli, 81. 3. Steenbock, H., Sell, M. T., and Buell, M. V., J. Biol. Chem., 1921, xlvil, 89. lt THE DETERMINATION OF CRESOL BY THE PHENOL REAGENT OF FOLIN AND DENIS. By ROBERT M. CHAPIN. (From the Biochemic Division, Bureau of Animal Industry, United States Department of Agriculture, Washington.) (Received for publication, May 9, 1921.) At first sight the colorimetric method of Folin and Denis! promised to dispel most of the difficulties and uncertainties previously attending the determination of phenols in low concen- trations. They and others immediately applied it to urine, feces, blood, etc., with apparently gratifying results. But a critical survey of the literature reveals that too little of the rela- tively unattractive foundation work has been executed. The temptation has been to utilize the method for the solution of certain problems without taking time for close scrutiny of its reliability under the operating conditions. That a rather large number and wide variety of substances may develop color with the reagent has been shown by Gortner and Holm? and by Levine.? It may be doubted whether adequate measures have been taken to exclude non-phenolic substances in some reported work. But even if the color developed has been in all cases entirely due to “phenols,” very little attention has been given to fundamental quantitative relations. For example, Folin and Denis determined the phenolic substances in urine as if they were exclusively phenol even though they state that ‘the most important phenol quantitatively is in fact paracresol.” The phenol reagent diluted with water yields a clear yellow solution. If there be added an excess of alkali or of an alkaline salt, even sodium acetate, the yellow color fades at a rate propor- ! Folin, O., and Denis, W., J. Biol. Chem., 1915, xxii, 305, 309; 1916, xxvi, 507. * Gortner, R. A., and Holm, G. E., J. Am. Chem. Soc., 1920, xlii, 1678. 3 Levine, V. C., Science, 1920, lii, 612. 309 310 Cresol by the Phenol Reagent tional to the degree of alkalinity. The final result is a colorless liquid, provided no traces of reducing impurities have gained access. Slightly alkaline mixtures remain clear; strongly alkaline ones soon deposit a considerable quantity of a white precipitate. The loss of yellow color marks the degradation of the active substance in the reagent, for at the end it no longer affords color with either phenol or sodium sulfite. Ifa phenol be present when the neutral- izing agent is added the primary product of the reaction with the phenol is green. The green will change to blue at a rate proportional to the alkalinity and the temperature. The nature of the phenol is also of considerable influence. The primary products from m-cresol and p-cresol change color most readily, followed by that from phenol, while that from o-cresol lags far behind. The change in all cases is effected completely and quickly by sodium sulfite, as noted by Benedict and Theis.4 Excessive alkali diminishes the intensity and permanence of the color, but in varying degree with different phenols. If it is merely a matter of measuring a single phenol against the same phenol as a standard, no very strict regulation of conditions may be necessary, provided they be maintained closely parallel in sample and standard. But when a phenol or a mixture of phenols is to be measured against an entirely different standard phenol then it becomes important to select the set of conditions which will yield the most uniform and dependable results. EXPERIMENTAL. Assuming a certain appropriate weight of a given phenol in a certain final volume, it is necessary to determine (a) the proper quantity of reagent, (b) the proper nature, quantity, and reaction period of the alkali, (c) the effect of developing or stabilizing agents, and (d) the color factor for the phenol against a chosen standard phenol under the conditions finally selected as standard. Stock solutions were prepared by weight from pure dry samples of phenol and the three cresols. These were freshly diluted for use and such aliquots were introduced into 100 ce. volumetric flasks as would yield colors closely approximating that yielded by 0.5 mg. of phenol under the same conditions of treatment, 4 Benedict, S. R., and Theis, R. C., J. Biol. Chem., 1918, xxxvi, 95. R. M. Chapin alt which constituted the standard. All the work was done at room temperature, 22—29°C. Color comparisons were made in the Duboseq colorimeter, the standard being set at 20 and first read against itself as recommended by Folin and Denis. Folin and Denis bettered their original reagent in accordance with a formula attributed to Bell. Wu® has made some further improvements, and his formula has been used in the present work. Preliminary experiments showed 3 cc. of reagent to be ample for the development of maximum color. The alkali previously used has been sodium carbonate. As Experiment 1 will show, sodium bicarbonate yields heavier colors and more consistent factors and is sufficiently rapid in action. The mixture of each phenol with the reagent was diluted to 63 cc., 5 gm. of pure powdered NaHCO; were added and the flask was swirled until solution was complete (about 2 minutes). The colors were developed by the addition of 5 cc. of a 10 per cent solution of anhydrous sodium sulfite. Benedict and Theis merely state that sulfite is to be added “after the sodium car- bonate.” But since sulfite itself will reduce the reagent with production of a deep blue (Wu), it obviously must not be added until sufficient time has elapsed to insure complete inactivation of excess reagent. Experiments showed that in absence of phenol complete inactivation was effected by 5 gm. of NaHCO; in 20 minutes. Therefore, all tests were allowed to stand one-half hour after solution of bicarbonate before sulfite was added. The contents of the flasks were then made to volume, mixed, and filtered, the first 25 cc. of the filtrate being discarded. Filtration may not be necessary under ordinary circumstances, for the only undissolved matter present appears to arise from traces of impuri- ties in the reagent. But if filtration is omitted the flasks should be allowed to stand 15 minutes after addition of sulfite to insure sufficient time for its action. Colorimetric comparisons were executed immediately after filtration. The colors are probably fairly stable, but even so, the colorimetric comparison is so simple and rapid a process that a cogent excuse for delaying it is hardly likely to arise. ’ Wu, H., J. Biol. Chem., 1920, xliii, 189. 312 Cresol by the Phenol Reagent Experiment 1.—In comparison with the method above described as standard, tests were run in which 10 ee. of a solution of sodium carbonate of specific gravity 1.15 (16 gm. of Na2CO; per 100 ec.) were employed in place of sodium bicarbonate. The colors obtained were approximately the following fractions of the colors yielded by the standard method: Phenol, 0.90; o-cresol, 0.78; m-cresol, 0.83; p-cresol, 0.79. That is, a given weight of o-cresol, for example, yielded only 78 per cent as deep a color with sod'um carbonate as it yielded with sodium bicarbonate. It is evident that the cresols are affected in different degree from phenol. TABLE I. Weight of cresol. Bes t2 Sovai cht of phenol. Weight of | Weight of | Weight of |Corrected| Average o-cresol. | m-cresol. | p-cresol. | readings. | factor. =e RSP eal hee 0.595 21.0 21.2 1.181 Series A. Temperature, 22- 0.633 18.2 24°. Phenol, 0.532 mg. 18.0 1.077 0.648 | 20.2 20.1 1.227 (| 0.526 19.6 19.9 1.193 | Series B. Temperature, 28- | | 0.456 20.7 29°. Phenol, 0.435 mg..... | 213 | ee | 0.529 | 19.6 { 20.1) |-1:207 Final average factors......... | 1.19 1.09 {222 Experiment 2.—In determining the color factor for each cresol by the standard method, two independent series of stock solutions were prepared, and two independent runs were made on each series. The results are reported in Table I. Each reading given is the average of at least three readings actually taken, and is corrected to be comparable to the standard phenol set at 20 on the same scale. molecular weight cresol The theoretical factor is 1.15. Taking molecular weight phenol commercial cresol as a 35:40:25 mixture of the isomers, the empirical factor would be 1.16. —" R. M. Chapin 313 Experiment 3.—To investigate the factor for xylenol, solutions were prepared from selected clean and colorless crystals of purchased material. Only one test was made. The factors were as follows: o-xylenol (1:2:4), 1.63; p-xylenol (1:4:2), 1.29. The theoretical molecular factor is 1.30. It appears, therefore, that if the composition of the cresol be known to the extent that it is either predominantly a certain one of the isomers or the ordinary commercial mixture, and the proper indicated factor be employed, the results need not be in error by more than 1 part in 20. The method has shown considerable practical value in this laboratory for the determination of phenolic preservatives in serums, etc. Whenever a sample of such a product has evinced abnormal physical or biologie characteristics it has here become customary to run a determination for its content of preservative. Elvove® distilled the highly diluted serum from a large flask to minimize interference by foaming, and applied Millon’s reagent to the distillate. For a number of reasons the present phenol reagent is preferable to Millon’s reagent. The writer has not attempted to dispense with distillation, for it has been necessary that the results reported should be free from suspicion. But it has been found that silicotungstic acid? is an effective precipitant for the substances which cause foaming, and therefore enables the distillation to be conducted more conveniently. To 1 cc. of serum in a 300 cc. flask are added 125 ce. of water, 4 cc. of 1:3 (by volume) H:SOu,, 4 ec. of a 12 per cent solution of silicotungstic acid, and a fragment or two of hot pumice. The flask is connected to a nearly vertical condenser by a three-bend tube. The contents are slowly brought to boiling, and the distillate is received in a 200 cc. volumetric flask. When the latter is half full, 7. e. when 100 cc. has been distilled off, the flame is withdrawn and 100 cc. more water are added to the distillation flask, then distil- lation is resumed and continued until the receiving flask is filled nearly to the mark. Of the distillate, made to the mark and mixed, either 20 or 50 cec., depending on whether phenol or cresol is the preservative probably used, are brought into a 6 Hlvove, E., Bull. Hyg. Lab., U. S. P. H., No. 110, 1917, 25. ‘This substance is now rather extensively used in alkaloidal work, particularly for the determination of nicotine, and should be procurable without difficulty. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 314 Cresol by the Phenol Reagent 100 ce. flask and the determination is executed according to the standard method previously given. Readings on the colorimeter should naturally be not too far from 20. Otherwise the more strongly colored solution should be properly diluted before com- parison. Again, the latter procedure should be not trusted to too great an extent so that it may be necessary to repeat the determination on a more appropriate volume of distillate. If it is further necessary to distinguish between phenol and cresol the method previously described by the writer’ may be utilized as a qualitative test. -Into a test-tube are brought 10 ce. of the distillate, and in a similar tube a colorimetrically equivalent quantity of the standard phenol, diluted to 10 cc. To each tube are added 5 cc. of the special Millon reagent, and the tubes are heated as described. A comparison of the resulting colors in the tubes will plainly reveal whether the preservative em- ployed has been essentially phenol or cresol. SUMMARY. It is shown that the quality and intensity of color developed by a phenol with the reagent of Folin and Denis is dependent both upon the nature of the phenol and the composition of the medium. The conditions affording colors of maximum intensity and most consistently comparable when developed from differing phenols have been determined. Accurate quantitative comparison between different phenols demands an empirical factor. By use of prescribed empirical factors and adherence to standard working conditions, the cresols may be determined with: fair accuracy as ‘‘total cresol.” The determination of phenolic preservative in serums is described. 8 Chapin, R. M., J. Ind. and Eng. Chem., 1920, xii, 771. THE EFFECT OF HYDROCHLORIC ACID INGESTION UPON THE COMPOSITION OF THE URINE IN MAN. By RAYMOND L. STEHLE anp ARTHUR C. McCARTY. (From the Laboratory of Physiological Chemistry, School of Medicine, Uni- versity of Pennsylvania, Philadelphia.) (Received for publication, May 12, 1921.) The data presented herewith are in a way an extension of an experiment reported in 1915 which concerned the effect of the administration of hydrochloric acid on the excretion of Na, K, Mg, and NH; in the urine and feces of dogs (1). Increases in the amounts of all these substances were noted in the urine. The augmentation was especially noteworthy in the case of ammonia and almost negligible in the cases of calcium and mag- nesium. However, the data showed the calcium and magnesium content of the feces to be increased while sodium and potassium were unaffected. Not long after the above results were published a paper by Givens (2) appeared in which the author concluded that his data failed to demonstrate any unequivocal effect of hydrochloric acid on the calcium and magnesium content of the feces of dogs. In view of the conflicting data it must be admitted that the question is still an open one but an examination of the respective sets of data will show that those of Givens show considerable lack of evenness. In Table II on Diet B, Dog M does show a higher Ca excretion during the acid period than during the control period but instead of making the obvious interpretation that the acid was the cause Givens offers the explanation that ‘‘it seems more plausible to assume that in the fore period . . . . the animal was trying to store up some lime after its depletion by the former low lime régime.” ‘‘Former” refers to a previous experimental period. Neither is it at all obvious why, in the same table, the 315 316 Hydrochlorie Acid Ingestion excretion of Ca on the highest Ca diet falls short of the quantity excreted during the preceding acid period. This applies to the experiment with Dog N as well, but in this instance the Ca excre- tion during the period of highest Ca intake is even lower than that in a period of lower lime intake. The problem as far as the dog is concerned has some rather difficult aspects. In order to overcome the difficulty resulting from the impossibility of separating the feces of the experimental periods sharply it is necessary to have long experimental periods. The diet must, of course, be uniform throughout the experiment and consequently of a nature so attractive that it will at no time be refused. As far as the dog is concerned this means that meat must be the principal constituent. Meat, however, is well suited to defeat the object desired since the ammonia resulting from its metabolism at once becomes available for the neutralization of any acid administered. Another possible difficulty presents itself in trying to determine where any bases excreted in response to acid administration will be found. It is not necessarily true that they need be in the feces corresponding to the day on which the acid was administered. Conceivably, they may be found in the feces of an earlier day depending upon what part of the alimentary tract is concerned with the excretion. The present communication deals with the effect of hydro- chlorie acid ingestion upon the urinary composition in man. Advantage was taken of the experience gained in the earlier experiments and the diet so arranged as to make any variations as prominent as possible. It was ample calorically, but low in protein and sodium chloride. The components were rice, French fried potatoes, bananas, wheat bread, jam, butter, apple pie, and figs. None of the food was salted. The quantities were as uniform from day to day as careful measurement could make them and the only variation throughout the 7 days the diet was adhered to consisted in the ingestion of 3.65 gm. of hydrochloric acid in the form of tenth normal acid daily during the last 3 days. It was divided into three approximately equal portions and was drunk just before meals. R. L. Stehle and A. C. McCarty 317 Analytical Methods. For the estimation of Ca and Mg the procedure developed by McCrudden (3) was employed. Na and K were first isolated as the mixed sulfates and the potassium was then separated as the cobaltic nitrite compound. The yellow precipitate was then oxidized with a standard permanganate solution. The details of the process were those described by Drushel (4). Ammonia was TABLE I. =e Day of | Total Chlo- NH Naas | Kas ; Individual. ae N. ee 3 | Nacl.| Kel | C2 Mg |Hs3PO,s|} Ph A. C.M. 3 | 6.85 | 4.22) 0.294) 3.56 | 4.61/0.058/0.104| 1.11] 6.70 4 | 6.59 | 5.70} 0.256) 2.55 | 3.80/0.075/0.115| 1.18] 6.75 5 | 7.95 | 10.47] 0.559) 5.19 | 8.74/0.090)0.181| 1.64) 6.28 6 | 7.34} 9.36} 0.663) 4.09 | 6.26/0.090)0.159) 1.74) 5.27 ¢@ | 6.97) 41°69) 0.731) 4.11 | 7.17|0.113|0.161) 1..69)-5.05 aS: 3 | 6.27 | 4.38} 0.399) 3.60 | 2.70/0.072/0.106) 0.85] 5.86 4 | 6.16 | 5.03) 0.300) 3.70 | 4.10/0.100/0.094| 0.97) 6.44 5 | 6.42 | 8.73] 0.515} 4.60 | 5.26)0.141/0.142| 1.27) 5.89 6 | 6.29 | 9.76) 0.730) 4.29 | 5.96/0.123/0.135) 1.53) 5.05 7 | 6.29 9.63) 0.758) 3.29 | 5.90/0.171/0.122| 1.64) 5.00 TABLE II. A.C. M. R.L.S NaCl equivalent of increased NH; excretion.......... 1.294 1.091 NaCl “ . me Na agg Dek Beas 1.41 0.41 NaCl Se oe K ih tase ie 2.49 1.81 NaCl ; % + Ca cisg axe ere 0.090 0.172 NaCl od as = Mg he Boe ee 0.282 0.161 NaCl S ‘s Se csi SE Ge Ay Un ege Oia 0.340 0.271 EOE) IRIE tl hale ice Le ee oe 5.906 3.915 determined by aeration, chlorides by the Volhard-Arnold method, and phosphates by titration with uranium acetate. The hydrogen ion concentration was measured electrometrically. The effect of the acid on some of the urinary constituents was as given in Table I. The sodium chloride equivalent of the 3.65 gm. of hydrochloric acid taken daily is 5.83 gm. If the sodium chloride equivalents 318 Hydrochloric Acid Ingestion of the increstsed NH3, Na, K, Ca, Mg, and H+ concentrations are calculated the results are as given in Table II. The changes in H ion concentration are taken into account here for the following reason. The pH of the foreperiod shows that there must have been practically equal quantities of primary and secondary phosphates present. The pH of the acid period shows that practically all of the phosphate must have been present in the primary form. Consequently, while the conversion of the secondary phosphate into primary does not involve any change in the amount of phosphate excreted it does signify that a portion of the hydrochloric acid has been neutralized and must be taken into account in the attempt to account for the hydrochloric acid ingested. If it be assumed that the increased phosphoric acid excretion is due to the conversion of disodium phosphate of the blood and tissues into monosodium phosphate, then for every atom of. sodium excreted as sodium chloride which came from the disodium phosphate in question there will have been excreted an extra atom of sodium in the form of monosodium phosphate. Hence from the sum of the sodium chloride increases given above one atom of sodium for every extra molecule of phosphoric acid must be subtracted. A.C. M. R. L.S. NaCl equivalent of increased NH;, Na, K, Ca, Mg, ANGSHSReXCHOMONMer ee ere icone ole ti -s.2 bee eke 5.906 3.915 Na excreted as NaH2PO, and not indicative of acid NULLA PALL OMe se er ea oe 5 Graienelecls nc Gale eee 0.328 0.340 NaCl equivalent of acid neutralizing factors.......... 5.578 3.575 In the case of A. C. M. 96 per cent of the ingested acid is accounted for while in the case of R. L. S. the fate of only 61 per cent is apparent. Both of these percentages are undoubtedly influenced by physiological factors, the control of which was impossible, and it is probably true that the mean of the two would approach the true average value more closely than does either of them alone. This would leave about 22 per cent of the acid to be accounted for. The results of similar experiments on ‘. eee, R. L. Stehle and A. C. McCarty 319 the dog mentioned at the beginning indicate that increased excre- tion of caletum and magnesium by the intestine may be a factor, but no data are available in the present instance to indicate the extent to which this may be true. If the bases of the body are drawn upon in any condition of acidosis in the same way that they have been drawn upon by the hydrochloric acid in the present experiments then it is interesting to see how the drain could be met to the best advantage. A simple calculation based upon the figures of A. C. M. in Table II shows that for every 3.65 gm. of hydrochloric acid (which is equivalent to 10.4 gm. of B-hydroxybutyric acid) there would be required in order to balance the increased NH; formation and to meet the loss of K, Na, and H;POx,, 3.70 gm. of KHCOs, 0.98 gm. of KxHPOu, and 3.88 gm. of NaHCOs. The calcium excretion via the urine is affected to a negligible extent but if the fecal excretion is affected in man to the same extent that it is in the dog as shown in the experiment previously referred to (1) then the above materials should be supplemented by a small quantity of some suitable calcium compound. It may be, however, that under conditions of alkali administration the effect on calcium excretion would be of no consequence. CONCLUSIONS. Data are presented which show that the ingestion of hydro- chloric acid causes an increased excretion of potassium, sodium, ammonia, phosphoric acid, and hydrogen ions. BIBLIOGRAPHY. 1. Stehle, R. L., J. Biol. Chem., 1917, xxxi, 461. 2. Givens, M. H., J. Biol. Chem., 1918, xxxv, 241. 3. McCrudden, F. H., J. Biol. Chem., 1909-10, vii, 83. 4. Drushel, W. A., Am. J. Sc., 1908, xxvi, series 4, 555. ie. a ay =p ipAut<: , -)e(eeie tr gt aC an 8 UL ae ivi oe ivy, eae ae etek" : if, Pie vi Pp raeel: Heer": - 0 eet Cee os ee? a ee ¥ ire el vey FLA , ‘eb Haw . 5 ’ Prelit } Port: Wed ke 19 A freee is pee ATT hoe a al = 2 é a Asay re ees || iment pia sie Sa eal Vic, ee CIP ee ee ota (pel walt 54 Suite (ii iaBa Ae! amps LR Da tien Part, alr ien sae Tew ante add awit Alenwta we (ie oe halen val e ‘ THE DISTRIBUTION OF CALCIUM AND PHOSPHORIC ACID IN THE BLOOD OF NORMAL CHILDREN.* By MARTHA R. JONES anp LILLIAN L. NYE. (From the Department of Pediatrics, University of California Medical School, San Francisco.) (Received for publication, May 3, 1921.) The acid-base equilibrium of the body is a subject which has been much discussed, and concerning which many theories have been advanced. A review of the literature reveals a large volume of uncorrelated data, observations having been made on one or another constituent of the blood or urine under conditions which were in no way comparable. So far as we are aware no studies have been made on the distribution of calcium and the various compounds of phosphoric acid in corpuscles and plasma on the same sample of blood. It seemed to us that a knowledge of the relation of these substances to each other might throw some light on the mechanism of acid-base regulation which would be of value in certain pathological conditions. This paper is a report on a series of observations made on the blood of normal children rang- ing in age from 4 weeks to 14 years which we shall use as a basis of comparison for other work now in progress. Technique. All the children under 3 years of age and a few of the older ones were from the wards of the University of California Hospital.! They were either surgical or convalescent cases and presumably had normal metabolism. The others were secured from a nearby orphanage and were apparently in good physical condition. * Part of the expense of this investigation was borne by a grant from the William H. Crocker fund for research in pediatrics. 1 We wish to express our thanks to Dr. Bradford F. Dearing of the hos- pital staff for collecting the blood for us and for his interest and cooperation throughout this investigation. 321 322 Blood of Normal Children Collection of Blood.—The blood of children over 18 months of age was col- lected before breakfast, about 15 hours after the last meal. That of chil- dren under 18 months was collected about 11 hours after the last feeding. 1 drop of a saturated solution of sodium citrate to every 5 ec. of blood was used to prevent coagulation. When possible, the blood was taken from the median arm vein. In the younger children it was usually taken from the external jugular, and in babies from the longitudinal sinus. From 25 to 30 ce. were drawn through a hollow needle into a clean, dry syringe, and of this, about 15 ec. were introduced by means of a short piece of glass tubing into a graduated centrifuge tube containing 3 drops of the citrate solution and 1 ce. of paraffin oil. Care was exercised to introduce the blood beneath the surface of the oil so as to avoid a possible exchange of acids and bases between the corpuscles and plasma. The remainder of the blood was quickly introduced into a test-tube containing 2 or 3 drops of the citrate solution and was used for the determinations on whole blood. Hematocrit Determinations.—Immediately after the collection of the blood the plasma and corpuscles were separated by centrifugation at about 4,000 revolutions per minute for 20 minutes and the hematocrit reading taken. In order to secure accurate values it was found necessary to recali- brate all our centrifuge tubes. After removing the oil and plasma as com- pletely as possible, the corpuscles were washed with 0.9 per cent NaCl solu- tion and centrifugated as before. The volume of cells was again noted to insure a constant reading. Sundstroem and Bloor (1) suggest the use of a capillary tube for determining the percentage of cells, claiming that the readings taken in this way differ from those obtained in the graduated centrifuge tube. We found this to be true. Since, however, our hema- tocrit determinations were used primarily to check our work by computing the values in whole blood from those found in corpuscles and plasma, we necessarily used the readings of the centrifuge tube. Dilutions.—In order to economize in material as well as time, we made dilutions which could be used for both the calcium and phosphoric acid determinations. The following were found satisfactory: (1) About 7 cc. of well mixed whole blood were laked with an equal volume of distilled water; (2) 3 ee. of corpuscles were laked with 9 ce. of distilled water; and (3) about 6 ec. of plasma were diluted with an equal volume of distilled water. Phosphoric Acid Determinations.—Bloor’s (2) nephelometriec method with a few minor changes was used. Determinations were made on total, lipoid, and inorganic phosphoric acids in whole blood, corpuscles, and plasma. So called ‘‘other forms” of phosphoric acid were calculated by subtracting the sum of the lipoid and inorganic values from the total. The whole blood determinations were made merely as a check on our work, and in every case agreed with the calculated value within the limits of experimental error. For the determination of lipoid phosphoric acid, 3 cc. of each - of the above dilutions of whole blood (1:1), corpuscles (1:3), and plasma (1:1) were used. The solutions were added drop by drop M. R. Jones and L. L. Nye 2a to the aleohol-ether mixture in 25 ce. volumetric flasks and treated as directed by Bloor. Since the phosphoric acid content of the extract was twice as great as that of Bloor’s, 5 ec. of the filtrate were used instead of 10 cc. In this way there was not only a large saving in alcohol and ether, but the evaporation of the smaller volume of liquid required much less time. For the determination of inorganic phosphoric acid in whole blood, corpuscles, and plasma, 1 cc. of each of the dilutions as given above was added dropwise to 9 cc. of the acid ammonium sulfate solution and treated as directed. For the determination of total phosphoric acid the dilutions were as follows: (1) 38 ec. of the 1:1 dilution of whole blood, made up to 10 ce.; (2) 1 ce. of the 1:3 dilution of corpuscles, made up to 5 ee.; and (8) plasma, 1:1 dilution. 1 cc. of each of the above dilutions was used. The solutions were introduced into Pyrex digestion tubes (1 by 10 inches) and evaporated to dryness on a water bath. This obviated the neces- sity of using glass beads during the digestion. Ordinary Pyrex tubes were found unsatisfactory, the strong acid mixture very quickly eating out the bottom and probably introducing an error into the determination. We succeeded in having satisfactory tubes made out of a special grade of Pyrex tubing. Instead of using the microburners with an improvised fume absorber as suggested by Bloor, we supported our tubes on wire gauze in the Kjeldahl apparatus where a large number of digestions could be carried on at one time. We found it convenient to digest all the total and lipoid phos- phates (six tubes) at the same time and to carry them through the various steps together, using only one standard (0.006 mg. H;PO; per cc.), as a rule, for the nephelometric readings. For the inor- ganic phosphoric acid a standard containing 0.006 mg. of H;PO, was also used. In starting our work we experienced considerable difficulty on account of a precipitate which flocked out of the strychnine-molybdate solution when no phosphoric acid was pres- ent. This was found to be due to an insufficiency of strychnine sulfate for the amount of sodium molybdate used. No further difficulties were encountered and the remainder of the procedure was as given by Bloor. In order to secure the greatest accuracy possible we recalibrated all our pipettes and volumetric flasks 324 Blood of Normal Children and used only one pipette of each volume throughout the series. In this way our actual and calculated values were in remarkably close agreement. Calcium Determinations.—Lyman’s (3) nephelometric method with a few modifications was used. 5 cc. of the whole blood (1:1), corpusecle (1:3), and plasma (1:1) dilutions were added drop by drop to 20 ce. of the trichloroacetic acid solution in 100 ec. Erlenmeyer flasks, filtered, and 10 cc. of each filtrate used for the determinations. If care is exercised, two 10 cc. portions can be obtained from each filtrate, enabling one to run duplicates if necessary. In using this method it is essential that the reactions be just right, else the calcium is not precipitated. To insure this we standardized our solutions and determined the end-points with utmost care, using alizarin rather than methyl orange for an indi- eator. Instead of shaking the solutions for 10 minutes as directed by Lyman, we allowed ours to stand in the refrigerator over night. By using sharply pointed centrifuge tubes and decanting the super- natant liquid quickly, we got better results than by pipetting the liquid from less pointed tubes. As was done in the case of phos- phoric acid, the whole blood, corpuscle, and plasma solutions were carried through the various steps together. A standard containing 0.1 mg. of calcium was used, and was invariably checked against itself before the readings on the unknown solutions were taken. In order to determine the accuracy of the method, known quan- tities of calcium were added to blood and blank solutions, and in all cases were recovered quantitatively. In addition, many determinations which seemed extraordinarily high were checked by means of a potassium permanganate titration method, the principle of which was essentially the same as that of Lyman’s except that the precipitate of calcium oxalate was dissolved in N sulfuric acid and titrated with 0.01 N permanganate solution. After making determinations on about twenty samples of blood with both methods, we finally adopted Lyman’s, since, with the titration method, our whole blood, corpuscle, and plasma readings frequently failed to check, while with the latter, our actual and calculated values practically always agreed within the limits of experimental error. We attribute the lack of agreement between the results secured with the former method to the fact that the end-point with 0.01 N potassium permanganate is very difficult M. R. Jones and L. L. Nye 329 to obtain, and 1 drop too much or too little may introduce an error of 20 per cent in the final value, the percentage of course, depending upon the volume of permanganate used in the titration. Alkali Reserve-—The CO.-combining power of plasma was deter- mined by the Van Slyke-Cullen (4) method without change. The results of the analyses are given in Tables I and II and are expressed in mg. of calerum and phosphoric acid per 100 cc. of whole blood, corpuscles, and plasma. Certain values which seemed exceptionally high were not included in the average. To be consistent, if the value of a constituent was not included in the average for corpuscles, the values of that same constituent were omitted from the averages for whole blood and plasma. DISCUSSION. The distribution of caletum and the various compounds of phosphoric acid was studied in thirty-four normal children, seven- teen boys and seventeen girls. In general, the values for phos- phoric acid are comparable to those reported by Bloor (5) and others. The total phosphoric acid values of corpuscles averaged 257 mg. per 100 cc. in boys, and 255 mg. in girls as compared with Bloor’s values of 248 mg. for men and 249 mg. for women. On the whole, the averages for boys tend to run higher than those for girls. This is especially striking in the lipoid phosphoric acid content of the corpuscles, the average for boys being 65.7 mg. per 100 ee. with variations from 36 to 84, and for girls, 55.8 mg. with variations from 33 to 72. Bloor’s averages were 57.0 and 56.6 mg. for men and women, respectively. Another striking difference between the values reported for adults and children is found in the inorganic phosphoric acid content of corpuscles. For men, Bloor found an average of 18.7 mg. per 100 ce. and for women, 15.7 mg. Our values for boys ranged from 6.5 to 20.9 with an average of 12.1, while those for girls varied from 5.7 to 26.0 mg. with an average of 10.3. The inorganic phosphoric acid content shows a greater percentage variation than that of any phosphorus com- pound in the corpuscles, or approximately 221 per cent in boys and 356 per cent in girls. In the plasma the higher values of the total and lipoid phos- phoric acids are again noted in the boys. 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(0° OE |0'S22 6°02 |2°9 |P 2E IO OIL | Sf! , F & GI |¢'se [g'I¢ €' Il G'0-(0'9 (0°92 |SITE |E°7Es |L°S |O°ZL |0'OSE |2°06 [S'S OOF [€'9ET | "N “V | ‘sth e & It |. 8 4'27 OTT 2S (€ 6 ITO |T'8 {8 Fe |0'é O'S |0 PES °C (0 OOT | “S “@ |sourg I *Suy A tod bu bu “Bu bu Bu “Bu bu Bu bu bu bu bu bu bu bu E a8 aS ‘olues |‘o1ues | ‘prod aun ‘orues | ‘prod wea ae a *[Boul E me ee “10 ~10U] ty ag “IQ | ~10uy “rT OE “10 ~i0uy nT Tae *s[]90 , : gout pod ee “BUISeT ‘soposnd109, “poo[q }o"UM SENS Sey eas : 09 (OT tod wintoyeD ‘09 OT 10d *Od*H “S]UD) JoULON {0 poolg ay? ur spunodwop piuy o10ydsoyd pun wnrapwg fo woyngriysrg 2Y,] | ‘TT AIAVL 327 328 Blood of Normal Children 41.1, while‘in girls the range was from 27 to 47 mg., the average being 36.6, as contrasted with Bloor’s values of 32 and 36.2 mg. for men and women, respectively. In boys, the lipoid phosphoric acid content of plasma ranged from 20 to 40 mg., the average being 32 or approximately 50 per cent higher than Bloor found in men. A possible explanation of the higher values in boys than in girls or adults might be found in their greater muscular activity. The difference between the values found in girls and women is not so marked. For the former, the average was 27.7 mg. with varia- tions from 20 to 37.5, and for the latter, 24.9mg. The inorganic phosphoric acid values of plasma were approximately the same in boys and girls, being 9.8 and 8.8 mg. per 100 cc., respectively. The variations in the boys did not equal 100 per cent and those in the girls did not exceed 150 per cent which is in striking contrast to the percentage variation of the inorganic phosphoric acid con- tent of the corpuscles. Marriott and Howland (6) using a differ- ent method, found the inorganic phosphorus content of the serum to vary from 1 to 3.5 mg. (approximately 3.2 to 11.2 mg. H3PO.) per 100 cc. in normal infants, and Denis and Minot (7) using Bloor’s method found a similar range in adults suffering from arious diseases other than nephritis and cardiorenal conditions. It should be noted that the sum of the inorganic and lipoid phos- phoric acid values in plasma is equal to the total. Bloor found a small amount of an unknown phosphoric acid compound (up to 10 per cent of the total) in plasma. This value was obtained by subtracting that of the inorganic phosphoric acid from the acid- soluble. We did not make determinations on the acid-soluble fraction, but as the sum of the inorganic and lipoid phosphoric acid values is equal to the total, within the limits of experimental error, the presence of this substance in an appreciable quantity seems doubtful. However, in certain pathological conditions which we are now investigating, we have found considerable quantities of the so called “organic”? phosphorus. In many cases it is interesting to note the approximate equilibrium of the inor- ganic phosphoric acid between corpuscles and plasma. This is contrary to Bloor’s findings in men and women which show a much higher concentration in the corpuscles than in the plasma. We are not able to explain the significance of the differences between boys and girls and between children and adults. ed ek ed M. R. Jones and L. L. Nye 329 Until recently, it has generally been conceded that little or no calcium exists in the corpuscles. Howland and Marriott (8) found about half as much calcium in normal whole blood as in serum, from which they concluded that there was no calcium in the cor- puscles and advocated the use of serum or plasma for calcium determinations. Many references to their work have been found in the literature. Hammarsten (9) cites the work of Gryns, Képpe, Hamburger, and others showing that blood cells are impermeable to calcium and magnesium, although Schmidt (10) in 1850 claimed that corpuscles contain a considerable amount of this metal. Recent investigations of Cowie and Calhoun (11) and others have confirmed Schmidt’s contention by showing that calcium is present in corpuscles in appreciable quantities. Cowie and Calhoun used Lyman’s method and made many determinations on a few cases, the values in one man being as follows: whole blood, 8.9 mg.; cor- puscles, 4.26 mg.; and plasma, 12.07 mg. per 100 cc. We were not able to ascertain whether the determinations were made on the same or different samples of blood. Brown, MacLachlan, and Simpson (12) also using Lyman’s method found an average of 9.5 mg. of calcium per 100 cc. of whole blood in eighteen normal infants, which is in close agreement with our value of 9.4 mg. Lyman reported an average of 6.1 mg. per 100 cc. of whole blood for men and 7.1 mg. for women. One woman had a value of 9.4 mg. The great majority of investigators have used serum or plasma for their determinations, the reported values of 9 to 11 mg. agreeing well with our average of 10 mg. per 100 cc. of plasma. The most striking feature of this investigation is the large amount of calcium found in the corpuscles, in many cases the content being equal to or greater than that in the plasma. As was found in the case of inorganic phosphoric acid, the greatest percentage variation of the calcium content occurred in the corpuscles, the boys showing wider limits than the girls. One boy, age 4 years, had an extremely low concentration in the blood, his values being: whole blood, 5.6 mg.; corpuscles, 5.6 mg.; and plasma, 5.5 mg. per 100 cc. His phosphoric acid values were well within the normal range. Neither age (within the above range) nor sex appears to be a factor in the calcium concentration of the blood or its distri- bution between plasma and corpuscles. Meigs, Blatherwick, and Cary (13) showed that in heifers the calcium content of plasma THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 330 Blood of Normal Children tends to become lower with advancing age up to 6 months. In some studies on the new-born now in progress, we have found the average calcium values of five babies under 14 hours of age to be as follows: whole blood, 8.7 mg.; corpuscles, 5.9 mg.; and plasma, 12.6 mg. per 100 ce. While we have not yet sufficient data from which we can draw conclusions, it appears that the cal- cium content of plasma is higher at birth than in later life, the average value for corpuscles being markedly less than that in older children. The plasma of thirty-two children had an average CO.-com- bining power of 51.8 volumes per cent. Sawyer, Stevens, and Bauman (14) report a plasma carbonate value of 54 volumes per cent in children between the ages of 4 and 8 years. Schloss and Stetson (15) found a range of from 46.1 to 76.1 volumes per cent in normal infants. Only one of our values (39.5) was less than Schloss and Stetson’s lower limit, and we consider this too low to be within the normal range. It is interesting to note that in this case the calcium content of plasma is lower than the average and that of the corpuscles is considerably higher. SUMMARY. The alkali reserve of plasma and the distribution of calcium and the various compounds of phosphoric acid in the blood were studied in thirty-four normal children whose ages ranged from 4 weeks to 14 years. From the above data it appears that the blood corpuscles are richer in all types of phosphoric acid compounds than plasma. The amount of unknown phosphoric acid in plasma is negligible, if any, while in corpuscles it averages approximately 70 per cent of the total. In general, the values for boys averaged slightly higher than those for girls. The lipoid phosphoric acid content of corpuscles averaged 17.7 per cent higher in boys than in girls, while the plasma value in boys was 16.6 per cent higher than that in girls. The inorganic phosphoric acid content of corpuscles showed the great- est percentage variation of all the phosphorus compounds of the blood. M. R. Jones and L. L. Nye 331 The average calcium content of corpuscles was found to be slightly less than that of the plasma, the values in mg. per 100 ce. being as follows: whole blood, 9.4 mg.; corpuscles, 8.7 mg.; plasma, 10.0 mg. A relation between the calcium and phosphoric acid contents of the blood is not apparent. The CO.-combining power of the plasma averaged 51.8 volumes per cent in thirty-two children. No relation between the alkali reserve and the concentration of calcium and phosphoric acid in the blood can be established. BIBLIOGRAPHY. - . Sundstroem, E. S8., and Bloor, W. R., J. Biol. Chem., 1920-21, xlv, 153. . Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 33. Lyman, H., J. Biol. Chem., 1917, xxix, 169. Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1917, xxx, 289. . Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 49. . Marriott, W. McK., and Howland, J., Arch. Int. Med., 1916, xviii, 708. Denis, W., and Minot, A. 8., Arch. Int. Med., 1920, xxvi, 99. . Howland, J., and Marriott, W. McK., Quart. J. Med., 1917-18, xi, 289. . Hammarsten, Text-book of physiological chemistry, New York, 1908, 195. . Schmidt, C., quoted in Mathews, A. P., Physiological chemistry, New York, 1915, 462. . Cowie, D. M., and Calhoun, H. A., J. Biol. Chem., 1919, xxxvii, 505. . Brown, A., McLachlan, I. F., and Simpson, R., Am. J. Dis. Child., 1920, xix, 413. 13. Meigs, E. B., Blatherwick, N. R., and Cary, C. A., J. Biol. Chem., 1919, xxxvui, 1. 14. Sawyer, M., Stevens, F. A., and Bauman, L., Am. J. Dis. Child., 1918, Rav epills 15. Schloss, O. M., and Stetson, R. E., Am. J. Dis. Child., 1917, xiii, 218. OCONMSOMPR We — oO re rr b — ND f ae Ale ' . bea eaLy ta Py et Be *, 4 ye <—y 4 55 tod ia wik &% v os ; i. f 7 i re Biba: yits 1 Vil Saar = hia Fae fiptalag ay J eae | — i oo oo Le ee " r iti he aA ; PAL heteah i Pp Lied | > | : - : ; " — eS > = 7 STUDIES OF AUTOLYSIS. VII. AUTOLYSIS OF BRAIN. By CHARLES A. GIBSON, FREDA UMBREIT, anp H. C. BRADLEY. (From the Laboratory of Physiological Chemistry, University of Wisconsin, Madison.) (Received for publication, May 10, 1921.) Brain undergoes autolytic decomposition very slightly when measured by amino-acid production and when compared with most other tissues. The evidence of the reaction even under the most favorable conditions is so slight that it may easily be over- looked, or be incorrectly interpreted. It is because of this that the brain proteins are assumed (1) to be peculiarly resistant to autolytic changes and not subject to those rather rapid fluctuations of mass which occur for example in the liver, and in other organs. The peculiar stability of brain and nerve proteins thus appears to relate itself to those functions of the brain which seem to demand a peculiarly stable physical structure, the functions of memory, habit, instinct, ete. While there can be no question about the stability of the protein framework of the brain cells, it does not necessarily follow that the autolytic mechanism is wanting, or is strikingly different from that of other organs. The outstanding fact of stability and per- manence of brain cell structure may equally well be explained by the peculiarly perfect protection against local acidosis which the large blood supply of the brain insures. The respiratory center is itself nerve tissue. It is characterized by its sensitiveness to H ion changes, to COs and O content (2-8). By its regulation the blood always supplies the brain with oxygen sufficient to pre- vent asphyxial reactions from developing, except under pathologi- eal conditions. With abundance of oxygen supplied, there is also very perfect CO: elimination, and a supply of buffer salts which tends to prevent the development of local acidosis. In short, the regulation of CO, elimination, of constant H ion level, and of oxygen supplied the body is very largely effected by a portion of the central nervous system itself. Conditions giving rise to acido- 333 334 Studies of Autolysis. VII sis of the liver, a group of muscles, or the kidney may be without influence on the brain, or on tissue respiration as a whole. On the other hand any condition of the brain tending toward acidosis, asphyxia, faulty CO, elimination, or rising H ion concentration, will result in an immediate respiratory and circulatory response affecting the entire body. The brain may be said to protect itself from asphyxia and acidosis, and incidentally in so doing to protect the rest of the body. If the brain tissue is lke other tis- sues it will not exhibit autolytic changes as long as it is so perfectly protected against the conditions under which alone autolysis can go on. Clinically we have much evidence to support the idea that atrophic changes do go on in the brain, just as they do in other tissues. We have the familiar losses of consciousness in brain anemias or asphyxias, rapidly progressing to more permanent functional losses and death where the condition is at all prolonged. General acidosis frequently leads to coma from which no measure of relief can save the patient, suggesting a progressive degenerative change in the central nervous system of an irreversible character. Local pressures in the brain lead to paralyses, and these may become permanent as a result of irreversible atrophic changes. The pathologist recognizes and correlates functional losses with brain or nerve degeneration. These degenerations are perfectly analogous to the atrophies of other tissues. They suggest that under favorable conditions autolysis may proceed quite rapidly, and in the delicate and essential protein structures of the brain cells produce irreversible losses and obliterations of a framework upon which the unique characteristics of the central nervous tis- sue depend. It appeared worth while, therefore, to review and repeat such significant work as has been reported on brain autolysis and to compare the autolytie reaction of brain with that of other tissues. Levene and Stookey (9) showed in 1903 that brain autolysis pro- duces such characteristic protein decomposition products as pep- tones, amino compounds, and free NH3;, while the coagulable pro- tein fraction diminishes. They found that acids accelerate this process, while alkalies inhibit it. Their experiments were few, however, and confined to the demonstration of the fact of brain autolysis, which had not previously been convincingly shown. Gibson, Umbreit, and Bradley 335 EXPERIMENTAL PART. The experimental technique is exactly like that described in previous papers (10). 50 gm. of brain tissue, finely ground, are made up to 250 ce. with water and toluene. 25 cc. samples are taken, made up to 100 ce. with trichloroacetic acid to precipitate the proteins and 25 ce. aliquots of the filtrates titrated by the formol method for amino-acids. The titra- tion figures are small and the errors due to end-points are proportionately large. By matching end-point colors with a standard and by using small burettes graduated in 0.01 ec., the titration errors have been kept as low as possible and the figures, though actually small, are nevertheless char- acteristic and significant. : A few typical examples, selected from the large number of exper- iments performed, are presented. From Tables I to V and Fig. 1 we are able to conclude that brain pulp autolyzes normally as do other tissues in vitro. The TABLE I. Sheep Brain. 0.20 N amino-acids. Net No. Condition. Days. gain in 33 days. 0 2 3 10 33 ar ce. || cc. | ce. || cc. | cc. ce. We Control oa ee ee oe 0.40/0.55|0.65)/0.85)1.25) 0.85 II oe 190 OlS Nera @ reser ee 0.40)0.55|0.68/1.00)1.50) 1.10 IU ne $50. O2sNaEL Gl erere.. sc 0.40/0.57/0.65/1.15)1.45} 1.05 IV .s TO OL NUELC leer ee bee 0.40)0.50)0.65|0.70)0.90) 0.50 V ss + 3 gm. NasHPO,..... 0.40)0.55)0.60/0.80)1.05| 0.65 TABLE II. Calf Brain. 0.20 N amino-acids. SS Net No. Condition. Days. gain in 30 days. 0 1 5 36 ac: cc ce cc. cc cc MalinControleiaeee we he seo ees oo 0.25|0.35/0.60/0.60) 0.35 II e IMBOROM NTE Clsss eee. 0.25|0.40/0.90)1.40} 1.15 III oe SePROROD ENG ERC ise ae EE a fs 0.25)0.40/0.80)1.380} 1.05 lV i SOLON NaOH pee i Re ...-|0.25]0.25|0.35/0.45| 0.20 V fire een CAO Osh: See. hiss 0.25,0.40|0.45|0.60) 0.35 336 I No. No. Studies of Autolysis. VII TABLE III. Calves Brains. 0.20 N amino-acids. Net gain in 30 Condition. Days. days. 0 1 2 5 10 20 30 10 30 Te 1 aE See RR mee | co.'| cc. | cen eee eee Control:..-) pea eee 0.3 0.35)0.37|0. 40/0..45 0.50)0.55\0.20)0.25 re in 0.005 n HCl. .| 0.3/0.4 |0.55 0.65)0.80/0.90)1.00,0.60)0.70 “ “ 0.01 nw HCl..| 0.3/0.4 |0.60)0.65/1.00]1.05]1.40/0.80)1.10 re “10:02 Sw LCL 0231024: '0.70.0.75/1.00/1.40|1.60]1.10/1.30 ce “ 0.04 nw HCl..| 0.3/0.25|0.300.33/0.40/0.40/0.40/0.10/0.10 oc “ 0.1 w HCl..} 0.3/0.2510.30/0.30/0.35/0.45/0.55/0.15/0.25 “ “< WakiG@s;.2 2. 0.3)0.35 0.35/0.35 0.20/0.45 0.50)0.15/0.20 “s TAKEN One eee: 0.3/0.35,0.35)0.35 0.40/0.4010.50/0. 1010.20 TABLE IV. Calves Brains. 0.20 N amino-acids. be ae t 3 ondition Days. see 0 1 3 15 cc. ce. cc. Pee ce. GWontrole ere remeticer | fe eee ses cee 0.25)0.40,0.50)0.50) 0.25 & IT BOA SN MELE po x54 he eck 0.25|0.60)0.55;0.60) 0.45 od Soe (O04 SS? 18K Ole eee ee et 0.25)0.50'0.70:0.80) 0.65 ae SSRs Ue 0 a 0.25)0.40|0.50)0.45| 0.20 se HS Ber Os 0) a 0.25)0.40,0.40'0.40) 0.15 =f alee ay Cie Np) 81) 21 Oe 0.25,0.35/0.300.30) 0.05 es aS TO pp Oe ol hls OO) be ee 0.25/0.40/0.30/0..35) 0.10 TABLE V. Sheep Brain, Foreign Protein, and Acid. 0.20 N amino-acids. ———————————— SS Digestion Condition. Days. Net gain. | of foreign protein. 0 1 3 7 15 ce, cc. cc. cc. cc. ce Control 22 eee 0.2510.50,0.500.60.0.60| 0.35 sf in 0.04 w acid... .|0.25 0.60)0. 6510.65 0.70) 0.45 ; Ps “ peptone......./0.75)1.90/2.10)2.30/2.40) 1.65 1.25 te A, e aerd 10.70 1.40/1.30/1.50)1.70 0.95 0.50 + “Cel abies ee 0.40 0.70/0.90,1.00)1.10 0.70 0.25 e “ “ acid 0.40)1.201.50 1.60/1.90 1.50 1.05 Gibson, Umbreit, and Bradley 337 amount of protein in brain is small, approximately 7 per cent of the total, while in muscle it approximates 18 to 20 per cent, and in liver 15 per cent or more. It is not surprising, therefore, that the figures for brain autolysis should be much smaller than those for liver, muscle, and other tissues. The addition of acid increases the speed and extent of autolysis of brain, but the amount required for maximum digestion is smaller than for tissues like the liver. 4 i agSnCSeeeEeE ————— pitt SeSeeeeeseeeees _- ERE eee ~ — sage ee ae Lt 0 a LO 20 30 DAYS This is correlated evidently with the protein content, the more protein present the more acid can be added before the H ion rises to the point of inhibiting the reaction. With liver, sufficient acid to make the digesting mixture 0.04 to 0.1 N gives maximum autolysis. With brain an acidity of 0.02 N is optimum. Foreign proteins such as gelatin and peptone, which are digested by the autolytic enzymes of the liver, are also found to digest in 338 Studies of Autolysis. VII the brain mixtures. It is of interest to note that peptone digests: better in the nearly neutral control than it does in the rather strongly acid brei used, while gelatin digests much better in the acid medium. The stability of brain structure depends not on any lack of proteolytic enzymes in the cells, but upon the fact that normally the brain proteins are not available substrata for the enzymes. They become available under the conditions that make liver pro- teins available for autolytic hydrolysis; namely, acidosis within the cells. That local acidoses do not frequently occur in brain tissue in spite of its large CO. production, is due we believe to the exceptionally large blood supply to that organ, and to its ability to modify the respiratory and circulatory rate so as to prevent any accumulation of CO, or other acid. By its extreme sensi- tiveness to increased H ion concentration and CO, and by its position as master tissue of the body it automatically prevents just those conditions from arising within itself which would eventuate in its own autolytic disintegration. SUMMARY. 1. Brain tissue autolyzes in the same way as other tissues thus far examined though quantitatively on a smaller scale comparable to the low protein content of brain tissue. 2. The speed and extent of the proteolysis is determined by the H ion concentration of the mixture. In alkaline or neutral media autolysis is inhibited. It is increased in proportion to the acid added. 3. The optimum acidity for autolysis of brain tissue is about 0.02 n, or much less than that for liver, kidney, and other epi- thelial tissues. The small amount of acid required corresponds to the small amount of protein present and made available for digestion by the addition of acid. 4. Brain cells contain proteolytic enzymes which digest such added substrata as gelatin or peptone, as in other gland structures. 5. The permanence of the protein structures of the brain appears to depend on the very perfect protection against asphyxia and CO, accumulation, which its large blood supply and its con- trol of respiration insures. When asphyxia and acidosis do develop in brain tissue, it autolyzes like other tissues. Gibson, Umbreit, and Bradley 339 6. The autolytic disintegration of the delicate protein structures of brain tissue appears to be an irreversible phenomenon, and is accompanied by loss of such characteristic functions as memory, habit, motor control, and consciousness. > Or Oo BIBLIOGRAPHY. . . Mathews, A. P., Text-book of physiological chemistry, New York, 3rd edition, 1920, 590. . Gasser, H. S., and Loevenhart, A.S., J. Pharmacol. and Exp. Therap., 1913-14, v, 239. . Haldane, J. S., and Priestly, J. G., J. Physiol., 1905, xxxii, 225. . Hasselbach, K. A., Biochem. Z., 1912, xlvi, 403. . Winterstein, H., Arch. ges. Physiol., 1911, cxxxviii, 167. . Campbell, J. M. H., Douglas, C. G., Haldane, J. S., and Hobson, F. G., J. Physiol., 1913, xlvi, 301. . Douglas, C. G., Ergebn. Physiol., 1914, xiv, 338. . Stewart, G. N., Guthrie, C. C., Burns, R. L., and Pike, F. H., J. Ezp. Med., 1906, viii, 289. . Levene, P. A., and Stookey, L. B., J. Med. Research, 1903-04, x, 217. . Bradley, H. C., and Taylor, J., J. Biol. Chem., 1916, xxv, 261. A STUDY OF THE CATALASE REACTION. By SERGIUS MORGULIS. (From the Department of Biochemistry, University of Nebraska, College of Medicine, Omaha.) (Received for publication, May 2, 1921.) The significance of catalase in the living organism is still an open question. Only one fact has thus far been incontestably established; namely, that catalase liberates molecular oxygen from hydrogen peroxide, though it fails to do so with similarly constituted organic compounds like ethyl hydrogen peroxide. In a sense catalase lends itself admirably to the investigation of the dynamics of its action. The course of the reaction can be easily and directly timed and measured by the evolution of oxygen. Furthermore, the interference generally caused through an ac- cumulation of products of the reaction is absent here since, ex- cept for very minute traces of oxygen remaining in solution, the gaseous by-product is quickly driven off, especially when the mixture is vigorously shaken. Me All the experiments reported in this paper were performed with a crude catalase preparation from liver! The finely ground pulp of beef liver was extracted with chloroform-water over night and the liquid was strained through cloth to remove solid particles. Enough alcohol was then added to this liquid to make a 50 per cent mixture, when a massy precipitate forms on standing. This precipitated material is collected on a hardened filter paper and the excess fluid removed by suction. The material is then dried in the air and ground to a fine powder in a mortar. The powder possesses strong catalase action. During several months the 1In some experiments with kidney extracts which were preliminary to this research the author enjoined valuable aid from Dr. V. HE. Levine (Science, lii, 612). 341 342 Catalase Reaction reactivity of the preparation has apparently remained unchanged. A more highly purified sample of catalase can be prepared from this powder by reprecipitating a watery extract with an equal volume of alcohol. Since repeated precipitation causes great loss of the catalase, the crude preparation was used exclusively in these experiments. A definite quantity of this dry powder was weighed out (usually 0.5 gm.) and extracted with 200 ce. of chloroform-water. A water-clear extract is obtained which, when kept in an ice chest, retains its strength undiminished for a considerable time. The extract was neutralized with 0.01 nN NaOH to make its pH = 7.0. The hydrogen ion concentra- tion in all these experiments has been determined colorimetri- cally with the aid of the Hynson, Westcott and Dunning stand- ards. The relative amount of catalase was measured by the number of ec. of the extract employed. The hydrogen peroxide used in the experiments was the grade known under the trade name of “Oakland Dioxogen.” Inas- much as the hydrogen peroxide is strongly acid it was first neutra- lized with NaOH and its hydrogen ion concentration adjusted to a pH somewhat below 7.0. The reason for this procedure is that the hydrogen peroxide decomposes spontaneously when it is neutral or alkaline in reaction. On this account also just enough peroxide was prepared for the day’s work; a fresh supply being made for each occasion. The strength of the hydrogen peroxide was standardized by titrating against 0.1 N potassium permanganate, and expressed in terms of its oxygen content. When a series of experiments lasted several hours the titer of the hydrogen peroxide was again checked up at the end of the experiment. II. The apparatus in which the catalase activity was measured (Fig. 1) consisted of three parts: (1) a shaker driven by an elec- tric motor; (2) an Erlenmeyer flask in which the reaction between the catalase and the hydrogen peroxide took place; and (3) a eudiometer tube in which the gas was collected for measurement. The volume of gas was reduced to standard conditions of tem- perature and pressure, and all the data recorded in the paper represent the corrected volume. The catalase preparation was S. Morgulis 343 measured into the Erlenmeyer flask and enough water added to secure the desired relative concentration. In some experi- ments the same volume was maintained throughout the entire series. The hydrogen peroxide was measured into a glass cup provided with asyphon. The cup was suspended from the bottom of a rubber stopper with which the Erlenmeyer flask was ¢losed. The stopper bore a plunger which could be easily pushed down into the cup by pressing on the rod extending to the outside. The descent of the plunger raised the level of the liquid in the cup and started the syphon which drained the contents in about 10 seconds. As soon as the cup was emptied the shaking was re, a started by turning the motor switch. The evolution of the gas was recorded at the end of every 5 minutes, and the shaking continued until no more gas was given off. Usually experiments required about one-half to three-quarters of an hour for com- pletion. III. To insure its stability the commercial hydrogen peroxide is strongly acidified. When this acid hydrogen peroxide is used a serious error is introduced into the experiments, which seems to have been the case in the older experiments on catalase. Even when hydrogen peroxide is made neutral to Congo red, the perox- ide is still strongly acid as far as its hydrogen ion concentration 344 Catalase Reaction is concerned. Certainly, in a series of experiments where the quantity of hydrogen peroxide is varied, the range of variation in hydrogen ion concentration may be very great. To avoid such a possibility, all our experiments were conducted at a definite hydrogen ion concentration. This end was accomplished either with the aid of appropriate phosphate buffer mixtures, or by bringing both the hydrogen peroxide and the catalase solution to a definite pH by adding 0.01 N NaOH. The objection to the use of the buffer is that, especially at a pH over 7.0, there is danger of the hydrogen peroxide being broken up by it. While within the short time of the catalase experiment this danger is greatly minimized, nevertheless, the second method was used in prefer- ence except where a wide range of pH values was required. The first thing, of course, was to determine the optimum hy- drogen ion concentration for the catalase reaction. Experi- ments were performed in which the only variable factor was the pH of the mixture, adjusted by means of phosphate buffers. The pH of the mixture was invariably checked up at the close of each experiment. The series reported below has been made with 5 cc. of the catalase extract and hydrogen peroxide sufficient to yield 173 ee. of oxygen (determined by titration); 20 ec. of the appropriate buffer mixture were used in each experiment. The initial hydro- gen peroxide concentration was 0.31 gram-molecular in all these experiments. For reasons which will become apparent from sub- sequent discussion the quantity of hydrogen peroxide and catalase was so adjusted that the curve of the oxygen evolution followed very closely the isotherm of a bimolecular reaction fe at a-—2z It is obvious from these experiments that the optimum condi- tion for the catalase reaction is at neutrality (pH = 7.0). As the acidity of the medium increases, both the velocity of the re- action and the total amount of peroxide decomposed diminish somewhat at first, but when the pH is below 6.0 the decrease becomes very noticeable. Thus, at pH = 6.4 the reaction is 98 per cent complete, but at pH = 5.8 it is 88 per cent, and at pH = 5.2 it is only 70 per cent of that at the pH = 7.0. In- S. Morgulis 345 creasing the hydroxyl concentration (pH = 7.2 to 8.3) has ap- parently little or no effect on the amount of hydrogen peroxide decomposed, though the reaction velocity is somewhat diminished. TABLE I. Oxygen evolved. pH Time: jg fem ee | KK 1 eee Observed. Calculated. a eae Al eee ce. sey: oe per cent 5 50.7 44.5 48 10 (ond 70.8 44 5.2 15 88.9 88.1 41 55 20 94.0 100.4 34 23 95.3 109.2 31 40 5 62.5 56.5 66 10 90.2 85.2 63 as) 105.2 102.5 60 5.8 20 Ses 114.1 55 69 PAE abteeil 122E5 48 31 119.7 129.8 42 56 5 62.9 63.5 66 10 93.6 92.9 68 15 113.4 109.8 73 6.4 20 124.2 120.8 74 76 25 128.3 128.6 66 29 ST, 134.3 63 33 1B. 7/ 137.0 58 67 5 65.1 65.8 70 10 95.3 95.3 71 15 114.3 112.4 75 fo 20 WA a4 122.8 19 79 25 130.9 130.4 ae, 29 133.9 135.9 69 34 136.1 140.2 63 71 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 346 Catalase Reaction \ TABLE I—Concluded. pH Time. Observed. min. ce. 5 60.3 10 89.9 15 108.2 1.2 20 119.4 : 25 126.2 31 130.0 5 60.1 10 88.2 15 108.0 15D 20 120.6 743) 128.3 30 132.8 35 135.2 Ehret sh 10 86.4 15 107.8 8.3 20 120.0 25 127.6 80 ike ON 13300 35 136.1 The fact of particular significance, however, is that while the reaction runs true to the course of a bimolecular curve within the range of hydrogen ion concentrations of from 6.4 to 8.3 (and about three-quarters of the peroxide undergoes decomposition), the reaction deviates from this course as the acidity exceeds pH = 64. Although pH = 7 has been found to be the optimum hydrogen ion concentration for the catalase reaction, the experiments were made in a medium slightly on the acid side (pH = 6.7 to 6.9) Oxygen evolved. Calculated. cc. 59.7 88.8 106.1 117.4 125.4 131.2 60.5 89.6 106.9 118.2 126.3 132.2 136.9 60.5 89.6 106.9 118.2 126.3 132.2 136.9 | K X 105 per cent 62 63 64 64 62 56 62 H202 decomposed. per cent 75 78 79 S. Morgulis 347 in order to guard against spontaneous decomposition of the hy- drogen peroxide which happens most readily at pH = 7.0 and above. Vis An examination of the effect of changing the quantity or the concentration of the hydrogen peroxide on the catalase reaction brings to light a number of interesting points. When a series of experiments is performed with a constant quantity of catalase it becomes at once apparent that, as the hydrogen peroxide increases, the reaction proceeds more slowly. Furthermore, ex- cept where there is a considerable excess of catalase, only a cer- tain and variable portion of the peroxide will be decomposed. In- deed, with every increase in the amount of hydrogen peroxide there is a diminution in the proportion which is decomposed with the liberation of its oxygen. This will be seen from a series of experiments with a constant amount of catalase and a variable amount of hydrogen peroxide, recorded graphically in Fig. 2. These experiments were made with 5 cc. of the catalase prepara- tion. The total volume of the mixture was 50 cc., and the re- action of the mixture was maintained at pH = 6.9 throughout the series. The temperature in this as well as all other experi- ments was 20-21°C. In this series the hydrogen peroxide used represented a range of variation of from 45 to 226 cc. of available oxygen, as determined by titration with permanganate. Since the final volume was the same in all experiments, the concentra- tion of the peroxide varied from 0.08 to 0.40 gram-molecular. The smallest concentration was so much below the catalase ca- pacity that it has been left out of the graphic record. With a gram-molecular concentration of 0.16 (7.e., with 90 ce. of oxygen available) 94.2 per cent of the hydrogen peroxide was decom- posed in 12 minutes when the reaction came to an end. With an increase in the concentration up to 0.32 gram-molecular the amount of oxygen evolved gradually rises but the proportion of hydrogen peroxide decomposed diminishes. Thus, while the amount of oxygen liberated increases from 85 to 122.7 ec. the percentage of hydrogen peroxide decomposed diminishes from 94.2 to 68.2 per cent. When the concentration of the hydrogen peroxide is greater than 0.32 gram-molecular, the quantity of of oxygen Ce. 348 Catalase Reaction oxygen actually set free in the reaction begins to decline. At concentrations below 0.16 gram-molecular the catalase is so much 100 190 15 Per cent of H,O. decomposed 0 016 024 052 040 G-M. CONC. Fic. 2. Effect of increasing quantities of hydrogen peroxide on the catalase reaction. *—* Ce. of oxygen available. °—° Cec. of oxygen liberated. X—>X Per cent of hydrogen peroxide decomposed. in excess that the hydrogen peroxide is completely decomposed. Considering the regularity of the curve, it is reasonable to assume a pre ee S. Morgulis 349 ‘that with hydrogen peroxide equivalent to 85 ce. of oxygen the decomposition would be 100 per cent complete. The decline in the absolute quantity of oxygen liberated with _the same amount of catalase requires further consideration. This may have been either due to an increase in the amount of hydrogen peroxide or to its greater concentration. It might be supposed, for instance, that the peroxide contains some impurity which is too negligible when the quantity of peroxide is small, but when more of this is used there is also enough of the injurious substance to retard and depress the reaction. Such a view, however, is untenable because it can be shown that the depressing effect may be produced by the same quantity of hydrogen peroxide when its concentration is increased. Thus in an experiment with the same amounts of peroxide and of catalase the volume was so adjusted that in one case the peroxide was in a concentration of 0.18 and in the other of 0.36 gram-molecular. In the former case the reaction ended with the production of 87.3 cc. of oxygen, while in the latter, only 78.2 cc. were set free. On the other hand, it must be emphasized that the change in concentration of the peroxide is not the only and effective factor in depressing the reaction. Even with a constant concentration of peroxide, as soon as the hydrogen peroxide quantity has been increased be- yond a certain point, the reaction is progressively depressed. Vv. The depressing influence exercised by hydrogen peroxide on the catalase reaction has been invariably observed also by the older investigators of the subject. It has been generally noted that the amount of oxygen evolved diminishes, but the effect was attributed by them to a destruction of the catalase through oxidation by the excess of hydrogen peroxide. Indeed, the fear of oxidizing the catalase with the hydrogen peroxide was so predominant in the earlier work on catalase that the reaction was carried out at very low temperatures (0°C.) and with very dilute hydrogen peroxide solutions. This has occasioned certain errors in interpretation to which we shall return later. In the older experiments on catalase no heed was paid to the hydrogen ion concentration of the reaction medium. Since peroxide is 350 Catalase Reaction usually strongly acid, and the relative acidity would increase with the increase in the quantity of the hydrogen peroxide em- ployed, it is not improbable that the very great depression ob- served under these conditions was primarily the effect of greater. acidity. The considerable depression (a loss of 30 per cent in catalase activity) brought on by a change in the reaction of the medium only from pH 7 to 5.2 has already been demonstrated in the foregoing. Experiments in which the depressing effect was produced by — merely increasing the concentration of the peroxide without actu- ally changing its quantity convinced us that the apprehension that the catalase may be oxidized by the peroxide is entirely unfounded. But to test this matter further a series of experi- ments was performed in which instead of a small excess of hy- TABLE II. Hydrogen peroxide. Oxygen evolved in minutes. H,02 decom- ; Cosh: Oxygen. 10 20 30 40 posed. mol ce. cc: cc. ce. cc. per cent 0.2 2, '53),-199 75.8 85.8 91.2 82.1 0.4 224 49.6 60.7 74.9 76.6 34.2 0.8 448 30.3 43.7 ja.2 ihe: 13.6 LG 896 23.0 29.9 30.3 34.1 3.8 2.4 1,344 19.5 23.0 231 1.8 drogen peroxide very large excesses were used. These experi- ments prove definitely the inadequacy of the idea of the des- truction of the catalase through oxidation ‘with hydrogen perox- ide. In the experiments reported below hydrogen peroxide of such strength was used that it bleached and even blistered the skin of the hands. One could naturally expect that a few milli- grams of catalase would be quickly and completely destroyed by so powerful an oxidizing reagent. This, however, was not the case, and the reaction, though much retarded and suppressed, continued for a considerable length of time, as can be seen from the data in Table II. It is obvious from these results, therefore, that the diminished catalase activity which occurs when the hydrogen peroxide is increased beyond a certain optimum quantity can be accounted S. Morgulis 351 for neither on the assumption that it is due to some incidental impurity, nor that the catalase undergoes destructive oxidation. Two things, however, can be regarded as proven: First, that when the quantity of hydrogen peroxide is above a certain minimum, which the catalase can break up completely, the amount of oxygen evolved gradually rises to a maximum point with an increase in the amount of hydrogen peroxide. In a number of experi- ments it has been established that with our liver catalase prepara- tion the maximum oxygen evolution occurs when 65 to 70 per cent of the total hydrogen peroxide is decomposed. Secondly, when the maximum evolution of oxygen has been reached, further increases in the quantity of the hydrogen peroxide, even when not considerable, cause a falling off in the amount of oxygen set free in the reaction. This fact permits apparently of only one interpretation; namely, that we are dealing with a reversible reaction. This view will receive further support when we con- sider the nature of the catalase reaction. VI. The question naturally presented itself whether or not all the catalase is used up in the reaction when 65 to 70 per cent of the hydrogen peroxide breaks up setting free its oxygen. The following experiments were made with this in view. 5 ce. of the liver catalase solution were allowed to react with a quantity of hydrogen peroxide equivalent to 173 cc. of oxygen. In 30 minutes the reaction was complete when 120.1 cc. of oxygen were set free. In other words, 69 per cent of the hydrogen peroxide was de- composed. If free catalase were still present in the mixture it would be impossible to explain why the remaining 52.9 ce. failed to be liberated. Indeed, when another 5 cc. of catalase are now added to the system from which no more oxygen came off, the previously undecomposed quantity breaks up very rapidly. In 8 minutes 52.1 ce. of oxygen are given off which is practically the theoretically expected amount. We must, therefore, con- clude that when the reaction came to a stop with the evolution of 120.1 cc. of oxygen that there was no more active catalase pres- ent in the system. The experiment, under precisely the same conditions, was then repeated with a certain modification. If 352 Catalase Reaction a certain amount of catalase can decompose only a definite quantity of hydrogen peroxide and is thereby itself used up, will this happen also when the reaction occurs in more than a single stage? 5 ec. of the catalase preparation were again allowed to react but this time with only about half the quantity of hydrogen perox- ide (92 ec. of Os). The reaction went off much quicker than before, and in 10 minutes it was all over while practically the en- tire theoretical amount of oxygen was set free (91.7 ec.). More hydrogen peroxide was then added to bring the total amount of available oxygen to 180 cc. of oxygen. Upon the addition of the second quantity of hydrogen peroxide the reaction started up once more, this time, however, much more slowly, and in 15 minutes 26.4 ec. were liberated. Altogether, therefore, 118.1 ec. of oxygen were given off in the two consecutive stages, which compares very favorably with the former result, when the entire peroxide quantity reacted at once with the catalase. The some- what smaller amount of gas formed may perhaps be due to the fact that in the second experiment there was about 5 per cent more peroxide than before. The experiment was then tried with still another variation. Starting with a quantity of hydrogen peroxide equivalent to 180 ec. of oxygen it was made to react with only 3 cc. of the catalase preparation. The reaction ran to completion in 29 minutes with the liberation of 71.2 cc. of oxygen. There was still, therefore, an excess of 108.8 cc. present. When now 2 more ce. of the catalase were added to the mixture, the reaction commenced again but at a very slow rate and ceased in 25 minutes when 52.3 cc. of oxygen were liberated. Thus, 123.5 ec. of oxygen were produced, or 69 per cent of the peroxide decomposed, when 5 ec. of catalase were used though the reaction occurred now in two stages. The conclusion seems, therefore, justifiable that the catalase is used up in the reaction, and that it reacts with a de- finite quantity of hydrogen peroxide. The reaction is essentially the same as that between an acid and a base, or perhaps more correctly as between an alcohol and a fatty acid. It likewise seems highly probable from our evidence that when the hydrogen peroxide is 65 to 70 per cent decomposed the liver catalase is’ completely used up. S. Morgulis 353 Wis The study of the relation between varying amounts of catalase and the quantity of oxygen liberated from a definite amount of hydrogen peroxide gains particular interest in the light of the 15 06 08 1.0 1.2 1 | Relative amount of catalase Fig. 3. *—e* Relative amount of oxygen set free. °—° Relative con- centration of catalase. experiments just discussed. In the preceding series of experi- ments using a constant catalase quantity and different amounts of hydrogen peroxide it was found that, at any rate within a limited range of variations of the peroxide, there is a regular 354 Catalase Reaction 1 1 1 1 Relative amount of catalase Fic. 4. *—* Relative amount of oxygen set free. centration of catalase. o—o Relative con- S. Morgulis 355 increase in the oxygen set free. When, however, a constant quantity of peroxide is employed it is found that the degree of its decomposition depends upon the absolute quantity of catalase and that the quantity of oxygen liberated is directly proportional to the catalase. The direct relationship between the varying amounts of catalase and the oxygen given off from a definite O67 = 083 10 125 Relative amount of catalase Fic. 5. *—* Relative amount of oxygen set free. °—° Relative con- centration of catalase. quantity of hydrogen peroxide is demonstrated by a number of experiments which are graphically recorded in Figs. 3, 4, and 5. These experiments represent a variety of conditions, the quantity and concentration of the catalase varying either sep- arately or simultaneously, while the concentration of the peroxide also is either changed or kept constant. The curves are plotted with the relative quantities of catalase as abscissze and the corres- 356 Catalase Reaction ponding relative quantities of oxygen set free at the time the re- action has been completed as ordinates. The relative concen- tration of the catalase is also indicated in each graph. It is clear from these experiments that there is a direct pro- portionality between the amount of catalase and the amount of oxygen it produces from a given quantity of hydrogen peroxide. This proportionality holds only for the absolute quantity of catalase,? changes in concentration alone having no direct effect - on the oxygen set free. This furnishes, therefore, additional evidence for the view that the catalase reacts with a definite quantity of hydrogen peroxide. WITS The amount of hydrogen peroxide decomposed (or the amount of oxygen liberated) is a linear function of the quantity of catalase provided a constant amount of hydrogen peroxide is used in the experiments (its concentration need not be constant). With the same quantity of catalase, however, changing the quantity of hydrogen peroxide (or even changing its concentration beyond a certain point) will limit the catalase reaction to a greater or less extent. This, of course, is true only in the case when the hydrogen peroxide is already present in such excess that it is less than 65 per cent decomposed. Within the limits of complete decomposition (100 per cent) and two-thirds decomposition (65 to 70 per cent) the amount of oxygen set free from the hydrogen peroxide increases but not in direct proportion to the increase of its quantity. This can be seen from the data recorded in the first part of Table III as well as from the graph in Fig. 6. On the other hand, when the decomposition of the hydrogen peroxide used falls below 65 per cent the amount of oxygen produced by the same quantity of catalase diminishes with every increase in the hydrogen peroxide. The data in the second part of Table III demonstrate this point. Plotting the results with the rela- tive quantity of hydrogen peroxide as the abscisse and the 2 This rule holds true only when the quantity of catalase is neither too small nor too large for the amount of hydrogen peroxide employed. In these events the disturbing factors already discussed dominate the reaction and there is no longer a direct proportionality between the catalase and the quantity of oxygen liberated from the hydrogen peroxide. S. Morgulis aot relative amount of oxygen formed by the catalase as well as the per cent of decomposition of the peroxide as ordinates, we find that the curve of the oxygen evolution has a smaller slope for every new increase in the amount of hydrogen peroxide employed. The greatest diminution in the evolution of oxygen from hy- drogen peroxide by a certain quantity of catalase occurs, therefore, with the relatively smaller excess of peroxide. This fact is signifi- cant because if the diminution were due to an oxidation of the catalase a proportionately greater destruction of it could be ex- pected with the greater amount of hydrogen peroxide. TABLE III. Hydrogen peroxide. | Oxygen evolved from H202. Oxygen available. Relative amount. Relative amount. Absolute amount. ce. cc. 90 1.00 1.00 85 113 1.26 1.14 97 135 1.50 122 104.5 158 bis) 3d 116 181 2.00 1.43 12250 eZ 1.00 1.00 92.0 168 1.50 0.90 82.1 224 2.00 0.83 76.6 448 4.00 0.66 60.8 896 8.00 0.37 34.1 1,344 12.00 0.26 748) Uf IX. The rate of the catalase reaction can be determined by com- paring the lengths of time required to liberate a definite quantity of oxygen under different conditions (see Osterhout). The re- sults of a number of experiments were plotted and the time neces- sary to produce 25, 50, 75, or 100 cc. of oxygen was measured from the graphs. It is understood, of course, that the reaction velocity is the reciprocal of the time. In Table IV the results are given of a number of tests performed under varied experimental con- ditions. This study extended to the effect upon the reaction rate of constant and varying quantities of catalase (with or with- 358 Catalase Reaction 10 126 15 1.76 2.0 Relative amount of hydrogen peroxide Fia. 6. *—* Relative amount of oxygen set free. °—° Per cent of hydrogen peroxide decomposed. 1 D2 H 8 12 Relative amount of hydrogen peroxide Fig. 7. *—* Relative amount of oxygen set free. °—° Per cent of hydrogen peroxide decomposed. S. Morgulis , 359 out change of concentration), and of constant and variable quan- tities of hydrogen peroxide (with or without changing its concen- tration). TABLE IV. Catalase. Hydrogen peroxide. Time required to evolve oxygen. Relativ' Saeniy: concen poseaay| ORVEPAD)| 25 ce. | 50 ce.” |W tbrea. #|hlMdredt ce. a mol ce. min. aint min. min. 5 120 0.16 90 1.20 3.60 8.5 = 5 1.0 0.20 113 150 4.00 8.0 — 5 126 0.24 136 1.70 4.25 8.6 18.1 5 1.0 0.28 158 1.85 4.75 8.9 15.8 5 1.0 0.32 181 1.80 4.50 8.8 15.8 5 1.0 0.36 203 1.85 4.60 8.7 15.4 ry 1.0 0.40 226 2.00 6.00 ais 21.7 9 0.67 Ontz 100 3.2 9.3 — 9 0.80 0.14 100 2.9 ? Patt (0) 9 1.00 0.18 100 29 8.5 28.0 9 i as 0.24 100 2.9 8.9 27.8 9 2.00 0.36 100 33 9.5 — 6.75 1.0 0.13 112 1.60 4.10 8.6 5.63 1.0 0.16 112 190 So. 20 11.9 4.50 1.0 0.20 112 2h 8.20 19.2 3.75 1.0 0.25 112 3.50 11.40 — 3.00 1.0 0.30 112 5.50 25.00 = 3 0.6 0.20 108 DEO eae DAV? -- 4 0.8 0.26 145 2.0 5.0 ORG 22.4 5 1.0 0.32 180 eS 4.0 8.5 15.0 6 2 0.39 217 10533 3) 5.8 9.5 7 1.4 0.42 254 2, 2.9 5351) 9.0 3 0.6 0.32 181 4.25 10.75 — — 4 0.8 0.32 181 1.90 4.85 10.1 21.6 5 1.0 0.32 181 10) 4.10 8.6 157 6 te? 0.32 181 0.75 2.30 4.5 7.6 Zz 1.4 0.32 181 | 0.45 1.60}; — gs Increasing the quantity of hydrogen peroxide (also its concen- tration), while the catalase remains the same, causes a certain amount of retardation of the reaction particularly during the 360 | Catalase Reaction early stages.. Thus, in the first experiment the hydrogen perox- ide quantity increased from an equivalent of 90 cc. to that of 203 cc. of available oxygen, while the hydrogen peroxide was thus more than doubled, the reaction velocity diminished somewhat, and only 1 minute more was required to liberate 50 ee. of oxygen (4.6 instead of 3.6 minutes). At a later stage in the reaction this difference disappears, and the time required to set free 75 ec. of oxygen is practically the same (about 8.5 minutes). However, when the concentration of the hydrogen peroxide reaches 0.4 gram-molecular the slowing up of the reaction becomes very pronounced and persists through the entire reaction. When the experiment is performed with varying relative con- centrations of catalase (maintaining a constant quantity) while keeping the same quantity of hydrogen peroxide (second experi- ment), we find that the rate of the reaction remains practically unaltered. To decide whether the constancy of the reaction velocity is due to the fact that the quantity of the hydrogen per- oxide or that of the catalase is the same, an experiment was per- formed in which the relative concentration of the catalase was maintained unchanged through the series (third experiment) while the absolute quantities varied from 3.0 to 6.75 ec. of the extract. The quantity of hydrogen peroxide in the meantime was kept constant. Under these circumstances the rate of the reaction (reciprocal of the time) was found to vary directly with the catalase quantity. The concentration of the catalase is evi- dently of no particular consequence so far as the reaction velocity is concerned. Two other experiments were made with varying quantities of catalase while both the concentration and quantity of the hydrogen peroxide either varied or were kept constant. Plotting the velocities against the quantities (also the concen- trations) of the catalase (Figs. 8 and 9), these results corroborate further the findings of the previous experiments demonstrating definitely that the reaction rate depends directly on the quantity of catalase used, while the effect of the hydrogen peroxide is to limit the rate. This conclusion seems, therefore, a corollary to that derived from the study of the rdle played respectively by the catalase and the hydrogen peroxide in the reaction. S. Morgulis 361 XxX. The catalase reaction is generally regarded as belonging to the monomolecular order, only one substance—hydrogen peroxide— undergoing decomposition. Though it is true that the reaction may follow the monomolecular curve, the widely accepted idea that from a dynamic standpoint the catalase reaction is of this 0.40 oy s Relative velocity of reaction G-m. Cone. Hz 02 3 4 5 6 Cec. Quantity of catalase. Fic. 8. Effect of changes in catalase on reaction velocity. ®*—®*25 ce. of oxygen set free. °—° 50 cc. of oxygen set free. XK—X 75 cc. of oxygen set free. particular type requires drastic revision. The evidence presented in this paper goes to show that not only does the hydrogen perox- ide disappear but that the catalase as well is used up in the course of the reaction, and that a definite quantitative relation exists between the interacting catalase and hydrogen peroxide. Furthermore, when the time relations of the evolution of oxygen, 362 Catalase Reaction S B ~ ba 4 R ie) 2. > a= z : : é 5 € 3 S a 0.32 3 4 5 6 T Cc, Quantity of catalase Fic. 9. Effect of changes in catalase on reaction velocity. The quan- tity and concentration of hydrogen peroxide is unchanged. *—®*25 ce. of oxygen set free. °—° 50 ce. of oxygen set free. S. Morgulis 363 resulting from the reaction of catalase with hydrogen peroxide, are studied under different experimental conditions, and the re- action is followed not for a limited space of time but until the reaction stops, it is soon discovered that it does not invariably follow the monomolecular isotherm. As will be shown presently, the reaction under certain conditions is bimolecular and even one and a half molecular, while under other conditions it does not conform to either of these types. In fact, the course of the reaction seems to depend solely on the quantitative relation be- tween the available catalase and the hydrogen peroxide. Where the former is in excess, the hydrogen peroxide will be completely decomposed; on the contrary, when the hydrogen peroxide is in excess, a greater or less proportion of it will undergo decomposi- tion. Barring from the present discussion either extreme, 7.e. cases where there is too great an excess of catalase or of hydrogen peroxide, for which I was unable to find a simple mathematical expression of the reaction, we will confine our attention only to such instances where the decomposition of the hydrogen perox- ide ranges from about 65 to 100 per cent of the quantity used. Within this limited range the reaction does not run true to any one particular dynamic formula. When 95 to 100 per cent of the oxygen available in the hydrogen peroxide is liberated, the reaction is unquestionably monomolecular, and the reaction coefhi- cients can be determined from the well known formula, K = : - log = When the catalase is present in large excess, and this can usually be detected immediately by the extreme vigor and velocity of the reaction, this no longer applies, while there also occur other changes which will be discussed separately in a forthcoming paper. The fact, however, has been definitely established that in the presence of a small excess of catalase the reaction runs true to the monomolecular course. Herein we find the reason why the older observers were led to believe that the reaction is of this particular order. Guided by the erron- eous notion that catalase may be destroyed through oxidation by hydrogen peroxide, they experimented with very dilute perox- ide solutions which were completely decomposed by the catalase. In other words, the experiments were confined chiefly to a par- ticular range of catalase-peroxide relationship, and this fact was 364 Catalase Reaction TABLE V. Catalase 5 ec. Total volume 50 ce. : | Hydrogen peroxide. | Oxygen Reaction H2O2 Bercce Gann evolved. coefficient.* decomposed. available. tration. cc mol min ce. Ky, X* 104 per cent 5 64.4 1092 90 0.16 10 81.6 1046 95 12 85.0 1030 1056 Ke X 10! 5 3.1 95 10 85.6 97 113 0.20 15 95.1 94 86 18 97.0 87 93 K; X 10° 5 58.5 107 10 82.5 113 135 0.24 15 96.5 121 76 20 103.3 116 24 104.5 112 5 56.7 67 10 81.6 66 15 99.5 a 158 0.28 20 110.4 72 73 25 115.2 67 27 115.9 69 5 56.5 50 10 81.6 46 15 me OOES 46 181 0.32 20 110.6 dt 68 25 118.0 42 30 122.0 39 34 122.7 44.7 *The designations Ki, Ke, and Ks; are employed as abbreviations for the coefficients of unimolecular, one and a half molecular, or bimolecular reactions, respectively. a S. Morgulis 365 TABLE V—Concluded. Hydrogen peroxide. Time Oxygen Reaction H202 Onveen Connene 5 evolved. coefficient. decomposed. available. tration. os mol min. Ges per cent 3) 56.0 10 82.9 15 100.5 203 0.36 20 SO 58 25 ea 30 119.0 5 47.7 10 69.4 15 86.6 226 0.40 20 97.8 49 25 105.2 30 109.1 33 109.7 responsible for the unqualified assumption that the catalase re- action is of this particular type. When, however, the catalase-hydrogen peroxide ratio is so adjusted that less than 95 per cent of the available oxygen is set free, the reaction is no longer monomolecular. Either by diminishing the quantity of catalase or by increasing the quantity of hydrogen peroxide any degree of decomposition may be se- cured, as was already expounded in the foregoing. When the decomposition is somewhere between 85 and 95 per cent of the hydrogen peroxide (the limits are not sharply defined) the course of the reaction follows the curve of a one and a half molecular, and the reaction coefficient can be determined from the formula, 1 Va—Vo-x K = 7: 5 / GE - When the decomposition is somewhere be- tween 70 and 80 per cent of the hydrogen peroxide used in the experiment the reaction runs true to the bimolecular course, , and K = aa applies within these limits. Since it has already been shown that when the decomposition of the hydrogen peroxide falls below 65 per cent the actual amount of oxygen set free by the catalase as well as the reaction velocity are considerably 366 Catalase Reaction diminished, ‘it is obvious that we are dealing not simply with a bimolecular reaction but also one that is apparently reversible.* - Before discussing this matter further a few experiments will be recorded in which either a constant quantity of catalase was used with varying amounts of hydrogen peroxide or a constant quantity of hydrogen peroxide while the catalase was varied. ? From theoretical considerations Yamazaki also comes to the con- clusion that depending upon the relative amounts of hydrogen peroxide and catalase the type of the reaction may shift from the bimolecular to the monomolecular. In his calculations he nevertheless employs the equation for a monomolecular reaction. This he does even where, as will be shown, the equation does not apply. Recalculating his data given in Table 76 (IV and V) where with 10 and 5 ce. of the catalase preparation, respectively, he obtained 90 and 75 per cent of decomposition of the hydrogen peroxide, it ean be shown that the reaction follows the equation of either a one and a half or of a bimolecular reaction as was also found to be the case with my preparation of liver catalase. I may add that since the paper had been sent to press I had occasion to experiment with catalase preparations from different sources and was able to substantiate the results in every instance. In the tabulation below the values of the constant (K) as found by Yamazaki with the aid of the monomolecular formula and those which I calculated in accordance with the bi-, or one and a half molecular equation are set down side by side. The point is brought out so clearly that no further comment is required. K Time. A xe (mono- | (himolec- ime molec- ( part 5 ec. catalase, 75 per cent de- 0 12.52 composition. 5.08} 12552 2.20 | 0.0159 | 0.00335 9.58 | 12.52 3.58 | 0.0145 | 0.00334 15.16 | 12.52 4.80 | 0.0114 | 0.00328 20.83 | 12.52 5.87 | 0.0114 | 0.00339 26.08 | 12.52 6.54 | 0.0088 | 0.00335 (One and a half molec- | ular) 10 ce. catalase, 90 per cent de- 0 13.33 composition. 4.03 | 13.33 | 3.24 | 0.0357 | 0.0102 13.11 | 13.33 8.72 | 0.0276 | 0.0113 18.46 | 13.33 | 9.18 | 0.0245 | 0.0119 23.86 | 13.33 | 10.11 | 0.0204 | 0.0118 S. Morgulis 367 The results under both kinds of conditions are the same. The experiments were all made under a uniform temperature (20-21°C.) and similar hydrogen ion concentration of the medium (pH = 6.7 to 6.9). TABLE VI. Catalase 4.5 cc. Hydrogen peroxide. qunie Oxygen Reaction H202 Gascon Ganson ; evolved. coefficient. decomposed. available. tration. ce. mol min. cc. Ke Os per cent 5 36.5 472 10 54.3 425 15 66.0 412 87 0.2 20 74.2 416 100 30 82.3 423 40 86.2 509 45 87.1 ee 443 K3 X 10° 5 36.7 88 10 53.5 82 15 67.0 89 112 0.2 20 75.8 93 82 30 85.8 97 40 91.2 98 45 92.0 eae 91 5 36.4 10 53.4 168 0.2 20 74.5 ee 30 80.7 35 82.1 10 49.6 20 67.6 ‘ 224 0.2 30 74.9 34 35 76.6 A review of these data shows that the results may be calculated in accordance with different dynamic formulas depending upon the degree of decomposition of the hydrogen peroxide effected in the reaction. The results of the four series of experiments are pre- sented diagrammatically in Fig. 10. 368 Catalase Reaction TABLE VII. - Hydrogen peroxide equivalent to 112 ce. Or. Catalase. Time. Oxygen evolved. Bee: de Prints edt ce min cc Ky, x 104 per cent 5 56.6 620 10 80.1 545 15 94.1 ; 531 6.75 20 102.1 559 100 25 106.3 ISsi lire 30 108.8 515 40 111.8 ee 548 Ke X 104 5 49.0 63 10 69.3 59 15 82.1 59 5.63 20 91.5 63 93 25 97.5 67 30 101.0 69 35 103.7 ae 61.7 K3 X 10° 5 37.4 90 10 55.1 88 15 67.4 90 3 20 76.1 95 4.5 25 80.7 92 79 30 83.7 88 35 85.8 84 45 88.0 mee j 90 » 10 47.1 SED 20 63.5 63 30 69.2 40 70.0 10 33.8 20 46.4 3.0 30 SRA 51 40 56.4 S. Morgulis 369 We can recognize (Fig. 10) three distinct zones in the dia- gram corresponding to 68 to 82 per cent decomposition, 88 to TABLE VIII. Hydrogen peroxide equivalent to 181 ec. of Oy. Catalase. Time. Oxygen evolved. (Ts min. ce. 5 88.7 10 131.0 15 15592 i 20 169.1 25 176.7 30 180.7 5 78.2 10 122 15 135.0 6 20 147.0 25 153.4 30 157.0 34 158.3 5 56.5 10 81.6 15 99.5 5 20 110.6 25 118.0 30 122.0 33 122.7 10 74.8 4 20 98.0 30 105.5 33 106.3 10 47.6 3 20 66.6 30 71.2 93, and 95 to 100 per cent. zone, and for this zone alone. Reaction coefficient. H202 decomposed. K, X 104 573 560 567 596 662 592 Ke X 104 49 46 49 49 47 44 47.3 K3 X 10° 50 46 46 44 42 39 44.7 per cent 100 88 68 59 39 A different formula applies for each We may consider, therefore, that the catalase reaction is bimolecular changing to monomo- 370 Catalase Reaction lecular, the’ middle zone merely representing the transition from the one to the other. Mellor recites a number of instances where the reaction shows a similar change. He says‘ that “the gradual approach of the velocity curves for the bimolecular reaction to the curves for a unimolecular reaction as the amount of one of the reacting components of the bimolecular reaction is increased, shows very clearly how the course of a bimolecular reaction might appear unimolecular when one of the reacting components is in excess.’”’ Among reactions of this kind Mellor mentions the 100% 90 Fie. 10. Diagram showing the order of reaction depending on the degree of decomposition of hydrogen peroxide. The shaded area represents the undecomposed fraction of hydrogen peroxide. reduction of potassium permanganate with an excess of oxalic acid; the action of an excess of hydrogen peroxide on hydriodic acid; etc. The catalase reaction seems to belong to the same category except that in this instance it is not the excess of hydrogen peroxide but of the catalase which shifts the reaction from the order of a bimolecular to that of a unimolecular. Evans in a very thorough study of the catalase reaction (which he regards simply as a catalytic decomposition of hydrogen perox- 4 Mellor, p. 42. S. Morgulis Sy) ide) observed that the reaction coefficients, calculated according to the formula an K (a—x), showed a peculiar behavior. In some instances the values of kK would form an ascending, some- times a descending series, and only occasionally did he obtain fairly constant values. He distinguished, therefore, three peri- ods: the rectilinear, the infralogarithmic, and the logarithmic, a terminology which does not seem happily chosen. The latter is the one when the reaction really follows the curve of a monomo- lecular reaction. Examining his data, I find that this condition was rarely met, and only when the substrate (H.O.) was used in very dilute solution while the amount of enzyme was “not very small.”’ In other words, when there was enough catalase to completely decompose the hydrogen peroxide. The three phases which Evans noted are all related to the catalase-peroxide ratio. I found that the same three phases occur no matter whether the reaction follows the monomolecular, bimolecular, or an inter- mediate course. Whenever the catalase is present in too great an excess the values of K (monomolecular) will continually in- crease. On the other hand, when there is less catalase than is necessary to decompose the entire amount of peroxide the values of K gradually diminish. This is likewise true when the values of K are calculated for a one and a half or for a bimolecular re- action. The change of phase is brought about very readily by the smallest alteration in quantity of either the catalase or the hydrogen peroxide. With a little experience it is possible to adjust the quantities so as to make the reaction proceed ac- cording to any of the three formulas employed in this paper for calculating the reaction coefficient. It is remarkable how little it is Necessary to change the quantities to produce appreciable differences in the values of K. Sometimes a change by a few drops of the catalase preparation was quite sufficient to bring this result about. When one bears in mind that with a decom- position of the hydrogen peroxide of about 75 to 80 per cent the reaction follows almost ideally the bimolecular curve, it is a simple matter indeed to adjust conditions so that the results of an entire experimental series can be made really comparable. In the study of the effect of the concentration of hydrogen ions upon the reaction it was already pointed out that the respective 372 Catalase Reaction quantities of both the catalase and the hydrogen peroxide were so chosen that the reaction was typically bimolecular. It is by virtue of this adjustment that it was possible to demonstrate in that series that increasing the hydrogen ion concentration not only limits the decomposition of hydrogen peroxide and slows up the reaction, but that the type of the reaction also changed and it no longer followed the course of a bimolecular curve when the pH fell below 6.0. 100 ~J on 3 of oxygen set free >; | C. Diese 5-20) 25°" 30 49 Min. Fre. 11. Table VII, Experiment 1. *—e Experimental results. °—°Cal- culated results. In Figs. 11, 12, and 13, the experimental curves are plotted together with the theoretical curve calculated with the aid of the average value of K for each respective experiment. The similar- ity of the two curves requires no further comment. Before closing the paper it may be well to point out the bearing of the results upon the question of the technique of catalase de- terminations. The use of very large quantities of hydrogen peroxide (equivalent to 500 or 600 cc. oxygen), as is practised S. Morgulis 373 commonly in researches on catalase, is objectionable because the depressing effect is great unless very large amounts of the catalase preparation are employed. There is no obvious. advantage in working with such tremendous quantities. On the other hand, when an attempt is made to compare the catalase activity of preparations of presumably different strengths, the depressing effect will be much greater in the case of the weaker sample and under such conditions the tendency will be to exaggerate the 15 s of oxygen set free Ce Or Ss “Oe 20°25, 35Min. Fig. 12. Table VII, Experiment 2. *—* Experimental results. °—° Cal- culated results. differences. In fact the entire method of comparing several samples of catalase on the basis of the amount of oxygen which they respectively liberate from hydrogen peroxide is of question- able accuracy. The comparison should instead be made between respective quantities of catalase preparation required to set free the same amount of oxygen from a given quantity of hydrogen peroxide. It is further advisable to adjust the reaction to follow some definite course (a 75 per cent decomposition of the hydrogen peroxide is a very good basis). Although the oxygen formation 374 Catalase Reaction is a linear function of the quantity of catalase, this rule does not hold true when either the catalase or the hydrogen peroxide are in great excess. When, however, the catalase is varied to produce a certain degree of decomposition with the same quantity of perox- ide, the catalase strengths will be inversely proportional to the quantities used for the tests. The method of estimating the catalase strength followed in most investigations on catalase is so crude and untenable from a chemical standpoint that one naturally is reluctant to accept the conelusions drawn from 100 a of oxygen set free Gc yf a 1 aT eT 35 45 Min. Fria. 13. Table VII, Experiment 3, *—* Experimental results. °—°? Cal- culated results. those researches, especially where the conclusions are of far reaching significance. Another important matter is the fact that in the catalase ex- periments the reaction is allowed to go on for a set length of time, usually 10 or 15 minutes. The reasons for determining relative reaction speeds from the time periods required to effect a given result, rather than from the results effected in a given time period have been fully discussed by Osterhout, and need not be repeated. It should be noted, however, that when the ons S. Morgulis 375 reaction is permitted to run for 10 minutes, only 50 or perhaps 60 per cent. of the reaction is completed. For comparative pur- poses, it may be argued, this will not matter, and this might be true if the reaction were always brought to the same end-point. This is not likely to be the case. If, on the other hand, the re- action is so vigorous and rapid as to be completed within the 10 or 15 minutes it is practically certain that there is a great excess of catalase. In this event, of course, the oxygen evolution will again fail to give a correct measure of the relative catalase strengths inasmuch as there is no means of determining whether or not the relative excess is the same in each instance. Little credence can therefore be given to results of catalase experiments unless very large differences are demonstrated. BIBLIOGRAPHY. Euler, H., Beitr. chem. Physiol. u. Path., 1906, vii, 1. Evans, C. A. L., Biochem. J., 1907, 11, 133. Hampton, H. C., and Baas-Becking, L. G. M., J. Gen. Physiol., 1920, 1, 635. Iscovesco, H., Compt. rend. Soc. biol., 1905, lvii, 1055. Mellor, J. W., Chemical statics and dynamics, London, 1904. Michaelis, L., and Pechstein, H., Biochem. Z., 1913, liii, 320. Osterhout, W. J. V., Science, 1918, xlviii, 172. Senter, G., Z. physik. Chem., 1903, xliv, 257. Sorensen, S. P. L., Ergebn. Physiol., 1912, xii, 393. - Yamazaki, E., Tohoku Imperial Univ., Scient. Rep., 1920, liii, No. 13. oe eel iting aie 1 ied ‘s sie grein a fa 8 hi i: ES ee " ye Le een iS -aiat Be ee ae ob lanes cnn dae, Oe Ric tice . ay ta oie PS vrtrety edi ti i ep LS He Ef eie “Wit ee es) ea THE RELATION OF THE MIGRATION OF IONS BETWEEN CELLS AND PLASMA TO THE TRANSPORT OF CARBON DIOXIDE.* By EDWARD A. DOISY anp EMILY P. EATON. (From the Laboratory of Biological Chemistry, Washington University Medical School, St. Louis.) (Received for publication, May 28, 1921.) Due to the interest attached to the relationship of the chloride- bicarbonate equilibrium (Henderson, 1921) to the transport of carbon dioxide by the blood, we are publishing some of our data on this subject. Van Slyke and Cullen (1917) showed that the plasma of whole blood which had been shaken with varying proportions of carbon dioxide had lost an amount of chloride which was sufficient to account for about two-thirds of its increase in bicarbonate. More recently, Fridericia (1920) has published data which show that the loss of chloride more nearly accounts for the gain in bicarbonate. Henderson and coworkers (1920) found that only 60 per cent of the increase of bicarbonate was due to the migration of chlorine. A study of this subject and permeability of cells in general were uppermost in mind when one of us started the adaptation of methods of inorganic analysis 3 years ago.! At that time the existing methods of determining sodium and potassium were laborious and required excessive amounts of material. We are now able to make a study of the permeability of cells to the more important anions and cations on small samples of blood. * The data in this paper are taken from a thesis presented by Miss Eaton in partial fulfillment of the requirements for the degree of Master of Science, Washington University, 1921. 1 A study of methods of analysis of inorganic substances of biochemical importance was begun in the fall of 1918 by R. D. Bell and the senior author of this paper. Procedures for the determination of sodium and phosphorus have been published. Complete details of the systematic inorganic analyses of blood will soon be submitted to the Journal for publication. 377 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL, XLVII, NO. 2 378 Ion Migration between Cells and Plasma Practically all the investigators in this field have admitted the permeability of red cells to hydrochloric acid when the blood is treated with carbon dioxide. According to Hamburger (1916) and coworkers this effect is extended to include sulfates, phos- phates, sodium, potassium, and calcium. A number of investi- gators (Giirber, 1895) have failed to find an exchange of cations between cells and plasma. Lately, Van Slyke (1921) and Hen- derson and coworkers (1920) have stated that the migration of cations probably plays no part in the transport of carbon dioxide. While we are not in a position to state emphatically that there is no transfer, our analytical data show clearly that quantitatively the shift, if it occurs at all, zs very small. Methods. For our investigation of this problem we have devised a scheme of inorganic analysis which permits the determination of sodium, potassium, chlorine, and phosphorus on 10 ec. of serum. An additional quantity of 3 cc. is necessary for bicarbonate deter- minations. Briefly the method is: Transfer 10 cc. of serum to a 100 ce. flask containing about 70 cc. of distilled water. Add while shaking 10 ec. of 20 per cent trichloroacetic acid.27 Make to mark and mix well. After 30 minutes filter through dry paper. Since the filtration is rather slow, it is essential that both the funnel and flask be covered to avoid evaporation. Usually we obtain about 83 cc. of filtrate of which the following amounts are used for the determinations named below. As may be seen from Table I we have utilized only 50 ce. of our filtrate; the remainder may be used for a repetition of doubtful analyses. We expect to extend this scheme to the determination of calcium and magnesium. 2 We had already worked out independently the use of trichloroacetic acid for chloride analysis when Smith’s (1921) paper appeared. Our results on these filtrates agree very closely with values obtained on picric acid filtrates from blood plasma. Invariably, however, the latter pro- cedure gives results, which are 2 or 3 per cent higher on whole blood. We have not found time to study this intensively and are still a little un- decided which method is the more nearly. correct. E. A. Doisy and E. P. Eaton 379 Treatment of the Blood—lIn order to avoid as far as possible the effect of change in strength (as an acid) of the hemoglobin in passing from the oxidized to reduced form, we have generally maintained in our saturators a sufficient amount of oxygen to keep essentially all of the hemoglobin as oxyhemoglobin. When the higher tensions of carbon dioxide were used, oxygen was added from a tank to maintain its concentration between 20 and 21 per cent. Barcroft (1914) has shown that about 13 per cent of oxygen is sufficient to keep over 90 per cent of the hemoglobin in the oxidized form in the presence of 11 per cent of carbon dioxide. We think that 20 per cent oxygen was sufficient to prevent the reduction of more than 10 per cent of the oxyhemo- globin in all of our experiments in which the gas mixture contained not more than 20 per cent carbon dioxide. TABLE I. Volume Analysis. of filtrate Method. used. cc. SOGIUNME . er oo5s 10 Sodium cesium bismuth nitrite, Doisy-Bell (1921). Potassium...... 20 Cobalti-nitrite. Chlorme.. 2... 10 McLean-Van Slyke solutions (1915). Phosphorus..... 10 Colorimetric, Bell-Doisy (1920). FROIN eee 50 Beef blood was defibrinated and filtered through several layers of gauze, and 60 cc. samples were measured out into a series of 23 liter bottles. At least 6 liters of the gas mixtures were passed through each bottle and the openings closed with pinch-cocks. The inlet tube extended nearly to the bottom of the bottle, the outlet being flush with the stopper. As our chief aim was to study the shift of inorganic ions from serum to corpuscles under higher tensions of COs, we did not analyze the gas mixtures in the saturators in any except the last experiment. These analyses revealed the fact that 6 liters were insufficient to bring the gas to even the approximate per cent of CO, given in our tables. Our 20 per cent CO, mixture probably sufficed to make the gas in the saturator not over 15 per cent CO:. The bottles were 380 Ion Migration between Cells and Plasma rotated to secure a maximum surface for the rapid attainment of equilibrium. After agitation for about 20 minutes the blood was drawn off under oil into centrifuge tubes. For the determination of corpuscle volume, we used graduated tubes of 6 mm. bore. All samples of the blood were centrifuged simultaneously for 30 minutes at 3,500 R.p.M. immediately after equilibration. Our centrifuge was occasionally tested to show that this speed was actually attained. The volume of corpuscles was read and the tubes were again centrifuged for 15 minutes. If comparison showed that any appreciable decrease had occurred the tubes were centrifuged again. The serum was immediately removed for analysis. The various analyses were started and the bicarbonate deter- minations finished on the same day that the blood was taken. One of us (E. P. E.) determined the chlorides; the other (E. A. D.), the bicarbonates. In general, the bicarbonate determinations were made accord- ing to the technique described by Van Slyke and coworkers (1919), using a standard pH 7.2 and _ phenolsulfonephthalein. It seemed preferable to titrate to a standard pH than to use the various gas mixtures required for this purpose if the Van Slyke- Cullen (1917) method were used. This point is mainly theoretical in nature, but will assume some importance in the larger changes of bicarbonate concentration. We are giving one of our experiments in its entirety at this point to aid in the discussion of our results. Other similar data are given in the protocols to strengthen our conclusions. Corpuscle Volume——By reference to Table II it can be seen that the vélume of corpuscles increased with increasing tensions of COs. This effect which was emphasized by Hamburger (1916) has not been confirmed by Joffe and Poulton (1920). The reason for their exceedingly variable results is not clear to us. In none of our experiments have we failed to find an increase in corpuscle volume with increasing tensions of CO:. This is probably due to the fact that the osmotic pressure of the cell contents is increas- ing more rapidly than that of the serum. The serum does not show a net gain of ions; for each molecule of NaHCO; gained a molecule of HCl is lost. The corpuscles, however, gain this molecule of HCl. " E. A. Doisy and E. P. Eaton (Gaheszo | TABLE II. Experiment 6.—Defibrinated beef blood equilibrated with various air- carbon dioxide mixtures at 25°C. Centrifuged for 45 minutes. No hemolysis. Bicarbonate by titration. Figures in column headed, COs, signify the amount of carbon dioxide in the 6 liters of gas passed through the saturators. NaCl 7 tr NaHCO = = petiole Corpuscless|>—cn5c0 = ae Na oA 100) K ae 100 | P Sa 100 blood. Per 100 ce. Goneen: tion. : ; be i per cent mg. mol mol mg. mg. mg. 3 42.9 623 0.1068 0.0293 322 20.9 7.9 10 43.1 610 0.1043 0.0317 319 PANE) 7.8 20 43.5 602 0.1030 0.0345 326 PAV GS 7.8 100 45.5 564 0.0965 0.0431 345 22.9 7.8 Correction for change in volume of corpuscles. 571 10 per cent ~~ X 0.0293 = 0.0294 m NaHCO; expected. 569 X 0.1068 = 0.1071 m NaCl * 20 per cent ut X 0.0293 = 0.0296 m NaHCO; ss X 0.1068 = 0.1079 m NaCl e 100 per cent a X 0.0293 = 0.0307 m NaHCO; ee X 0.1068 = 0.1119 m NaCl a Effect of increasing CO: content of air from: 3 to 10 per cent 3 to 20 per cent 3 to 100 per cent BCl BHCOs3 BCl BHCOs3 BCl BHCOs mol mol mol mol mol mol Before increase. 0.1071 0.0317} 0.1079) 0.0345) 0.1119) 0.0431 After eg 0.1043} 0.0294) 0.1030) 0.0296) 0.0965) 0.0307 —0.0028} +0.0023) —0.0049) +0.0049) —0.0154)+0.0124 This molecule of hydrochloric acid probably reacts with the alkali oxyhemoglobinate in much the same way that the carbonic acid does. HCl = Cibo" BHbO = Bt + HbO- If Bt + Cl- + HHbO 382 lon Migration between Cells and Plasma Instead of two we now have three units which exert osmotic pressure. This means a real gain and quite naturally water passes from the serum into the cells to equalize the osmotic pressure on both sides of the membrane. When the hydrogen ion concentration passes the isoelectric point of oxyhemoglobin we have a very similar phenomenon. There is no net gain of ions in the serum but the increase continues in the cell due to the hydrochloric acid gained. This acid probably reacts with a molecule of hemoglobin which is now functioning as a base. Whereas, previously we had only an undissociated hemoglobin molecule, we now have two ions. HbO + HCl = HbO.HC1 = HbO.Ht + Cl— These schematic equations are intended to express only the increase in osmotic pressure of the cells. We do not intend that they shall convey our idea of the linkages between acids or alkalies and proteins. Other factors such as alterations of the degree of association of hemoglobin molecules may influence the changes of osmotic pressure. It seems evident to us that the cause of swelling of the corpuscles is the increase of osmotic pressure within the cell. We should then expect water to pass in until both the serum and cell contents have the same osmotic pressure. The volume of serum per 100 ce. of blood is now less than formerly. All its constituents, provided no migration has occurred, should be present in greater concentration. According to this view, it is essential that cor- rections be introduced in our calculations for the change in volume of the corpuscles. Reference to Table II shows that at 20 per cent COs, the cor- puscle volume has increased 0.6 cc. per 100 ec. of whole blood. This amounts to slightly more than 1 per cent increase in the concentration of the serum salts. The serum volume at 3 per cent CO» is 57.1 c¢.; at 20 per cent CO» it is 56.5 ec. Consequently the concentration of sodium, potassium, bicarbonate, chlorine, and phosphorus at 3 per cent CO. must be multiplied by 571 565 to give the expected concentration at 20 per cent. E. A. Doisy and E. P. Eaton 383 = X 0.0293 = 0.0296 m NaHCO; expected value. oul X 0.1068 = 0.1079 m NaCl “ es 565 . CO . a a Gain in base by loss of HCl, 0.1079 — 0.1030 = 0.0049 m Be as Na es, 0.0345 — 0.0296 = 0.0049 m We have purposely taken an experiment where the agreement is better than usual. If we have failed to correct for the volume change of corpuscles the loss in chloride would account for only 73 per cent of the gain in bicarbonate. Gain in base by loss of HCl, 0.1068 — 0.1030 = 0.0038 mM SS SvassNakiC@Os: 0.0345 — 0.0293 = 0.0052 m The foregoing calculations demonstrate the error caused by failure to correct for the loss of water from the serum. The discrepancy, of course, will be much larger in experiments where the corpuscle volume change is larger; 7.e., at 100 per cent COs. It is evident that mathematically the accurate determination of corpuscle volume is of great importance if one wishes to study the true relationship between the direct and indirect methods of determination of increased base in plasma. For instance, if the volume of corpuscles found had been 44.1 per cent instead of the 43.5 per cent then our calculations would have been: = X 0.0293 = 0.0299 m NaHCO; expected value. rfl = ce ce Faq X 0.1068 = 0.1091 m NaCl Gain in base by loss of HCl, 0.1091 — 0.1030 = 0.0061 m sry SOOT Stas Naklic@Os- 0.0345 — 0.0299 = 0.0046 mM From a very close agreement between the values, an error of about 1 per cent in determination of plasma volume produces results which are rather widely divergent. The foregoing series of calculations illustrates why the pre- viously published figures have shown that the shift of hydro- chloric acid freed an amount of base equivalent to only about two-thirds or three-fourths of the increase of bicarbonate. When we started our work we fully expected to find the remainder accounted for by a passage outward of sodium from the corpuscles. 384 Ion Migration between Cells and Plasma However, we now feel that such an explanation is entirely superfluous. These results make it appear more than probable that in as far as the plasma functions as a carrier of carbon dioxide (Joffe and Poulton, 1920; Smith, Means, and Woodwell, 1921; and Fridericia, 1920) the transport is based entirely (providing the [H+] of the plasma remains constant) upon the passage of hydro- chlorie acid back and forth across the cell membrane. That this mechanism is possible is closely related to the preeminent buffer value of hemoglobin due to its change of dissociation constant in passing from oxyhemoglobin to reduced hemoglobin. Permeability of Blood Corpuscles. We have found it quite easy to repeat the demonstration of the permeability of cells to the chloride ion. Naturally one would expect this effect to be extended to include other anions. How- ever, when one considers the concentration of the other inorganic anions in serum (P=0.002 mM; S=0.002 Mm) it is evident how little importance they would probably have in the transport of carbon dioxide. Though we have not attempted to decide upon the permeability to the sulfate ion, we have made a few colorimetric determina- tions of inorganic phosphate. Although nearly all of our results point to a migration of phosphate they are not decisive in that most of the differences are within the possible experimental error. Our one gravimetric determination indicates that no shift of inorganic phosphate occurs. We are very loth to draw any conclusion from this but will extend our work in this direction. With respect to a shift of cations, Hamburger (1916) concluded from the results of a few experiments that carbon dioxide causes a passage of potassium into the cells and of sodium outward into the plasma. Previous workers (Giirber, 1895) had failed to detect any shift of cations but Hamburger attributed this to a lack of suitable methods of analysis. We fully expected to detect a transfer of both sodium and potassium. Quantitatively, Hamburger found that shaking horse blood with 20 volumes per cent of CO, (20 ec. of pure CO» to 80 ce. of blood) caused about 17 per cent of the potassium to E. A. Doisy and E. P. Eaton 385 pass into the corpuscles and a 6 per cent increase of the sodium of the serum. While our experiments were not conducted exactly as Hamburger’s were, we have a few data on blood treated in a comparable manner. We have shaken beef blood with 40 or 50 volumes of gas, containing proportions of CO, varying from 5 to 100 per cent. In practically no case has any shift of either potassium or sodium been found. We consider that our determinations of sodium have more significance than those of potassium. In case our duplicate results varied by more than 2 per cent other analyses were made. In general the values reported are the mean of two results which varied by less than 2 per cent. Such being the case a variation of 2 per cent of the value found from that expected may be con- sidered a real shift. In some of our later experiments a deviation of 1 per cent may be taken as an exchange of sodium. By ref-. erence to the protocols it can be seen that only very infrequently does a shift of sodium seem to occur. In view of its rare occurrence and the difficulty of obtaining filter paper, etc., free from sodium we are inclined to ascribe this to experimental error. Our determinations of potassium are less reliable than those of sodium. However, it is possible that any variation greater than from 2 to 3 per cent from the expected value is a real transfer of potassium. This rarely occurs. We feel that our results lead to the conclusion that blood cells are permeable to the chloride ion but impermeable to both sodium and potassium. Collip (1921) has eves published data which also point in this direction. DISCUSSION. According to Van Slyke’s (1921) recent review, hemoglobin seems to be by far the most important factor in the transport of carbon dioxide from the tissues to the lungs. As it is non-diffu- sible an auxiliary reaction is utilized to assist the plasma to take up or give off carbon dioxide. This mechanism is a shifting back and forth across the cell membrane of hydrochloric acid. As we picture the process it is dependent upon a very slightly varying hydrogen ion concentration of the plasma. In the capillaries where the tension of carbon dioxide is high the hydrogen ion concentration of the plasma tends to increase. 386 Ion Migration between Cells and Plasma This produces the shift of HCl to the cells. At the same time there is the reduction of the oxyhemoglobin which alone would TABLE III. Chloride Bicarbonate Equilibrium. Increase of base Plasma chloride. Plasma bicarbonate. determined by. Experiment pgs SSS —_ SS — SSIES 2 pleas Concen- Concen- | Concen- | Concen- Goneeay Congens tration tration tration tration ees ee gain in i found. expected. found. | expected. chloride.'| NaHiGGs. Ba eee (ee I ee ae ee 1 5 0.0332 0.0072 10 0.0327 | 0.0336 | 0.0081 | 0.0073 | 0.0009 | 0.0008 20 0.0316 | 0.0344 | 0.0098 | 0.0075 | 0.0028 | 0.0023 2 5 0.0287 0.0112 20 0.0280 | 0.0292 | 0.0126 | 0.0114 | 0.0012 | 0.0012 3 3 0.0366 0.0097 15 0.0340 | 0.0370 | 0.0131 | 0.0098 | 0.0030 | 0.0033 4 3 Ost 0.0274 10 0.1081 0.1136 | 0.0311 | 0.0280 | 0.0055 | 0.0031 20 0.1063 0.1141 | 0.0352 | 0.0281 | 0.0078 | 0.0071 5 3 0.1090 0.0294 10 0.1071 0.1098 | 0.0318 | 0.0296 | 0.0027 | 0.0022 20 0.1043 0.1107 | 0.0366 | 0.0298 | 0.0064 | 0.0068 100* 0.0977 0.1139 | 0.0444 | 0.0307 | 0.0162 | 0.0137 6 3 0.1068 ; 0.0293 10 0.1043 | 0.1071 | 0.0317 | 0.0294 | 0.0028 | 0.0023 20 0.1030 0.1079 | 0.0345 | 0.0296 | 0.0049 | 0.0049 100* 0.0965 0.1119 | 0.0431 | 0.0307 | 0.0154 | 0.0124 if 3 0.1076 0.0270 14 0.1020 | 0.1098 | 0.0351 | 0.0276 | 0.0078 | 0.0075 21 0.0998 | 0.1111 | 0.0380 | 0.0279 | 0.0113 | 0.0101 37* 0.0976 | 0.1131 | 0.0408 | 0.0284 | 0.0155 | 0.0124 80* 0.0960 | 0.1155 | 0.0431 | 0.0285 | 0.0175 | 0.0146 * Oxygen was considerably less than 20 per cent. produce a more alkaline reaction within the cells. The two effects are normally so well balanced that there is practically no alteration in the [H+] of the blood. E. A. Doisy and E. P. Eaton 387 In the lungs the reverse process occurs; namely, a loss of carbon dioxide from the plasma, oxygenation of the hemoglobin, and a shift of hydrochloric acid back to the plasma. Our experiments were undertaken with the hope of being able to clear up the transfer of anions and cations back and forth across the cell membrane. In Table III we have grouped together our experimental data on the loss of chloride and gain of bicarbonate in the serum. Over what might be called a very extreme physio- logical range (3 to 15 per cent CO.) we find that the loss of serum chloride adequately accounts for the gain in bicarbonate in experiments conducted at room temperature (25°C.) When much higher percentages of carbon dioxide (50 to 100 per cent) were used this equivalence was not found. The loss of chloride invariably exceeded the gain in bicarbonate. We have no expla- nation to offer for this circumstance. The important feature to us is the equivalence over physiological ranges of carbon dioxide tensions. In the one experiment in which the gas mixtures were analyzed our data agree with those of Hasselbalech and Warburg (1918) rather than with those of Haggard and Henderson (1920). We find that the shift of chloride with the accompanying increase of bicarbonate continues beyond 280 mm. of CO,. Variable factors of temperature and species should be mentioned. These changes do occur to a greater extent for a given increase of CO, at the lower than at the higher tensions. In Table IV we have placed our results on the shift of phos- phate, sodium, and potassium. With the possible exception of phosphate, our data indicate an impermeability of the cell mem- brane to these ions under our experimental conditions. We do not mean to give the impression that the corpuscles are impermeable under all circumstances. Such a condition seems to be rather improbable. However, we do think that migration of sodium and potassium plays no part in the transport of carbon dioxide. The permeability of cells to both phosphates and sulfates has not been settled. Our data with respect to the former are not conclusive. In view of some recent comments on the ash of plasma (Mel- lanby and Thomas, 1920) and the possibility of sodium proteinates we are grouping some of our data in Table V to show the excess of cations over anions. 388 Ion Migration between Cells and Plasma TABLE IV. \ Effect of Various Carbon Dioxide Tensions on the Migration of Sodium, Potassium, and Phosphorus. Experiment Dieter Na per 100 ce. K per 100 ce. P per 100 ce. oe blood. Found. | Expected. Found. Expected.| Found. | Expected. Wik ae | a ma a a) 2 5 249 78.9 20 253 253 80.5 80.3 3 3 150 15 151 152 4 3 336 20.7 7.4 10 336 340 7.4 7.3 20 350 345 21.6 PP ike 7.6 5 33 oon Pavel 8.3 10 331 329 PAL SI 21.2 Seo 8.3 20 3B 332 PAL A 21.4 8.3 8.4 100* 345 341 ee) 22.0 8.3 8.7 6 3 O22 20.9 7.9 10 319 323 21.3 21.0 7.8 7.9 20 326 326 Pb | PHL AIL 7.8 8.0 100* 345 337 22.9 | 21.9 fs 8.3 % 7 3 339 24.4 totief 14 347 346 { 21 355 350 8.6 9.0 Sie 365 356 PASAT Doni 8.6 9.1 s0* 355 358 g6e8" 5 Jere | . * Oxygen less than 20 per cent. i TABLE V. Molecular Concentration of Anions and Cations in Serum of Beef Blood in Equilibrium with 3 Per Cent Carbon Dioxide. Sum of Excess the of anions. cations. Experi- Sum of “= . Bicarbo- | Phos- pes os z atic: a iu pha 4 0.1461) 0.0053) 0.1514) 0.1112 | 0.0275 | 0.0024 | 0.1411 | 0.0103 5 0.1422) 0.0054) 0.1476) 0.1090 | 0.0294 | 0.0027 | 0.1411 | 0.0065 6 0.1401) 0.0053) 0.1454) 0.1068 | 0.0293 | 0.0025 | 0.1386 | 0.0068 7 0.1474) 0.0062) 0.1536) 0.1076 | 0.0270 | 0.0028 | 0.1374 | 0.0162 . E. A. Doisy and E. P. Eaton 389 Although we did not determine the sulfate, calcium, or magne- sium of plasma, we venture to tabulate our figures for the other known inorganic cations and anions. De Boer (1917), considers the normal SO; value to be about 20 mg. per 100 ce. which amounts to a molar concentration of 0.0021. Mean values for: caletum = 0.0027 m; magnesium=0.0010 m. Normally, then, we have a considerable excess of cations which presumably in accord with L. J. Henderson (1908) and others are combined with the proteins. We have unaccounted for, except in this way, about 7 per cent of the bases of serum. It is evident from the table that the chloride concentration is equal to about 75 per cent of that of sodium. We have found in our analysis of human blood that a marked deviation of chloride from the normal is accompanied by a corresponding change of sodium. CONCLUSIONS. In vitro experiments on beef blood equilibrated with various tensions of CO, show the following points: (1) Equivalence of loss of chloride to gain in bicarbonate of serum. Though a migration of phosphate may occur, it is quantitatively of little importance in the transport of carbon dioxide; (2) non-trans- ference of either sodium or potassium from cells to serum; and (3) a marked increase of corpuscle volume with increasing tensions of carbon dioxide. PROTOCOLS. Under the column headed, COs, we mean only the amount of carbon dioxide that was present in the 6 liters of gas passed through the saturators. The actual per cent of COs in the satu- rator did not nearly equal that of the entering gas. Unless otherwise stated this mixture contained between 20 and 21 per cent of oxygen. In Experiment 7 the amount of CO: in the satu- rators was determined. In order to diminish discrepancies due to analytical errors, artificial serums were employed in Experiments 1, 2, and 3. We had hoped to make the molar concentration of bicarbonate equal that of the chloride. This was unsuccessful. These data are included because of the close approximation of decrease of chloride to increase of bicarbonate. 390 Ion Migration between Cells and Plasma The data in the protocols are used for the preparation of the tables. In each case the necessary corrections are applied as in the example given in Table IT. Experiment 1.—Defibrinated beef blood centrifuged and serum removed. An artificial isotonic serum, containing protein, glucose, potassium chlor- ide, and bicarbonate was added. Equilibrated at 25°C. Considerable hemolysis in artificial serum. Serum bicarbonate by the Van Slyke-Cullen method. NaCl = of miele bios) |) Commumcles.. | 2 ee Per 100 ce. Concentration. COz2 per cent per cent mq. mol mol 5 55:1 194 0.0332 0.0072 10 55.6 191 0.0327 0.0081 20 56.7 185 0.0316 0.0098 Experiment 2.—Defibrinated beef blood centrifuged and serum removed. An artificial serum, containing serum proteins, glucose, and disodium phos- phate was added. Equilibration at 25°C. Plasma bicarbonate by the Van Slyke-Cullen method. Phosphorus determination gravimetrically: the weighing of MgNH,PO,- 6H20 as described by Jones (1916) being used. ee | blood. Per 100 | Concen- tion. per 100 ce. | Der 100 Concen- ee tration. ce: tration. COz2 per cent| per cent mg. mol mol mg. mg. mol 5 49.1 168 | 0.0287 | 0.0112 249 78.9 | 0.0254 20 | 50.0 164 0.0280 0.0126 250 80.5 0.0259 Experiment 3.—Defibrinated sheep blood. Whole blood centrifuged and serum removed. The same volume of an artificial isotonic serum, containing the serum proteins, glucose, and inorganic salts was added. The concentration of the sodium salts was made small in order to detect any part that its transfer might play in the attainment of equilibrium. Equilibration at 25°C. Considerable hemolysis occurred. Plasma bi- carbonate by the Van Slyke-Cullen method. NaCl : NaHCO; Treatment of Na Corpuscles. concentra- whole blood. 3 : er 100 cc. Per 100 ce. Popbente: tion. 4 COz per cent per cent mg. mol mol mg. 3 56.3 214 0.0366 0.0097 150 15 56.8 199 0.0340 0.0131 151 EK. A. Doisy and E. P. Eaton 391 Experiment 4.—Defibrinated beef blood. Equilibration at 25°C. No hemolysis. Bicarbonate by titration. facptment 5 ; a Rene Na K Pp of whole orpuscles. concentra- 100 ce. 100 ce. 100 ce. blood: it ee Concen- ton: per ce. |per ec.|per ee ‘CO2 per cent| per cent mg. mol mol mg. mg. mg. 3 38.4 650 Oetih 0.0274 336 20.7 LA 10 39.7 632 0.1081 0.0311 336 7.4 20 40.0 | 622 0.1063 0.0352 350 21.6 t.3 The volume change of corpuscles at 10 per cent CO, seemed to us pe- culiarly large in comparison with the 20 per cent. Upon the assumption that there is no exchange of nitrogen, we ran Kjeldahl determinations on the serum. The figures found follow. CO2 N per 100 ce. per cent per cent 3 1.017 10 1.030 When these results were used to correct the chloride and bicarbonate figures we obtained the following results. Gain in base by loss of HCl, 0.1127 — 0.1081 = 0.0046 m a <<. as Nalco; 0.0311 — 0.0278 = 0.0033 mM The figures from our usual method of calculation are: by loss of HCl, — 0.0055 mM; by gain of NaHCO; ,+ 0.0031 m. Experiment 5.—Defibrinated beef blood. No hemolysis. Bicarbonate by titration. ‘Treatment — NaHCOsz + - = N K P pr uole Corpuscles. ae ii) || Caacare aig per 100 ec. | per 100 ce. |per 100 ce. ce. tration. Rei per cent mg. mol mol mg. mg. mg 3 40.9 637 | 0.1090 | 0.0294 Bil 2a 8.3 10 41.2 626 | 0.1071 | 0.0318 331 Pala 8.3 20 41.8 610 | 0.1043 | 0.0366 332 PAA 8.3 100 43.4 571 | 0.0977 | 0.0444 345 23.8 8.3 Experiment 7.—Defibrinated beef blood. No hemolysis. Bicarbonate by titration. Equilibrated with the following gas mixtures. CO2 Oz per cent per cent 3 20-21 14 20-21 21 20-21 o7 13.0 80 4.0 392 Ion Migration between Cells and Plasma NaCl Sasa a ; lest Na K Pp of whole | Corpuscles.. —————-—————| c - blood. P ere: Coneen- an per 100 ec. | per 100 ce. |per 100 ce. shales t per cent mg. mol mol mg. mg. mg. 3 46.2 629 0.1076 0.0270 339 24.4 8.7 14 47.3 596 0.1020 0.0351 347 21 47.9 584 0.0998 0.0380 355 8.6 37 48.8 570 0.0976 0.0408 365 20.0 8.6 80 49.0 561 0.0960 0.0431 patsts) 26.3 Addendum. We are indebted to Dr. Donald D. Van Slyke for calling our at- tention to a point in our paper which perhaps needs further explanation. The point concerns the fact that as stated on page 380 we have determined the shift of ions occasioned by different tensions of CO., but in all cases have determined the bicarbonate by titration to an arbitrarily fixed and constant pH and have thus excluded from consideration the additional shift of base from serum proteins to carbonic acid which doubtless occurs with changing pH. With Dr. Van Slyke’s consent we append his comment on this point, with which we agree. “There are two types of reactions with plasma by which increase in [H.CO,] causes increase in [BHCOg]; v7z., H.CO; + BC] = BHCO; + HCl (1) the HCl being transported into the blood cells; and H.CO; + B Protein = BHCO; + H Protein (2) (Van Slyke, 1921). Of these two reactions, the titration to the ‘constant end-point measures only the first. The results there- fore indicate that the above chloride reaction accounts for prac- tically all the bicarbonate change due to migration of ions; 2.e., that Cl- is the only ion other than HCO;~ that plays a significant part in the acid-base shift between cells and plasma. The results do not indicate the relationship between the amount of BHCO, formed as the result of this migration and the amount formed by reactions with plasma buffers of the type exemplified above by Reaction (2).”’ E. A. Doisy and E. P. Eaton 393 BIBLIOGRAPHY. Barcroft, J., The respiratory function of the blood, Cambridge, 1914, 58. Bell, R. D., and Doisy, E. A., J. Biol. Chem., 1920, xliv, 55. de Boer, 8., J. Physiol., 1917, li, 211. Collip, J. B., J. Biol. Chem., 1921, xlvi, 61. Doisy, E. A., and Bell, R. D., J. Biol. Chem., 1921, xlv, 318. Fridericia, L. S., J. Biol. Chem., 1920, xlii, 245. Girber, A., Jahresb. Tierchem., 1895, xxv, 164. Haggard, H. W., and Henderson, Y., J. Biol. Chem., 1920, xlv, 189. Hamburger, H. J., Wien. med. Woch., 1916, lvi, 519, in this paper the author refers to earlier papers on this subject. Hasselbalch, Kk. A., and Warburg, E. J., Biochem. Z., 1918, lxxxvi, 410. Henderson, L. J., Am. J. Physiol., 1908, xxi, 169. Henderson, L. J., J. Biol. Chem., 1921, xlvi, 411. - Joffe, J., and Poulton, E. P., J. Physiol., 1920, liv, 129. Jones, W., J. Biol. Chem., 1916, xxv, 87. McLean, F. C., Murray, H. A., Jr., and Henderson, L. J., Proc. Soc. Exp. Biol. and Med., 1919-20, xvii, 180. McLean, F. C., and Van Slyke, D. D., J. Biol. Chem., 1915, xxi, 361. Mellanby, J., and Thomas, C. J., J. Physiol., 1920, liv, 178. Smith, M., J. Biol. Chem., 1921, xlv, 437. Smith, L. W., Means, J. H., and Woodwell, M. N., J. Biol. Chem., 1921, xlv, 245. Van Slyke, D. D., Physiol. Reviews, 1921, i, 141. Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 1917, xxx, 289. Van Slyke, D. D., Stillman, E., and Cullen, G. E., J. Biol. Chem., 1919, Xxxviil, 167. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 he hw 4 } ‘ %! a | 7 = ‘ i 248 - + = a 4 . ‘ 1 ‘ . A i - 2 1 ' i . -~ i EXPERIMENTAL RICKETS IN RATS. II. THE FAILURE OF RATS TO DEVELOP RICKETS ON A DIET DEFICIENT IN VITAMINE A.* By A. F. HESS, G. F. McCANN, anp A. M. PAPPENHEIMER. (From the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York.) Puates 1 To 3. (Received for publication, May 28,.1921.) The most recent hypothesis regarding the etiology of infantile rickets places it in the group of deficiency diseases resulting from a lack of vitamine. This hypothesis was advanced from a theoret- ical standpoint by Funk, and later was sustained by Mellanby as the result of experiments on dogs (1). The latter believes he has proved that when puppies are deprived of the so called fat-soluble vitamine (vitamine A) they develop rickets. The conclusions of Mellanby were accepted by the committee on accessory food factors of Great Britain (2) appointed by the Medi- cal Research Committee and the Lister Institute.t This report considers the fat-soluble factor, or a factor with similar distri- bution, synonymous with the antirachitic factor, and presents a table of antirachitic foods of three grades of potency. In‘a previous paper one of the authors has shown that infants do not develop rickets when on a diet containing a minimal amount of this factor, but that on the contrary they not infrequently develop rickets in spite of receiving food rich in this vitamine, and concluded, therefore, that this unidentified factor could not be regarded as the antirachitic vitamine, and that it does * Read in abstract before the Society of Experimental Biology and Medicine, May 18, 1921. 1 Experimental evidence against the contention of Mellanby that the fat-soluble vitamine is the determining factor in rickets, has been pre- sented by Paton, Findlay, and Watson (Brit. Med. J., 1918, ii, 625), and by Paton and Watson (Brit. J. Exp. Path., 1921, ii, 75). 395 396 Experimental Rickets in Rats. II not exert a:controlling influence on the development of this dis- order (3). More recently McCollum, Park, and their associates (4) have stated that in their experience rats fed on certain diets deficient in the so called fat-soluble A or in both that substance and calcium develop ‘‘a condition identical with the rickets of human beings.” In a paper just published (5) they express the opinion that the fat-soluble A vitamine is not the sole cause of rickets, but that it cannot be excluded as an etiological factor in the production of this disorder. The following investigation concerns itself with the effect on young rats of a diet markedly deficient in the fat-soluble vitamine. That lesions similar to those of infantile rickets can be induced in young rats has been shown recently by the work of Shipley and his associates (5) and by Sherman and Pappenheimer (6). For our purpose rats weighing about 30 gm. were placed on a basal ration (Diet C) similar to that employed by Drummond and Coward (7): Wasein: Fak bere tetas Aeiesl. on. bb sladare . ore ee ee 21 per cent TRA CET SE AEC NS. career ee siciecercis,o:s,«\e,0°sloha, 03 ARE coe Gy fae Bey BREUER RET ee Nes corn nau oss a0 « vo: 2 «8 Bi to ae CHICeO ed RAE ASA ION SSL GS 6 SHEE Ree ns SEE 52 ty fetes Boe SVIGH ST eee etree SN oie o's elnete edie Solt OR EERE eRe 60 mg The casein was extracted with about twice its volume of cold alcohol by means of slow filtration which lasted 3 to 4 hours, until the last washings came through colorless. It was then ex- tracted with ether (using about twice the volume of casein) for about 48 hours, and employing two changes of ether. The salt mixture was that used by Osborne and Mendel. Yeast was the Harris extract, prepared according to the formula of Osborne and Wakeman (8). One group of animals was also given orange juice, 0.5 ce. daily. Both the yeast and the orange juice were not incorporated in the food, but given separately, so as to make certain that they were consumed in full amount. In addition to this ration, which was complete except for an almost total lack of the fat-soluble vitamine, we employed Diet B, which was complete in all respects, 6 per cent of butter fat having re- placed an equivalent amount of Crisco. In some instances Diet B was given practically throughout the experimental period, in order to allow of a comparison of growth and health between 397 Hess, McCann, and Pappenheimer (+) 42 PEPPL SeA 4dipnq 4aIpP esoymM oO, CLO4 INOY ot Oh aain€ eBueio moun J 491 jurrsyoq ——— avin aBudio YAM (Dd, 41d 4uaoyog ----- 4 HT. WIG yoduroy ——— 1 shop ov 09? og (spotsed 404s 404 asin€ abueso -99 oo uar6 asam Leo“be‘ce‘ze ‘lt ‘L'9 ‘son ) J9INF JONVYO LAOHLIM ool CLT |S fae: - — LAI LNGIDI30 ANINVLIA 378M10S-LV4 ai. 4 bas / T LUVHD E 71 ——— = (11) a : = | ip . ] ass eas ~ eo = = 091 aan oz | : / Ciera ef oi |) = = cir f Oh ony po S / % 2 fs o0z = | Z a) Lael es fo Mw) Ds) & Is (SY Be Sov x se) q 0 Sup St x Ses . AG c | 7 i 4. dav ool oz fa ae 4 oot SWwVvuS 398 Experimental Rickets in Rats. II a complete ration and one deficient in the fat-soluble vitamine (Chart 3); in other instances Diet B was substituted where the deficient diet had been maintained for months (Chart 1). The accompanying charts and tables illustrate our results. It will be seen that the rats on this deficient diet failed to gain in weight after a period of about 2 months, that somewhat later they began to lose in weight, and that they invariably died prema- turely. This is clearly illustrated by the graphs of Charts 1 and 2. The corresponding tables (Tables I and II) show that these rats in almost all instances showed lesions of keratitis, which is considered distinctive of this vitamine deficiency. These are the two criteria which are accepted as pathognomonic of a lack of the fat-soluble factor, growth failure and keratitis, so that this diet must be judged to have been markedly lacking in this essen- tial factor. That such was the case is evident from a survey of Chart 3, which differs from the preceding groups (Charts 1 and 2) merely in that butter was added to the dietary. Here growth invariably was excellent. This distinction is well exem- plified also in Chart 1 and Table IV, which show the sharp rise in the growth curve and the rapid cure of the ophthalmia when 6 per cent of butter was substituted for an equal amount of Crisco. One group of rats on the deficient dietary was given 0.5 ce. of orange juice daily, whereas the other group received no anti- scorbutie foodstuff. These groups were constituted in order to determine whether a lack of antiscorbutic still further retarded growth and favored the development of the eye lesions. As is well known, opinion is divided as to whether rats require antiscor- butic vitamine or whether they thrive normally when deprived of this factor. On adding orange juice a definite and prompt increase in weight was brought about in some instances, but this gain did not persist for long periods. In general, it may be stated that the curves in Chart 2, representing rats which received orange juice do not appear superior to those in Chart 1, where this antiscorbutic was lacking. Ophthalmia occurred less frequently and with less severity among the rats which received orange juice. This was more noticeable clinically than on pathological examination; among the eleven rats which received no orange juice, only one failed to develop ophthalmia during life (Table I), 399 Hess, McCann, and Pappenheimer ‘UOI{eUIUTeX OIdOOsOIOIUL Aq PoULIGUOH y “OU NT “(eIqnop) “(T) stated *sopoy -So0[VUI}SOJUT “puv[s Are] ]Ixvurqns pue sunt jo woTpoosul oATYVANddng , w1uouNeud -oyouorq {('J) sriypoAg ‘sus puvw ping uly} UIe}UOD soUT}seq, UT y STPIOYORIY oynoy x STJIIYSVS oTyI9yy yr ‘fou -ply UL. Sossoosqe [ROOT “UOTIOJUT 9poOysad ‘SIPI[OO «= OLS VY ALIOWIO FT *[BUIION 4 SIPIZCIOY ON « SIPIPVIOY YYSITS A190 ‘stytArTyounluoy 4 SUOISO] [BUITUT] Wad es el LATA y STIWATQOUNTMOD 4 OUON, # PIM, -Gloy f1oo[N [BouLoD , WOISO] [BUTUT YY « STT}BIOY ON * 5) 4 SIFIPEIOY FYBITS |,."0UON “SUOISo] I9YIO “elurpeyyy do “UOI}LUTUIEXO [BoITO[OYZB “ABp UILE1 9ATLVBON “Avp UHI 9AT}VIGO N ‘ouraIn quopning “BOYLIVIC, ‘ourin Apoolg "BOY IIVICT “BOY IVICG, ‘ayBpnxe Apoolg ‘ssoug -jnd 7 ) ” ”? ‘oyepnxe Apoolg “‘ssouqgnd ‘oyepnxe — guoyning ‘ssouygng "IBID ‘ssouygnd pu ”) ) ‘ayepnxe yuopning ‘ssouygnd pur eyepnxe . Juepning S}04O1Y ‘oyy] Sump SUOTPIOJUT ‘ayt] SuLINp sok 06 Lb ot | oF 96 oF 8 i sel | & e9 | OF cg 18 dL a3 3 a A 08 L Eth ae $ yeIp . ON to sAucy | TS ‘oon ¢ abunig ON “aig quavyoad ourumpjir.,A a1qnjog-19q ‘T AIadVL aT Experimental Rickets in Rats. 400 ‘por{ ON ‘“Avp puzgy ‘oaryedou ‘Kvi-xX | ‘UOIpBUIUTBXS dIdoosoOIOIU AQ PoUIGUO/ fp ee eS ee eee - JOAT] Ul SOSOIOIU [BOOT | 4 SIFIFVIOF qysys AIDA | «5, “BIPSUL SI}1FO “IBID | «6S 9§ + 89p0}zse0 [BUIPSOFUT ‘sIpATpoUN[UOS | 5, ‘ayepnxe Apoolg | €¢ 1g * 9) “SOU IIerCT ‘Tea | 6S 8% »BOU100 ‘aUON | JO UOT}RIz[gUI ,, 4 OUON ‘ayepnxe Apoo[g | SOT 1Z ‘ssouy -jnd foyepnxo yuojning | ZS8T 12 4 SISBPOOTGOUOIE * ” ” * ‘IBID | STL 1% : ‘ssouygnd ‘QUO N 4 SIFTPLIOT YSIS | x5, pus oyepnxe yuojning | 691 91 ‘staAjod [wuor JO WOTPOOJUT OIVISVIVT "STI}IZVION ON | x9, “STYIULY YY ‘IvaIQ | SOT el ‘Bun JO Ssossoos ‘ssouyynd -qe {fssoasqe Areypixeuqng * a Ss ‘eoyIIviq, | puw eyBpnxe yus[Ming | PeT ral “('T) S140 JueTning * ” ” # 99 “BIPOUL BETO ” 16 Il “BIGOT s « OUON 4 STHPOIOY YSIS | x, :BOYLIGI, ‘IBID | IT 6 ‘Bou , sruoumoud aarzeinddng | -100 Jo Aywovdo ep ‘ssouygnd pus oyepnxg| TST c ‘stAjod “SIPIUIYL jeuor ut sjsko d1zISBIvg 4 SIFTPVIOT YYSITG | +5, {iva pojpoojuy "IvaIO | SIT e “mol - ‘yoou Ul 199[N Burpwoyy ‘QUON |,,OUON | Jopun 1007 /) ‘ayepnxe yysIS | 6IT ed *suOIsa] 19440 “erurpeqyydoO *SPOHOY a eS ee ee pecel ‘Osl sump soAny BuLmMp suopoojyuy “UOLFBULUIBXO [BOITO[OYyB : ————— SSS ‘one abunig Yun aig quavyag eupunjzpA 219"19S"IPA “Tl WTA VL 401 Hess, McCann, and Pappenheimer ”? "OUON “T[NOTBO Te oteoK *gapoysoo [eurnsoqyuy “oUON ”? ”) “OUON ? ”? #5 “QUON ” “VOY IIVIC ‘oul ul sng ” “BOYLIVIC, *SUOISO, IOqAO| viUT~EqyYdO] ‘syoxo1yY ‘UOIPLUIMIBXA [BOISO[OyyeT ‘OsT] duLimp SUOTJOOJUT *10}JNG SULATODAL OTIT AA “AVP UIFOT OF 4996 ‘ssouggnd puv oyepnxo Apoolg “ACP UFOT ‘shup g Ul pony | 04 YIZ6 2epnx9 Apoolg ” : ms gd “IVOTO “IVOTD "AUP YI69T OF ‘sAup fF Ul pony | YIPPl UO oyepnxe yuo[NInNg "(UISPT) op ‘hep ouo oyepnxea Apoolg mutts) 1) “IBID ‘qolp JWLOYop UO OTT A ‘ajt] SuLMp soAnt POT FOL 02 FL 69T 691 GLI shinp “7901p quolyop uo pollog “901P uo skep [830.L ‘TH ATaAVL ‘aon abunig ypu ‘vayjng fo uoyrppy fq panojjog ‘aig quavyag uo por.sag buoT ‘ON [Bes 402 Experimental Rickets in Rats. II whereas among the fourteen which did receive an addition of orange juice, in seven the eyes were at all times normal (‘Table IT). In this connection it should be borne in mind that some prelimi- nary observations of Osborne and Mendel (9) indicated that orange juice contains traces of the fat-soluble vitamine. Probably a deficiency of the antiscorbutic factor intensifies the alteration of the cells brought about by other nutritive deficien- TABLE IV. Control Rats on Full Diet for Entire, or Almost Entire, Period. ; PS Gall ee ‘ 4 Pathological examination. Beet | cae gel eral ene || Taleo a det | Riekete[OPbehal| Ober With orange juice. 15 114} l1lithto | Clear. None. | None. | None. 33rd day. 18 148 0 $s Rhinitis. + s . 90 148 0 “ “ “ “ce 25 179 0 “ x * “ 26 131 6th to “2 # fe Re 30th day. 388 114 0 “ce “ x “ 48 137 0 SS tae rb ey Without orange juice. 1 121 | 1ithto | Slight exu- None. | None. | None. 44th day. date on 89th day. 10 117 || Atthto |) Clear. ce a4 e 44th day. 35 114 6th to ee is sé ¢ 42nd day. * Confirmed by microscopic examination. cies. In experiments on scurvy in guinea pigs ophthalmia and keratitis have been observed by us. Similar lesions have been reported in the course of human scurvy (10). In this connection it may be mentioned that conjunctivitis, unaccompanied by puffiness of the lids, occurs not infrequently among white rats, especially where they have not been kept in the dark. ot on 09 09 ool tral) onl Bulan tv ‘(¥) yO Aatyng fo UolWtppe ue UdsAlG siam sper BOUL [> arin? aGueio ynoypim J faq pud1>14a2q— (a2¢o)aoin{ abucdo UM D 4o1 fuera ---- A. FIT ajaiduroy ckapog —— | ading JONVYO HLIM IFId IND1ID1430 WINVLIA J1gMmos-Lv4 @ LYVHD ean 4 09 0g 00 ot Oh! 403 | ee MB We A Se Fy Simo BAY on FAT-SOLUBLE VITAMINE ADEQUATE DIET “B” Hess, McCann, and Pappenheimer 405 Pathological Examination. As regards gross changes the skeletal system was found normal in all respects. In no instances were there the deformation of the thorax, the beading and angulation of the costochondral junctions, the multiple infractions surrounded by masses of callus, which form so striking a feature of rickets in rats. The long bones also gave evidence of no undue pliability, curvature, or epiphyseal enlargement. Under the microscope,? however, cer- tain deviations from the normal picture were found quite regularly. As will be evident from the accompanying description and illus- trations, these lesions were in no way suggestive of rickets but were correlated rather with the deficient growth of the animals. The zone of proliferating cartilage was narrow; the columns rarely exceeded four cells in depth, and frequently were composed of not more than two or three cells. The matrix of the cartilage in this zone of proliferation was invariably densely calcified, not only the capsules of the cartilage cells but the tissue between them taking a deep purplish blue stain indicative of calcium. This was confirmed in some of the preparations by applying von Kossa’s silver nitrate method. The most striking departure from the normal was found in the subchondral zone. Instead of a series of stout trabecule showing an orderly parallel arrangement about the calcified car- tilage matrix, and directed in the long axis of the rib, the primary spongiosa was defective. In many cases it was represented by two or three delicate and irregularly disposed trabecule leaving the calcified cartilage exposed over a large portion of the chondro- costal line; in others there was a continuous thin transverse plate of fully calcified bone, limiting the cartilage and fusing with the calcified matrix. The osteoblasts, surrounding these trabeculze or lying against the subchondral bone plate, were inconspicuous in contrast to their swollen appearance in actively proliferating bone. Osteoid tissue was conspicuously absent here as also along the completely calcified cortical layer. 2 After fixation in Muller formol, ribs were decalcified for 5 to 15 days in Muller’s fluid, embedded in paraffin and stained with hematoxylin eosin. The examination of the long bones has not yet been completed. 406 Experimental Rickets in Rats. II The trabecule of the secondary spongiosa were often better developed, having been formed during the period of more active growth. The marrow usually was of the normal cellular type. TABLE VY. Histological Picture of Costochondral Junctions of Rats Fed Complete and Incomplete Diets. Zone of prolif- erating cartil- age. Primary spongiosa, Secondary spongiosa. Cortex. Complete diet. 24 cells deep. Regular = ar- rangement. Matrix calci- fied. Stout trabecule corresponding to columns of cartilage cells, parallel align- ment. Well developed. Narrow osteoid margin in young growing rats; invisible in older ani- mals. Fat-soluble A deficient diet. 14 cells deep. Columns often slightly sepa- rated. Matrix - calcified. Trabecule re- duced in num- ber and size, irregular in ar- rangement, often fused into thin plate, completely calcified. Well developed in some cases, deficient in others. No visible os- teoid margin. No infractions. Rachitie diet. 1-20 cells deep. Irregular pro- longations into metaphysis. Matrix uncal- cified. Composed chiefly of osteoid, with core of calcified cartilage or bone. Dense tissue with nar- row vascular spaces. Dense trabecule with wide oste- oid margins. Irregular broad subperiosteal and endosteal osteoid mar- gins. Frequent infractions, with masses of cartilaginous and osteoid cal- lus. In some rats which had acute suppurative infections in other tissues (submaxillary gland, kidneys), there was an unusual pro- portion of polymorphonuclear leucocytes among the marrow ele- = Hess, McCann, and Pappenheimer 407 ments. In a few preparations the marrow immediately about the cartilage was depleted of blood-forming elements, and com- posed predominantly of pale polyhedral cells, often forming multinucleated masses about the cartilage. The features which differentiate the bones of these animals maintained on fat-soluble vitamine deficient diet from those of rats on an adequate diet and pursuing normal growth as well as well as from those with rachitic lesions, are brought out in Table V. From the foregoing comparison it is clear that the skeletal changes in rats on a fat-soluble deficient diet are in no respect suggestive of rickets, but may be interpreted as due to an inactive osteogenesis. This is what might be expected in view of the sta- tionary or declining weight and arrested growth. The ocular lesions have been so thoroughly studied by Stephen- son and Clark (11), and by Wason (12) that detailed reference need not be made to our experiences. The histological changes were such as have been described by these observers. Of the 25 rats comprised in Tables I and II it will be noted that in 5 the corneze were normal on microscopic examination. Incidental infections were encountered in a large proportion of the rats; notably, submaxillary abscesses, suppurative infec- tions -of the urinary tract, and bronchiectases. Infestation with the cestode, Hymenolepis murina, was also extremely common. Vesical calculi, described by Osborne and Mendel (13) in rats deprived of the fat-soluble vitamine, were encountered at autopsy in one instance (Rat 24). In this animal it was associated with pyelitis. Its occurrence in this connection is probably due in part to the susceptibility to infections induced by a diet lacking in this vitamine. CONCLUSION. Young rats receiving a diet complete except for a lack of the fat-soluble vitamine invariably failed to grow and generally de- veloped keratitis. The keratitis developed less frequently when the ration included orange juice. If this diet is continued for a period of months the animals die, either of inanition or, more often, of some intercurrent infection. The skeletons of such rats show no gross changes whatsoever. Microscopic examination 408 Experimental Rickets in Rats. II of the bones of 22 rats on a ration of this character presented definite signs of a lack of active osteogenesis, but in no instance lesions resembling rickets. In view of these results and their conformity with our previous experience in regard to infantile rickets, we are of the opinion that this vitamine cannot be regarded as the antirachitic vitamine, and that, if the diet is otherwise adequate, its deficiency does not bring about rickets. BIBLIOGRAPHY. 1. Mellanby, E., Lancet, 1919, i, 407. 2. Hopkins, F. G., and Chick, H., Lancet, 1919, ii, 28. 3. Hess, A. F., and Unger, L. J., J. Am. Med. Assn., 1920, lxxiv, 217. 4. Shipley, P. G., Park, E. A., McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1921, xlv, 343. 5. Shipley, P. G., Park, E. A., McCollum, E. V., and Simmonds, N., Bull. Johns Hopkins Hosp., 1921, xxxii, 160. 6. Sherman, H. C., and Pappenheimer, A. M., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 193. 7. Drummond, J. C., and Coward, K. H., Biochem. J., 1920, xiv, 661. 8. Osborne, T. B., and Wakeman, A. J., J. Biol. Chem., 1919, xl, 383. 9. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1920, xlii, 465. 10. Zlocisti, T., Klin. Monatsbl. Augenh., 1917, lix, 572. 11. Stephenson, M., and Clark, A. B., Biochem. J., 1920, xiv, 502. 12. Wason, I. M., J. Am. Med. Assn., 1921, lxxvi, 908. 13. Osborne, T. B., and Mendel, L. B., J. Am. Med. Assn., 1917, lxix, 32. EXPLANATION OF PLATES. PuatTeE 1. Fic. 1. Normal rib. Rat 82. Weight 158 gm. 52 days on complete diet; continuous growth and gain in weight. Decalcified 3 days in Mul- ler’s fluid. Fic. 2. Rib of Rat 17. 139 days on fat-soluble A deficient diet. Maxi- mum weight 118 gm. Terminal weight 71 gm. Decalcified 10 days in Muller’s fluid. Zone of preparatory calcification is very narrow, not exceeding three cells in depth. Cartilage cells separated by densely calcified plate. Pri- mary spongiosa represented by continuous bony plate, without definite trabeculae. Cortex thick, without visible osteoid margin. Osteoblasts inconspicuous. Hess, McCann, and Pappenheimer 409 PLATE 2. Fic. 3. Rib of Rat 44. 82 days on fat-soluble A deficient diet. Maxi- mum weight 140 gm. Terminal weight 125 gm. Decalcified 5 days in Muller’s fluid. Zone of preparatory calcification not over four cells in depth, regular, matrix completely calcified. Osteogenesis at epiphysis is fairly active. Spongiosa composed of coarse trabecule, with absent or inconspicuous osteoid margin. Cortex broad and completely calcified. Marrow cellular, not fibrous. Fig. 4. Rib of Rat 58. 34 days on rickets-producing diet. Marked rachitic lesions. Note increased width and irregularity of proliferative cartilage, absence of calcium deposition, great excess of osteoid in region of metaphysis and about cortex. Decalcified 5 days in Muller’s fluid. PLATE 3. Fig. 5. Rat 16. 175 days on fat-soluble A deficient diet. X-ray. Absence of rachitic lesions at upper epiphysis of tibia. Fig. 6. Rat 59. 41 days on rickets-producing diet. X-ray, showing rachitic changes in upper epiphysis of tibia. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 . é > Ps = ‘ 2 ; : x 7 ee ee ‘ ‘ es ~ + , / Me: ids ? oe 3 ‘ aan techs 5? Or) ty. 84 522 OEE we se a j 7 ~ v - ‘ LU r * i) eee ee eee ee Se et PEA est) ~ - _ be ¢ ~ <— * te . «= a 7 — ’ 7 bend 5A ee oe x \ , ee i a = ‘ : F s ae m > ’ THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVI1. PEARES: aa ah sia oats “Ge ns egg a - 4 peak re See : = (Hess, McCann, and Pappenheimer: Experimental rickets in rats. II.) “4/0 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII. PEATE 2: Fia. 4. (Hess, McCann, and Pappenheimer: Experimental rickets in rats. IT.) THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII. PEATE S- ays pce Mae egg bss Frag: 6. (Hess, McCann, and Pappenheimer: Experimental rickets in rats. II.) Pull Si i ( METHODS OF EXTRACTING AND CONCENTRATING . VITAMINES A, B, AND C, TOGETHER WITH AN APPARATUS FOR REDUCING MILK, FRUIT JUICES, AND OTHER FLUIDS TO A POWDER WITHOUT DESTRUC- TION OF VITAMINES. By J. F. McCLENDON: (From the Laboratory of Physiological Chemistry, the University of Minnesota Medical School, Minneapolis.) (Received for publication, May 16, 1921.) He As the population increases, the question of food supply be- comes more urgent, and the vitamines being necessary food constituents need to be conserved. It is therefore important to develop methods for extracting them from plants that are not considered edible and for preserving them in foodstuffs that re- quire storage. Except for the perfection of the drying appa- ratus, the methods here described, were worked out in 1919-20. Methods A, B, and C, are especially devised for the vitamines, respectively A, B, and C. Each product is, in reality, a mix- ture of vitamines with one predominating. By these methods the vitamines are extracted with very little loss, and concentrated without the use of very expensive reagents. The unique features of the methods are believed to be; first, the extraction of vitamine A from green leaves or fruit skins by the use of high pressure (after moistening with alcohol). Second, the separation of the resinous and lipoid material from the water- soluble portion in extracting vitamine B by increasing the H ion concentration up to the isoelectric point of these colloids; and third, the removal of the sugars from the B and C extracts by fermentation with baker’s yeast. All processes are carried out in the absence of oxygen, and the drying is done very quickly. In making the A extract, green leaves or fruit skins may be used. These are dried, preferably in the absence of oxygen, 411 412 Extraction and Concentration of Vitamines and then ground to a powder. This powder is moistened with alcohol by being thrown into a container half filled with boiling 95 per cent alcohol and allowed to remain without application of heat for 24 hours. The mass is then placed in a strong canvas bag and subjected to a pressure of 5,000 pounds to the square inch ina suitable press. The press-cake may be ground in a mill and reextracted in the same manner. The press-juice is dried in the apparatus described below. If, however, it is de- sired to recover the solvent, a preliminary concentration may be done in a vacuum pan. The product may be a sticky powder when absolutely dry, but absorbs water from the air, and be- comes pasty. It contains resinous or fatty substances. It is of a waxy consistency when made from spinach and absolutely dry, but is hygroscopic. The preparation of the water-soluble B is made from wheat germ or other foodstuff, such as yeast, which is rich in this vitam- ine. The wheat germ is treated in the same manner as the green leaves up to the pressing out of the extract, except that 80 per cent alcohol is used. The press-cake is ground in a mill and extracted again in the same manner. The press-juices are concentrated in a vacuum pan to about one-tenth of the original volume or until the first precipitate starts to form. An equal volume of water is now added and hydrochloric acid is added slowly and with stirring until a bulky and sticky precipitate forms. This is filtered off and the precipitate washed with dis- - tilled water and the washings are added to the filtrate. The precipitate is dried and extracted with benzene in any convenient extraction apparatus and the benzene is evaporated. The residue contains some of the wheat oil. The filtrate is brought to pH=4 to 5 and is fermented until the reducing sugar is lowered to about 1 per cent of its original value. The yeast is filtered off and added to the next batch of wheat germ and the filtrate is evaporated to dryness. In both the filtrate and the benzene extract, the water- soluble B is in quite concentrated form and there is practically no loss except the portion absorbed by the yeast and which would be recovered in the next batch. The water-soluble C is extracted from fruits or tomatoes. If oranges are used they are pressed and the juice is run into vessels previously filled with carbon dioxide so as to exclude the J. F. McClendon 413 oxygen of the air, and baker’s yeast added and a cover kept on so that no air enters. The fermentation is allowed to proceed at room temperature until the reducing sugar is lowered to about 1 per cent of its original value which requires about 24 to 48 hours. The juice is then filtered with the exclusion of air and is condensed by spraying (in the apparatus described below) in the absence of oxygen, until the volume is reduced to about one- twentieth of the original volume. It is then thrown into four volumes or more of 95 per cent alcohol and the precipitate thus formed is filtered off and the filtrate sprayed and reduced to dry- ness with the same precautions. These three preparations may be designated A, B, and C. Preparation A contains sufficient fat-soluble A vitamine so that about 0.05 to 0.1 gm. daily added to a ration free from fat-soluble A will produce normal growthinarat. It also contains consider- able water-soluble B. Preparation B is very rich in water-soluble B vitamine so that a very small dose of either the lipoid or water-soluble fraction will cure a pigeon of polyneuritis. The lipoid fraction will re- vive the hypodynamic heart of the turtle, but the water-soluble fraction is ineffective. Both fractions are effective growth stimulants. Preparation C contains sufficient antiscorbutic vitamine so that about 5 to 10 cg. a day will prevent scurvy in a guinea pig _on a basic diet supposedly free from water-soluble C for a period of about 2 months. Preparation C was in one instance purified further by the removal of citric acid, but since it is highly hygroscopic and deteriorates more rapidly (when moist) when not acid, further work on this preparation was postponed awaiting the perfection of the drying apparatus. The tests for the concentration of vitamines were made on rats, guinea pigs, and pigeons. The rats and guinea pigs were kept in metabolism cages, one unit of a 10 compartment cage being shown in Fig. 1. In case metabolic data were not kept, the pan and beaker under the cage were removed and a newspaper placed under the cage to catch the excreta. Drinking water was always present in the bottle. The basic diet for the guinea pigs was equal parts of white flour and alfalfa meal, and for the rats 414 Extraction and Concentration of Vitamines was 10 per cent vitamine-free casein, 6 per cent dehydrated sea water, and 84 per cent white flour. In case vitamine A was investigated the rat diet contained 5 per cent dry Fleischmann’s yeast and in case vitamine B was investigated the rat diet con- tained 1 gm. of butter fat per day. II. The Drying Apparatus. Many forms of drying apparatus were tried, such as evapora- tion at low temperature in a rotating drum by a blast of air, vacuum pans through which a small stream of carbon dioxide and nitrogen passed, atomizer sprays in flue-gas, and evaporation in front of an electric fan at room temperature. None of these methods was efficient, and, therefore, the following method was devised. The apparatus, Fig. 2, consists of a chamber near or at the bottom of which is the flue-gas exhaust, and in the center of the ceiling of which is the flue-gas and spray intake. The spray or distributor is rotated on the same shaft with the fan propelling the flue-gas at a speed of 5,000 revolutions a minute by an elec- tric motor. The milk or other liquid passes down from a tank through a cock which regulates its flow into the hollow shaft of this apparatus. The furnace is built on top of the chamber. 1 Some of the tests were made by Dr. M. A. Shillington, Helen Brenton, H. D. Reineke, and E. Dunlap, and these workers may publish details of their tests elsewhere. J. F. McClendon 415 It is constructed of fire-brick plastered with fire-clay, and has two flues that may be closed with valves, one extending upward, and the other closed by nichrome wire gauze and extending down- ward through an opening in the center of the ceilng. The ap- paratus propelling the flue-gas and making the spray is shown in Stack Valve + Nichrome Wire Gauze __. Milk Powder... Hires 2: detail in Fig. 3. It revolves on a steel tube that serves as a shaft and which is driven by a pulley at the top. Inside this steel tube is an aluminum or brass tube, which touches the steel tube only at the extreme top and bottom. Between the steel tube and the aluminum tube is an air space to prevent the passage of heat 416 Extraction and Concentration of Vitamines Milk Intake “Aluminum Tube | Atel Tube -;- Water Cup | Ni ae a Fan Blade Ceilin § . Aluminum Distributors ea. Milk Exit Fig. 3. J. F. McClendon 417 into the milk or other liquid being sprayed. Above the aluminum tube is fixed a nozzle which spurts a stream of the milk into the aluminum tube. The milk passes down the aluminum tube by gravity and out through a series of holes bored through the aluminum tube near the bottom, whence it strikes the surface of two cone-shaped aluminum distributors and spreads out on their surfaces and is thrown off from the periphery, which is cut into numerous saw-tooth-like points. From each point drops of liquid are thrown off by the rotation of the apparatus, pro- ducing a fine spray. On the steel tube, just above the aluminum distributors is a hollow drum which serves to prevent eddy cur- Distributor Bre. 4. rents and retard passage of heat to the steel tube. Above the drum is a fan with a hollow rim. This fan forces the flue-gas downward, cutting the spray at right angles and rapidly evaporat- ing the water. In order to prevent undue heating of bearings in which the steel tube rotates, an air space is provided between the flue-gas intake and the stationary tube in the ends of which the bearings are located. Also a deep groove or “cup”’ filled with water is provided on the lower end of this stationary tube. The aluminum distributor and fan are shown in perspective in Fig. 4. The hollow rim of the fan is useful to prevent burning of some of the spray that might be drawn against the rim of the fan by eddy currents. The portion of the flue right above the 418 Extraction and Concentration of Vitamines fan is made of wire screen plastered on both sides with asbestos furnace cement. In order to prevent the presence of oxygen in the flue-gas the amount of air must be reduced to a point at which some carbon monoxide persists. This carbon monoxide will form a crust of carbonyl on metallic surfaces, which will peel off and drop in the chamber. It is, therefore, best to have all metal coming in contact with the heated flue-gas made of nichrome, which seems more resistant than other metals or alloys, and the amount of metal should be reduced as much as possible. Therefore, all stationary metal parts exposed to flue-gas are covered with asbestos furnace cement. In operating the ap- paratus the furnace is filled with fairly large pieces of charcoal or coke, and this is ignited by means of illuminating gas. The valve in the flue or stack that extends upward is opened until the heat of the furnace is sufficient to drive off volatile substances in the fuel. The valve in the stack is then closed and the valve in the flue extending downward into the chamber is opened and the motor is set going, thus driving the hot flue-gas into the chamber. When the chamber is heated to about 150° at the top a little water is passed downward through the aluminum tube in order to cool it. Then the fluid from the tank is allowed to pass slowly into the aluminum tube. The rate of flow of this fluid is regulated by the cock so that the temperature at the bottom of the chamber remains at about 80°. The temperature is greater at the top than at the bottom, and, if the walls are cooled, a con- densation of moisture will take place within the chamber. It is therefore desirable to have the walls of non-conducting material, or else have the space surrounding these walls filled with the exhaust gas. It is desirable to have a series of thermometers with bulbs extending through the wall into the chamber at inter- vals from the top to the bottom. By reading these thermometers the flow of the fluid can be so regulated that the spray will always be reduced to a powder at the lowest possible temperature. The incoming flue-gas may be about 1,000°, but is instantly cooled by coming in contact with the spray. It is necessary, however, for the temperature within the chamber to be high enough to pre- vent recondensation of the moisture evaporated. Just what tem- perature that will be depends on the exact temperature of the flue-gas before meeting the spray and on whether there is any J. F. MeClendon 419 loss of heat through the walls or floor of the chamber. The temperature must, in any case, be higher than that required by warm-air-drying systems, but this disadvantage seems to be com- pensated by the greater stability of the vitamine in the absence of oxygen. In case milk is sprayed the absence of oxygen pre- vents the oxidation of the fat. As to how well this apparatus will work on a very large scale needs to be determined, but as an experimental apparatus, it has the great advantage that the cost of installation is com- paratively small and changes are easily made. The necessity of steam boilers, steam radiators, and condensers is obviated. The greatest loss is the heat that passes out in driving off the vol- atile constituents of the fuel before starting the spray, but this heat could be used for other purposes. Also the exhaust gas, if not saturated with moisture may be used for drying vitamine- containing products on tray dryers. So far this apparatus has been used for drying the vitamine extracts described above and also for drying orange juice and milk. The dimensions of this apparatus as it now stands are as fol- lows: The chamber is 8 feet square and 12 feet high, the aluminum distributor is 3 inches in diameter, and the fan 6 inches in diameter inside the rim. The flue is also 6 inches in diameter. A + horse power motor is used. The size of the motor could be greatly decreased by the use of ball bearings replacing the Babbit metal bearings in which the steel tube rotates. The main difficulty experienced has been the burning of some of the spray around the edge of the flue-gas intake. Practically no spray sticks to the walls if the temperature is maintained high enough. Incase it is desired merely to condense the spray, there is considerable condensation of moisture on the walls, and care should be taken that this does not drip into the condensed spray which falls into enamelled pans on the floor. Very little dust in the form of ashes need pass into the chamber if the apparatus is operated properly. The fan acts as a centrifugal dust separator. Two methods have been tried to improve the dust separation. One is the passage of gas through an asbestos filter, and the other is the modification of the fan so that it forms a more efficient cen- trifugal dust separator. The latter method is open to the objec- tion that the dust accumulating in the fan must be periodically 420 Extraction and Concentration of Vitamines removed or else it may throw the fan out of balance. The com- plete combustion of the fuel in the furnace is desirable. Ap- parently the nichrome wire gauze acts as a catalyzer when heated red hot. It is, however, necessary to have the bed of coals deep enough so that some carbon monoxide is formed in order to ob- viate the danger of an excess of oxygen. Apparatus for the analyses of flue-gas might be installed if the drying apparatus were used on a large scale. While operating the apparatus as described above, the powder is kept at about 80° until the end of the operation. If it is de- sired to keep the powder cool, the flue-gas outlet may be made a few feet above the bottom, and the chamber below this outlet kept filled with cold CO, or other gas. The flue-gas escaping from the outlet may be cooled and dried and used to fill the lower portion of the chamber. If this cool dry flue-gas is warmed slightly it may be used to complete the drying of the spray in case this was not accomplished by the time the spray reached the level of the flue-gas exit. The powder may be transferred to a vacuum drier to complete the drying. Previous condensation of milk or other liquid to be sprayed is a disadvantage and may cause it to be thrown onto the walls. Condensation increases viscosity and increases the size of the particles of powder resulting from the spray. These large powder grains dry superficially, but if then placed in a hermetically sealed container, the moisture from the inside of the grains ap- pears on their surfaces. If such powder, soon after being formed, is placed in a vacuum desiccator, it may be completely dried without coalescence of the grains. Addenda.—Owing to the hygroscopic nature of orange power the drying apparatus has been modified as follows: The floor of the drying chamber is made of a box 1 foot high, the top of which is made of muslin. Air dried over CaCl, is forced into the box and comes slowly through the muslin, thus completing the drying of the powder that falls on top and keeping it cool. HEMATO-RESPIRATORY FUNCTIONS. XII. RESPIRATION AND BLOOD ALKALI DURING CARBON MON- OXIDE ASPHYXIA. By HOWARD W. HAGGARD anp YANDELL HENDERSON. (Investigations performed for the United States Bureau of Mines* in the Laboratory of Applied Physiology, Yale University, New Haven.) (Received for publication, May 17, 1921.) Carbon monoxide asphyxia has long been accepted as involving the typical condition of acidosis. The decreased oxygen-carrying power of the blood was supposed to result in incomplete combus- tion in the tissues and a production of organic acids. ‘The increase of lactic acid in the blood and urine (Araki (1) and Ryffel (2)) was thus explained. Without this foundation of correct observa- tion, but incorrect inference, the theory of acidosis in a wide variety of conditions would probably never have attained the hold which it now has on current thought. It is true, as found by Saiki and Wakayama (3), that under carbon monoxide asphyxia the blood alkali is greatly decreased. But, as we shall show, this decrease is not of acidotic origin. In previous papers (4, 5, 6) we have demonstrated that a decrease of blood alkali may be induced in two almost diametrically opposite ways: (1) the acidotic process, and (2) the acapnial process. In the acidotie process strong acids find their way into the blood, partialiy neutralize the NaHCO; of the plasma, and overload the corpuscles with acid. A differential test of this condition may be carried out by causing the subject to inhale air containing 6 or 8 per cent CO,. If the acid intoxication is extreme, and the inhalation is pushed, the animal is soon killed by the excessive acidity thus induced in the blood. A normal animal is not harmed. In the acapnial process, on the other hand, various influences and conditions excite the respiratory center through agencies * Published by permission of the Director. 421 422 Hemato-Respiratory Functions. XII other than merease of Cy. This results in overbreathing and an excessive elimination of CO, which leaves the blood abnormally alkaline. A gradual compensatory disappearance of alkali from the blood follows. Subjects in this condition respond favorably to inhalations of CO.. Such inhalations not only overcome the alkalosis, but also rapidly recall alkali to the blood. It will be seen in the experiments here to be reported that it is the acapnial, and not the acidotic, process which comes into play. In a con- dition of acidotic origin the administration of sodium bicarbonate should be beneficial. It should be injurious in acapnia; and, as we find, it is injurious and even fatal in carbon monoxide asphyxia. All the experiments were carried out upon dogs. Enough illuminating gas, containing about 25 per cent carbon monoxide, was mixed with air in a large spirometer to afford the desired concentration of carbon monoxide, namely 15 to 45 parts in 10,000 of air, that is 0.15 to 0.45 per cent. A mask was made air-tight over the animal’s head with adhesive plaster. An in- spiratory valve and tube led from the spirometer to the mask, and an expiratory valve from the mask to the outside air, or at intervals to a wet gas meter of low resistance. Such conditions are quite comfortable and painless. The femoral artery was exposed under cocaine, and blood samples were taken at intervals. Part of each sample was analyzed, and part was equilibrated with three tensions of CO, in air and analyzed, as in our previous work. The following protocol, of which the data are given in Experiment 1 and Fig. 1, is typical. The subject breathed air containing 0.25 per cent of carbon monoxide until death resulted after 237 minutes. The volume of respiration gradually increased nearly threefold. The blood alkali, or CO.-combining power, fell gradually, but more slowly and to a less degree than the res- piration increased. Consequently, the CO, content of the arterial blood, as the table in the protocol shows, was relatively so much reduced that the ratio HeCO;: NaHCO;, and presumably there- fore the Cy, was constantly subnormal. There was no acidosis, but a marked alkalosis until near the end when respiration was as usual depressed. The most complete distinction between the acidotiec and acap- nial processes is to be gained by plotting the results of a series H. W. Haggard and Y. Henderson 423 of blood gas analyses in a CO, diagram. Such a diagram for this experiment is shown in Fig. 1. The dissociation curves are drawn from the data of the protocol and the position of the arterial blood on each curve is indicated by a dot. The abscissze in such a diagram are proportional to the alveolar tension of CO, and therefore to the content of HCO; in the arterial blood. The ordinates express the combined CO, or blood alkali. The diagonal Experiment 1.—Dog, female, 11 kilos. From 12 o’clock on the animal breathed air containing 0.25 per cent carbon monoxide from a large gaso- meter through a mask and valves. Blood drawn from the femoral artery under cocaine; part analyzed for CO: content, remainder equilibrated with 40, 72, and 18 mm. CO: at body temperature and analyzed. Blood equilibrated Volume art 9 Arterial blood. Gerre Time. Poon | ee reeee a, We eee oe (Char per 18 40 72 CO:z CO2 COz 8). meee em. | eae ems | ea a Peo eae ys eo. ae nok ve vol. Pa per cent|per cent|per cent|per cent|per cent|per cent 1 | 11.45] 4.5] 23 38 48 38 2.8 |-35.3 | 0.79 | 0.63 12.25 | 5.2} 23 38 48 38 2.8 | 35.3 | 0.79 | 0.63 2 it Os) 6.1) 23 37 46 3l 2.0 | 29.0 | 0.69 | 0.55 1.40 | 6.3 2.00} 7.4] 18 32 42 25 1.7 | 23.3 | 0.73 | 0.58 2.25] 8.0 2.45 | 9.0] 18 33 41 24 1.5 | 22.5 | 0.70 | 0.56 3 SHOU IO | 17 30 41 18 1.1 | 16.9 | 0.69 | 0.55 4 A OOeP=FFeOe)" 13 25 32 14 1.2 | 12.8 | 0.93 | 0.74 Gasps, apnea, death. The data of this experiment are expressed graphically in Fig. 1 and are discussed in its legend. line OC expresses the normal Cy or pH, since for every point in this line the relation of abscissa : ordinate is constant. Thus whenever the arterial point falls to the left of the OC line the ratio H,CO3;: NaHCO; is below normal, and overbreathing, acap- nia, and alkalosis are indicated. This condition prevailed throughout this experiment until the terminal depression of breathing set in. That these conditions are induced and de- termined by the volume of breathing is shown by the observa- tions noted on the curve for the respiration (Resp.) in the figure. £499 4a] sauin/o/\ role rat aa REGS EsGOeReo sey Nice ist ee seas hee ere ry MERRDGEESR So EUG ele alny SERBS ReURe ee Twa hese Coe ee eee 7 0G ee ESE PERS REESE PERSP SaSao OF Sonn aaaee Bete tt tte tea aa aaa Ree ew 25 BR S00C00000 POR0RRESRen dwasc == SSeS R ee BZER Eee wo Poin ~1 KUO S CC Eeaaael eal IG CeCe BEE 70 20 30 40 50 60 70 Tension CQ, in tr. Fie. 1. The CO» diagram (5) plotted from the data of Experiment 1. The lowering of the dissociation curve from 1 to 4, expresses the decrease of blood alkali. The line OC expresses the normal Cy of this animal. The deviation of the arterial points (*) to the left of the OC line on Curves 2 and 3 indicates that the excessive breathing is producing acapnia and alkalosis. Only during the terminal depression of breathing does a devia- tion to right, or toward acidosis, occur. The volumes of breathing (Resp.) as estimated from the arterial blood and as measured directly coincide in the first 3 sets of observations but deviate slightly from agreement in the fourth (* and X on curve marked Resp. ). 424 H. W. Haggard and Y. Henderson 425 The protocols of several other experiments are here given in abbreviated form. In addition to the points brought out in Experiment 1, some of these experiments throw light on additional matters. Thus in Experiments 2 and 3 the animals were removed from the gas-containing atmosphere before death, and allowed to recover. During the period of recovery respiration was for a time markedly depressed. A similar depression has been ob- ‘served by us previously in experiments following true acid in- toxication. It causes the ratio HeCO3;: NaHCO; to rise much above normal. Such an acidosis is, we think, to be regarded as a restorative process; it is an effort of the body to recall its alkali into use in the blood. We are inclined to believe that acidosis, that is a spontaneous high ratio of Hx.CO;: NaHCOs, or high Cy, always has this meaning. In other words alkalosis is an effort, and the normal method of the organism to lower the blood alkali and acidosis is Nature’s method of calling more alkali into use (Experiments 2-5). Experiment 2.—Dog, male, 16 kilos. From 12 o’clock on the animal breathed air containing 0.17 per cent carbon monoxide. At 6 o’clock it was in coma. It was then removed to fresh air, and recovered spontane- ously. The blood alkali was estimated by equilibrating the blood with 40 mm. CO: at body temperature and analyzing for COo. The volume of respiration is expressed in liters per minute. Normal. Breathing 0.17 carbon monoxide. peor AEG J Ra eee Hie 4o |) 123) 2502 10 3200)" 42 00\" 6200 8.00 Respiration..... 6.0 6.0 8.3 OROe | MM ZO RMS Mie aO 4.0 Blood alkali.....| 41 40 36 30 32 28 26 32 Arterial CO:2....| 40 40 | 26 24 25 18 17 30 Experiment 3.—Dog, male, 11 kilos. Conditions similar to Experiment 2. Inhaled 0.16 per cent carbon monoxide from 12 to 9 o’eclock. Then in coma, removed to fresh air, recovered spontaneously. Normal. Breathing 0.16 carbon monoxide. Recovering. amen. a5. 3 11.45 | 1.15) 2.15} 4.10) 5.10} 8.00} 9.00 9.30/10.50 12.00 Respiration... 5.8 SOR G55) | eames: |) S820 2" Oa|) 726 | SzOniGs9 Blood alkali...| 47 47 - |45 43 39 36 36 45 44 44 Arterial CO:s..| 48 43 43 39 32 29 28 44 |43 43 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 426 Hemato-Respiratory Functions. XII Experiment 4.—Dog, female, 7 kilos. Inhaled 0.49 per cent carbon monoxide from 12 o’clock on. Respiration failed at 12.40, and the heart stopped beating at 12.46. Breathing 0.49 Normal. per cent carbon Dying. monoxide. TMC... Seo po oe a ee eee 11.45 12.36 12.38 12.42 12.46 Bloodvalkaliz cs. eee eee 49 31 31 30 29 Arterial ‘(COS a eee 48 19 lv 21 26 Experiment 5.—Dog, female, 7 kilos. Inhaled 0.45 per cent carbon monoxide for 44 minutes. Then 60 cc. of 2 per cent solution of sodium bicarbonate were injected intravenously. Tetany developed, respiration ceased, and death followed. Breathing 0.45 Dyi ft Normal. pereeny corner Nalco, nM La sVo\3. Sete Paral inc 4.4 5.4 Boe aa eee 1145s 12.43 12.46 Blocdvalkah reer oe 50 38 75 Arterial Os-. sheet mes eee 51 20 52 Note that in these experiments the arterial CO, leads the blood alkali downward. Thus augmented breathing induces acapnia and alkalosis. In Experiment 5 an intravenous injection of a moderate amount of NaHCO; caused death in respiratory failure and tetany. This experiment affords an instructive contrast to some other experi- ments recently published by one of us (7) in which it was found that when a dog remains in an atmosphere of carbon monoxide until death the presence of CO, (7 per cent) distinctly prolongs life. By tending to neutralize the alkalosis, the CO. prevents the occurence of apnea vera, due to excessive loss of CO», which is the common mode of death in carbon monoxide asphyxia. Oxygen Consumption and Respiratory Quotient under Carbon ' Monoxide Asphyzxia. It might be supposed, if the current theory of asphyxial, or rather anoxemic, acidosis were correct, that during carbon mon- oxide poisoning the oxygen consumption by the body as a whole = H. W. Haggard and Y. Henderson 427 would begin to fall (owing to lactic and other organic acids escaping oxidation) coincidentally with overbreathing. In Ex- periments 6 and 7 this is seen not to be the case. The respira- tory quotient rises greatly, but the oxygen consumption, instead of falling, actually increases in one experiment almost to the end, due probably to the muscular exertion of the respiratory movements. Experiment 6.—Dog, male, 7 kilos. The oxygen metabolism and respir- atory quotient were obtained by placing the dog in an air-tight glass cham- ber of 300 liters capacity (minus a number of liters equal to the weight of the dog in kilos). An electric fan circulated the air in the chamber and a spray of water on the top of the chamber kept the temperature at 22°C. At intervals gas samples were drawn from the chamber and analyzed for oxygen and CO. The decrease of oxygen (1.7 per cent per hour) and increase of CO: were sufficient for calculation of the metabolism and respiratory quotient, but insufficient to affect the condition of the animal appreciably. After obtaining the normal figures for oxygen consumption, CO: output, and respiratory quotient, fresh air plus carbon monoxide to the desired concentration were run into the chamber and the measurements continued until death. : Air in chamber. Oz con- Respira- Time. Remarks. eee eS a ee | sumiption tory Op CO> per minute.| quotient. min. per cent per cent coe oan. () Dog in chamber. 20.91 0.04 , 15 Quiet. 20.50 0.41 74.5 0.80 30 Me 20.08 0.73 76.0 0.76 47 Moving. 19.58 Las 90.4 0.79 60 Quiet. | 19.18 1.44 13.9 0.77 The chamber was then thoroughly ventilated with fresh air, closed again, and a charge of 1,450 ce. of pure carbon monoxide was run in. 0 Gas run in. 20.83 0.05 15 Quiet. 20.48 0.35 72.6 0.75 30 f 19.94 0.77 89.0 0.86 39 Slightly excited. 19.37 ie 103.5 0.95 45 Occasional gasps. 19.14 1.69 104.4 1.60 52 Frequent 19-00. 1.74 50.6 1.10 53 Respiration failed. 428 Hemato-Respiratory Functions. XII Experiment ?.—Dog, male, 14 kilos. Conditions as in Experiment 6. ! Air in chamber. Os con- Respira- Time. Remarks. | eee i earmntion: tory O» j co, per minute.|} quotient. 2 min, per cent per cent ee. 0 Dog in chamber. | 20.90 0.05 30 Dog quiet. 19.75 0.98 110 0.76 45 a 19.15 1.44 112 0.79 The chamber was then thoroughly ventilated with fresh air, closed again, and 1,500 ec. of pure carbon monoxide were run in. 0 Gas run in. | 20.85 | 0.06 30 Dog quiet. | 19.64 | 0.96 | 106 0.81 45 Dyspnea. | 18.94 1.56 118 0.96 62 os Pealsesl PARAS) 102 1.90 69 Respiration failed. | Overbreathing and Acapnia as the Cause of Low Blood Alkali. It will be seen that the foregoing observations afford no support whatever to the idea that the lactic acid formed in the body during carbon monoxide asphyxia is the cause of the hyperpnea, or of the decrease of blood alkali. On the contrary the data are in complete _ accord with the view expressed in previous papers from this labora- tory; and with the conclusions reached by Haldane and his collaborators (8) that asphyxia causes not acidosis but alkalosis. Macleod (9) suggests the view, which we had put forward (6, 10) that the lactic acid which is formed in such conditions is to be regarded, not as causing acidosis, but rather as assisting in neu- tralizing the alkalosis. Evidently carbon monoxide asphyxia in- duces overbreathing and a considerable degree of acapnia, a conclusion supported by the ill effects of injection of alkali in Experiment 5. The beneficial effects of inhalation of 6 to 10 per cent CQO, in dogs after carbon monoxide asphyxia have re- cently been described by us (11). With Coburn (12) we have demonstrated the same beneficial effects on men in the depression following prolonged etherizations, in which condition also acapnia is a factor. The view now receiving general assent, that asphyxia may cause overbreathing and acapnia, is decidedly at variance with H. W. Haggard and Y. Henderson 429 the theory that the Cy of the blood is the sole chemical factor in the control of breathing. Evidently the oxygen supply is also a factor. But merely to say that ‘“‘oxygen deficiency stimulates respiration”’ leaves wide open the question as to how the de- ficiency. acts. We need some substance, chemical process, or physiological mechanism to take the place heretofore erroneously assigned to lactic acid as a respiratory stimulant. It must how- ever be a substance (if it is a substance at all) which does not act through an acid property, but as a respiratory stimulant of another order. There are such substances, but for the most part they have not been demonstrated to occur in the body. Ethyl ether in small amounts is a powerful respiratory excitant (13), and in unskillful or prolonged anesthesia may cause pro- found acapnia. Hydrogen sulfide, as we have found in ex- periments as yet unpublished, is even more potent. Indeed, inhalation of minute amounts of H.S has in our hands proved capable of inducing the most intense hyperpnea, and subsequent fatal apnea vera. That this is not an effect of H.S as an acid is demonstrated by the fact that intravenous injections of Na.S are equally effective. We have recently been engaged in trying to identify in the blood some sulfur compound readily altered by oxygen, which might play the role of the hypothetical respiratory stimulant which we have called ‘‘respiratory X.” As yet, how- ever, our results in this search are inconclusive. The problem is clear only on one point: Oxygen deficiency does not stimu- late respiration through the formation of a substance with acid properties. Carbon Monoxide Asphyxia afier Section of the Vag. It seems: well established, or at least accepted, that CO: and Cy influence respiration through action on the respiratory center itself (14). It occurred to us that perhaps oxygen deficiency, or anoxemia, stimulates not the center itself, but rather the vagus endings in the lungs. The probabilities are, of course, strongly against this hypothesis (which we are now investigating, how- ever), and we mention it new only because it has led to an obser- vation which is of critical importance here. Thus among other tests of this idea, we have carried out experiments on the influence . 430 Hemato-Respiratory Functions. XII of carbon monoxide asphyxia upon respiration and blood alkali in dogs in which the vagi were cut. The striking result of this operation (Experiments 8 and 9) is that the hyperpnea and dyspnea which are ordinarily seen under carbon monoxide asphyxia (Experiments 1 to 7) are. entirely Experiment 8.—Dog, male, 16 kilos. Under local anesthesia (cocaine) the trachea was exposed, cannulated, and connected with double valves, so that the expired air passed into a recording spirometer. Blood samples were obtained from the femoral artery. After the normal volume of respiration in liters per minute, and the arterial COz had been determined, the vagi were cut, and the minute volume of respiration was again deter- mined. Then the inspiratory valve was connected to a large spirometer containing 0.5 per cent carbon monoxide in air, which the animal inhaled until death occurred 50 minutes later. The hemoglobin of the blood was then found to be combined with carbon monoxide to the extent of 76 per cent. Normal. | Vagi cut. Breathing 0.5 per cent CO. ENS XS ON IRE eee che Soo Oe 0 15 25 |385 |45 155 |65 {75 Respiration, liters .........; 3.1 3.6 | 3.4] 3.2) 3.5] 3.4! 2.8] 0 Arterial CO2......- ee Ate ae 48 46 48 Note that in this and the following experiment, in marked contrast to all the preceding experiments, there was no hyperpnea and no fall of blood alkali. Experiment 9.—Dog, male, 12 kilos. Conditions similar to Experiment 8. At death HbCO = 72 per cent. Normal. | Vagi cut. Breathing 0.5 per cent CO. ING 70a eee a hee 0 20 (25 |38 |45 |60 |70 80 Respiration, ifers,.. ace os .- 2.6. | 4.1) 3.2) 2.9) 3.7] 2.8) 2.6 0 Arterial COpsgos see eee 40 38 | Death. lacking after section of the vagi; and the blood alkali remains practically unaltered throughout the whole course of asphyxia, and even up to death. It is not necessary in the present con- nection to assign to the vagus section, or to consider here, any effect beyond the prevention of excessive breathing. This pre- vents the loss of CO, (acapnia) and the usual compensatory fall H. W. Haggard and Y. Henderson 431 of blood alkali. If the acidotic process were involved in asphyxia the blood alkali would fall as well without hyperpnea as with it. Evidently therefore, oxygen deficiency as such does not cause a production of acid in the tissues, or at least, no passage of such acids into the blood, for the blood alkali is not at all or only slightly decreased. This demonstration is so clear-cut a disproof of the theory of anoxemic acidosis under carbon monoxide as to bring into serious doubt the possibility of such an acidosis under any con- dition. The papers from this laboratory (15) a few years ago, and from other laboratories more recently in which the reasoning has been based upon the assumption that such a condition may occur, will require fundamental reconsideration. Evidently the increase of acidity in tissues to which the circulation is obstructed is wholly due to accumulation of H,CO3 (16), and not at all to neutralization of alkali by lactic and other strong acids. When an increase of lactic acid, or rather lactate, occurs it indicates as above shown, not acidosis, but alkalosis. CONCLUSIONS. Carbon monoxide asphyxia induces, not acidosis, but alkalosis. The lowering of blood alkali is due to the acapnial, not the acidotic, process. The anoxemia induces excessive breathing (up to 300 per cent or more), and the decrease of blood alkali is an attempt, at compensation. The rate of oxygen consumption is scarcely, if at all, decreased until death is imminent, but the respiratory quotient may be more than doubled. After section of the vagi, on the contrary, anoxemia due to carbon monoxide causes no overbreathing, and no distinct lowering of blood alkali, even up to death. This fact, appears to be a decisive demonstration that oxygen deficiency itself does not directly cause in the tissues and blood an increased production of organic acids. BIBLIOGRAPHY. 1. Araki, T., Z. physiol. Chem., 1891, xv, 335, 546; 1892, xvi, 201, 453; 1894, xix, 422. 2. Ryffel, J. H., J. Physiol., 1909-10, xxxix, p. v, ix, xxix. 3. Saiki, T., and Wakayama, G., Z. physiol. Chem., 1901-02, xxxiv, 96. 432 Hemato-Respiratory Functions. XII . Henderson, Y., and Haggard, H. W., J. Biol. Chem., 1918, xxxiii, 333, 345, 355, 365. . Haggard, H. W., and Henderson, Y., J. Biol. Chem., 1919, xxxix, 163. . Haggard, H. W., and Henderson, Y., J. Biol. Chem., 1920, xliii, 3, 15. Henderson, Y., 1920, xliii, 29. . Haggard, H. W., Am. J. Physiol., 1921 (in press). . Haldane, J. S., Kellas, A. M., and Kennaway, E. L., J. Physiol., 1919- 20, liii, 181. . Macleod, J. J. R., Am. J. Physiol., 1921, lv, 184. . Henderson, Y., Science, 1919, xlix, 431. . Henderson, Y., and Haggard, H. W., J. Pharmacol. and Exp. Therap., 1920, xvi, 11. . Henderson, Y., Haggard, H. W., and Coburn, R. C., J. Am. Med. Assn., 1920, lxxiv, 783. . Henderson, Y., and Searbrough, M. M., Am. J. Physiol., 1910, xxvi, 260. Bryant, J.,and Henderson, Y., J. Am. Med. Assn., 1915, Ixv, 1. . Haldane, J. S., Respiration, New Haven, 1921. . Henderson, Y., Am. J. Physiol., 1910-11, xxvu, 152; J. Am. Med. Assn., 1916, Ixvul, 580. . Haggard, H. W., and Henderson, Y., J. Biol. Chem., 1919, xxxviii, 77. gO ae ANTIKETOGENESIS. I. AN IN VITRO ANALOGY .* By PHILIP A. SHAFFER. (From the Laboratory of Biological Chemistry, Washington University Medical School, St. Louis.) (Received for publication, May 31, 1921.) ’ For many years the writer has been interested in the “acetone bodies,’”’ and various papers dealing with these substances have been published from this laboratory. Our main interest has been in the problems of intermediary metabolism underlying the formation of the acetone bodies; but until about 2 years ago we were not able to secure the sort of evidence which seemed to us essential for the adoption of a conception held in common with certain other workers on the subject, and our progress on these problems was slow. The missing evidence has now been found and with this encouragement the subject of ketogenesis and of the mechanism of antiketogenesis has been taken up and is being developed from several directions. It has long been common knowledge that if any human subject fasts, or merely omits carbohydrate from his food for a few days, acetone appears in his breath, and acetone and acetoacetic and 6-hydroxybutyric acids are excreted in his urine. The same substances are excreted in larger amounts by subjects of severe diabetes and are responsible for the acidosis and coma in this disease. The precursors of the acetone bodies are known to be chiefly the fats and certain of the amino-acids of protein. And the fact, * A preliminary report of the experiments described in this paper was presented in December, 1919, before the American Society of Biological Chemists; and their application to the determination of the ketogenic antiketogenic balance in man was presented before the Federation of American Societies for Experimental Biology in December, 1920 (J. Biol. Chem., 1921, xlvi, p. vi). 433 434 Antiketogenesis. I. first pointed out by Hirschfeld (1) in 1895, and since abundantly confirmed, that they appear when the amount of carbohydrate catabolized is small, demonstrates a relationship between carbo- hydrate metabolism and the production or the avoidance of production, of the acetone bodies. A legion of workers in the past two or three decades has tested a great variety of substances as to their effect in causing or preventing acetone body production and many of these substances have been definitely classed as either “ketogenetic” or ‘antiketogenetic.” But efforts to explain the character of the action of antiketogenetic substances in preventing acetone body formation have not been successful and althéugh there are many hypotheses, there is at present little direct evidence to indicate the mechanism of the reactions which may be involved. As first suggested by Geelmuyden (2) in 1904, the most likely explanation would appear to bea definite chemical reaction between some one of the acetone bodies, or one of their precursors, and glucose, or a product of its catabolism. The detailed hypotheses of Woodyatt (3) and Ringer (4) as well as of Geelmuyden are based upon this conception, but the reactions proposed by these inves- tigators have not been established and, perhaps in consequence, the underlying conception has not been developed and applied as it deserves. These hypotheses will be again referred to in the following paper. With the feeling that it should be possible to find an analogy in the test-tube to the antiketogenic action of glucose in the body the writer from time to time has sought for evidence of an effect by glucose upon the oxidation of fatty acids and of the individual acetone bodies by hydrogen peroxide. The results were uni- formly negative! until the combination of acetoacetie acid and glucose in alkaline solutions was tried. With this combination the results are striking. When hydrogen peroxide is added to such a mixture the acetoacetic acid disappears rather rapidly even at room temperature, the rate of disappearance increasing 1 Experiments of a similar character reported by Witzemann (5) led him to the opinion (6) that the action of glucose in antiketogenesis is to ‘“‘spnare’’ butyric acid from oxidation and thus prevent the formation of acetone bodies. But other evidence is opposed to such a view of the nature of antiketogenesis. Penk =p P. A. Shaffer 435 ' with the amount of glucose and the alkalinity. In strongly alkaline solution (N NaOH) at body temperature and in the presence of an excess of glucose and hydrogen peroxide the oxi- dation is complete in a few hours, while under the same conditions except for the absence of glucose, 24 hours or longer are required. In less alkaline solutions the rate of the reaction is slower, though equally striking; while in neutral or acid solution the effect of glucose is absent. With acetone, hydroxybutyric acid, or butyric acid a similar reaction has not been found. Glucose thus exhibits, in alkaline solution in vitro, a “‘ketolytic” action in hastening the oxidation of acetoacetic acid which would appear to be anal- ogous to its ‘‘antiketogenic”’ action in the body. Among other antiketogenic substances so far tried, glycerol and fructose are ketolytic while lactic acid has no such action. While the degree of alkalinity found to be necessary does not, of course, exist in the body, and one cannot therefore suppose _ the in vitro and in vivo reactions to be identical, there are facts suggesting that the alkalinity may concern primarily the “ dissoci- ation” or preliminary decomposition of glucose rather than other stages of the reaction, perhaps the same effect to which Woodyatt (7) has directed attention in comparing. the action of alkali on sugar with the action of the pancreatic hormone. At. any rate, it seems to us that the “ketolytic” action of glucose may profit- ably be studied on the assumption that it is similar to the reaction taking place in the body. And on the other hand, we are inclined to accept the existence of the in vitro reaction as strong evidence in favor of the view that antiketogenesis is to be explained by a chemical reaction between ketogenic and antiketogenic substances. In the following paper we attempt to apply this conception. In the present paper we shall present experiments which illus- trate the ketolytic action of glucose, fructose, and glycerol, and which bring out the effect of alkalinity and temperature upon the reaction. A discussion of the products of the reaction and its chemical mechanism will be postponed to a later paper. EXPERIMENTAL. Acetoacetic acid was prepared from the ethyl ester by saponi- fication with 1 or 2 N NaOH at room temperature for 24 to 36 hours, or in the incubator over night. The solution was cooled, 436 Antiketogenesis. I acidified with sulfuric acid to Congo red paper, and extracted three to six times with a half volume of ether in a separatory funnel. The ether layer was separated and shaken in a second funnel with a small volume of water containing a few drops of alizarin red and kept alkaline by the addition of NaOH. The keto-acid is quickly and completely absorbed as sodium salt by the water, and the same ether is thus used repeatedly for extrac- tion of the saponified ester solution. The alkaline solution of the salt is aerated by a strong air current for some hours to remove acetone, is filtered, and kept in the ice box. The acid is gradually decomposed into acetone and carbon dioxide, but in alkaline solution at room temperature or below, the rate of decomposition is slow, amounting to less than 1 per cent per day (Engfeldt (8)). When the solutions are boiled, preferably in acid solution, the keto-acid is rapidly and quantitatively decomposed into acetone which may be determined in the distillate by the iodine titration. In alkaline solution hydrogen peroxide alone very slowly oxidizes acetoacetic acid at ordinary temperatures. At boiling temperature, during distillation the oxidation is more rapid but the rapid decomposition of the acid into acetone, which is quite resistant to peroxide and quickly distills off, prevents great loss. Oxidation during distillation may be wholly avoided by aeidi- fying the mixture before distillation, when neither acetoacetic acid nor acetone is attacked by hydrogen peroxide, and the yield of acetone is quantitative. The procedure usually followed in the experiments described below was to mix known amounts of sodium or calcium acetoace- tate in slightly alkaline solution, sodium hydroxide, hydrogen peroxide, and glucose, some one or more of the components being omitted in various controls. All the mixtures were diluted to the same volume, usually 500 or 1,000 cc., the flasks stoppered and placed at about the same temperature. In the early experiments the solutions were at once heated to boiling and distilled. Under these circumstances the reaction takes place only until, as the temperature rises, the acetoacetic acid is decomposed into acetone and carbon dioxide. In the later experiments the solutions were allowed to stand at room temperature or in the incubator and from time to time portions were withdrawn and the acetoacetic acid remaining, re P. A. Shaffer 437 together with small amounts of acetone formed by the slow ketone decompositon, was determined as acetone after distillation from the acid solution. As stated above the reaction does not take place in acid solution; but since volatile acids and small amounts of hydrogen peroxide may pass into the distillate from acid solutions, the first distillate was usually redistilled from alkaline solution (NaOH) or after the addition of sodium peroxide to remove possible aldehydes. In either case the peroxide is decomposed, and is without action upon acetone. Experiment 11.—One of the early experiments is recorded in Table I. 50 ce. of an approximately 0.4 mol calcium acetoacetate solution were placed in each of four flasks, to which were added solutions of glucose and NaOH as stated in the table, and the mixtures were diluted to 100 cc. After 3 days at room temperature, 5 cc. portions were diluted to 300 ce. and distilled, after adding (a) 25 ec. of 5N H2SOu, (b) 25 ec. of 3 per cent H.O2 and NaOH to make the solution 0.25 nN, and (c) H2O2 and 0.5 Nn NaOH. TABLE I. — Effect of Glucose and Alkali on the Oxidation of Acetoacetic Acid by H20:. Experiment 11. Solutions mixed and distilled after 3 days at 20+°C. Redistilled distillates after adding Na2O». In 100 ce. of solution. First distillation from: 5 Z z Solution = 4 s ie a No. Ca aceto- : 5 Z fe} o3 oe eoctate Glucose. NaOH = = S < Acetoacetic acid as acetone. Found. | Decomposed in distillation. | | p’ millimols | millimols | millimols mg. | mg. La mg. oes mg. ee ik 20 0 0 1,114 | 16 | 1.4) 138)12.5) 118/10.6 II 20 20 0 1,128 | 60 | 5.3, 46040.8) 694/61.5 Ill 20 0 20 1,084 | 72 | 6.6] 220|20.3} IV 20 20 | 20 | 1,064 | a2 | 6.7| 492 46.2 The results allow the following conclusions. (1) There was little, if any, disappearance of acetoacetic acid on standing at room temperature with glucose (in absence of peroxide) in either 438 Antiketogenesis. I neutral or alkaline solution. (2) On distillation in the presence of hydrogen peroxide in ‘‘neutral” solution, 1.4 to 6.7 per cent of the keto-acid was oxidized, while a much larger amount dis- appeared on boiling with peroxide in alkaline solution. In the absence of glucose, the loss was 10 to 20 per cent, but in the presence of glucose 40 to 61 per cent of the acetoacetic acid disappeared. Since the keto-acid quickly decomposes into acetone, which as other experiments show is not similarly destroyed by glucose and peroxide, 30 to 40 per cent of the keto-acid was decomposed through the presence of an equimolecular amount of glucose within the 5 or 10 minutes taken to warm the solution to boiling and to split off acetone from those molecules of the acid which had not already been converted into a different substance. Other experiments of the same kind but with larger amounts of glucose showed almost complete decomposition of the acid. Experiment 14. (Table II) Distillation of Ca Acetoacetate, Glucose, and H202.—50 cc. of acetoacetate solution (equivalent to 0.7 millimol or 41 mg. of acetone) + 25 ce. of 5 Nn NaOH (= 0.4 N) + 11.5 ce. of 3 per cent H.O: (= 10 millimols) total volume 300 cc. Glucose added as below, heated slowly, and distilled 25 minutes. Distillates redistilled after acidi- fying with H.SOx,. TABLE II. Experiment 14. In 300 cc. of Acetoacetic acid oxidized. oe | 0.4 peat a ti 0.7 milli Acet % Nou ESR Gets. ad _| Corrected for effect of H2O2 alone. 10 millimols H202 Total. and glucose: Total. Glucose. ee ee millimols mq. per cent millimols per cent mols it None. 36 12 II 0.5 18 | 56 OFSleaa 43 0.6 iil ih) 5 88 | O752 >] 76 0.5 IV 2.5 2 95 | Od) 83 0.24 V 5.0 4 90 |- 0355R5 78 OcDE The results show that under the relatively unfavorable conditions during heating to boiling and distillation an excess of glucose accomplishes the decomposition of nearly all of the acetoacetic acid, and that 1 millimol of glucose is as effective (for 0.7 millimol of acetoacetate) as a larger amount. OOOO EEE EEE OOO ————<_ ee eee ee P. A. Shaffer 439 Experiment 18. (Table IIIT).—This experiment shows a marked increase in the ketolytic action of glucose with increasing alkalinity of the mixture. 25 cc. of Ca acetoacetate solution (= 0.7 millimol or 41 mg. of acetone) were added to 1 millimol of glucose and 10 millimols of H.O2 in a volume of 250 ec., and after adding NaOH to give the final alkalinity stated, the solutions were distilled and the distillates redis- tilled from dilute H2SO,. TABLE III. Experiment 18. Immediate distillation. In 250 ce. of solution: Acetoacetate oxidized 0.7 millimols Ca acetoacetate. Alkalinity Acetone as acetone. 1.0 ee glucose. NaOH. found. 10.0 Fa H202: Total. N mg. mg. per cent ‘ i + Neutral. 41 II 0.01 36 5 12 Ill 0.10 28 13 32 IV 0.20 13 28 68 V 0.5 @ 34 83 Experiment 34. (Table IV) Ketolytic Action of Glucose at 20°C.—After mixing as stated in Table IV the solutions stood in stoppered flasks at room temperature (20+°C.) for 16 hours, when they were acidified with H.2SO, and distilled. In acid solution both oxidation by peroxide and the effect of glucose are avoided, and the results represent therefore the reac- tion which took place at room temperature. The distillates were redistilled from Na2Oo. TABLE IV. Experiment 34. 16 hours at 20°C. ee ease ee oe eee | acetone pana] Gee mg. per cent 1 Control, no H2O2, no glucose. 73 II 22 millimols H2O2, no glucose. 67 8 MTS (22 H.02 + 2.5 millimols 19 74 glucose. Experiment 44. (Table V).—Solutions were mixed as stated below. After standing at room temperature (20+°C.) for 48 hours, 50 ec. of each were acidified, diluted, and distilled. Distillates were redistilled from NaOH to remove volatile acids and peroxide. 440 Antiketogenesis. I TABLE V. Experiment 44. In 500 ce. of 0.6 N NaOH: After 48 hours at 20°C. Solution Acetoacetic oxidized No. Gtstes Waa corrected for H2O: alone. sontate: Glucose. H202 Werk: For 1 mol glucose. millimols millimols millimols mg. mol I 20 feiss II 20 100 592 10.0 Ill 20 100 958 Sin 0f IV 20 4 100 360 4.0 1.0 V 20 10 100 126 6.3 0.6 VI 20 20 100 118 6.4 0.3 TABLE VI. Experiments 45 and 54. 24 hours at 20°C. Acetoacetate oxidized. Acetone Soluti 8} ) lution paaeto Gilgcose: HO found after ve Corrected for H2O2. Total. | Ee ae millimols millimols | mittimols | mg. millimols | 0.5 n NaOH I 6.6 0 0 384 0 1ul 6.6 0 100 259 Daley IOUS 6.6 145) 100 204 346 7 IL (0 0.8 IV 6.6 2.50 100 167 3.74 1.59 0.6 V 6.6 3.75 100 125 4.46 Bar 0.6 VI 6.6 5.0 100 107 At 2.62 0.5 VII 6.6 10.0 100 39 5.95 3.8 0.4 1.0 n NaOH I 6.0 0 oD 301 II 6.0 3.0 35 142 2.74 0.91 III 6.0 50 35 117 | 3.18 0.63 From the results of this experiment it appears that if allowance be made for the amount of acetoacetic acid oxidized by peroxide in the absence of glucose, glucose may accomplish the transfor- oa aa es a ——“«™ P. A. Shaffer 44] mation of an equimolecular amount of acetoacetic acid when reacting upon a large excess of the keto-acid (Solution IV). In the mixtures with a larger relative amount of glucose, its apparent effect is less, due in part to the error in assuming the full effect of peroxide alone in these solutions. It may be noted also that a large excess of glucose, even without peroxide (Solution III) caused some disappearance of keto-acid. This has been occasionally noted at high alkalinity, but has not been further investigated. Experiments 45 and 54. (Table VI).—The procedure was the same as in Experiment 44. The solutions stood 24 hours at room temperature (20+°C). Experiment 56.—After mixing the solutions as stated in Table VII, they stood at 30+°C. for 20 hours, after which portions of each were dis- tilled from sulfuric acid, and the distillates redistilled from NaOH. TABLE VII. Experiment 56. Acetoacetic oxidized in 20 hrs. Saree Ge sett | Gtocoee..| sda gene Corrected for 10s Total. Per mol | glucose. | millimols | millimols | mittimots | mg. millimols | millimols | In 500 ce. of 0.25 n NaOH: I | u 0 0 | 668 II 11 0 100 412 4.42 Wie i 1 100 78 10.15 5.72 | 0.52 In 500 cc. of 0.5 Nn NaOH: IV 11 0 0 632 Ve 11 0 100 366 4.5 VI 11 11 LOOM 420 10.5 6.0 0.54 Experiment 59. (Table VIII).—The results of this experiment are plotted in curves in Fig. 1, which shows graphically the relative rate of the decomposition of acetoacetic acid under these conditions, in the presence and absence of glucose. Experiment 65. (Table IX).—Rate of reaction at 304°C. The details are given in Table IX and are shown in the form of curves in Figs. 2 and 3. Attention may be called to the following points: (a) the slow oxidation of _acetoacetic acid in the form of its sodium salt in 0.25 n NaOH by H2O2 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 2 442 Antiketogenesis. I TABLE VIII. Experiment 59. Na acetoacetate + glucose + H2O2 at 25+ °C. eee In 1,000 ce. of 0.2 N NaOH: Acetone by distillation. ie RoPtor Glucose. H202 Start 3 hrs. 6 hrs. 22 hrs. millimols | millimols | millimols I 16.8 0 0 968 932 912 . 956 Il 16.8 16.5 0 944 930 908 892 Ill 16.8 0 100 1,024 942 962 810 IV 16.8 8.2 100 1,068 936 772 386 V ' 16.8 16.5 100 996 804 588 140 Wel 16.8 33 100 1,036 722 352 148 Sodium acelo acetate + HQ, 0.2N Nebt of 225°C. a fa mle F (16) il ieee fo 12 { /6 Fic. 1. Experiment 59, showing rate of ketolytic action in 0.2 n NaOH at 25+°C., by 0.5, 1.0, and 2.0 molecular equivalents of glucose. at 30°C.; (b) the increasing speed of its disappearance with increasing amounts of added glucose and with increasing alkalinity (Fig. 2); and ~ (c) the fact that an equimolecular amount of glucose (Solution V) was required to accomplish almost complete decomposition, indicating a 1:1 molecular relationship between glucose and acetoacetice acid in the reac- tion. The slope of the curve with one-half molecular equivalent of glu- cose is slightly greater than expected and is perhaps due to error in the P. A. Shaffer 443 determination, or the glucose solution added was possibly slightly stronger than was supposed. Fig. 3 shows the marked increase in the speed of the reaction with increasing alkalinity (Solutions III, V, X, and XI). TABLE IX. Experiment 65. Sodium acetoacetate and glucose at 30+ °C. At end of period solutions were acidified and distilled. Redistilled from NaOH. ota, |r nO0Oee: O25 N NaOH: | Action nioxidized acetosestets alters Repo N °: t. | Glu- 12 25 60 125 235 420 sepetes cone: H202 | min. | min. | min. | min. | min. | min. | 242": j milli- milli- | milli- mols mols mols I 20 | 0 0 1,204 1,206) 1,208 1,148 II 20 | 20 0 1,204 1,196| 1,196 1,148 fies 204) 0! | 150 1,212 1,198| 1,172] 1,120] 1,020 i | 20 |\10 «| 150 1,216 1,164| 1,054} 812} 244? Vv 20 |20 | 150 1,198 1,128} 916} 524, 32 BaP |. 20.'|-40,. | 150 1,112 1,004} 696] 182] 38 VII | 20 |20D,| 150 1,118 844, 612; 352) 39 30 150 300 min min. min viit| 20 |20D,| 300 | 1,180] 1,112] 1,088] 1,018 942 Ix | 20 |10D.| 300 | 1,196] 1,154] 1,128) 1,052 976 In 0.55 NaOH: st ead lain. x | 20 | 20 | 150 | 1,209] 1,096 4ss| 128] 26 In n NaOH. x1 | 20 | 20 | 150[1,204 [1,068] | 138] 56| 28 Another point of great interest is shown in this experiment. It will be seen that in Mixtures IV, V, and VI (Experiment 65, and Fig. 2) in which neutral, fresh solutions of glucose were added simultaneous with the peroxide, there was an interval of an hour or less before the decomposition or disappearance of acetoacetic acid began, after which, as shown by the shape of the curves, the rate of the reaction increased to a maximum. If, however, the glucose is first allowed to stand for some hours with alkali, during which time various rearrangements and ‘‘dissociations”’ 444 Antiketogenesis. I of glucose take place, as shown by the well known work of van Eckenstein, de Bruyn, and Nef (9), the reaction with acetoacetic acid and peroxide begins at once when the last two are added. In Mixtures VII, VIII, and IX of Experiment 65 the; glucose used had been previously ‘dissociated’ by standing at room temperature for 16 hours in about 0.3 Nn NaOH (D, in Solution odumaceloacelele 20 mil pod 7 JoV@0 Ci bot Fic. 2. Experiment 65, showing rate of ketolytic action of ordinary glucose as compared with the faster rate with glucose previously “‘dis- sociated”’ by treatment with alkali. VII) and 0.6 n NaOH (D, in Solutions VIII and IX). For at least 5, hours the rate of reaction of Solution VIII exceeded the rate with double the amount of ordinary glucose, Solution VI, after which time the rate falls off, but continues to exceed that of Solution V containing the same amount of ‘‘undissociated” glucose. The same phenomenon is observed in Solutions VIII P. A. Shaffer 445 and IX but in these cases the initial rate more quickly falls off, perhaps due to an injurious effect of the stronger alkali (0.6 N) on the glucose. The results in these experiments are less striking and their curves are omitted from the figure. Similar experi- ments have been repeated a number of times with essentially the same result. The rate of the oxidation of glucose alone, by peroxide, is likewise affected by previous treatment with alkali, and there appears to be no doubt that the rate of “dissociation” by alkali is one of the limiting factors determining the rate of the reaction with acetoacetie acid. eo 2 aA 6 & fo Fie. 3. Experiment 65, showing increasing rate of ketolytic action with increasing alkalinity. It is evident from this behavior that glucose must be either first “dissociated” or transformed into some other more active substance before it takes part in this reaction. This fact is at least suggestive when it is recalled that glucose is not antiketo- genic in the severe diabetic who is unable to metabolize it. One is tempted to wonder whether the diabetic could use glucose after it had been “dissociated” for him by appropriate treatment with alkali. Further consideration of this matter will be reserved for a later paper. 446 Antiketogenesis. I Ketolytic Action of Glycerol, Fructose, and Lactic Acid. The results given below in Tables X, XI, and XII, and in Figs. 4 and 5, representing Experiments 63, 68, and 69 show that fructose and glycerol have marked ketolytic action, similar to and perhaps even exceeding that of glucose. Both are antiketo- genic when fed to diabetics. Lactic acid on the other hand ap- pears not to be capable of affecting the decomposition of acetoace- tic acid under the conditions of the zn vitro experiments. Further experiments with these and other substances are in progress and will be reported in a later paper. TABLE X. Experiment 63. Na Acetoacetate + Fructose + H20, at 30°C. In 250 cc. 0.5 N NaOH: Acetone by distillation from H2SOs,and redistillation from NaOH. Solution) | No. Ae Total for 250 ce. acetoacetate.| Fructose. H202 Start 13 hrs 3? hrs millimols | millimols | millimols I 5 5 100 238 61 39 II 5 0 100 235 226 214 Tit 0 5 100 11 TABLE XI. Experiment 68. Ketolytic Action of Glycerol and Lactic Acid at 38°C. Teel (ioice monnnl NAOH: Acetone by distillation from H2SOx Solaues | DilenttallNdlacctoncetate, and redistillation from NaOH. oC: 200 ** ‘* H»eOe and: eS SS 33 hrs. 55 hrs. 234 hrs. I Control, H2O2 alone. (1,090) | (1,030) (550) EE 20 mol glucose. 221 82 93 III 20 “ glycerol. 535 148 54 IV 20 “ lactic acid. 985 834 442 TABLE XII. Experiment 69. Ketolytic Action of Lactic Acid at 38°C. Acetone by distillation from H2SO4 and In 1,000 ce. of normal NaOH: redistillation from NaOH. Bolukion 20 cc. mM Na acetoacetate + ; 200 ec. M H2O2, and: 1 hr. | 2 hrs. 3% hrs. 19 hrs. ° Control, H2O2 alone. 15122 982 547 20 m lactic acid. | 1,148 Auli ly/ 1,068 640 a a P. A. Shaffer 447 =) Sociol aceloacerlale | NV Nadhi af 36°C.| } mol Tacke ach Fig. 4. Experiments 68 and 69, showing rate of ketolytic action of glu- cose and glycerol, and absence of such action by lactic acid, in normal NaOH at 38°C. pista tf ia) ee Nanas eno ae ae HER i (ia cli ladle ba fa/ celine by dishilayay EEE aol 4 Fia. 5. Experiment 63, showing rapid ketolytic action by fructose in 0.5 n NaOH at 30+°C. joo To 448 Antiketogenesis. I SUMMARY. The foregoing experiments demonstrate that the oxidation of glucose in alkaline solution by hydrogen. peroxide accomplishes the disappearance of acetoacetic acid if the latter be present in the solution. Acetoacetic acid in the absenée of glucose or other “ketolytic”’ substance is oxidized very slowly by hydrogen perox- ide, but its disappearance is rapid even at room temperature if glucose is simultaneously oxidized. Fructose and glycerol exert the same effect as glucose, while lactic acid is without such action. The rate of the “‘ketolytic action” is increased with alkalinity, temperature, and amount of glucose or other ketolytic substance. ° The rate of the reaction appears to be determined primarily by the rate of the “dissociation” or the conversion of glucose by alkali into a derivative which is then oxidized. The inference seems justified that it is some intermediate oxidation product of glucose which combines with acetoacetic acid, the compound being then further oxidized. The details of the reaction and its products will be considered in a separate paper. The phenomenon is believed to be an zn vitro analogy to the action of glucose and of similar substances in abolishing or pre- venting the formation (accumulation) of acetoacetic acid and the related acetone and 6-hydroxybutyric acid in man. BIBLIOGRAPHY. . Hirsehfeld, F., Z. klin. Med., 1895, xxviii, 176. . Geelmuyden, H. C., Z. physiol. Chem., 1904, xli, 135. . Woodyatt, R. T., J. Am. Med. Assn., 1910, lv, 2109. . Ringer, A. I., J. Biol. Chem., 1914, xvii, 107. . Witzemann, E. J., J. Biol. Chem., 1918, xxxv, 83. . Witzemann, E. J., J. Am. Chem. Soc., 1917, xxxix, 2660. s WOO Atty heen ee EOLC Hem. Oly, sxx. On . Engfeldt, N. O., ‘‘Beitrage zur Kenntnisse der Bio Chemie der Aceton- korper,’’ Lund, 1920, 80. . Nef, J. U., Ann. Chem., 1913, cece, 204. ANanrhwWN tc ANTIKETOGENESIS. II. THE KETOGENIC ANTIKETOGENIC BALANCE IN MAN. By PHILIP A. SHAFFER. (From the Laboratory of Biological Chemistry, Washington University Medical School, St. Louis.) (Received for publication, May 31, 1921.) In the preceding paper (1) it has been shown that glucose and related substances such as fructose and glycerol exert a marked effect upon the oxidation of acetoacetic acid by hydrogen perox- ide in alkaline solution. At room temperature or in the incubator, and in the absence of glucose, alkali salts of acetoacetic acid are only very slowly oxidized by hydrogen peroxide; but if glucose is present the disappearance of the keto-acid is rapid, the rate increasing with the temperature, with increasing alkalinity of the solution, and with increasing amounts of glucose. Although the details of the reactions are not yet fully known, it is evident that a chemical reaction occurs between some derivative of glucose and acetoacetic acid, involving definite molecular quanti- ties of each substance. It is likewise evident that this ‘‘keto- lytic” action of glucose in vitro is strikingly similar to the long known “‘antiketogenic”’ action of glucose and related substances in preventing the appearance of the acetone bodies in man. AI- though the degree of alkalinity used in the experiments is not approached in the body, the cells probably possess, as pointed out by Woodyatt (2), some means of producing a similar effect. As explained in the preceding paper, it was the search for a chemi- cal explanation of ‘‘antiketogenesis’”’ which brought to light the behavior of glucose there described. The fact that the ketolytic action of glucose in vitro is a chemical reaction suggests that the effect of carbohydrate in preventing or abolishing ketonemia in man is also the result of a definite chemical reaction in the tissues. The latter conception is by no means new as will be shown by the review of the literature, but the various hypotheses in which 449 450 Antiketogenesis. IT such conceptions have been advanced have not been established, and there is at present no accepted explanation of the mechanism of antiketogenesis. The literature on the subject under discussion is so large that its detailed and complete review will not be attempted, but brief reference to a few of the more important investigations and hypotheses concerning antiketogenic action is necessary. The relationship between acetonuria and carbohydrate starva- tion, first pointed out by Hirschfeld (3) and Rosenfeld (4) has been abundantly established. All of the various conditions in which acetone and the related two acids are excreted appear to have in common the fact that abnormally small amounts of carbohydrate are being burned. The formation of acetoacetic and $-hydroxybutyriec acids does not represent a qualitative dis- turbance of metabolism; since both are readily oxidized when given to man and animals (5-7), it is more reasonable to believe that they represent normal intermediates in metabolism, which, in the absence of sufficient carbohydrate combustion, escape further oxidation (Geelmuyden,8). When they accumulate in the tissues, acetone, which is not a normal intermediate, is formed by decomposition of acetoacetic acid (8). Geelmuyden (8) in 1904 first advanced the hypothesis that carbohydrate and acetone bodies react in their intermediary metabolism by a synthesis which is necessary for the further decomposition of the acetone bodies; and that failing this synthesis their accumulation and excretion result. He assumed a conjugation with glycuronic acid as explaining the character of the reaction, and called it “‘die Hypothese von der chemischen Interferenz der intermediaren Stoffwechselproducte.”’ In a later paper, Geelmuyden (8) reit- erates this view, but no experimental evidence was adduced as to the qualitative or quantitative aspects of the chemical reac- tions between the acetone bodies and the derivative of glucose. Rosenfeld (9) proposed the less definite conception that carbohy- drate serves to catalize or set off the oxidation of fats, ‘‘Der Ziindstoff fiir die Fette sind die Kohlenhydrate.”’ That such ideas failed to take root is indicated by the remark of von Noorden (10), who, in his Handbuch concludes the dis- cussion of the action of carbohydrate in preventing acetone body formation with the statement, ‘Wie die Kohlenhydrate und antiketogenen Substanzen es bewirken. ... wissen wir nicht.” a ‘ ss 2 6.6 6.9 5) 100° a 9 iil fs) 6 100m Ee 5 9.8 It is evident that the border-line of ketonuria lies between the conditions in the two periods, the first showing small but distinctly abnormal amounts of acetone bodies, whereas with the larger amount of sugar, the acetone excretion is even smaller than ob- served by Lang when on full mixed diets. The ketogenic anti- ketogenic balance and the ratio point to the conclusicn that the 1Lang (29), p. 463. P. A. Shaffer 461 acetone bodies first appear when the molecular ratio, as here calcu- lated, exceeds 1. In the period when ketonuria existed the ratio is 1.25, while it is 0.88 when ketonuria was absent. The total energy exchange is estimated by Lang at 2,500 calories. On this assumption we may calculate the materials burned on the last day of each period as follows. calories 3rd day. 9.7 gm. of urine N X 26.5 = 257 60.0 gm. of sucrose = UH 515 2,500 - 515 = 1,985 calories = 213 gm. of fat burned. calories 6th day. 9.8 gm. of urine N X 26.5 = 260 100.0 gm. of sucrose 380 640 2,500 — 640 = 1,860 calories = 200 gm. of fat burned. Antiketo- Ketogenic. pene: Ratio. millimols | millimols s 3rd day, 60 gm. of sugar. ING, Le Teta Bek 3.9 pee Bene De enc o.oo cae 97 194 LIE ie: OIE fa 10 e e OCee eee ORS ones 734 121 Sucrose, 60 gm. (63 gm. invert sugar)....... 350 TROSFEY Ih Pee eA ea a Ee Oc 831 665 25 6th day, 100 gm. of sugar. FIN OE Sis rn Sos ae Sa Sh a BN 8 98 196 Gas OR OTN occ c/s sea seen yee aka: SAN cs ose 688 114 Sucrose, 100 gm. (105 gm. invert sugar)..... 584 UT ROSES Ie eos ae BC oT II, Tk OO eee 786 894 0.88 Data of Ascoli and Preti (80).—A normal man, weighing at the start about 59 kilos and whose total energy requirement the authors estimated at 33 calories per kilo, was maintained on fat and protein for a period of several months. With changes in the amounts of food protein and carbohydrate the acetone body excretion varied from about 1 to 13 gm. expressed as hydroxy- butyric acid. 462 Antiketogenesis. IIT Toward the end of the experiment increasing amounts of car- bohydrate were added and with 45 gm. the excretion of acetone fell to 130 mg. and of hydroxybutyric acid to 100 mg. or less per day. Taking this period as representing the border-line of ketosis, I have calculated the ketogenic antiketogenic balance (day of February 9) as follows. 54 kilos X 33 calories = 1,782 calories for total energy exchange Urine nitrogen, 12.2 gm. X 26.5 calories = 323 calories from protein : 1,459 calories from fat and CH 45 gm. of glucose X 3.76 calories = 169 calories 1,290 calories from fat 1,290 + 9.4 = 139 gm. of fat burned. Antiketo- Ketogenic. ea Ratio. millimols | millimols & A a 2 PA at ROG Aas oid one | Se ete pe 122 244 Wat slip he tp Me ooo. 2. ows ee 477 79 RICO RC wane ene cs a 5a eae 250 IPG} NDI is ence eaves Gh Shon 4 OD eo 599 573 1.04 For the preceding day, (February 8) with 30 gm. of glucose added to the diet, and when 220 mg. of acetone and about 200 mg. of hydroxybutyric acid were excreted, the balance may be calculated as follows. calories Total 1,782 13 gm. of urine, N X 26.5 = 345 1,427 30 gm. of glucose X 3.76 mec) From fat = 1,324 + 9.4 = 141 gm. of fat. Ketogenic. eee Ratio. millimols | millimols x N, 13 gm... <> «ccs ace ee ok ee | 180 260 Fat, VAT gm’... |. ooo ee rete asic 483 80 Glucose; 30° gm). 3:54) ee ee ee 167 Total, ....s.5.03 605.002 ane ioe a 613 507 1.21 — P. A. Shaffer 463 It would appear from these data that the mixture being metabo- lized when the subject was on the border-line of ketonuria was such as to supply approximately equimolecular amounts of keto- genic and antiketogenic substances. Dietary of Eskimos.—Reference is occasionally made to the Eskimo as illustrating an acquired ability to oxidize large amounts of fat with little or no carbohydrate. But when one calculates the ketogenic antiketogenic balance of their dietaries the ex- planation of their reputed tolerance for fat becomes evident. The calculation by Krogh (31) of the data collected by Rink may be cited as representing such dietaries, according to which the average daily diet of an adult Eskimo contains 282 gm. of pro- tein, 135 gm. of fat, and 54 gm. of carbohydrate. The metabolism of such a mixture would not be expected to lead to acetone body production, the ratio being only 0.7. Even without all carbohydrate, the subject could probably remain practically free from acetonuria, the antiketogenic value of the protein at least equalling the ketogenic value of protein and fat, as shown below. Eskimo Dietary (Krogh). Antiketo- Ketogenie. pean Ratio. gm. gm. K molecule molecule A BROLIN oo A CIn a —745.5) Oa. Nene oe 0.453 0.906 AGRA SOB OU. cys «ae, oatn oc So Tee as 0.452 0.077 Warbolyanrate. O4ems-.. | 25.02. eee ne 0.300 “RORY Bie ee te arnt a ee Re es 2 0.905 1.283 0.7 Subject, H. S. K.—We have carried out experiments similar to those cited from the literature, with in general the same re- sults. Only one need be included in this presentation, and this will be stated briefly. The subject was a patient in the surgical service of the Barnes Hospital and was studied in the Metabolism Ward over a period while awaiting operation for a suspected brain tumor. His complaint was gradual loss of vision; no ab- normality of metabolism was detected, and as far as the subject of this paper is concerned, the patient may be considered normal. Age 56, body weight 82 kilos. He remained in bed throughout —— — ~ 80°0 ‘qoIp [eyrdsoyy ST ss 81°'0 | 60} S89 LIP G6 S| -9LT 0g9 GPS 88 CL GOT | 9S 8°8 |8P0‘?| G2 6LT | SP LT 5 98°0 | OT | &F9 OSE £6 00z SF9 SS OOT | €9 FOI | $9 |(0°OT)|SFO‘Z| $9 6LT | OS 9T » DO. | te L-|. ¥SS II 86 GIG Z89 PLS SOT | 88 GLI | 69 8'OT [80'S] 8 62ZT | OS CT» $L°0 |86'°T | 6LE OST 69 O9T L8¥ LOF O08 LG GGL | TS 8 |POr‘T! 22 $9 0Z (‘Sty G°LT) FT “990q mo | | sem | wm | | ws | oe | | | ws | wo |owe | we | | we | uo | uo Bee on | HO | Wa | ois | Tn| wea | he | HO | sea | oe BE HO | ‘38a | oly Bord O1gey aaa ee 078d 8.8 ‘orues0zOy UY *o1UeT0JO ST “pozljoqeqoyy > o “qoIq aT Te 464 ‘MSH walang ‘Il WIAVL P. A. Shaffer 465 the experiment. The resting metabolism was determined daily with the Benedict spirometer unit apparatus (not the portable), by Dr. L. P. Gay, to whom J am indebted for the results. After about 60 hours fasting, the patient was given for 2 days a diet representing about 10 per cent over his resting metabolism and containing about 10 per cent of the total calories in the form each of carbohydrate and protein, and 80 per cent as fat. It was expected that this diet would cause the continued excretion of small amounts of acetone bodies, which proved to be the case. The urines were collected in short periods and each analyzed for total nitrogen, acetone + acetoacetic acid, and hydroxy- butyric acid. On the 3rd day of the diet at 5.30 p.m., and on the 4th day additional starch was given and the total acetone body excretion dropped immediately from about 20 mg. of acetone per hous to about 4 mg. per hour. The results are summarized in Table Ii. In making the cal- culations of the ketogenic balance the following assumptions are made: (1) That the total energy exchange for the day was 10 per cent higher than the average determined rate of the resting metabolism; (2) that stored glycogen was exhausted and not available; (8) that the carbohydrate fed was burned; and (4) that the protein metabolized is represented by the nitrogen excretion. The results show that the ketogenic ratio at the time of small but definite acetone body excretion was with this case, like the preceding subjects, in the neighborhood of 1. If one reviews the data calculated from the experiments of Zeller, Lang, Ascoli and Preti, the Eskimo dietary, and the ex- periment with subject H. 8. K. it is found that metabolism of the mixtures of fat, carbohydrate, and protein in the proportions shown in Table III led to little or no acetone body excretion, and therefore, are examples of metabolic mixtures at the border- line of ketosis and ketonuria. From these data one is led to draw the conclusions: (1) that the production and excretion of acetone bodies are dependent upon the relative amounts of protein, fat, and carbohydrate in the mixture being metabolized at the time; (2) that, as here cal- culated, the border-line molecular ratio between ketogenic and 466 Antiketogenesis. IT antiketogenic factors is 1; and (8) that such diets as supply from carbohydrate at least 10 per cent, from protein about 10 per cent, and from fat not more than 80 per cent of the energy required by the subject, will produce little or no ketonuria. If the argument being developed in this paper is sound, it should be possible to apply the method of calculation not merely to the border-line of ketosis but to results from cases of extreme acidosis and ketonuria as well. I have made a number of such calcula- TABLE III. Calculated Metabolic Mixture near Threshold of Ketonuria. Per cent of total calories from: nee ydroxy- Author or subject. SSS eS Ratio. butyric Fat. Protein. Caos es ; K + Ti gm Zellerst® att wes setes 82 4 14 1.23 aa LAY eae Sis ieee ae eye 80 10 10 25 0.36 75 10 15 0.88 0 Ascoli and Preti......... 72 18 10 1.0 == 053 74 19 6 eZ +0.6 Eskimo dietary.......... 48 44 8 0.7 0(?) Ss ee ee 3 a eee 78 14 8 1828 0.74 78 14 8 iiss Oni do 13 13 1.0 0.36 74 11 15 0.9 + 0.18 tions. In most instances they show what is already indicated by a comparison of the = ratios and the amounts of total acetone bodies excreted, as given above in Table III; namely, that although the border-line of ketosis falls approximately at a ratio of 1, when the ratio is greater than 1 the excretion expected from the computation often greatly exceeds the amounts actually found. For example, when subject H. S. K. excreted about 0.7 gm. of total hydroxybutyric acid he had a calculated ratio of 1.30, and if the calculations and underlying assumptions were altogether P. A. Shaffer 467 correct he should have excreted an amount of hydroxybutyric acid corresponding to the excess of ketogenic over antiketogenic molecules, which on this day amounted to 158 millimols, or 0.158 X 104 = 16.4 gm. total hydroxybutyric acid. The realiza- tion thus falls short of the expectation and indicates that some of the assumptions are wrong. The explanation may, of course, be that some ketogenic substance may be oxidized without the intervention of ketolytic substance, but such a conclusion should be accepted only as a last resort, and if it is not possible to inter- pret the results without such additional assumption. The necessary corrections and modifications will doubtless re- quire extended investigation and it is not proposed to make such an attempt here; but it may be stated that the corrections required appear to be necessary rather for the relative values assigned to the antiketogenic factors and not for the ketogenic, which latter there is reason to believe are approximately correct. As evidence for the last statement and in general confirmation of our hypothesis a recalculation is presented of the very inter- esting case of severest diabetes, ‘Cyril K,”’ studied and reported in admirable detail by Gephart, Aub, Du Bois, and Lusk (32). Severest Diabetes. “Cyril K” of Gephart, Aub, Du Bois, and Lusk. During a few days this subject was a total diabetic and the authors state that ‘during this period . . . . all the glu- cose derivable from protein and all the beta-oxybutyric acid formed as an end-product of the oxidation of fat is completely eliminated in the urine.’”’ They further state that ‘‘a consider- able origin of beta-oxybutyric acid from (amino acids of protein) is not indicated.”” But according to my calculations of their data, the amounts of total hydroxybutyric acid excreted corre- spond fairly well with the expectations based on the assumptions, stated earlier in this paper, as to the ketogenic value of protein as well as fat. . Due to the fact that the subject during several days metabolized very little of the sugar-forming material from glycerol as well as from protein, he was thus almost wholly deprived of antiketogenic effect, and in consequence virtually all of the ketogenic mole- 468 Antiketogenesis. IIT cules catabolized appeared as acetone and acetoacetic and hydroxybutyric acids. And since antiketogenic effect is almost absent, errors in its calculation are also nearly excluded and we are enabled to test the accuracy of the computation of ketogenic factors alone. The results are on the whole quite in harmony with our estimates of the relative ketogenic values of fat and protein. The statement above quoted from Lusk and his coworkers, as to the origin of hydroxybutyric acid from protein, is based upon calculations from the respiratory quotients, which may be not wholly reliable in severe acidosis. For this reason it has seemed preferable in my computations of these data not to depend upon the respiratory quotients. The method of making the calculations may be illustrated by the following example repre- senting the first day (December 15) on which the subject was studied in the calorimeter. The hourly metabolism is given as 81.9 calories which for 24 hours would be 1,966 calories. The total acetone body excretion was about 80 gm. of hydroxybutyric acid, (70.9 gm. of hydroxy- butyric + 5.61 gm. of acetone and diacetic acid) allowing for the excretion of a few gm. of acetone in the breath. The caloric value of this amount, 80 4.69 = 375 calories, is added to the heat produced, giving 2,341 calories. The heat produced from the non-carbohydrate quota of protein is, 36.4 gm. of urine nitrogen x 12.97 calories = 472 calories leaving 1,869 calories derived from. the combustion of fat and carbohydrate, including glucose from protein. The amount of sugar burned is estimated as follows: Urine glucose — food CH = glucose excreted from protein and glycerol 167.9 gm. — 24 gm. = 143.9 gm. Glucose possible from protein = 36.4 X 3.6 = 131 gm. “Extra glucose,’’ from glycerol = 12.9 gm. According to this calculation the subject failed to use, and excreted, not only all of the glucose from protein but 12.9 gm. from glycerol. The caloric equivalent of the latter must be added to 1,869 giving 1,917 calories, equivalent to 203 gm. of fat which was metabolized (incompletely). From these data the ketogenie balance is tabulated. P. A. Shaffer 469 Antiketo- Remarks. Ketogenic. eerie millimols | millimols N, 36.4 gm... 364 0 All glucose excreted. (C15 Baaeeemeee 0 0 No food C H burned. Fat, 203 gm.. 696 45 116—(12.9 X 5.56) for glucose excreted from glycerol. Ou a oe... « 1,060 — 45 = 1,015 excess ketogenic. 1.015 X 104 = 105 gm. of total hydroxybutyrie acid expected. About 80 gm. were actually excreted. It will be noted that glucose corresponding to all of the fat catabolized is 21 gm. of which 12.9 gm. or 61 per cent were excreted. This condition continued during the next 2 days after which, with the onset of fasting, there was a sudden, though small, increase in the sugar-burning power which, coinciding with the fall in total metabolism and the amount of fat burned, resulted in the gradual approach toward ketogenic balance, until on the last day only 11 gm. of hydroxybutyric acid were excreted. The details of the calculations for other days are given in Table IV and from this the figures representing the ketogenic balance are brought together in Table V. From these data it will be seen that the basis used for the calculation of the total. ketogenic and antiketogenic factors accounts approximately for the total hy- TABLE IV. Results from Severe Diabetic, ‘‘Cyril K.”’ aa. 2be. 5 os eae ey pet’ + ’ : . : 3) Prete Gwe Wixi Heat aie ie be pres ‘ai a, pee Se ae a on Aue Pi... ae Rave _ Son ceit Siiieahe Orn | wipe i wet ie ’ rt ws hws ; YSN ey. ee vid Tr atau TT rr Mh oer fe” Aiea i F Wali “ti ch ane in ll ee é rie Ore ae B= 4 i, ~ A SIMPLE TECHNIQUE FOR THE DETERMINATION OF CALCIUM AND MAGNESIUM IN SMALL AMOUNTS OF SERUM. By BENJAMIN KRAMER anp FREDERICK F. TISDALL. (From the Department of Pediatrics, the Johns Hopkins University, Baltimore. ) (Received for publication, June 1, 1921.) Method. 1 or 2 cc. of serum are measured into an ordinary 15 cc. gradua- ted centrifuge tube containing 2 ec. of water. 1 cc. of a saturated solution of ammonium oxalate is then added and the sample mixed. The mixture is allowed to stand for half an hour. The volume is thenmade up to 6 cc. with distilled water and the sample again mixed. The tube is centrifuged at 1,300 revolutions per minute for 10 minutes. All but 0.3 cc. of the supernatant fluid is syphoned off. The precipitate is mixed with the remaining fluid and 2 per cent ammonia (2 ec. of concentrated ammonia to 98 ec. of water) is added up to 4 ec. and mixed. The tube is then centrifuged for 5 minutes. All but 0.3 ec. of the supernatant fluid is again syphoned off. The last step is repeated twice, making three washings in all. The tube is centrifuged for 5 minutes after each washing. After the last washing the superna- tant fluid is syphoned off. The crystals are suspended in the residual liquid and dissolved in 2 ec. of approximately N sulfuric acid, heated in the water bath for a few minutes, and then titrated to a definite pink color, which persists for at least 1 minute, with 0.01 N potassium permanganate. This is delivered from a micro-burette graduated in 0.02 cc. Calculations. The number of cc. of 0.01 N potassium permanganate used — the blank (volume of 0.01 N potassium permanganate required to produce the same intensity of color in an equal volume of water) X 0.2 = the number of mg. of calcium in the sample. = 475 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 3 476 Determination of Ca and Mg in Serum Reagents. The only reagent that must be quantitatively accurate is the 0.01 n sodium oxalate (Sérensen). This is used to standardize the permanganate. An 0.1 N sodium oxalate is prepared in the usual way. Sclution of the oxalate is facilitated by the addition of 5 ce. of concentrated sulfuric acid. The solution is then diluted 10 times to make an 0.01 N sodium oxalate. The latter solution will remain unchanged for several months; the former indefinitely. The other reagents are prepared in the usual way. The Magnesium Determination. 5 ee. of the supernatant fluid from the calcium determination corresponding to 1.66 cc. of serum are measured into a 30 ce. beaker, 1 ec. of (NH4)sHPO, solution is added and then 2 ce. of concentrated ammonia. The next day the sample is filtered through a well packed Gooch crucible, washed ten times with 5 ce. of 10 parts of concentrated ammonia to 90 parts of water, then twice with 95 per cent alcohol made alkaline with ammonia. The crucible is returned to the beaker and dried for a few minutes at 80° C. in the oven. 10 ec. of 0.01 Nn HCl are added to the crucible and after a few hours the entire material is transferred to a test-tube, centrifuged, and 5 ee. of the supernatant fluid are measured into a flat bottomed colorimeter tube graduated for 10 ce., which contains 2 cc. of the iron thiocyanate solution. The volume is then made up to 10 ee. with 0.01 n HCl, a rubber stopper inserted, and the fluid mixed. A series of standards is prepared by adding varying amounts of a known NH,MgPO, solution to the thiocyanate solution and bringing the volume up to 10 cc. as in the unknown samples. The color is compared by looking through the entire length of the liquid column against a white background. Calculation—The calculation is the same as in the original method: Reading (cc. of standard solution) X 0.01 X 2 X 5 X50 = mg. of magnesium in 100 cc. of serum when 2 ce. of serum are used. Preparation of Reagents. Ammonium Magnesium Phosphate Standard —This solution is made by dissolving 0.102 gm. of air-dried magnesium ammonium B. Kramer and F. F. Tisdall 477 phosphate (MgNH,PO;.6H:O) in 100 cc. of 0.1 nN hydrochloric acid and diluting to 1 liter with water. Of this solution 1 ce. is equivalent to 0.01 mg. of magnesium. Magnesium ammonium phosphate loses water of crystallization when heated and must therefore be dried atroomtemperature. Commercial preparations of the salt are generally unreliable; it should be prepared by precipitation of pure solutions.' Ammonium Phosphate Solution—This solution is made as follows: 25 gm. of (NH,)sHPO;, are dissolved in 250 cc. of H.O. 25 ec. of concentrated ammonia are added and the mixture is allowed to stand over night. The following day it is filtered, the filtrate is boiled to remove the excess of ammonia, cooled, and made up to 250 cc. This solution is diluted 5 times with water. The Ferric Thiocyanate Solution—The ferric thiocyanate solution is made from two solutions which are mixed an hour before use. Solution A is 0.3 per cent ammonium thiocyanate. Solution B is 0.3 per cent ferric chloride, made up from the salt with its contained water of crystallization, adding a few drops of acid, if necessary, to clear the solution. 5 cc. portions of Solutions A and B are mixed and the whole is diluted to 40 cc. with water. 10 Per Cent Ammonia.—100 ce. of concentrated ammonia are diluted to 1 liter. Protocols. Solutions of blood salts containing calcium and magnesium in concentrations comparable to those found in serum and as high as 30 mg. of phosphorus per 100 cc. of solution in the form of o-phosphoric acid have been analyzed by this method and the calcium recovered within 5 per cent of the amount actually present (Table I). . Table II gives the results of a series of comparative determina- tions on normal and pathological sera performed by this method and by another method previously described by us, the accuracy of which has been established (1). Table III gives the results of the estimation of magnesium in the supernatant fluid derived from the determination by either calclum method. The figures obtained show excellent agreement. 1 Jones, W., J. Biol. Chem., 1916, xxv, 87. 478 Determination of Ca and,Mg in Serum t DISCUSSION. ’ The use of ammonium oxalate for the precipitation of calcium is a well known procedure. Its application for the direct determi- nation of calcium in unashed serum was used by Pribram (2) as. early as 1871 and more recently by both de Waard (3) and Clark TABLE I. 4 Analyses of Samples of Solution B. | Calcium. Specimen No. abit = Found. Present. Pree mg. 1 0.102 2 0.098 3 0.096 EVEL OREN. don eee 0.098 0.100 = | = 4 0.198 5 0.206 6 0.200 A Veratketbg Sots 0.202 0.200 7 0.294 S 0.290 9 0.302 Average...... Oat 2. 0.296 0.300 Composition of Solution B. BOUL LAR vee ete eis 5... es SIRs Seen eee 7.739 gm. pipe ies OOPS R48. ee renee 2.005 “ oo | A Oe AR ", 0.453 “ SSM. 5s: Ae cos as + Seas weg ard ee 0.250 “ Wie8Og 7 Eee ec see. es es oe 0.189 “ Concentrated "aiGlieaes 2.5... 0255s et OVO ce: pe Water t0.. sch wee eee oot cs oe -1,000.0 cf ; re i * wae (4). The former has reported only a few determinations on cow’s serum. These are 10 to 15 per cent lower thantheresults — obtained by Meigs, Blatherwick, and Cary (5), Halverson and Bergeim (6), and Marriott and Howland (7) on material derived — from similar sources. Thus de Waard reports the pola F | B. Kramer and F. F. Tisdall 479 83 mg. of calcium per 100 cc. of cow’s serum whereas all the investigators above mentioned have found approximately 10 mg. in the same volume of serum. Clark (4) has briefly described methods for the direct determi- nation of calcium in plasma and whole blood, using ammonium TABLE II. Comparative Calcium Determinations on Serum by the Simplified Technique and the Authors’ Original Method. Ca per 100 cc. of serum. Specimen No. Diagnosis. Simplified method, | AUthors’ original mg mg 1 Normal adult. 10.0 9.6 2 a . 10.5 10.6 ; 3 tf ss 9.6 9.2 | 4 a 9.6 9.5 5 *i ‘ 9.9 9.9 - 6 ss 9.5 9.7 7 3 10.3 10.1 8 i § 10.3 10.3 9 ef = 9.8 10.1 10 y o 10.0 10.0 Y ilil “11 years old. Qa 9.7 i, Sy" <9) 10.5 Wii ail 13 Scurvy. 10.2 Dae 14 Diabetes mellitus. il ey eee TORS 15 Pyelonephritis. 10.9 10.3 16 Rickets. 9.8 9.5 il S 8.0 eal . 18 ue 7.8 7.4 ae cee ee Tetany. 7.5 on) ) eee 20 a Ts: aot, 21 ae 6.2 6.3 ie ~ OT, bao _ Chronie nephritis. 708! jks bre it. No figures are published to ae the claims made or these methods. Ina subsequent communication dealing with . of the administration of calcium salts to rabbits, results for the calcium content of plasma and whole blood methods used are not described. 480 Determination of Ca and Mg in Serum In a subsequent paper we shall discuss in detail the sources of error in the different methods that have been described for the determination of calcium in serum, plasma, and blood. In the — ; present communication we merely desire to place on record a simplified technique for the quantitative determination of this element in small amounts of serum which has been in use in this laboratory for almost a year and has given satisfactory results when tested on a large series of both normal and pathological sera. Some of these results are published in Table II. TABLE Il. Determination of Magnesium in Serum. Me per 100 ce. of serum. Name. Age. Diagnosis. aa EEE Simplified | Authors’ origi- method. nal method. mg. mg. E. B. 1 yr. Normal. 1.8 “2.8 L. B. 1p Tetany (treated). 1.8 1.8 iby e 8 tie # a} 2.4 2.1 oy ® Adult. Normal. 2a Zine S. G. R. ae 4 Dee 2.1 | OPah ceed be fd 2.1 Pal, B. K. ns ee oT 1.9 1 ep Oered Do 3 - PA Dae B. B. : 6 23 2.3 G. M. 2 yrs. Rickets. 2.2 2.2 R. G. 18 mos. Epilepsy. 1.8 1.8 CSD 14.7% Scurvy. 3.0 2.8 CONCLUSIONS. A simple and rapid en for the seieeies a pa ion » % determination of magnesium. These methods are within + 5 per cent of the amount of calcium anc present.” 2 Dr. von Meysenbug (personal communication) has on calcium method with that of Lyman and obtained excellent ag B. Kramer and F. F. Tisdall 481 BIBLIOGRAPHY. . Kramer, B., and Tisdall, F. F., Bull. Johns Hopkins Hosp., 1921, xxxii, 44. . Pribram, R., Berichte iiber die Verhandlungen der Kéniglich. Siichsi- schen Gesellschaft der Wissenschaften zu Leipzig, Math.-phys. Classe, 1871, xxiii, 279. . de Waard, D. J., Biochem. Z., 1919, xevii, 186. . Clark, G. W., Proc. Soc. Exp. Biol. and Med., 1919-20, xvii, 136; J. Biol. Chem., 1920, xliii, 89. - Meigs, E. B., Blatherwick, N. R., and Cary, C. A., J. Biol. Chem., 1919, rSegiin Ie . Halverson, J. O., and Bergeim, O., J. Biol. Chem., 1917, xxxii, 159. . Marriott, W. McK., and Howland, J., J. Biol. Chem., 1917, xxxii, 233. i rele } eae ¥ r : * i ra ' ft 2 OP . a, oe VITAMINE STUDIES. VIII. THE EFFECT OF HEAT AND OXIDATION UPON THE ANTISCORBUTIC VITAMINE.* By R. ADAMS DUTCHER, H. M. HARSHAW, anp J. S. HALL. (From the Section of Animal Nutrition, Division of Agricultural Bio- chemistry, University of Minnesota, University Farm, St. Paul.) (Received for publication, June 7, 1921.) During the summer of 1918 experiments were conducted in this laboratory which indicated that rhubarb juice and orange juice could be boiled for 15 minutes without appearing to lose their antiscorbutic potency. At the time, we were inclined to the belief that the natural acidity of these juices acted in a protective manner. Delf,! working with juices extracted from fresh cabbages, Swedish turnips, and oranges, made similar observations She suggested that the stability of the antiscorbutic vitamine, to temperatures above 100°C., might be due to the absence of air. Other workers ? *.4* have studied the influence of acidity and alkalinity upon the stability of the antiscorbutic vitamine and are in general agreement that the vitamine is stable in acid solution, while alkalinity tends to destroy it. Rossi® has shown that guinea pigs develop scurvy and die in about 20 days when subsisting upon a diet of hay and oats which * Published with the approval of the Director as Paper No. 262, Journal Series, Minnesota Agricultural Experiment Station. A portion of the expense of this investigation was defrayed by a grant from the Graduate School, University of Minnesota. 1 Delf, E. M., Biochem. J., 1920, xiv, 211. ? Weill, E., and Mouriquand, G., J. physiol. et path. gén., 1918, xvii, 849. 3 Hess, A. F., and Unger, L. J., J. Biol. Chem., 1919, xxxviii, 293. 4 Harden, A., and Zilva, 8. 8., Lancet, 1918, ii, 320. > Lamer, V. K., Campbell, H. L., and Sherman, H. C., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 122. ® Rossi, G., Arch. fisiol., 1918, xvi, 125, cited in Chem. Abstr., 1920, xiv, 1844, 483 484 Vitamine Studies. VIII has been subjected to open sterilization. When the same materials are sterilized in hermetically sealed jars guinea pigs grow well and remain healthy for 60 days or more. Recently, Hess and Unger’ and Zilva*® have shown in a more conclusive manner that oxidation has a decidedly destructive action upon solutions containing the antiscorbutie vitamine. The last mentioned writer also observed that carbon dioxide did not } inactivate lemon juice, while air possessed a very destructive action. This agrees with our findings with regard to the influence or carbon dioxide and air upon the antiscorbutic potency of heated milks.° Ellis, Steenbock, and Hart!® have shown that the antiscorbutic vitamine is susceptible to oxidizing agents although they were unable to prevent its destruction in cabbage when this foodstuff was desiccated for 35 hours at 65°C. in an atmosphere of carbon dioxide. The last mentioned observation is difficult to explain unless the destructive action was brought about by intracellular oxidation during the drying process. 9 5 EXPERIMENTAL. The experimental technique in this experiment was similar to that described in previous papers published from this laboratory, with the exception that the scorbutic diet consisted of unhulled oats (60 per cent) and chopped alfalfa hay (40 per cent). The alfalfa hay was autoclaved for 30 minutes at 15 pounds pressure and dried before mixing with the oats. 3 ec. of orange juice, treated in various ways, as indicated in Table I, were fed (daily) to each animal. Hydrogen peroxide was used as the oxidizing agent. In all cases, where hydrogen peroxide was used, 10 ec. of hydro- gen peroxide (3% per cent) were added for each 100 ec. of fresh orange juice. Those samples of orange juice which did not receive Hess, A. F., and Unger, L. J., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 143. 8 Zilva, S. S., Lancet, 1921, i, 478. * Anderson, E.V., Dutcher, R. A., Eckles, C. H., and Wilbur, J. W., Science, 1920-21, liii, 446. 10 Ellis, N. R., Steenbock, H., and Hart, E. B., J. Biol. Chem., 1921, xlvi, 367. Dutcher, Harshaw, and Hall 485 treatment with hydrogen peroxide received 10 cc. of distilled water in order to make all samples comparable as to volume. The pasteurized samples (63°C.) were heated in closed flasks in a water bath, while the boiled samples were boiled under reflux condensers to prevent loss by evaporation. All samples were prepared at the same time and under the same conditions. Suf- ficient quantities were prepared at one time to last for 1 week. After treatment all samples were cooled in running water and placed in a refrigerator. The containers were made of Pyrex glass, tightly stoppered to exclude the air. TABLE I. Experimental Diets. mental Diet. I | Basal diet + 3 cc. of raw orange juice. II ‘i “+3 “ “ orange juice + H.O, (room temperature). Ill eae eee “heated for 30 minutes (63°C.). IV ee epee “+. HO, heated for 30 min- utes (63°C.). V Basal diet + 3 cc. of orange juice boiled for 30 minutes. VI ie es a ef ‘4 ‘““ + H,O, boiled for 30 minutes. DISCUSSION. The results of the experiments are indicated in Charts I, II, and III. All the animals receiving 3 cc. of raw orange juice (Group I, Chart I) grew well and No. 357 gave birth to healthy twins. Group II (Chart I), which received orange juice treated with hydrogen peroxide at room temperature, did not grow quite as well as those in Group I and they were in much poorer physical condition, tending to softness or flabbmess. Animals 366 and 367 also gave birth to twins but the young were born dead in both instances. Comparison of the animals in Groups III and IV (Chart II) shows that pasteurization of the orange juice (Group III) had no detrimental effect, for the growth curves are as good as those which received the unheated raw orange juice (Group I). 486 Vitamine Studies. VIII Cuart II. Dutcher, Harshaw, and Hall 487 It would appear, therefore, that heat alone at 63°C. does not destroy the antiscorbutic vitamine. AIl the animals in Group IV, which had received orange juice that had been treated with hydrogen peroxide at 63°C., died with pronounced scurvy symp- toms and lesions. These animals died within 3 to 6 weeks (average 4 weeks), while those in Group III were in excellent condition at the end of 2 months. Animal 362 (Group III) also gave birth to healthy twins, indicating that pasteurization had no harmful effect. ae cae errr arg { IN Cuart III. The growth curves of the animals in Group V (Chart III) indi- eate that boiling for 30 minutes has no destructive action upon the antiscorbutic vitamine, for all of the animals were in excellent physical condition at the end of the experimental period. The influence of hydrogen peroxide during the boiling process, however, is very marked. All the animals of Group VI (Chart III) died with scurvy in less than 30 days. This would indicate that the speed of the oxidative destruction of the vitamine is hastened as the temperature increases, appearing to conform to the recog- nized chemical laws regarding the influence of heat on chemical reactions. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 3 488 Vitamine Studies. VIIT SUMMARY AND CONCLUSIONS. 1. The antiscorbutic vitamine is not destroyed by heating at pasteurization temperature (63°C.) for 30 minutes in closed vessels or by boiling (100°C.) for 30 minutes under reflux condensers. 2. Hydrogen peroxide possesses some destructive action when added to orange juice at room temperature and the destructive action is increased when the orange juice-hydrogen peroxide mix- ture is heated at 63 and 100°C, 3. The antiscorbutic properties of orange Juice are susceptible to oxidation but, in the absence of oxidizing agents, are stable to heat up to the boiling temperature of orange juice. x ae “2 A SIMPLE LABORATORY GAS METER AND AN IM- PROVED HALDANE GAS ANALYSIS APPARATUS. By H. 8S. NEWCOMER. (From the Laboratory of the Henry Phipps Institute, Philadelphia.) (Received for publication, May 23, 1921.) Carpenter (1) has described in detail the methods which are available for the study of respiratory exchange in man. Of these the simplest is that of Douglas which depends upon the collection of a specimen of expired air in a rubber bag and the determination of its CO, and Oy. percentages in a Haldane gas analysis apparatus (2). Since the publication of Carpenter’s work, several types of apparatus have appeared. They all determine the calorie production by methods of measuring the reduction in the oxygen volume in a closed system which includes the patient’s respiratory tract. The methods, while simple, are not always satisfactory. In so far as they are limited to the determination of oxygen consumption, they fail to give as much information as is frequently desired concerning the gaseous meta- bolism. This paper describes some improvements in apparatus to be used in connection with methods similar to those of Douglas. For the collection of expired air, one may use in addition to a suitable face mask and valves, either a calibrated spirometer (3) or a properly constructed rubber bag. A spirometer is less portable and more expensive. The rubber bag designed by Douglas furnishes an easy and portable means of collecting the expired air. For the measurement of the volume of its contents, it is customary to use a wet meter of the Bohr type. The Danish meters are very expensive, but those made in this country, while less elegant, are very reliable. They maintain vapor saturation of the expired air but there is no data available as to the error which they introduce by the absorption of the CO, from the air 489 490) Basal Metabolism Apparatus passed. They are not very portable and to read correctly must have the water level closely adjusted. In the case of the 745 cubic foot size, the volume read is 1 per cent low for each ;', inch rise of the water level above the mark set. The meters have a slow rate of flow and if the rate be more than about 4 cubic foot per minute there results a rise in the water level producing a contrac- BGiail. tion in the volume reading which may readily amount to 4 per cent. It is easy by increasing the rate, to cause the air to bubble through the meter and increase this error to 20 per cent or more. An inexpensive and portable meter may be adapted from the ordinary five light gas service meter (Fig.1). This meter has two soft oiled leather bellows in closed compartments, and works as indem reciprocating engine to rotate a vertical shaft at the H. S. Newcomer 491 rate of one revolution per } cubic foot of gas passed. A worm drive from this operates the dial. The top of the meter may be removed, or the meter purchased without a top, and a graduated circle and hand placed on the vertical shaft. It then reads about 3,540 ce. per revolution. The factory adjustment of the meter is made to within about 1.0 per cent of this figure. The meter may be calibrated by displacing air from a large bottle with tap water. The calibration of two such meters was done by delivering air from a 10 cubic foot standard meter prover of the spirometer type in one of the laboratories of the United Gas Improvement Com- pany. The air was delivered under pressures varying from 0.5 to to 2.5 mm. of Hg. The speed varied from 2 to 4 cubic feet a minute. A test performed. with a standard cubic foot bottle, correct to one part in ten thousand and delivering air much slower, gave the same readings as the slowest of the above speeds. During numerous consecutive runs the variation in the meter reading was at any one pressure never more than two parts in one thousand. There was a variation due to pressure changes. Up to a speed of 2 cubic feet per minute the readings were constant. At 3 cubic feet per minute (1.6 mm. of Hg) the reading for 10 cubic feet (corrected) was 0.6 per cent low and at 4 cubic feet per minute (2.5 mm. of Hg) the reading was 1.0 per cent low. When cali- brated at low speeds and used at higher speeds the meter reading can therefore be increased in these proportions. The decreased readings at higher pressures do not seem to be due to leakage through the reciprocating valves of the meter. If the zero of the dial on the main shaft be taken as the top with the meter facing the operator, then Fig. 2 represents the calibration curves of two of these meters when run in series with a Bohr meter. The hand does not run evenly around the cirele but varies by as much as 180 ce. from the expected value. The correction curves for the two instruments are about alike and the unevenness is a part of the design of the apparatus. A calibration curve of this form will correct for this error and such a correction may be applied in case the meter is used under condi- tions in which such a difference is of importance. For metabolism experiments in which the total volume is large compared with 180 ec., this error may be neglected. It is of about the order of magni- tude of the error in reading the position of a spirometer bell. 492 Basal Metabolism Apparatus The expired air is saturated with water vapor and is at a rela- tively high temperature. If it is, however, allowed to cool before being measured care must be taken that the meter is not warmer than the air for in passing through it will be warmed and no longer be vapor-saturated. To obviate this, on admitting the air to the meter, it may be passed through a water bottle slightly warmer than the meter. The temperature of the air whose volume is measured by the meter had best be taken by hanging a ther- mometer in the exit tube. The thermometer should reach its end-point quickly. | For the analysis of the expired air it is customary to use the portable gas analysis apparatus devised by Haldane (Fig. 3). The construction of this, as it is ordinarily obtained, is crude. It may be much improved by constructing a more adequate rack for the mercury leveling bulb and adding a similar one for the potash bulb. The various parts are usually connected by pieces of rubber tubing. These dry out and in any event are subject to leaks which are always suspected as possible sources of error. The principal glass parts may be blown in one piece so that there is only connecting rubber tubing below liquid levels.- It is advisable to place a mereury trap between the burette and potash bulb, a feature which frequently saves muchtime. An additional improve- ne H. S. Neweomer 493 ment is to place all the glass parts in a water bath instead of only the burette and its control. The apparatus is then less sensitive to currents of air producing temperature changes. Since it may be desirable to fill the gas burette to exactly 10 ce., it is necessary Fie. 3. to have several graduations on the potash burette, about five at millimeter intervals, one of which may serve as an initial reading. The water bath may be constructed of a copper-lined box faced in front with plate glass, using rubber tubing as a washer between 494 Basal Metabolism Apparatus the copper flange and the glass. The two racks and pinions may then be placed one at each side of the instrument and the connec- tions to the leveling bulbs brought out through corks inserted in thimbles in the walls of the bath. These rather simple improve- ments do much to make the apparatus easier to work with. SUMMARY. There is described a simple, accurate, and relatively inexpensive laboratory gas meter, being an adaptation of the common five light service meter. Some improvements in the Haldane gas analysis apparatus are described. BIBLIOGRAPHY. . Carpenter, T. M., Carnegie Inst. Washington, Pub. 216, 1915, 67. . Haldane, J. S., Methods of air analysis, London and Philadelphia, 1912. . Boothby, W. M., and Sandiford, I., Laboratory manual of the technic of basal metabolic rate determinations, Philadelphia, 1920. NT oe 1The meter with dials attached and the modified Haldane apparatus may be obtained from the Arthur H. Thomas Co., Philadelphia. LIPASE STUDIES. I. THE HYDROLYSIS OF THE ESTERS OF SOME DICARBOXYLIC ACIDS BY THE LIPASE OF THE LIVER, By ADAM A. CHRISTMAN anp HOWARD B. LEWIS. (From the Laboratory of Physiological Chemistry of the University of Illinois, Urbana.) (Received for publication, May 24, 1921.) The hydrolysis of the esters of the dicarboxylic fatty acids, malonic and succinic, by lipase has not been the subject of careful study. Doyen and Morel (1) observed that serum lipase caused hydrolysis of diethyl succinate, but the results are difficult of . interpretation from a quantitative standpoint. Kastle (2) stated that diethyl succinate was readily hydrolyzed by liver lipase, but cited no quantitative data as to the extent of hydrolysis. He also observed that the metallic salts of the monoethyl] esters of the dicarboxylic acids, e.g. sodium ethyl succinate, were not split by the lipase of the liver. He considered that the inactivity of lipase toward this class of compounds was due to the fact that they are ionized and that lipase is unable to attack ionized compounds. Morel and Terroine (3) undertook a comparative study of the hydrolysis of the diethyl esters of malonic, succinic, glutaric, suberic, and sebacic acids by the pancreatic juice of the dog, and concluded that the ease of hydrolysis increased with the increase of molecular weight of the ester up to glutaric acid. The maximum hydrolyses obtained were 2.6 per cent for ethyl malonate and 12.9 per cent for ethyl succinate. It is difficult to interpret their results inasmuch as no very definite details as to the amounts of ester and enzyme used or the dilution employed are given. It would appear, however, that the concentration of ester was such that a considerable part of it must have remained in suspension or emulsion. Moreover, the time (6 to 7 hours in most experiments) was too short to allow the reaction to reach an equilibrium, especially with the high concentration of ester in 495 496 Lipase the :eaction mixture. To obtain optimum conditions for the action of lipase, it is desirable that the concentration of the ester be such that complete solution is effected and that the dilution be sufficiently high to prevent inhibition of the action of the enzyme by the acid resulting from the hydrolysis of the ester. In the experiments to be reported in the present paper the hydrolysis of diethyl malonate and diethyl succinate by the lipase of hog liver has been studied. Every attempt has been made to observe the optimum conditions for lipolytic activity in order to obtain the maximum hydrolysis possible. This has been effected by the use of dilute solutions (0.05 to 0.0125 nN) and by frequent retitrations to neutralize the acidity developed which might inhibit the activity of the enzyme. The results obtained have confirmed the work of Kastle (2) on the stability of the metallic salts of the monoethy] esters of the dicarboxylic acids toward lipase. They indicate, however, that the hydrolysis of the diethyl esters studied can proceed only to the removal of one ethyl group and that the monoethy] esters as well as their metallic salts are stable toward lipase. EXPERIMENTAL. Preparation of Lipase-—The lipase was prepared from fresh hog liver by the use of glycerol as an extraction agent according to the method of Kanitz (4). The liver was obtained directly from the slaughter house and the extraction made within 1 hour after the death of the animal. After thorough admixture the extract was allowed to stand 2 months and then strained through cheese-cloth for use in the experiments. The use of a glycerol extract is advantageous since the glycerol not only furnishes a medium in which bacterial action is inhibited but also fails to extract fats and lipoids which by their autolysis might obscure the acidity developed by hydrolysis of the esters under investi- gation. Our observations are in agreement with the results of Simonds (5) who has reported that glycerol extracts of liver may retain their ester-splitting power unimpaired for some months. The Esters—The purity of the diethyl malonate and succinate used as substrates was checked by determinations of their boiling: points and saponification values The potassium ethyl malonate was a pure white crystalline compound. The preparation of the eee A. A. Christman and H. B. Lewis 497 acid ethyl malonate presented some difficulties. The procedure finally adopted was as follows. 4.25 gm. of pure potassium ethyl malonate were carefully weighed out and dissolved in a small volume of water, and the calculated amount of concentrated sulfuric acid was added. This was sufficient to convert the potassium salt to the monoethyl ester. After the addition of the acid the mixture was thoroughly extracted with twice its volume of ether and the extraction repeated twice. The com- bined ether extracts were then washed repeatedly with small amounts of water to remove traces of sulfates from the ether layer. After the evaporation of the ether’ at room temperature there remained a light-colored oily residue. This was carefully washed intoa volumetric flask and made up to a liter with distilled water. - No test for sulfates was obtained in this solution. For the analysis of the monoethyl ester prepared as described 25 cc. of the solution were titrated with 0.09818 n sodium hydrox- ide using phenolphthalein as an indicator, and the saponification value of this neutralized solution determined in the usual manner. Results (averages of closely agreeing duplicate determinations) of the analysis of two different preparations are given. 25 ec. of solution required 4.10 ce. of 0.09818 n NaOH for the neutraliza- tion of the free acidity and 4.20 ec. of alkali for the subsequent saponifica- tion of the ethyl group. 25 ce. of the solution required 4.00 cc. of 0.09818 n NaOH for the neutral- ization of the free acidity and 4.20 cc. of alkali for subsequent saponification of the ethyl group. These analytical results check satisfactorily with the theoret- ical for acid ethyl malonate, which would require that the alkali used for the neutralization of the free acid group should be equal to that used for the saponification of the ethyl group. It was computed also from these analyses that the normality of the solution of acid ethyl malonate was approximately 0.033 N. Determination of the Action of the Lipase on the Esters.—All hydrolyses were carried out at room temperature. To 25 cc. of the ester (usually in 0.05 N concentration) was added 0.5 ce. of the strained glycerol extract of liver and after incubation for the desired period of time, the acidity developed was titrated with 0.09818 nN sodium hydroxide with phenolphthalein as indicator. 498 | Lipase The flasks were arranged in pairs and one pair titrated after 15 minutes, a Second pair after 30 minutes, ete. Each pair of flasks was also retitrated at the intervals shown in the tables and the figures given represent the total volume of standard alkali required for neutralization. The figures presented in the tables are the averages of check determinations from which the blanks due to the acidity of the extract and of the esters have been subtracted. When ethyl propionate or butyrate has been used as substrate! it has been possible to obtain a hydrolysis of 85 to 90 per cent in 7 hours with the above procedure. The significance of the tables will perhaps be made more evident by the following explanation. In Table I with 0.05 n ester, under the heading 1 hour, the figures 3.80, 3.30, 2.75, and 1.70 ce. are given. The figure 1.70 represents the average acidity developed in two flasks which were first titrated at the end of an hour. The figure 2.75 represents the average total acidity of two flasks which were first titrated at the end of a period of 45 minutes and again 15 minutes later at the end of an hour. Simi- larly 3.30 ce. of the standard alkali were required to neutralize the acidity in two flasks which were first titrated at the end of a period of 30 minutes, and then at 15 minute intervals at the end of periods of 45 and 60 minutes. It will be noted that the rate of hydrolysis is increased by neutralization of the acidity as it develops. Thus at the end of an hour in the ease just discussed, the acidity produced was equivalent to 1.70 ec. of the standard alkali in the flasks not previously titrated, while the total acidities developed in those flasks in which 2, 3, and 4 titrations had been made during the same period were 2.75, 3.30, and 3.80 cc., respec- tively. This increased rate of hydrolysis continues up to the point of equilibrium and no further increase is then noted. Thus with 0.0125 Nn ester (Table I) the equilibrium point (1.35-1.40 ec.) is reached in 1 hour when the neutralization of acidity has been made at 15 minute intervals from the beginning of the experiment, and in 3 hours when no previous neutralization has occurred. 1 Unpublished data. A. A. Christman and H. B. Lewis 499 TABLE I. Comparative hydrolysis of 0.05, 0.025, 0.01666, and 0.0125 n solutions of diethyl succinate by lipase of hog liver, expressed in ce. of 0.09818 n NaOH required for neutralization of acid formed. 25 ee. portions of the ester solution were employed. For complete saponification 12.73, 6.36, 4.24, and 3.18 cc. of 0.09818 n NaOH, respectively, are required. Time 15 min.} 30 niin.| 45 min. ihr) sobs: | 3 hrs. | 5 hrs. | 7hrs. | 20 hrs. Naeem Standard NaOH required for neutralization of acidity. cc. cc. ce. cc. cc. cc. cc. cc. ce. 0.70* | 2.00 | 3.10 | 3.80 | 5.45 | 5.65 | 5.65 | 5.65 | 5.65 | £20. |) 2270 | 3.305).5.554))5.75 | 5275. (5.75) 5.75 | 1.65 | 2.75 | 4.80 | 5.60 | 5.65 | 5.65 | 5.65 | 1.70 | 4.75 | 5.60 | 5.60 | 5.60 | 5.60 | 2.90 | 5.60 | 5.60 | 5.60 | 5.60| 0.05 Nn 3.60 | 5.60 | 5.60 | 5.60 | 4.60 | 5.75 | 5.75 | 4.80 | 5.45 | 5.75 | 0.55 | 1.45 | 2.10-| 2.45 | 2.70 | 2.70 | 2.75 | 2.75 | 2.75 | 0.60 | 1.50 | 1.90 | 2.65 | 2.70 | 2.70 | 2.70 | 2.70 | 0.85 | 1.35 | 2760 | 2.65 | 2.65 | 2.65 | 2.65 | 0.60 | 2.05 | 2.45-| 2.50 | 2.50 | 2.50 1.65 | 2.65 | 2.65 | 2.65 | 2.65| 0.025 n 230 2:70. |, 2-70. | 2. 70° 2250} 2.70).| 2.70 | 2.45 | 2.80 | | 2.70 | 0.55 | 1.25 | 1.60'| 1.80 | 1.80 | 1.85 | 1.85 | 1.85 | 0.75 | 1.35 | 1.75 | 1.75 | 1.75 | 1.85 | 1.85 | 1.10 | 1.75 | 1.85 | 1.85 | 1.85 | 1.85 1.10) hO) |. 80: |) 1.80 (0.01666 Nn P 1.70 | 1.95 | 1.95 | 1.95 | | 1.75 | 1.85 | 1.85 | | 1.70 | 1.85 | | 1.75 | | * In all the tables, the last figure in each vertical column represents the amount of alkali required for neutralization of the acidity developed during the period represented. Each figure in the horizontal column to the right of the first figure represents this amount of alkali plus the additional amounts of alkali required for retitration at the intervals indicated. For further explanation of this and succeeding tables see the text. ~- 500 Lipase TABLE I—Continued. Time 15 min.| 30 min.|45 min.} lhr. | 2hrs. | 3hrs. | 5hrs. | 7 hrs. | 20 hrs. oa ce. ce. ce. | ce. ce. ce. cc. CC. ce. 0.30 0.80 | 1.15 | 1.35 | 1.40 | 1.40 | 1.45 | 1.45 0.50 | 1.00 | 1.30 | 1.35 | 1.85 | 1.40 | 1.45 ‘0.65 | 1.15 |.1.40 | 1.40 | 1.40 | 1.40 0.80 | 1.45 | 1.45 | 1.45 | 1.45 0.0125 n 1.20 | 1.45 | 1.45 | 1.45 1.40 | 1.45 | 1.45 1.40 |.1.50 1.45 DISCUSSION. In Table I are presented the data obtained from the hydrolysis of solutions of diethyl succinate in concentrations ranging from 0.05 to 0.0125 N by the lipase of the liver. It will be noted that regardless of the dilution of ester employed in no case was the hydrolysis greater than 50 per cent of the theoretical. Since with the esters of the monocarboxylic fatty acids (e.g., ethyl propionate) hydrolysis was nearly complete under the same conditions this seemed to indicate either that at this stage equi- librium had been reached in the reaction or that the reaction was more nearly complete than appeared and that definite products were formed other than succinic acid and ethyl alcohol. Further consideration of the results showed that the percentage of hydrol- ysis in all the various dilutions closely approximated 90 per cent of the theoretical provided only one ethyl group was removed from the diethyl ester by the action of the lipase. The fact that equilibrium was reached at approximately the same point in each dilution would further indicate that a condition of equilibrium between diethyl succinate on the one hand and ethyl alcohol and succinic acid on the other at the point where about 45 per cent hydrolysis had occurred had not been reached. If the reaction were as incomplete as this would indicate, change in the dilution of the substrate should alter the station of equilibrium and the percentage hydrolysis increase with increasing dilution. Thus Taylor (6) has shown in his studies on the action of the — lipase of the castor bean upon triacetin that under like conditions | | A. A. Christman and H. B. Lewis 501 a variation in the concentration of ester from 0.5 to 2.0 per cent resulted in a lowering of the percentage of ester hydrolyzed from 86 to 70. Kastle, Johnston, and Elvove (7) have also maintained that the more dilute the solution the greater the rate of lipolysis. In the present experiments the equilibrium corresponding to about 90 per cent hydrolysis of diethyl to monoethyl succinate was reached in the higher dilutions in 2 to 3 hours and remained unchanged thereafter. The results of the experiments with diethyl malonate as sub- strate (Table II) are also in harmony with the theory that hydrol- ysis of the diethyl ester proceeds only to the removal of one TABLE II. Hydrolysis of 0.05 n diethyl malonate by lipase. 25 ce. of 0.05 N solu- tion of ester were used. For the complete saponification of this amount of ester 12.73 ce. of 0.09818 n NaOH are required. Time...15 min.| 30 min. | 45 min. 1hr. 2 hrs. 3 hrs. | 4 hrs. | 5 hrs. 7 hrs. Standard NaOH required for neutralization of acidity. cc. cc. j cc. cc. cc. ce. CC. cc. ce. 0.65 2 2.05 3.05 3.85 5.05 5.65 5.95 6.05 0.65 1.30 eed 3 (0 4.45 5.30 7d) 6.05 1.00 Pails 2.95 4.50 5.30 5.80 6.00 1.40 MPAS 3.85 4.80 5.40 5.80 1.60 3.30 4.30 5.10 5.80 PIETED 3.80 4.70 5.65 3.30 4.35 5.60 Sele 5.05 4.00 ethyl group. The hydrolysis progressed rapidly to the point which corresponded to 85 to 90 per cent cleavage of one ethyl group and remained nearly constant thereafter. In order to test the hypothesis that the second ethyl group of the diethyl ester could not be readily hydrolyzed by the lipase a study was made of the action of lipase on the acid ethyl malonate (Table III). No appreciable hydrolysis occurred in 24 hours. In order to determine whether this inactivity of the lipase was due to the inhibitory influence of the free acid group the action of lipase on potassium ethyl malonate was also investigated (Table IV). In confirmation of the work of Kastle (2) on sodium 502 Lipase ethyl succinate and other salts of monoethyl esters, no hydrolysis was observed. Inasmuch as both the acid ethyl ester and its potassium salt had been demonstrated to be stable toward the hydrolytic action of the lipase, a series of experiments was carried out to determine whether the acidity and the presence of potassium ions in the compound were the factors which inhibited or destroyed TABLE II, The action of lipase on acid ethyl malonate. (See text for the prepa- ration of the acid tester.) 25 cc. portions were used. Initial acidity due to the acid group of ester plus the acidity of the glycerol extract is equivalent to 4.30 ec. of 0.09818 n NaOH. This blank has not been deducted. Time 30 min. 1 hr. | 3 hrs. | 4 hrs. | 5 hrs. | 7 hrs. | 24 hrs. Standard NaOH required for neutralization of acidity. cc. cc. cc. cc. cc. cc. cc. 4.35 4.35 4.35 4.35 4.35 4.35 4.55 4.30 4.30 4.30 4.30 4.30 4.65 4.25 4.25 4.25 4,25 4.50 4.30 4.35 4.35 4.55 4.30 4.30 4.55 4.30 4.50 TABLE Iv. The action of lipase on potassium ethyl malonate. 25 ec. of 0.05N solution were used. : PAMELs oo poet 30 min. 1 hr. 2 hrs. 24 hrs. Standard NaOH required for neutralization of acidity. ce. cc. cc. cc. 0.00 0.00 0.00 0.10 0.05 0.05 0.15 0.05 0.15 0.05 the lipase. Solutions were prepared containing the same concen- tration of the potassium and acid ethyl malonates as used in the previous experiments and ethyl propionate added to each. The action of lipase on these 0.05 n solutions of ethyl propionate in the presence of potassium and acid ethyl malonates is presented in Table V. The potassium salt of the ester exerted no demon- A. A. Christman and H. B. Lewis 503 strable influence on the course of the hydrolysis, the reaction proceeding at the usual rate. When acid ethyl malonate was present in the solution, the results were otherwise. No appre- ciable splitting of the easily hydrolyzed ethyl propionate occurred in 4 hours and little in 24. These experiments would indicate that the acidity of the monoethyl ester of the dicarboxylic acids studied may be one factor which inhibits the action of lipase on this type of compounds. TABLE V. The action of lipase on 0.05 N solutions of ethy! propionate in the pres- ence of acid ethyl malonate and potassium ethyl malonate. 25 ce. por- tions were used. 12.73 cc. of 0.09818 Nn NaOH are required for complete saponification of this amount of ethyl propionate. 0.05 n ethyl propionate and potassium ethyl malonate. Time. .2 hrs. 4 hrs. 12 hrs. 17 hrs. 24 hrs. Standard NaOH required for neutralization of acidity. ce. cc. ec. ce. cc. 3.30 6.25 7.80 4.00 8.15 6.95 9.55 0.05 nN ethyl propionate and acid ethyl malonate.* 0.45 2.35 0.50 2.30 *The blank due to initial acidity of acid ethyl malonate has been sub- tracted. Since all the experimental evidence thus far obtained indicated that the lipase of the liver could split the diethyl esters of the series under investigation only to the monoethy] esters, an attempt was made to isolate and identify the monoethyl derivative among the products of the lipolytic action 3.75 cc. of ethyl malonate were added to water (100 cc.) with 5 ce. of the glycerol extract, and hydrolysis was allowed to proceed with frequent neutrali- zation of the acidity by 0.09818 n sodium hydroxide, until the acidity developed over a period of several hours became almost negligible. For complete hyrolysis of this amount of ester, 506 504 Lipase ec. of the standard alkali should have been required, but it was found that even with frequent retitrations the reaction reached an equilibrium when about 240 ec. of alkali had been added, a figure which is in rather striking confirmation of the experi- mental results obtained previously with smaller amounts of ester. The reaction mixture was then filtered and carefully extracted three times with ether. This should have removed any unchanged diethyl malonate which would have been present in considerable amount if malonie acid and ethyl alcohol had been the products and the reaction had reached an equilibrium at a hydrolysis of less than 50 per cent as indicated by the titration figures. If on the other hand, the products formed were acid ethyl malonate and ethyl aleohol, the amount of unchanged diethyl malonate should have been very slight. The ether extract was carefully evaporated at room temperature, and the residue dissolved in water. Aliquots of this solution contained no material: which could be saponified either by alkali or by the glycerol extract of ~ hog liver. This furnished evidence that no significant amounts of diethyl malonate remained after hydrolysis by lipase. The solution which remained after the ether extraction was acidified with sulfuric acid and repeatedly extracted with ether. The combined ether extracts were washed several times with water to remove traces of sulfates and sulfuric acid, and the ether removed by evaporation at room temperature. The oily residue of a light yellow color was dissolved in water and made up to 200 ce. No sulfates were detected in this solution. Quan- titative tests were carried out on aliquots in the manner similar to that outlined in another part of this paper for the analysis of the acid ethyl malonate prepared from potassium ethyl malonate. If the chief product of the reaction had been malonic acid, then no saponifiable material should have been present after neutral- ization of the free acid. If ethyl acid malonate had been formed, however, the alkali required for neutralization of the free acid and that for the subsequent saponification of the ethyl group should have been the same. Analysis showed that 25 ec. of the’ preparation described required 16.30 cc. of 0.09818 N sodium hydroxide for neutralization of the free acidity and 17.43 cc. for the subsequent saponification of the neutralized aliquot. Analyses of the products of another experiment carried out under similar el lh 2 ae , —— A. A. Christman and H. B. Lewis 505 conditions showed titrations of 16.25 and 18.17 cc., respectively. When the possible sources of error in an experiment of this sort are considered, these results may be considered satisfactory and to offer further proof in support of the theory that the monoethyl ester is the main product of the hydrolysis of the diethyl esters of succinic and malonic acids by the lipase of hog liver and that the monoethy] ester is hydrolyzed with difficulty, if at all. Further studies to determine whether lipases from other sources behave similarly are in progress. SUMMARY. On the basis of the acidity developed when the lipase of hog liver was allowed to act upon the diethyl esters of succine and _ malonic acids, it is considered that the reaction proceeded to an - equilibrium which corresponded to the removal of one ethyl group from the diethyl esters. A substance was obtained from the products of the reaction between diethyl malonate and lipase which gave on analysis figures which were in good agree- ment with those required for monoethyl malonate. Lipase of hog liver was not able to effect the cleavage of monoethyl malonate or potassium ethyl malonate. Ethyl propionate was hydrolyzed by lipase in the presence of the potassium salt of monoethyl malonate, but not in the presence of the monoethyl ester itself. BIBLIOGRAPHY. . Doyen, M., and Morel, A., Compt. rend. Soc. biol., 1903, lv, 682. . Kastle, J. H., Am. Chem. J., 1902, xxvii, 481. Morel, L., and Terroine, E., J. physiol. et path. gén., 1912, xiv, 58. . Kanitz, A., Z. physiol. Chem., 1905-06, xlvi, 482. . Simonds, J. P., Am. J. Physiol., 1919, xlviii, 141. . Taylor, A. E., J. Biol. Chem., 1906-07, ii, 87. . Kastle, J. H., Johnston, M. E., and Elvove, E., Am. Chem. J., 1904, 2OS0 OVA le NO oP WwW eH i =o ae : < aa: : “Ta ; STUDIES ON EXPERIMENTAL RICKETS. VIII. THE PRODUCTION OF RICKETS BY DIETS LOW IN PHOS- PHORUS AND FAT-SOLUBLE A. By E. V. McCOLLUM anv NINA SIMMONDS, (From the Laboratory of the Department of Chemical Hygiene, School of Hygiene and Public Health, the Johns Hopkins University, Baltimore. ) AND P. G. SHIPLEY anv E. A. PARK. (From the Department of Pediatrics, the Johns Hopkins University, Baltimore.) Puates 4 To 7. (Received for publication, June 20, 1921.) INTRODUCTION. In a brief preliminary report! we described a faulty ration and its effects on the skeleton of the growing rat, which corresponded in all essential details to rickets as that disease manifests itself in the skeleton of the human being. The faulty ration in ques- tion, No. 3127, was deficient in phosphorus and fat-soluble A.2 In other respects it was, apparently, satisfactorily constituted. It contained protein of good quality and adequate in amount. The calcium was furnished in the form of the carbonate in a propor- tion (2 per cent of the total ration) which was essentially’ the amount necessary for the best promotion of growth, longevity, and reproduction. We took pains to point out that, when the 1Read before the American Pediatric Society, Swampscott, Mass., June oe ODI: 2We are at present unwilling to commit ourselves to the view that the antixerophthalmic factor and the factor concerned in the ossification and growth of the skeleton are distinct, though we are prepared to acknowledge that such may be the case. We, therefore, use fat-soluble A to describe both the antixerophthlamic and antirachitic factors, assuming that the latter has a separate existence. 507 508 Studies on Experimental Rickets. VIII content of phosphorus in the faulty ration was raised through the substitution of 2.5 per cent CaHPO, for the 2 per cent CaCOs, osteoporosis and not rickets developed. We also called attention to the fact that, when a deficiency in calcium as well as in phos- phorus was created through the omission of the CaCQ ; from the faulty ration, a pathological condition developed in the skeleton which was entirely distinct from rickets. We showed further that, when cod liver oil was added to the faulty ration in an amount equal to 2 per cent by weight of the ration, the pathologi- cal condition which developed in the skeleton was osteoporosis and had no resemblance whatsoever to rickets. We believe the results obtained in the experiments cited to be of considerable significance. In the first place, the experiments showed that it was possible to produce a pathological condition in the rat un- questionably similar to the rickets of the human being through the diet alone. In the second place, they showed that rickets could be induced by means of a ration, the faults of which were clearly defined and sharply limited; v7z., deficiencies in phosphorus and fat-soluble A. In the third place, they indicate that deficiency of phosphorus in the ration insufficiently supplied with fat-soluble A would give rise to rickets only when calcium was present in a ratio considerably higher than the calcium- phosphate ratio which is optimal for ossification. The experi- ments indicated, therefore, that the development of rickets in this instance depended on the existence in the faulty ration of (1) a specific disproportion in the caleium-phosphate ratio, the phosphorus being low, the calcium, relatively speaking, high, and (2) an insufficiency of an organic substance contained in cod liver oil having a profound influence on the calcification of cartilage and the ossification of bone. In the present paper we wish to describe further the composi- tion of the faulty ration which produced the rachitic lesions above referred to, and to present in detail the anatomical evidence on which the statement of the production of rickets was based. We also wish to describe two other similarly constituted diets, one being the diet just referred to only slightly modified, and their effects on the growth and ossification of the skeleton in the rat. : a, McCollum, Simmonds, Shipley, and Park 509 Ration 3127. per cent Rolled oats’. >... «> NA eee ee eee ene eens 40.0 G21 10 © 690) ey ee 3 OV ERT fae end oe ie Oa A YP 10.0 Wilneariy lia tbern:.- 5 sin ert 2) it so ie ae a ee ieOhes ING Oecd cocks 5 OO ee ae aan Syren 40a) coke Ne eee 1.0 ES GS ee een. 2 5 UN ae eee 1 EER ET re oe Sak 10 CaCO; AEE a EAS ole Siena oaths ORO Og Oe e Soke OLE ea sc 7240) NE) OX GT UT 0s ae ee No Wi sree aie aber clade tap case one This mixture was markedly deficient in phosphorus and in fat- soluble A (antixerophthalmic substance) but was otherwise well constituted. Its proteins were abundant and of good quality; its content of calcium not far from the optimum. So far as ‘we can judge from experimental data available, the content of other inorganic constituents was satisfactory. Whileit did not furnish much above the actual requirement of the antineuritic substance, this factor could not be regarded as influencing in any way the well being of the animals. TABLE I. Data Concerning Rats of Lot 3127. : Weight 7 No.of rat [Age when | wtentpat | Paimom | Agest | Xerorh~ | sox. | aight days gm. days gm. 642 25 41* 24 49 = Q 4] 688 25 41 35 60 = of 48 689 25 41 35 60 ae i} 46 690 BS 41 35 60 ae rot 46 708 25 41 39 64 a5 oh 48 *The entire litter of rats composing Lot 3127 was weighed together. Each recorded weight in the column does not represent the exact weight of the individual but one-sixth the total weight of the litter. The sixth rat was partly eaten after being on the diet 35 days. Ration 3133 was closely similar to Ration 3127 except that the former contained 0.5 per cent of butter fat in place of an equivalent amount of dextrin. This supplied a small amount of the fat- soluble A (antixerophthalmic substance) but not sufficient to en- tirely protect the animals against ophthalmia. This addition of butter fat was not sufficient to enable them to grow, although more liberal amounts would have done so. We know from other 510 Studies on Experimental Rickets. VIII experiments, however, that they could not have been normal or have developed normal bones even with very liberal amounts of butter fat. The 0.5 per cent of butter fat added to their diet extended the lives of the animals to some extent. TABLE II. Data Concerning Rats of Lot 3133. w: Weight | j : : No. of rat. ufom diet,| When put | MGY°" | Geach: | chaina, | Sex. | Weight. days gm. days gm. 590 16 agri" 35 51 9 35° 639 16 29 43 59 Q 27 706 “16 29 59 75 o 67 717 16 29 61 77 Q 45 S11 16 29 91 107 a 60 860 16 29 98 114 ofl 39 *The entire litter of rats composing Lot 3133 was weighed together. Each recorded weight in the column does not represent the exact we'ght of the individual but one-eighth the total weight of the litter. Two rats disappeared from the cage; they were probably eaten. Ration 3133. percent TUOMCCMOSUS RAPE I: os. asccsine « Ss + «ee oe 40.0 re AUN yee ey i rere wine a's as ke ee 10.0 BMG PURE ee ets ces ax «ss os. say eq ae op er ae 7.0 IN Se ee Ae acca ae o's) eae eon iS ee 1.0 1 a Mab is oe Chr mee ee Ce ee Se ee 1.0 be DCR UT IS ee a kine edo sce Backes W shan Ee eee eee 38.5 CA Oe a ea ae Ee iS ns ook s FRE, nisin ee 259) Beer Dh. cee eee Nici ie 54 «nae obind oO ee 0.5 Ration 3143. | pe cent N12) ea oc St a er Rhee Sse UB) } MERZ Os ne: ee eR oe ac one a8 es SP era Steels! oso be 33.0 | Geldtin )-.). 2s) ees Pe lacs 1 eee eee 15.0 | Wheat ‘gliten=s2-ie ari ais sc) eon eh eee 15.0 Wal, 2)... sche ees esis Ss ape avin bide eee 1.0 A | OF: 8 0 Nae ee) ees, 52 Se SG) 7 v This diet contains nearly twice the optimal content of calcium and is decidedly below the optimum in its content of phosphorus and of fat-soluble A. Otherwise it is well constituted. On this McCollum, Simmonds, Shipley, and Park 511 food mixture young rats grow somewhat more than on the other diets described in the paper. They do not develop xerophthalmia but become very deformed and die early. When placed upon Ration 3127 the young rat lives for not more than 5 to 8 weeks. The growth momentum derived from the normal diet results in a preliminary gain. This soon ceases, however. The weight then remains stationary and declines toward the end of the experiment. The rats of Lot 3127 were 25 days old when confined to the faulty ration, and were killed after periods varying from 24 to 39 days. TABLE III. Data Concerning Rats of Lot 3143. No, of at Age when | when put | Payson | Ageat | Xeroph- | sox. | Weight. days gm. days gm. 571 50 64 50 100 ot 81 657 50 65 63 113 2 82 695 50 67 63 113 Q 82 831 45 Os weal 43 88 ot 62 879 40 60 60 100 of 65 914 40 55 65 105 rot 68 915 40 53 65 105 fof! 15 918 40 60 76 116 je) 60 919 28 56 69 97 Q 63 At autopsy the bodies were small and greatly emaciated. AlI- most no fat was present. The eyes were inflamed. The incisor teeth were brittle. The thorax was flattened at the sides, and in one or two animals was hollowed slightly along the line of junction of the cartilages and the ribs. In none of the animals, however, was it greatly deformed. The costochondral junctions themselves formed fusiform enlargements, most evident on the internal surface of the thorax. The enlargements were thrown into especial prominence if the thoracic wall was bent inwards. Fractures of the shaft of the ribs with callous formation situated usually not far behind the costochondral junctions were present in three of the animals. The lower end of the radius and ulna and the adja cent ends of the femur and tibia formed enlargements similar to those seen in the rachitis of human beings. Whenstripped of their 512 Studies on Experimental Rickets. VIII muscles the ends of all the long bones were found to be enlarged. Fractures of the long bones of the extremities were not noted. The strength of the tibia and femur was greatly reduced and both these bones and the ribs offered little resistance when cut. Examination of the cut surface of the femur or tibia showed that the junction of cartilage and shaft was not clearly defined and that below the cartilage there lay a pale yellow zone of not very great depth which proved when examined under the microscope to be a transi- tional zone containing a mixture of elements derived from cartilage and shaft. Examination of the cut surface further showed the cortex to be extremely thin and the bone marrow to have approxi- mately normal color. The following changes were noted when the bones were examined “Ander the microscope.’ The epiphyseal cartilage separating the nucleus of ossification from the shaft was in most places broader than in the healthy animal and at several points was continued in irregular blue-staining prolongations toward or actually to the ‘shaft. Its diaphyseal border did not end abruptly on coming in contact with the elements derived from the diaphysis but seemed to undergo a gradual transition into the latter. The zone of un- differentiated cartilage was represented by few cells. Almost at their beginning, close to the nucleus of ossification, the cells of the epiphyseal cartilage became arranged in columns or fascicles separated by rather more matrix than is seen in the bones of young rats on normal diets. Near their points of origin the cells com- posing the columns were flattened as if compressed in the long direction of the shaft and lay close to each other, but at a short distance from their points of origin became oval, round, or cu- boidal. As soon as the cells of the epiphyseal cartilage underwent the expansive change just referred to, the intercellular substance which separated the cell groups became greatly diminished, so that the columns appeared to lie close to each other. Coincidently, the cells seemed to lose to a certain degree their affinity for hematoxy- ‘The technique employed in these experiments was as follows: The bones were hardened in formaldehyde. They were cut after embedment in cel- loidin in sections 15 to 25 mikra thick and stained with hematoxylin and eosin. Control preparations were made with the freezing microtome and stained with hematoxylin and also silver nitrate after the method of von Kossa, McCollum, Simmonds, Shipley, and Park 513 lin with the result that both nucleus and cell body appeared more pale. The cell membrane appeared to be unusually thick. In general the nuclei were large, round, and well preserved. The columnar arrangement of cells was at some points maintained throughout the breadth of the cartilage, but at others became irregular or entirely lost as the metaphysis was approached, so that the cells seemed to bé irregularly jumbled together. Wher- ever the cartilage cells came in proximity to the marrow ele- ments they lost their blue color and were pale yellow. In many places the cartilage after losing its staining reaction ended abruptly in contact with the blood vessels derived from the marrow. At other places, however, the cartilage was continuous with the cartilage which formed the bulk of the transitional zone between cartilage and shaft, the rachitic metaphysis (Fig. 3). The metaphysis in which the elements derived from cartilage and shaft were blended in an irregular manner was not deep. Indeed in comparison with the metaphysis in the bones of the rats on Rations 3133 and 3143, it was exceedingly shallow. It was composed of the irregular prolongations of cartilage from the main mass which retained their blue stain, and of quantities of carti- lage cells which had lost their staining reaction to hematoxylin entirely and were in all stages of transition into osteoid. There were osteoid trabecule derived largely from cartilage and having a waxy appearance. The remains of calcified intercellular sub- stance staining deep blue and having a configuration like the cross-section of a honeycomb were formed in the metaphysis. The detailed description of the various elements composing the meta- physis will be found in the description of the histological findings of the rat on Ration 3133 (Fig. 4). The cortex of the shaft near the metaphysis was broad and com- posed of a thick interlacing network of trabecule having for the most part central cores of calcified material and borders of osteoid. As the middle of the bones was approached, however, the cortex became thin. The trabecule had a central core of calcified material but peripheral portions composed of osteoid. The os- teoid zones were fairly broad. In most of them a fibrillary arrange- ment could be made out. The bone corpuscles of the calcified portion were small and those in the osteoid borders appeared especially small and far separated from each other. They pre- 514 Studies on Experimental Rickets. VIII sented a variety of shapes. Some were round, some spindle- shaped, some irregular. Moreover, they were very unevenly distributed. Some resorptive phenomena could be recognized in the calcified portions of the trabeculae. The marrow appeared normal. The nucleus of ossification showed changes both in its cartilaginous elements and in the trabecule exactly analogous to those described in the epiphyseal cartilage, and in the shaft. The rats on Ration 3133 were younger (only 16 days old) when placed upon the faulty mixture than the rats of the lot on Ration 3127, and were kept on the faulty diet for longer periods of time, on the average 63 days. The shortest period was 35 days and the longest 98 days. The rats scarcely more than main- tained their initial weight on Ration 3133. They were small and poorly nourished. The eyes of most of them were inflamed. The thorax was considerably deformed. The costochondral junc- tion was considerably enlarged. In the majority of the animals they were displaced inwards and were greatly distorted. Frac- tures of the ribs were present with well marked callous formation. The knees, wrists, and ankles were considerably enlarged, as were the ends of all the long bones. The femurs and tibias cut with greatly diminished resistance. On section the enlargement of the ends of the long bones was especially evident and seemed to correspond to the cartilage and to a white zone between the carti- lage and bone marrow which on microscopic examination proved to be the rachitic transitional zone or metaphysis. The line of junction between the cartilage and shaft was indistinct. The shafts of the long bones were not enlarged. The spleen was not increased in size in the majority of the rats but was enlarged in some. The microscopic changes in the femurs and the tibias of the rats on this diet resembled those described in the bones of the rats on Ration 3127, but were considerably further advanced. The epiphyseal cartilage was irregularly increased in depth and seemed to merge with the metaphysis in a manner presently to be described. The breadth of the metaphysis was almost if not quite equal to the breadth of the epiphyseal cartilage. The shaft of the bone was broad where it bordered on the metaphysis but rapidly contracted so that the shaft of the bone as a whole did not seem to be enlarged (Fig. 2). -_—emmmmmemep Gai Terai hls ee oe rl _———s McCollum, Simmonds, Shipley, and Park 515 The undifferentiated cartilage was*represented by only a few scattered cells or cell clusters. Almost at their beginning the cartilage cells became arranged in short columns orrather groups of columns. These were more widely separated from each other by matrix than in the healthy animal; that is, the amount of matrix seemed to be considerably in excess of that found in the rat under normal conditions. The columns or bundles of carti- lage cells just referred to took their origin at somewhat different levels. They were bullet-shaped and the ends of the columns directed toward the nucleus of ossification were for the most part pointed. The cartilage cells composing them were flat, the long dimension running from side to side. Some of them had the same thickness throughout, others were spindle- or wedge-shaped. Many of them were curved like crescents, the concave side of the crescent opening diaphysealwards. The nuclei were in some instances centrally placed, in others at the side. The nuclei were long and thin, partaking of the shape of the cell, and took a very dark stain with hematoxylin. In some cells no cytoplasm could be seen; in all, the cytoplasm if present was scant and took a paler blue stain than the cell capsule or nucleus. The cell capsule was not clearly defined and could not be easily separated from the adjacent cell capsules or surrounding matrix. In most of the bundles the arrangement of the cartilage cells was quite irregular. The cells lay one upon the other, but not in continuous columns. One row of cartilage cells would appear to interdigitate with another or a third column would seem to be interpolated between two rows of cartilage cells. The matrix in the part of this portion of the proliferative cartilage near the nucleus of ossification stained blue with hematoxylin but as the hypertrophic zone of the cartilage was approached it began to lose its blue color and appeared yellow. As the metaphysis was approached the cells of the cartilage underwent a remarkable change. The cells forming the bundles suddenly expanded assuming globular, oval, or cuboidal shapes and at the same time began to lose their power to take the blue stain of the hematoxylin and soon ceased to take it altogether. The nuclei shared in the expansion of the cells, becoming large, round, and vesicular, and took a pale blue stain. The matrix between the cell bundles which had lost its blue color earlier 516 Studies on Experimental Rickets. VIIT than the cells themselves was stained a pale yellow after hema- toxylin and eosin. As the cells increased in size, the broad zones of matrix which separated them became somewhat de- creased in amount with the result that the columns lay closer to each other. In many places, however, the broad zone of matrix which separated the bundles of flat cells above continued to exist between the columns of swollen cells nearer the diaphysis. As the cartilage cells underwent the sudden enlargement and alteration in staining reaction their columnar arrangement tended to become more definite. In certain places in which the cartilage was prolonged in solid masses into the metaphysis, the cells tended to retain their affinity for hematoxylin. The mor- phology of the cells in these irregular prolongations will be dis- cussed later. Calezfication of the cartilage was entirely wanting in all the animals on this diet. The metaphysis varied considerably in its depth, and in its general character. In the majority of the animals it was deep. -In one rat, however, which weighed only 25 gm. when killed, it was shallow. In all the animals it was made up of the various ele- ments derived from cartilage and shaft which usually compose the metaphysis of a rachitic bone intermingled in an irregular manner. There were the irregular prolongations of the epiphyseal cartilage just referred to which retained their staining reactions to hema- toxylin. There was cartilage which had lost its staining reaction altogether and seemed to be in various stages of transformation into osteoid. Many blood vessels were present, filled with red blood cells and surrounded by marrow elements. There were remnants of calcified matrix and in some rats considerable amounts of fibrous tissue (Fig. 5). In all of the rats by far the greater part of the metaphysis was composed of the cartilage described as having lost its staining reactions entirely in the process of.transformation into osteoid. This cartilage appeared in the sections lightly colored as cream color or pale yellow with eosin. It had exactly the same staining reaction as the osteoid borders of the trabecule. These cartilage cells for the most part looked swollen and presented a_ great variation in size, shape, and general morphology and composed the main bulk of the metaphysis. Many were large, and round, oval, or cuboidal in shape. Some were pear-shaped and some rs ed McCollum, Simmonds, Shipley, and Park 517 flattened. The nuclei were for the most part large and round and stained pale blue but in some the nuclei were small and deep blue or pyenotic. The cell bodies were yellow like the surrounding matrix or were actually colorless. The capsules appeared to be exceedingly thin. They were colorless in the case of many of the cells and could be distinguished only with reduced light as a transparent circle surrounding the cell. In the case of other cells the capsules retained some of the blue stain. The cartilage cells which had thus lost their staining reaction were anything but uniform. A row of globular cells like those just described might be continuous with a group of small cells, the smallest perhaps no larger than osteoblasts with correspondingly small nuclei staining deeply with hematoxylin, or they might be continuous with cells having a fiat contour like those described in the cell bundles near the epiphyseal nucleus. Cells could be found which seemed to be fading away into a substance indistinguishable from the sur- rounding matrix. No nuclei seemed to be present in them, or the nuclei were so faintly stained as to be scarcely visible and the existence of the cells could be recognized only by the faint out- line of the cell body visible under reduced illumination. In general, the cells in the metaphysis near the cartilage tended to be disposed in columns which were continuous with the columns of the cartilage proper. On the diaphyseal side of the metaphysis, however, the columnar arrangement had very largely disappeared. The large globular forms of cartilage cell were more numerous near the epiphyseal cartilage. The small forms were especially common on the diaphyseal side. The degenerating forms, also much more frequent on the diaphyseal side, were especially numerous in the immediate neighborhood of the blood vessels. Some of the blood vessels seemed to be surrounded by matrix exactly like the osteoid covering the trabecule in its staining properties and almost devoid of nuclei. The amount of matrix in which the cartilage cells lay was present in much greater quanti- ty on the diaphyseal side of the metaphysis than on the side of the epiphyseal cartilage (Fig. 7). The cells of the cartilage which retained their staining property were for the most part those composing solid prolongations from the main mass of epiphyseal cartilage. Blood vessels did not penetrate them. The retention of their normal staining proper- 518 Studies on Experimental Rickets. VIII ties and a more nearly normal morphology may have been due to the fact that they happened to have been protected fromthe influences of the diaphyseal circulation. The cells composing these irregular prolongations, however, showed considerable mor- phological variation. Near the epiphyseal cartilage they were frequently indistinguishable from the large globular cells close to the bundles of flattened cells. As the diaphysis was approached, however, the cells perhaps assumed a flat shape and appearance exactly comparable to the cells forming the bundles at the be- ginning of the epiphyseal cartilage. Nearer the diaphysis they might again become large and round and finally they might again become flat or show evidence of partial calcification or break up into small groups composed of cells globular in form and much reduced in size, with clearly defined rather thick blue staining cell capsules and separated by an abundant matrix which had lost its staining reactions to hematoxylin altogether. Blood vessels penetrated the metaphysis from the shaft, and were found at the - level at which the sudden transition in the cartilage above des- cribed took place; 7.e., to the level at which the cartilage cells suddenly became swollen and lost their staining properties. This fact would seem to indicate that the changes in the cartilage might be initiated as the result of circulatory influences. Blood vessels of all sizes could be seen ramifying in an irregular manner through the diaphysis. For the most part, however, they were not large. The large vessels terminating in huge tufts so fre- quently seen in the bones of rachitic human beings were not present. The blood vessels were filled with red blood cells and were surrounded by marrow elements. In general, it would. seem that they penetrated the cartilage rather by insinuating themselves along the intercellular ground substance separating the columns than by destruction of the cartilage cells themselves. Occasionally, however, places could be found in which the red blood cells were present within the cartilage cell capsule. Evi- dently under the abnormal conditions induced by the diet the cartilage cells could be destroyed by the vascular elements, but in general tended to persist, undergoing a slow degeneration or change into hyaline substance or possibly a change of a metaplastic nature into osteoid or connective tissue. In general, the blood vessels had a delicate endothelial lining but groups of red blood ~ ay McCollum, Simmonds, Shipley, and Park 519 cells could be found lying in spaces in the metaphysis without evidence of surrounding endothelium. In general, the meta- physis did not appear to be disrupted by the vascular elements to the extent frequently seen in the bones of rachitic human beings. Though in none of the bones of the animals on this diet was there calcium deposition in the cartilage, in almost all there were irregular calcium deposits at one point or another in the meta- physis. In one animal a zone of calcium deposition extended completely across the metaphysis, bisecting it. The calcium deposits were delicate and for the most part completely sur- rounded the cartilage cells, having in sections an appearance like that seen in a cross-section of a honeycomb. The cartilage cells in the metaphysis encased in calcified matrix showed morphologi- cal changes and loss of staining reactions comparable to those in evidence in the cartilage of the metaphysis elsewhere. In some of the interstices of the calcified matrix red blood cells and other marrow elements could be seen. In one or two of the rats there was considerable connective tissue formation exactly similar to the fibrous tissue commonly seen in the rickets of human beings. As the shaft was approached the numbers of the blood vessels increased and the numbers of cells of diaphyseal origin became more numerous. Some of them bordering the blood vessels had the morphology of osteoblasts, others of connective tissue cells. Trabeculze devoid of any calci- fied matter but containing instead cores of cartilage cells which varied greatly in size and were in various stages of transformation or degeneration into osteoid were seen in the shaftward portion of the metaphysis. The junction of the diaphysis and metaphysis in most of the rats was marked by calcified intercellular substance indicating the level in the growing end of bone at which the effect of the faulty diet first became manifest. The cortex was thin and the trabecule were covered with osteoid. The cells of the osteoid investments were for the most part elon- gated, resembling connective tissue cells, and were widely separated from each other. The osteoid had a fibrillar arrangement. In some of the animals there were large osteoid formations on one side of the bone or the other under the periosteum. Few evi- dences of resorptive activity could be found. The marrow ele- ments appeared to be essentially normal (Fig. 6). 520 Studies on Experimental Rickets. VIII In experiments with the rats of Lot 3143 the normal diet was stopped and the faulty ration substituted at ages ranging from 28 to 50 days. The animals were allowed to live on the faulty ration for from 43 to 76 days. When placed upon the faulty ration they continued to gain slowly or remained stationary in weight. They never developed inflammation of the eyes (xeroph- thalmia) on account of the presence of sufficient protective sub- stance in the wheat and maize of the diet. If this diet is con- tinued over a sufficient length of time the animals die. Just before the end of their lives rats on this ration show a definite loss of control of the posterior extremities which results in a tottering gait. The trouble is progressive and finally results in a complete paralysis of the hind legs. This condition will be fully discussed in another communication. At autopsy they were small and emaciated. The incisor teeth were loose and so brittle as to be easily fractured. The thorax was greatly deformed. Its anteroposterior diameter was greatly increased as compared with its lateral diameter and the lower part of the sternum projected forward as in the pigeon breast deformity of human beings. At the line of the costochondral junctions there were deep grooves. When the thorax was opened and the interior examined the deformity was enormous. The costochondral junctions and shafts of the ribs met at acute angles, the apices of which pointed toward the vertebral column and were not farfromit. The costochondral junctions themselves were exceedingly large and some of those of the lower ribs were twisted into the most bizarre shapes. The posterior epiphyseo- diaphyseal junctions were enlarged and appeared like so many white beads on either side of the spinal column. Many frac- tures of the shafts of the ribs were present, marked by large white callous formations. The spinal column itself was bent in various curvatures. The wrists, knees, and ankles were greatly enlarged, as indeed were the ends of all the long bones. Even the scapule were deformed. Fractures of the tibia marked by angular deformity and huge callous formation were present in some of the rats at the junction of the upper third with the lower two-thirds. The description of the microscopic changes in the bones of the animals on this diet corresponds in its main features to the de-: scription already given of the microscopic changes in the rats on Sr McCollum, Simmonds, Shipley, and Park 521 Ration 3133. We shall limit the description of the microscopic changes, therefore, to a few observations. The enlargement of the ends of the long bones was due to an increased depth and prob- ably also to an increased breadth in the epiphyseal cartilage ‘and to the presence of an enormous metaphysis. The width of the metaphysis was equal to that of the epiphyseal cartilage; its depth was many times greater. The groups of flattened cartilage cells constituting the columnar zone of the cartilage were very widely separated from each other by hyaline matrix. It was impossible not to think that the amount of hyaline matrix throughout the cartilage was greatly increased. The hyaline matrix early lost its power to take the hematoxylin stain. As soon as the cartilage cells had undergone the expansive change fully described in the case of the rats on Ration 3133 they rapidly lost their normal staining properties and underwent the changes of a metaplastic and degenerative character already described. By far the greater part of the metaphysis was composed of cartilage or derivatives from the cartilage. The osteoid trabecule of the metaphysis in the main had a cartilaginous basis; that is, inalmost all of them cartilage cells in various stages of degeneration or meta- plasia could be identified. The cartilage in every animal of the group was entirely free from lime salt deposits. Some irregular lime salt deposition was present in the metaphysis of certain of the animals. The blood vessels which ramified through the meta- physis were in the main small. The cortex wasthin. It was com- posed of bone-containing scanty central cores of calcified material and with broad osteoid borders. The breadth of the osteoid borders was extreme. The bone corpuscles found in them were widely separated from each other by quantities of yellow osteoid material. The number of cells per unit of area was con- sequently greatly diminished. The nuclei were long, some were spindle-shaped. They resembled the nuclei of connective tissue cells much more closely than those of bone corpuscles. A fibrillar arrangement of the osteoid could be made out. Few osteoblasts or at least cells which could be identified as such could be found lining the osteoid trabecule. The evidences of resorption of the calcified portions of the trabecule were slight. The bone marrow appeared to be essentially normal (Fig. 1). 522 Studies on Experimental Rickets. VIII Osteoid production was extreme in the epiphyseal nuclei of ossification. The large calluses which were formed about healing fractures were composed entirely of osteoid tissue. DISCUSSION. The faulty rations, Nos. 3127 and 3133, containing 2 per cent added calcium carbonate, gave rise to pathological conditions in the skeleton essentially identical with those found in the human subjects of rickets. The rachitic lesions in the group of rats on Ration 3133 were more advanced than those in the group of animals on Ration 3127, presumably on account of the presence’ in Ration 3133 of 0.5 per cent of butter fat. This small quantity of butter fat, while insufficient to prevent the development of xerophthalmia, was sufficient to delay its advent and to diminish its rate of progress. It increased the duration of life and made possible, presumably, increased growth of the skeleton. The small amount of butter fat in Ration 3133 probably intensified the ‘rickets-producing properties of the diet through its stimulating effect on growth and its favorable influence on the duration of life. We have already called attention to the fact that butter fat as compared with cod liver oil is exceedingly low in its rickets- inhibiting properties.* Ration 3143 was derived in part from wheat and maize, which contained a certain amount of antixerophthalmic substance (more than was present in Ration 3133, which contained 0.5 per cent butter fat). Ration 3143 further differed from Rations 3127 and 3133 in having a higher calcium-phosphate ratio than either. It gave rise to the most extreme degree of rickets. The osteoid production and the metaplastic and degenerative changes in the cartilage exceeded anything ever seen in the bones of rachitic human beings. We have repeatedly observed that, if calcium carbonate in large quantities (3 to 6 per cent of the total ration) is added to a ration insufficiently supplied with the organic factor and only slightly or not at all deficient in its content of phosphorus, most pronounced changes occur at the growing ends of the long bones. The cartilage undergoes the degenerative and metaplastic ‘McCollum, E. V., Simmonds, N., Shipley, P. G., and Park, E. A., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii. ~McCollum, Simmonds, Shipley, and Park 523 changes already described, and fails to take up calcium phosphate with any regularity, or to take it up at all. The trabecule near the end of the shaft become surrounded with enormous quantities of osteoid and great quantities of osteoid trabecule largely derived from the cartilage develop between the cartilage and the shaft. The exaggerated character of the rachitic lesion brought about by Ration 3143 is explicable on the ground that the phosphate is extremely low and the calcium high (3 per cent of the total ration in the form of calcium carbonate), so that the calcium-phosphate ratio was exceedingly high. The quantity of the antixeroph- thalmic substance contained in Ration 3143 was sufficient to pre- vent effectually the development of xerophthalmia. The quantity of the antixerophthalmic substance contained in the diet, how- ever, was insufficient to exert, or incapable of exerting, any visible ‘inhibitory influence on the development of the rachitic lesions in the skeleton. Indeed, it seems not unlikely that the amount of the organic factor contained in the wheat and maize of Ration 3148 may have intensified the development of the rachitic changes by promoting the growth of the skeleton and increasing the duration of life of the animal, as did the small amount of butter fat in Ration 3133. ; In a preliminary communication? Sherman and Pappenheimer described the production of rickets in rats by means of diets containing patent flour, 95 per cent; calcium lactate, 3 per cent; and sodium chloride, 2 per cent, with and without the addition of 0.1 per cent ferric citrate. They also described the prevention of the rickets when potassium phosphate in amounts equaling 0.4 per cent of the total was added. They also observed that, when no calcium was added to their faulty rations, osteoporosis instead of rickets developed. Patent flour is one of the most deficient foods which enters into the human diet, being exceeded in this respect only by isolated foods such as starch, sugars, fats, or polished rice. Bolted flour is rather poor in protein and this is of rather poor quality. Itis very deficient in calcium, phosphorus, sodium, chlorine, iron, and possibly also in potassium. The only essential inorganic element which it probably contains in amount sufficient to meet the physiological needs of an animal is magne- 5Sherman, H. C., and Pappenheimer, A. M., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 193. 524 Studies on Experimental Rickets. VIII sium. Bolted flour is also very deficient in the antineuritic substance (water-soluble B) as shown by experiments on animals and by the frequent occurrence of beri-beri in man in Labrador and Newfoundland, where bread from this source is a principle article of food. It is exceedingly poor in fat-soluble A, and in the organic antirachitic factor. The lack of antiscorbutie sub- stance in flour is, we believe, a matter of little or no importance in the nutrition of the rat, since this species is capable of synthe- sizing this complex. The ration employed by Sherman and Pappenheimer was, therefore, deficient in fat-soluble A, water- soluble B, its protein content, potassium, phosphate, and water- soluble C. In the presence of so considerable a number of defects in the ration it was impossible to be sure which were operative in the production of the disease. In the light of our experience it seems probable that their results should be interpreted as follows: The development of the rachitic condition in their animals was due (1) to a disproportion in the calcium-phosphate ratio, the calcium being present in optimal proportion or in a proportion not far from the optimal (3 per cent of the lactate of caletum) and the phosphorus at the lowest possible level, and (2) to a deficiency of the preventive organic substance. When neither calcium nor phosphate was added, the ratio between these (in the patent wheat - flour) was more nearly the optimum than after the calcium addi- tion, and, as was to be expected, osteoporosis, not rickets, de- veloped. The animals needed (among other things) calectum very badly; yet we witness here the apparent paradox that meeting a physiological need and without exceeding it (i.e. by adding to the diet 3 per cent calcium lactate) more damage of a particular kind is done than would have resulted from permitting the pro- nounced calcium starvation in addition to the other deficiences of their experimental diet to continue. Apparently in the rat the profound disturbances in the deposition of lime salts in cartilage and bone and the changes in the cells of those tissues which give rise to the pathological complex known as rickets may be produced by disturbances in the diet of the optimal ratio between calcium and phosphorus in the absence of an amount of an organic substance contained in cod liver oil sufficient to prevent them. It would seem from the results of a large number of experiments, which will be pub- lished in detail soon, that in so far as calcium and phosphate are con- ————— el McCollum, Simmonds, Shipley, and Park 525 cerned, the physiological relation in the diet between the two is of infinitely greater importance in insuring normal calcification than the absolute amount of the salts themselves. In examining the microscopic preparations from the rats which served as the material for the experiments reported in this paper, we were again struck with the similarity of behavior of the cartilage cell and of the bone corpuscle under the abnormal conditions im- posed by the faulty diets. On coming into direct contact with the blood vessels the cartilage cell tended to undergo a gradual de- generation or transformation into a homogeneous substance indis- tinguishable except in its lack of a fibrillar arrangement from the osteoid zone surrounding the calcified trabecule. On coming into less direct contact with the vascular elements the cartilage cell tended to revert to its original state in the undifferentiated carti- lage, or to some intermediary state of development, or to undergo metaplasia into bone corpuscles, at least such as are found in the osteoid, or into connective tissue. In the process of reversion or of metaplasia of the cartilage cell in which it underwent a diminu- tion in size, lost its characteristic staining reaction to hematoxylin, ete., 1t surrounded itself with (manufactured), or was surrounded by, an increased amount of homogeneous matrix, which remained free from lime salt deposition. The bone corpuscles in contact with the vascular elements, 7.e. those of the osteoid borders of the trabeculae, underwent a diminution in size and a change in shape, and tended to revert, if they actually did not do so, to connective tissue cells. At the same time they surrounded them- selves with (manufactured), or were surrounded by, an excessive amount of matrix, which remained free from lime salt deposition. In our experience whatever the reaction of the osteoblasts to the abnormal condition of the faulty diet, the same reaction will be manifested by the cartilage cell. The processes of bone resorption so prominent in the skeleton of rats confined for periods of some duration to faulty diets low in calcium seemed to be held in abey- ance under the particular abnormal conditions obtaining in these experiments. Only slight evidences of resorptive activity in the calcified portions of the trabecule could be found. In the pres- ence of the particular disproportion in the calcium-phosphate ratio characterizing the faulty diets used in these experiments both osteoblast and cartilage cell seemed to undergo changes which made them particularly resistant to the destructive forces ordi- narily in operation. 526 Studies on Experimental Rickets. VIII EXPLANATION OF PLATES. PLATE 4, Figs. land 2. Photomicrographs of low magnification of sections from the distal end of the femur of rats on the diet of Lots 3148 and 3133. The photomicrographs show the appearance of bones affected with a very severe rickets. There are broad zones of osteoid tissue around the trabec- ule and the epiphyseal nucleus of ossification and the diaphysis. The cartilage is persistent to a marked degree, forming a wide metaphysis. It contains no ealeium salts and is invaded by blood vessels from the shaft. (Rats 679 and 811. Diet of Lots 3143 and 3133.) Objective—Leitz micro- summar F-5 full aperture. No ocular. PLATE :5. Fic. 3. This picture shows a condition which exactly simulates that found in mild cases of ricketsin man. The osteoid borders of the trabeculze in this bone are much narrower than those of the bones shown in Figs. 1 and 2. The cartilage is persistent and irregularly invaded. There is no zone of provisional calcification. The metaphysis of the bone is narrow _and contains small scattered deposits of calcium salts. This picture was taken with the same apparatus used to photograph Figs. 1 and 2. (Rat 642. Diet of Lot 3127.) Fic. 4. Toshow detail of the growing region of the bone in Fig. 3. Note the absence of provisional calcification and the irregular prolongation and invasion of the cartilage. The metaphysis, which is narrow, is made up of trabecule of osteoid, and the metaphysis contains irregular deposits of lime salts. Objective—Leitz acromatic No. 3. No ocular. PLATE 6. Fic. 5. Photomicrograph with the same apparatus used in making Fig. 4. This picture shows the metaphysis in detail of-a bone affected with exaggerated rickets. The very broad metaphysis is composed of osteoid trabecule, traversed by a few small marrow spaces, and blood vessels which have sprouted from the vascular tree of the diaphysis. Much of this osteoid tissue was the product of metaplasia of the cells of the epiphyseal cartilage, some of which may be seen embedded in it in a condition of transition into osteoid corpuscles. (Diet of Lot 3133.) - Fic. 6. High power photomicrograph. This picture shows calcified bone, OS, surrounded by a zone of osteoid tissue, OST. Note the lami- nation of the osteoid, the small size and wide separation of the osteoid corpuscles and the endothelioid appearance of the osteoblasts which sur- round the trabecule#, O. (Diet of Lot 3143.) Objective—Leitz acromatic No. 6. No ocular. McCollum, Simmonds, Shipley, and Park 527 PLATE 7. Fig. 7. This picture shows the cartilage cells in the metaphysis during their transformation into osteoid. In it the fusion of the cells into osteoid tissue in the immediate vicinity of an invading blood vessel is well shown. (Diet of Lot 3133.) Fig. 8. Section from the distal end of the femur of a rat (No. 819) on the diet of Lot 3137. In this diet 2 per cent of cod liver oil replaced 1.5 per cent of dextrin and 0.5 per cent of butter fat. This bone was in a condition of osteoporosis with essentially normal calcification. There isa provisional calcified zone and no metaphysis has been formed. Only physiological osteoid tissue is present. 4 IB B, (MeCollum, Simmonds, Shipley, and Park: Experimental rickets. VIII.) a THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII. PEATE (6: Fie. 4. (McCollum, Simmonds, Shipley, and Park: Experimental rickets. VIII.) ) 4) - THE JOURNAL OF BIOLOGICAL CHEMISTRY,VOL. XLVII. PLATE 6. TGsG: (McCollum, Simmonds, Shipley, and Park: Experimental rickets. VIII.) : 5279 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII. PEATE V7 Fie. 8. (MeCollum, Simmonds, Shipley, and Park: Experimental rickets. VIII.) ve THE DIFFUSIBLE CALCIUM OF THE BLOOD SERUM. I. A METHOD FOR ITS DETERMINATION. By L. von MEYSENBUG, A.M. PAPPENHEIMRER, T. F. ZUCKER, anp MARJORIE F. MURRAY. (From the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York.) (Received for publication, June 22, 1921.) It is now well recognized that the calcium in the plasma does not all exist in the form of simple solution. The first conclusive demonstration of a non-diffusible calcium we owe to Rona and Takahashi (1), who subjected serum to dialysis in which all inor- ganic salts except the calcium were fully compensated for in the dialyzing fluid outside the membrane, while calcium was present in varying amounts. By such means these authors showed that on the average only 65 per cent of the calcium in the serum was capable of diffusing through a membrane. Very interesting ex- periments on the state in which calcium occurs in serum were done by Cushny (2), who filtered ox serum through collodion membranes under 150 mm. mercury pressure. He found that, while sodium, potassium, and chlorine filtered as any ordinary solution would, a portion of the calcium washeld back. The non- filterable (colloidal) calcium represented 30 to 49 per cent of the total. This colloidal, non-diffusible calcium is looked upon as a pro- tein combination and while nothing is known reyarding its real nature, it seems definite enough to warrant further study. It seemed to us worth knowing whether in conditions of abnormal calcium metabolism, such as rickets or tetany, the amount of the diffusible and non-diffusible calcium might throw any light on the processes involved. Brinkman (3) and Brinkman and Van Dam (4) have recently published observations on the calcium ion concentration in serum and emphasize strongly the importance of 529 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 3 530 Caleium of the Blood Serum. I its exact maintenance. According to Brinkman, this mainte- nance of calcium ion concentration depends on the concentration of hydrogen ion and bicarbonate ion. It appeared to us that the amount of the total calcium in solution (diffusible, non-colloidal) might be influenced by differences in [H+] or [HCO;7], since it is perfectly well known that definitely acidifying the serum (as in Lyman’s method of calcium determination) results in the total calcium being present in true solution. In this paper we describe the technique of dialysis together with observations on variation in CO, tension under which the dialysis took place, while in the following paper we record the results ob- tained on the blood in rickets and experimental tetany. TABLE I. Showing Dialysis Complete in 24 hours. COz |Cainserum | Ca in dialy- . satura- i c sate in 4 cc. | Serum | Dialy- Prams, |dinigno.| $05, | |__| aa eae a mm. Hg.| Start.| End. | Start.| End. hr. mm. mg. mg. mg. mg. mg. |per cent Jan. 20, 24 46.2 |0.420/0.384/0.168/0. 236) 10.5 | 72 | Duplicates. 1921. 48 46.2 |0.420/0.382/0.168/0.226] 10.5 | 68 A.M.P. 24 48.3 |0.420/0.396)0.209|0. 248) 10.5 | 68 i 48 48.3 0.420 0.394'0. 209 0.246) 10.5 67 24 46.0 |0.420/0.414,0. 258/0. 256) 10.5 60 48 46.0 0.420/0. 408 (0. 258/0. 266 10.5 65 | In order to carry on our studies, we have devised a method of dialyzing serum against a buffer solution of the Ringer type, at the same time maintaining a constant CQ. tension in the dialyzing system. If serum be dialyzed against a calcium-free Ringer buffer solu- tion, there appears to be a progressive dissociation of the col- loidal calcium compound, so that calcium continues to diffuse out, until at the end of 7 days 90 per cent has passed into the dialysate. When, however, calcium is added to the dialyzing fluid (“‘compen- sation dialysis’’), equilibrium is obtained in 24 hours, as Table I shows. Our method will now be described in detail. von Meysenbug, Pappenheimer, Zucker, Murray 531 Details of Method. Collection of Blood for Dialysis.—Blood is obtained by venous puncture into 100 ce. centrifuge tubes, defibrinated by whipping, centrifuged, and the serum pipetted. If not used immediately, the serum is kept stoppered in the ice box without preservative. Dialyzing Fluid.—Fluid A, used in the early part of the work, was composed of equal volumes of double strength calcium-free Ringer’s solution, and M/375 primary and secondary phosphates, having an initial pH of 7.1. The composition of the double strength Ringer’s solution was: LEN SHE cle MS Oe Sd ee Se 36.0 gm JETS 5 OD) epee pee oe ho aie bean” | ea TR, Ae 10.08 “ LG eee we 2G ot a a rs ie 1680 Prstillledwias OMOm cee ee o ce cee eee 2,000. ee _ The composition of the phosphate solution was: iG Wahl S18 15) 2k Ok oO el a 2 a a ae Lecce MOEN ADE PO a ta: ssc 5 SAI lotetiotage sins Pens 6453.5" This was diluted 25 times, so that the final concentration of phosphorus was approximately that of inorganic phosphate of blood serum, namely 0.003 gm. per 100 cc. The hydrogen ion concentration of the Ringer phosphate solu- tion after saturation with 6 per cent CO, was found to be pH 7.4. Depression of freezing point = 0.708 (18 per cent hypertonic). Fluid B was based on analytical figures for normal human plas- ma obtained from Dr. Greenwald, to whom acknowledgment is hereby ‘made for placing at our disposal his unpublished data, and its composition is given in Table II. Depression of freezing point of this fluid was 0.585 (isotonic). This solution was made up double strength, so that it could be mixed with equal volumes of double strength CaCk solutions, as will be described under the method of compensation. ‘The figures in the table represent the value of the single strength solution, ready for use. Calcium figures are not given, for these vary with the compensation de- sired. It will be noted-that the values approximate closely those for normal human plasma, with the exception of the chlorine, which is here in excess of that found normally. The buffer action of this fluid is due mainly to its bicarbonate content. The curve of Fig. 1 illustrates the CO, dissociation of the fluid. 532 Calcium of the Blood Serum. I TABLE II. Composition of Dialyzing Fluid.* Molecular Compound. Cl Na K Mg Pp concentra-~ tion. per cent per cent per cent per cent | per cent | per cent NaHCOs, 252 mg.... 69.0 0.03 KH2PO,, 8.8 mg..... PAV? 2.0 | 0.00065 MgCl, 10.59 mg..... 7.89 Heat 0.0011 GI 228:6 me... o 275i: 14.1 15.5 0.00397 Wal 625 mg: ¢;. 255.5. 380.0 246.0 0.1069 Totalsy 65.2650 eee | 40199 315.0 | 18.02 Dele 2.0 *Based on analytical figures for human plasma obtained from Greenwald. PCO, mm. mercury 10 20 30 40 50 60 594 vol. per cent 2.2 3e1 347 4.8 6.0 6.9 1.5 1.9 Fic. 1. CO, dissociation curve for dialyzing fluid B containing cal- cium. shows curve of Fluid B. - - - - represents the relation between pH and CO, tension in cat’s blood, taken from Dale and Evans. is ene. te ————— << a von Meysenbug, Pappenheimer, Zucker, Murray 533 Dialyzing Sacs.—There was considerable difficulty in obtaining satisfactory sacs. Collodion membranes did not hold back pro- tein for a sufficient length of time, and there was a progressive passage of fluid into the sac, presumably an osmotic phenomenon. Later, parchment sacs were obtained. These were Schleicher and Schiill’s Diffusions-Hiilsen, No. 579A. They proved to be impermeable to protein after 7 days dialysis at ice box tempera- ture, and after 3 days at’ 48 to 52° C. A series of six sacs was tested for individual variations in permeability, using 0.9 per cent sodium chloride against distilled water. No significant differences in rate of diffusion of chlorine ion were found. Nor was there passage of fluid into the sac containing serum as determined by inserting a graduated capillary tube into the fluid within the sac. Before using, the sacs were soaked over night in 5 per cent hydrochloric acid and washed free of acid with distilled water, and allowed to dry in an open flask covered with filter paper. The same procedure was followed with the used sacs, so as to remove all possible traces of calcium in the sac wall. . Pipettes—To obtain greater accuracy, special pipettes to con- form to the requirements of the Bureau of Standards, were made for us by E. Leitz, Inc. These pipettes, measuring 4 cc. to the tip, and graduated in 7's cc. are straight with inside diameter of 5mm. They were used for measuring the serum and dialyzing fluid before and after dialysis. Dialyzing Tubes.—Special Pyrex glass tubes were made for this work, 33 X 1 inches. The parchment sacs were suspended in these by means of cotton threads, threaded through the upper rim of the sac. The threads were looped around glass rings encircling the upper end of the sac, so as to prevent the sac from leaning against the tube, and so allowing the dialyzing fluid to creep up the sac wall. Regulation of CO, Tension.—Before dialysis, both serum and dialyzing fluid were saturated with CO, of known tension, and this tension was maintained throughout the duration of the dialysis. CO.-air mixtures of any desired proportion were obtained by driving measured volumes of air and CO, through separate gas meters fitted with precision gauges, into a 20 liter rubber bag. After allowing time for diffusion, the CO, content of the mixture 534 Caleium of the Blood Serum. I was checked with a Fredericia apparatus. The composition as determined by analysis usually varied from the theoretical by less than 0.2 volume per cent. Where the error was greater than this, the bag was emptied and refilled. For analysis, a volume of approximately 1,500 ec. was passed through the apparatus. Saturation of the dialysate and of the serum was performed by exposing 5 ce. of fluid in a 500 ce. separatory funnel through which 1,500 ec. of the gas mixture were passed (over moist glass beads). The fluid was shaken for 2 minutes. Care was taken in trans- ferring the fluid to the dialyzing tubes to avoid unnecessary exposure to the air. Method of Compensation.—A calcium chloride solution was made up roughly to contain the same calcium concentration as serum. The calcium was quantitatively determined by Lyman’s method (5) and checked by the titration method. A quantity of the solution representing the desired amount of calcium was pipetted into the dialyzing tube and evaporated to dryness, first on the water bath and then in the oven, and cooled in the desiccator. The calcium-free dialyzing fluid was then added to the dried calcium chloride. This way of adding the calcium seemed of great advantage, in that any desired amount of the calcium chloride could be pipetted and dried. It was at once found, however, that a large error had crept in and that the analytical figures for the calcium in the dialyzing system were too high. The following experiment was then performed. 3 ce. of the calcium chloride solution (= 0.273 mg. of calcium) were pipetted into the Pyrex dialyzing tube and dried as described. The dry residue was then dissolved in 3 cc. of the dialyzing fluid which had been saturated with 6 per cent CQO.,-air mixture and gave a pH of 7.4. The clear solution was then analyzed for calcium. Duplicates so treated and analyzed gave these results. Tube A, from which 2.5 cc. of solution were taken for analysis, showed 0.248 mg. of calcium; the amount of calcium in the origi- nal calcium chloride solution was 0.228 mg. Tube B. In 2.6 cc. of the redissolved calcium chloride, 0.260 mg. of calcium was found; while 2.6 cc. of the original solution contained 0.237 mg. This experiment showed us the source of our error. Calcium, even in so small amount as 0.02 mg. (Tube A) and 0.023 mg. i von Meysenbug, Pappenheimer, Zucker, Murray 535 (Tube B) was dissolved from the glass during the drying process. So small an amount would hardly seem to vitiate any results, but when working with only 2 ec. of serum, which contains about 0.2 mg. of calcium, it introduces an error of 10 per cent. To avoid this error, the calcium was added by mixing with the dialyzing fluid B an equal volume of calcium chloride standard solution. These solutions were made up in four different concentrations: Solution A, to contain 40 per cent of the serum calcium, using 10.5 me. per 100 cc. as an average figure. Analysis, 2 ec., 0.168 me. Solution B, to contain 50 per cent of the serum calcium. Analysis, 2 ec., 0.210 meg. Solution C, to contain 60 per cent of the serum calcium. Analysis, 2 ¢e., 0.258 mg. Solution D, to contain 70 per cent of the serum calcium. Analysis, 2 ec., 0.297 mg. When these calcium chloride solutions (double strength) were added in equal volume to the double strength dialyzing fluid B, there was a slow precipitation of the calcium carbonate, and so the solutions were kept separate until immediately before being used. Saturation with the COQ.-air mixture prevented this precipitation. Technique of Dialysis.—4 ce. of saturated dialyzing fluid are pipetted from the separatory funnel into the dialyzing tube, which has been previously filled with the same CO, mixture as that used for saturation. It is then corked. The pH of the dialysate is determined with the excess of dialyzing fluid in the funnel by the colorimetric method of Levy, Rowntree, and Marriott (6). 4 ce. of the saturated serum are pipetted into a dry sae which is then suspended by the attached threads in the dialyzing fluid, but with the serum level above that of the outer fluid level. This is done because when the sac has become thoroughly moistened, it expands and the level inside falls, while that outside rises. The tube is then closed with a paraffined cork, through which passes a glass tube. The air in the tube is replaced by the COs-air mix- ture, an outlet being provided by a slit in the cork, and the tube sealed with paraffin. The duplicate tube having been set up in the same way, the two are placed in a tightly stoppered museum jar, which is also filled with air-gas mixture, and sealed with paraffin. Fig. 2 shows the apparatus set up. 536 Caleium of the Blood Serum. I Calcium Determinations.—As much of the serum and dialysate as it is possible to recover are pipetted (usually 3.5 to 3.6 cc.), the pH of the dialysate determined as before dialysis, and the caleitum in each is determined. The clear dialysate was treated in exactly the same manner as the dialyzed serum. In every case, where pro- tein had leaked through, the material was discarded. Ita. 2. Apparatus used in determination of diffusible serum calcium. Calculation of Results ——Yor this, the amount of calcium dialyzed, the calcium added to dialysate before dialysis, and the undialyzed serum calcium must be known. This is expressed in the formula (Ca in dialysate after dialysis 2) — Ca added to dialysate Diffusible Ca = tebe Basie \ Original serum Ca in amount used a von Meysenbug, Pappenheimer, Zucker, Murray 537 Tables III to V show the results obtained. It will be seen in Table III that in a range of CO, saturation of serum from 17 mm. of mercury tension to 62 mm. there is no alteration in the percentage of diffusible calcium of the serum. Table III also shows that a change in the hydrogen ion con- centration of dialysate from pH 7.6 before dialysis to pH 7.0 after dialysis exerts no influence on the percentage of diffusible calcium. TABLE III. Varying COz Tensions. 0 & pH of Ca in serum | Cain dialy- | 2 ES Tate anil 2am ‘dialysate. in 4 cc. sate in 4 ce. 3 Fy 5 ae es name, 2eR iby aes rks. O38 8) Be: | AL | start.| End. | Start.| End.] 45 | 85 ¢ mm. mg. | mg. | mg. | mg. psa mg. Feb. 2, | 28.0/7.6 |7.33/0.444/0.420/0. 258/0.278| 67 | 11.1] Dialyzing fluid 1921. was hypertonic. A.M.P. | 46.2/7.4 |7.1 |0.420/0.408/0.258/0.266| 65 | 10.5) Fluid A. 60.6/7.3 |7.0 |0.444/0.410)0. 258)0.292) 73 | 11.1 Feb. 21, | 17.6/7.6 |7.5 |0.440/0.448/0.295/0.296|] 67 | 11.0} Isotonic dialyz- 1921. ing fluid. , B.E.W. | 43.5/7.45/7.35/0. 440/0. 42410. 295/0.306} 72 | 11.0 62.0/7.2 |7.0 |0.440 0.295)0.300} 70 | 11.0) Fluid B. SUMMARY. A method is described for determining diffusible serum calcium. Values are given for normal human and normal dog serum. CONCLUSIONS. 1. The diffusible calcium of the serum of normal men and dogs was found to comprise from 60 to 70 per cent of the total serum calcium. 2. Varying the CO: saturation of the serum between 17 mm. mercury tension and 62 mm. does not alter this percentage. 538 Calcium of the Blood Serum. I TABLE IV. Dialyzable Calciwm of Normal Bloods. g pH of Ca in serum | Cain dialy- | © 3 |} 3 dialysate. in 4 cc. sate in 4 cc. a Os sh and | 3 mel ets Remarks. ame. aa = 8 =! | ae ie ee Start.| End. | Start.) End. an 83 1921 aT mg. | mg. | mg. | mg. | Pt, | mg. Jan. 20, | 46.2/7.4 |7.1 |0.420/0.408/0. 258/0. 266) 65 | 10.5 Fluid A A.M.P. | 28.0)/7.6 |7.33)/0.440/0.420/0.258/0.278] 67 | 11.1 i Feb. 8, | 46.3/7.4 |7.3 |0.42110.398/0.258/0.272| 67 | 10.5) Fluid B. B.C.K. Feb. 17, | 44.8|7.37/7.3 |0.412/0.424/0. 295/0. 288} 68 | 10.3 EW Feb. 21, | 43.5|7.45/7.35|0.440/0.424/0. 295/0.306} 72 | 11.0 E.E.W. 4 Mar. 12,) 45.5|7.43/7.25|0.416)0. 420\0.295/0.300| 73 | 10.4 Mrs. S. Feb. 28,| 45.2\7.4 |7.15|0.438)0.424/0.295|0. 296} 68 | 10.9] Considerable Dog 1. hemolysis. ’ Mar. 3, | 43.7/7.4 |7.0 |0.444/0.434/0.295|0.302| 69 | 11.1] Considerable Dog 2. hemolysis. Mar. 7, | 45.0|7.3 |7.2 |0.430/0.440/0. 295/0.276} 60 | 10.7| Slight Dog 3. hemolysis. Mar. 21, 45.0\7.4 |7.15|0.424/0.436/0.295/0.276| 61 | 10.6) No hemolysis. Dog 4. | Mar. 24,| 43.7/7.4 |7.15|0.408|0.384/0.254|0.268] 69 | 10.2} “ at Dog 5. | TABLE V. Duplicate Analyses. Experimental Tetany, Dog 6. Ca in serum in | Ca in dialysate Total Cain system eet — abies Serum Ca ste Ec Dialyz- Series NN. in 100 ce, : able Ca. | Start. | End. | Start. | End. Before.| After. | Differ- |, | eel an | oe | nes ee 1 0.248) 0.274) 0.210) 0.184; 6.1 0.458} 0.458} 0.0 63 2 0.248) 0.274) 0.210) 0.180} 6.1 0.458) 0.454) 0.004; 60 3 0.248) 0.218} 0.105) 0.128} 6.1 0.353) 0.346) 0.007; 61 1 0.248} 0.232) 0.105) 0.124; 6.1 0.353) 0.356) 0.003) 58 ; + von Meysenbug, Pappenheimer, Zucker, Murray 539 BIBLIOGRAPHY. . Rona, P., and Takahashi, D., Biochem. Z., 1911, xxxi, 336. . Cushny, A. R., J. Physiol., 1919-20, liii, 391. 3. Brinkman, R., Biochem. Z., 1919, xev, 101. 4. Brinkman, R., and Van Dam, E., Koninklijke Akad. v. Wetensch. t. Amst., Proceedings, 1919, xxii. 5. Lyman, H., J. Biol. Chem., 1917, xxix, 169. 6. Levy, R. L., Rowntree, L. G., and Marriott, W. McK., Arch. Int. Med., 1915, xvi, 389. Soy — 7 7 5 * = ‘ . ’ ‘ <, T 4 » A? ‘ . se . wee *. ; i . ' . i als ‘ i ~ 7 re . sei Ae aie fi a 4 y. eae, Vii oye i) Pipe) cy | iy in vic we aim i Pa “ =f rviLavrn if ‘Yer seve THE DIFFUSIBLE CALCIUM OF THE BLOOD SERUM. II. HUMAN RICKETS AND EXPERIMENTAL DOG TETANY. By L. yon MEYSENBUC ann G. F McCANN. (From the Department of Pathology, College of Physicians and Surgeons, Columbia University, New York.) (Received for publication, June 22, 1921.) In the preceding communication (1), there was reported in detail the technique employed in this work. It is our purpose in this paper to record the results of the study of the blood calcium in rickets and experimental tetany. Rickets. In the consideration of the pathology of rachitic bones and cartilage, there would seem to be four possibilities to account for the failure of proper ossification. First, that there is a defi- ciency of calcium in the diet or a failure of absorption of a sufficient supply; second, that there is an alteration of the form of the blood caleium, making it less available for the normal forma- tion of bone; third, that there are absent intermediary products, possibly of the ductless glands, possibly other salts, particularly phosphates, which are necessary for the deposition of calcium in the bones; and fourth, that there is an alteration, chemical or otherwise, in the osteoid tissue and provisional cartilage cells or matrix, such that calcium, even though it may be supplied in sufficient and proper form by the blood, cannot be taken up by these cells. Or there may be a combination of any or all of these possibilities. It is to the second that we have directed particular attention. Since the work of Howland and Marriott (2) in 1917, it has been known that there is a slight diminution of the serum calcium in rickets. They found in twenty-one cases an average of 9.4 mg. of Ca per 100 cc. of serum, whereas the normal is always 541 542 Caleium of the Blood Serum. II above 10 mg. Their lowest figure is 7.9 and in five other cases the Ca was less than 9 mg. We also have found a definite reduc- tion of the serum calcium in rickets. Thus, five cases showed: 9.8, 8.7, 8.5, 9.0, and 7.6 mg. of Ca per 100 ec. of serum, respec- tively. The electrical reactions of these cases were normal, except in the first two, which showed anodal hyperexcitability. The determination of the diffusible calcium in rachitic blood was carried out in precisely the same manner as that for normal blood described in our first paper. Attention is directed to Table I, which gives the analytical figures. Both cases presented clinically, as well as by x-rays of the long bones, evidences of active rickets. TABLE I. H of Ca in 4ce. of | Ca in 4 ec. of : CO2 dinlghdte, dislgnnte. pe le = Serum Total Ca in system. Dialyz- gg nea Ca per | eee ea BOO | Be | after| S| atter| 30"| Atver-|” - "| cee | After | Era mag Baby L. F. Age 11 months. mq. mg. mg. mg. mg. mg. mq. mg. |per cent 45.5 | 7.35 0. 295|0. 272|0.362/0.386) 9.0 |0.657\0.658/+0.001| 68 45.5 | 7.4 0. 295/0. 268|0.362 9.0 |0.657 66 45.5. | 7.4 0.210/0. 210|0.362 9.0 |0.572 58 Baby R. M. Age 12 months. 44.6 | 7.4 | 7.4 0.29510. 2440. 302/0.334 eG 0.597|0.578| 0.019 63 45.0 | 7.35) 7.3 |0.295/0.246/0.302 he6 65 * Blectrical reactions normal. Tetany. It is hardly necessary to state that the blood calcium in infantile tetany and experimental tetany has been found markedly reduced. MacCallum and Voegtlin (3), MacCallum and Vogel (4), Howland ~ and Marriott (2), Binger (5), and others are all agreed in this respect. MacCallum, Lambert, and Vogel (6) in 1914 made the following statement: “If tetany blood be dialyzed under exactly the same conditions as normal blood, it still loses a proportionate amount of the calcium, which would perhaps show that it is not especially — =-_- At, . L. von Meysenbug and G. F. McCann 548 the loss of diffusible calcium as contrasted with a non-diffusible form which is important in producing tetany.” We have sought in view of the increased CO.-combining power of the plasma in tetany, reported by some investigators, to correlate the hypothesis discussed in the previous paper (1) with the low calcium content of tetany blood. For this purpose, dogs were used, removing parathyroids, and in all but one case the thyroids as well. The CO,-combining power of the plasma in the tetany dogs was determined. Dog 4 had a preoperative combining power of 57.9 volumes per cent. During active tetany, it had fallen to 41.5 volumes per cent. Dog 6 showed practically no change. Dog 7 before operation showed 58 volumes percent. 2 days later, without tetany, the combining power had risen to 69 volumes percent. Later during a convulsion, it fell to 39 volumes per cent. In the presence of this definite acidosis, the diffusible calcium was still 66 per cent, so that it is apparent that Brinkman’s hypothesis does not apply to the diffusible calcium. Dog 4.—Female. Mar. 21. Blood was drawn under oil from jugular vein. The CO2-combining power of the oxalated plasma, saturated with alveolar air, was 57.9 volumes per cent (Van Slyke micro method). Blood was also taken for determination of dialyzable calcium. This was not citrated, but was defibrinated by whipping (Table II). Mar. 26. Under ether anesthesia, complete thyroparathyroidectomy was performed. Mar. 28. Dog showed twitching of various groups of muscles, and the reflexes were hyperactive. Respirations were deep and rapid. Blood drawn at 10.30 a.m., before anesthesia, showed a plasma COs-combining power of 41.5 volumes per cent. Dog was then etherized, and blood drawn and defibrinated as before for determination of dialyzable calcium (Table IT). Dog 6.—Male. Apr. 4. Blood plasma CO2-combining power was 57 volumes per cent, when saturated with 5.5 per cent CO2-air mixture. The serum calcium was 10.2 mg. per 100 ce. The dog was then etherized and complete thyroparathyroidectomy done. Apr. 6. Early in the morning the dog was found in convulsions, and he had been vomiting. Respiration and pulse rates were very rapid. Slight twitchings of the abdominal muscles could be felt, but reflexes were not obtainable. CO2-combining power was 54.1 volumes per cent when saturated with a 6.0 per cent CO»o-air mixture. Blood was taken for determination of dialyzable calcium, Table IIT. ' 544 Caleium of the Blood Serum. II TABLE II. Dog 4. Se EEE EEE i .of | Ca in 4 ce. of A CO: amet. ae Paty gen bho x Serum Total Ca in system. Dialos: i fecal ee Caper |e eS é a cc. Be- Differ- P-C0s.| Be-| AC] Be: | atter| Be | After. BE; | atter| Die Before operation. mg. mg. | mg. mg. mq. mg. mq. mg. per cent 45 17.4 |7.15/0.295/0.276\0.424| 0.436) 10.6 |0.710/0.712|+0.002} 61 45 |7.4 |7.15/0.295|0.274|0.424| Lost.| 10.6 {0.710} 59 45 |7.37/7.25/0. 258|0. 252/0.424! 0.440} 10.6 |0.682/0.692/+-0.010). 58 2 days after operation. Thyroparathyroidectomy. 45 |7.4 |7.25/0.210)0. 1840. 254! 0.282) 6.35 |0.464/0.466|/+0.002| 62 45 |7.42/7.2 |0.210/0. 180/0. 254) 0.282) 6.35 |0.464/0.462/|—0.002) 59 45 |7.4 7 25)0 105). 1360254 0.230) 6.35 |0.359'0.366/+0.007, 65 45 |7.4 |7.2 |0.105)0.114)0. 254) 0.244 6.35 |0.359|0.358/—0.001} 48* * This is an exceptional figure, possibly due to variation in the sac used. TABLE III. Dog 6. 2 days after operation. Thyroparathyroidectomy. Hof | Cain4ecc. of| Cain 4 cc. of : co eet omactes Aisin, Perini : Serum Total Ca in system. Dialyz- aries on per aye | c : ; Ose Bee || gee, | atten gee, | Attar)“ Seo ee mq. mg. mg. mg. mg. mg. mg. mq. per cent 45.7 |7.4 |7.15/0.210)0. 184/0.248\0.274; 6.1 |0.458/0.458) 0.0 63 45.7 |7.38/7.2 |0.210)0.180/0.248/0.274) 6.1 |0.45810.454;—0.004| 60 45.7 |7.4 |7.2 |0.105/0.128)0.248|0.218} 6.1 |0.353)0.346)—0.007 61 45.7 |7.42'7.2 |0. 105)0. 124\0. 248/0.232} 6.1 |0.353/0.356/+0.003) 58 TABLE IV. Dog. 7. 4 days after operation. Thyroparathyroidectomy. H of Ca in 4 ce. of | Ca in 4 cc. of 4 sea dinbvants: aaaates Aaah ¥ Serum Total Ca in system. Dialy Z- pee a . a i per Ape P-CO:. | Be- Be- oe - iffer- a ide: lore After Ba After a mq. mg. mg. mg. mg. mq. per cent 45 |7.43/7.3 |0.21 0.270\0.288| 6.7 |0.480\0.482/4+-0.002) 66 45 |7.42/7.25)0. ae 196\0.270/0.286) 6.7 |0.480/0.482/4+0.002) 67 45 |7.42/7.2 10.180 0. 186)0. 2700. 268 6.7 |0.450\0.454/+0.004 7 L. von Meysenbug and G. F. McCann 545 Dog. 7.—Male. Apr. 9. COs-combining power of plasma saturated with 6 per cent CO:-air mixture was 58 volumes per cent, and the serum calcium 10.8 mg. per 100 ec. Under ether anesthesia, the right thyroid, both upper parathyroids, and right lower parathyroid were removed. Apr. 11. Dog showed slightly hyperactive knee jerks, and a CO2-com- bining power of 69.2 volumes per cent (saturated with 6 per cent mixture). Apr. 12. Left thyroid and remaining parathyroid were removed. Apr. 13. At 9 a.m. the dog had a general convulsion. COzs-combining power of plasma, saturated with a 6 per cent CO:-air mixture was 39 volumes per cent. Dog was etherized and blood was drawn from the jugular vein by cannula and defibrinated for calcium determination, Table IV. Dog. 8.—Male. May 2. Plasma COs-combining power was 45 volumes per cent, and the serum calcium was 10.7 mg. per 100 cc. The dog was etherized and right thyroid, both right parathyroids, and left lower para- thyroid were removed. The parathyroids were calcified. TABLE V. Dog 8. 14 days after operation. Partial thyroparathyroidectomy. Hof |Cain4cc. of | Cain4ce. of ay. | CO2 inlvante: dialysate. ” een a Serum Total’ Ca in'system: Dialyz- pag oe per hie P-CO2. | Be-| Af- | Be- Be- Co eBe= Differ- os fore. ter. fore: After. fora! After. fore. After. ents: mg. mg. mg. mg. mg. mg. mg. mg. per cent 44.7 |7.4 |7.25/0.210)0. 214,0.336)/0.336| 8.4 |0.546/0.550)+0.004 65 44.7 |7.4 |7.3 |0.210/0. 218\0.336/0.346) 8.4 |0.546)0.564/+-0.018 67 45.5 |7.42/7.3 |0.127/0.182/0.336 8.4 Til 45.5 |7.4 |7.35)/0.127|/0,182/0.336 8.4 71 May 10. The dog showed slight twitching of abdominal muscles and spasticity of legs. Mayll. Serum calcium was 7.2 mg. per 100 ce. May 12. Condition noted on May 10 has persisted, but there have been no convulsions, nor active tetany. The dog was etherized and blood taken for calcium determination as in previous experiments, Table V. SUMMARY. In two cases of rickets, with serum calcium of 9.0 and 7.6mg. per 100 cc., the percentage of diffusible calcium was found to be between 58 and 70 per cent, within the range found in normal subjects. 5 546 Calcium of the Blood Serum. II In four cases of experimental tetany in dogs, similar percentages of dialyzable calcium were found, 58 to 71 per cent, with serum calciums of 6.1 to 8.4 mg. per 100 ce. Two of these dogs showed a reduced CQO,.-combining power of the plasma at the time the caleium determinations were made, showing that this form of acidosis does not affect the diffusible calcium. It, therefore, appears that, in so far as can be determined by 7n vitro experiment, the reduced serum calcium in experimental tetany is not due to a lowering of the diffusible as contrasted with the non-diffusible form. The proportion between the two remains constant in the presence of a reduced total. Also, this proportion does not chamee with varying CO.-combining powers of the plasma. CONCLUSIONS. The diffusible calcium of the serum in experimental tetany in the dog and human rickets is between 60 and 70 per cent of the - total serum calcium. BIBLIOGRAPHY. _ . von Meysenbug, L., Pappenheimer, A. M., Zucker, T. F., and Murray, M.L., J. Biol. Chem., 1921, xlvii, 529. . Howland, J., and Marriott, W. McK., Quart. J. Med., 1917-18, xi, 289. . MacCallum, W. G., and Voegtlin, C., J. Exp. Med., 1909, xi, 118. . MacCallum, W. G., and Vogel. K. M., J. Exp. Med., 1913, xviii, 618. . Binger, C., J. Pharmacol. and Exp. Therap., 1917, x, 105. . MacCallum. W G., Lambert. R. A., and Vogel, K. M., J. Exp. Med., 1914, xx. 149. Oa & W bd . THE OXYGEN DISSOCIATION OF HEMOGLOBIN, AND THE EFFECT OF ELECTROLYTES UPON IT. By EDWARD F. ADOLPH anp RONALD M. FERRY. (From the Chemical Laboratory, Harvard University, Cambridge.) (Received for publication, June 21, 1921.) Barcroft (1) has gathered extensive data on the dissociation of oxyhemoglobin. Plotting oxygen tension against percentage sat- uration with oxygen, he has drawn dissociation curves, and, by developing the aggregation theory in conjunction with Hill (2), he has sought to explain divergences in their form. Incidentally they have represented mathematically the curves for the dissocia- tion HbO, = Hb + Oy by a general equation kK = Hiblss1Oz[s " [O2]” [HbO»] and have determined constants which fit the equation in the case of every experimental curve. The data of Barcroft and Camis (3) have shown that in whole blood divergences in form of the curves are due to variations in the salt content of the blood from various species and individuals; but that, on the other hand, preparations of crystallized hemoglobin made at different times give sensibly similar curves. Barcroft and Roberts (4) have demonstrated that dialyzed hemoglobin gives curves which differ from those for crystallized hemoglobin, but which are identical for dialyzed preparations from different animals. These curves they have regarded as representative of pure hemoglobin. Barcroft and Means (5) have shown that car- bonic acid shifts the dissociation curve of dialyzed hemoglobin and have measured the shift quantitatively. Barcroft (1) and L. J. Henderson (6) have suggested that. hemo- globin in alkaline solution, or alkali hemoglobinate, hasasmaller dissociation constant for oxygen than has acid hemoglobin. And Henderson has by an indirect method calculated the ratio of two constants for the following equations in the case of whole blood: 547 548 Oxygen Dissociation of Hemoglobin J _ [HH]: [02] HHbO, = HHb + O- Kz = [HHbO,] e _ [BHb}-[0.] BHbO, = BHb + 0, Ks = TBHbO)] showing that Ky, is greater than Ks. Henderson concludes: “ .. 6. oxyhemoglobin must be a-stronger acid than re- eee hemoglobin. Of course it also follows that the salts of hemoglobin must have a greater affinity for oxygen than has acid hemoglobin itself.” To obtain direct experimental evidence upon this point, the effect of electrolytes and non-electrolytes on the dissociation curve of oxyhemoglobin was studied by us at the suggestion of Professor L. J. Henderson. Methods. Fresh defibrinated beef blood was centrifuged, and the corpuscles - thrice washed with 0.9 per cent sodium chloride solution. The corpuscular magma, placed in freshly made collodion sacs of about 100 cc. capacity, was dialyzed against running tap water for48 hours. Dialysis was continued on ice for one or more days against several changes of distilled water. The sacs were usually closed securely to diminish dilution of the contents. In order to rid the hemoglobin of metallic cations one of the following procedures, suggested by Professor Henderson, was usually adopted. Either (a) the solution was poured from the sacs and saturated with carbon dioxide, dialysis being continued against distilled water; or (b) the sacs were placed intermittently in distilled water saturated with carbon dioxide. In both cases the hemoglobin was finally dialyzed against pure distilled water. It was supposed that this treatment with carbonic acid would free hemoglobin from the cations at its isoelectric point, according to the equation BHb + H,.CO; = BHCO; + HHb. The freedom of the dialyzed solutions from electrolytes was roughly estimated by measuring conductivity at 25°C. The- lowest conductivity reached, 8 < 10-* reciprocal ohms, is the lowest conductivity reported in the literature when it is con- sidered that the concentration of the hemoglobin was 16 per cent. tarely did the conductivity exceed 3 10-4 reciprocal ohms, alti Re E. F. Adolph and R. M. Ferry 549 which is 4 per cent of that of whole blood, or equivalent to that of a 0.0027 m potassium chloride solution. The hemoglobin solutions contained, on the average, 14 percent hemoglobin, as measured by the oxygen combined with the hemo- globin after saturating it with pure oxygen at atmospheric pressure, and assuming a molecular weight of 16,700. Except for a diminu- tion in conductivity, no difference could be detected in the be- havior of solutions which had been treated with carbon dioxide during dialysis as compared with those not treated. The solutions of hemoglobin were equilibrated with mixtures of carbon dioxide-free hydrogen and of air, selected to give about 50 per cent saturation of the hemoglobin with oxygen. This was done in a rubber-stoppered bottle of 1 liter capacity, provided with two outlet tubes. Through the shorter, a sample of solution could be withdrawn without exposure to the outside air; the other was provided for gas sampling. 20 to 25 cc. of hemoglobin solution, evacuated by boiling with a water pump at 35 to 40°C., were run in from a pipette to the bottle, which had been previously filled with the gas mixture. Electrolytes and non-electrolytes, in solutions of suitable concentration were introduced through the rubber stopper from a graduated Luer syringe. The equilibrator bottle was then rotated continuously for 15 to 30 minutes in a thermostat at 38°C. The rotation was interrupted to adjust the pressure to that of the atmosphere. The same solution was often equilibrated five times. After equilibration the bottle was inverted in order to collect the solution in its neck, thereby presenting only a small surface to the gas above it. A gas sample of 10 cc. was then taken into an analyzer of the type described by Y. Henderson (7), and analyzed for carbon dioxide and oxygen. Some of the stock gas mixture was then admitted into the bottle at atmospheric pressure and room temperature, and a 2 cc. sample of solution run into a Mohr pipette. The oxygen content was determined by the blood-gas method of Van Slyke (8). A second sample of solution was satu- rated with pure oxygen at atmospheric pressure and room tempera- ture in a separatory funnel, and the oxygen capacity was deter- mined. This was determined once or twice in a series of suc- cessive equilibrations. Van Slyke’s method is not well suited to measuring the percentage saturation of hemoglobin with oxygen, 550 Oxygen Dissociation of Hemoglobin since two samples must be taken for each determination. The results of many workers seem to show that the quantities of oxygen found, though consistent among themselves, are too high. It was, however, the most available method. Great care was taken to correct calculations upon each analysis for dissolved gases, and to determine the errors due to manipulation and reagents. ince | | IXyYOen Tension in millimeters of mercur 20) ||. 30) eee ae ee Fra. 1. EXPERIMENTAL RESULTS. Dialyzed Hemoglobin.—Some 40 determinations of percentage - saturations were made upon 26 different solutions prepared from the blood of 4animals. These are plotted against oxygen tension MPLS E. F. Adolph and R. M. Ferry 551 in Fig. 1, and an arbitrary curve has been drawn through these points. It is a reflexed or S-shaped curve, and resembles Bohr’s (9) dissociation curve for crystallized hemoglobin. Blood samples from the same animal prepared simultaneously gave solutions exhibiting the most uniform results. There is, however, no close agreement among the other points, nor was any to be expected from the varying conditions. Most of the points to the right of the line were obtained in the latter part of the inves- tigation, and it is probable that these points are to be explained by the fact that the gas analyses occasionally revealed small ten- sions of carbon dioxide, 1 to 8 mm., which were reckoned in with the oxygen. The individual points bear no demonstrable relation to the purity of the hemoglobin as measured by conductivity, nor to the use of carbon dioxide during dialysis. The variations among corresponding points may be due in part to differences in hydrogen ion concentration, to very slight differences in the electrolyte content, and to bacterial action. None of these factors was ade- quately controlled. Addition of Acid.—Lactie acid and carbonic acid (Fig. 2, B and A) lowered the amount of oxygen combined at a given oxygen tension (30 mm.), and the effect was increased by increasing con- centrations of acid. This change was reversible, and could be nearly restored by the addition of an equivalent amount of alkali. Concentration of lactic acid greater than 0.015 m caused a gradual diminution of oxygen capacity due to an irreversible change in the hemoglobin. Barcroft and Means (5) have demonstrated this effect of car- bonic acid in varying concentrations upon dialyzed hemoglobin. Barcroft and Orbeli (10) have shown that lactic acid lowers the amount of oxygen taken up by whole blood. Addition of Alkali—Solutions of sodium hydroxide (Fig. 2, B) from 0.001 to 0.04 m caused more oxygen to be taken up by the hemoglobin at a given tension. This change was reversible upon the addition of an equivalent amount of acid. It incréased with the amount of alkali added. Similar results were obtained with disodium phesphate, NaszHPO,, from 0.01 to 0.1 m (Fig. 2, D). These results confirm those of Barcroft and Camis (3) with ammonia, disodium phosphate, and sodium bicarbonate added Oxygen Dissociation of Hemoglobin 552 27k 0e] EN W 100 proe 21998] W S000 a3 yeanyes;— e4yuad4 ad K. F. Adolph and R. M. Ferry 553 to crystallized hemoglobin; and those of Momose (11) with sodium hydroxide and ammonia added to whole blood. Addition of Neutral Salts.—Potassium and sodium chlorides at concentrations from 0.01 to 1.0 m reduced the oxygen bound at a given oxygen tension (Fig. 2, A and C). This effect increased with increasing concentration, at first rapidly and then more gradually. A solution containing both sodium and potassium chlorides had the same effect as a solution of either alone. Addition of Non-Electrolytes.—Neither urea from 0.1 to 1.0 M nor sucrose of 2 per cent (Fig. 2, D) affected the oxygen dissociation of the dialyzed hemoglobin solutions. These results confirm those for urea of Poulton and Ryffel (12) and of Momose (11) both of whom used whole blood. SUMMARY. Our results apparently differ from those of Barcroft in two res- pects. Our dialyzed solutions of hemoglobin give S-shaped curves comparable to those of Bohr (9) for crystallized hemoglobin. Barcroft’s hyperbolic curves for dialyzed hemoglobin were ob- tained when ammonium carbonate or hydroxide was added,! and we have obtained comparable points when other alkalies were added. Barcroft has recently! obtained curves similar to ours for dialyzed hemoglobin to which no alkali was added. Secondly, Barcroft (3) found that the addition of neutral salts to crystallized hemoglobin increased the amount of oxygen taken up by it at a given tension of oxygen. We have found the reverse to be the case with our samples of dialyzed hemoglobin. Very recently Barcroft! has made the same observation. Our results were obtained only at oxygen tensions of about 30 mm., and we are unable to predict the result at higher tensions. DISCUSSION. The chemical behavior of hemoglobin has been the subject of much speculation. The data of Barcroft (1); Douglas, Haldane, and Haldane (13); Roaf (14); and others, have been clearly inter- ‘ preted on the basis of Hill’s (2) aggregation theory as applied to 1We are greatly indebted to Mr.Barcroft for communicating the results of his recent experiments to one of us. 554 | Oxygen Dissociation of Hemoglobin the mass action principle. The implications of the aggregation theory, which we believe serves as a foundation for understanding the chemistry of hemoglobin, have been ably set forth (1, 2, 13). Up to the present time little has been said of the dependence of the equilibrium between oxygen and hemoglobin upon that be- tween hemoglobin and electrolytes as such. The present experi- ments suggest that the degree of electrolytic dissociation of hemoglobin, as of other proteins (15), is variable, and that it is markedly different for alkali hemoglobinates and for acid hemoglobin. ‘The effect of acid in diminishing the amount of oxygen or carbon monoxide bound by hemoglobin at a given ten- sion, the effect of alkali in increasing the saturation with oxygen at a given tension, and the fact that oxyhemoglobin behaves as if it were a more highly dissociated acid than reduced hemoglobin (6, 16, 17) give support to this view. Certain facts are explicable upon both hypotheses, such as: the influence of neutral salts, the absence of effects due to non-electro- _ lytes, and the increased osmotic pressure of hemoglobin in alka- line solution. It seems probable that the electrolytic dissociation of hemo- globin must be considered as supplementary to its aggregation in giving to hemoglobin its remarkable range of variation in physico- chemical activity. The conception of hemoglobin as an ionized substance is the only view so far suggested to account for the effects of acid and of alkali upon hemoglobin. Unfortunately the present data are inadequate for more than a qualitative discussion. It is hoped that work now in progress will give a quantitative definition of the effects due to ionic equilibria. We are indebted to Professor L. J. Henderson for much valuable aid and criticism. CONCLUSIONS. 1. A technique is described for dialyzing hemoglobin, depending upon its protein property of acting as a dissociable electrolyte. 2. Hemoglobin prepared by several procedures for dialysis gives S-shaped curves for oxygen dissociation. 3. Addition of alkali with formation of alkali hemoglobinate results in an increase of the oxygen bound by hemoglobin at a constant tension. K. F. Adolph and R. M. Ferry 550 4. Neutral salts even in small concentrations decrease the amount of oxygen bound. 5. Non-electrolytes have no effect upon the oxygen equilibrium. 6. The equilibrium between oxygen and hemoglobin is a func- tion of that between hemoglobin and electrolytes. BIBLIOGRAPHY. 1. Barcroft, J., The respiratory function of the blood, Cambridge, 1914. 2. Hill, A. V., J. Physiol., 1910, xl, p. iv. 3. Barcroft, J., and Camis, M., J. Physiol., 1909-10, xxxix, 118. 4. Barcroft, J., and Roberts, F., J. Physiol., 1909-10, xxxix, 143. 5. Barcroft, J., and Means, J. H., J. Physiol., 1913-14, xlvii, p. xxvii. 6. Henderson, L. J., J. Biol. Chem., 1920, xl, 401. 7. Henderson, Y., J. Biol. Chem., 1918, xxxiii, 31. 8. Van Slyke, D. D., J. Biol. Chem., 1918, xxxiii, 127. 9. Bohr, C., Centr. Physiol., 1903, xvii, 682. 10. Barcroft, J., and Orbeli, L., J. Physiol., 1910-11, xli, 355. 11. Momose, G., Biochem. J., 1915, ix, 485. 12. Poulton, E. P., and Ryffel, J. H., J. Physiol., 1913, xlvi, p. xlvii. 13. Douglas, C. G., Haldane, J. S., and Haldane, J. B.S., J. Physiol., 1912, xliv, 275. 14. Roaf, H. E., J. Physiol., 1909, xxxviii, p. i. 15. Loeb, J., J. Gen. Physiol., 1918-19, i, 559. 16. Christiansen, J., Douglas, C. G., and Haldane, J. 8., J. Physiol., 1914, xlviil, 244. 17. Parsons, T. R., J. Physiol., 1917, li, 440. ANIMAL CALORIMETRY. SEVENTEENTH PAPER. THE INFLUENCE OF COLLOIDAL IRON ON THE BASAL METABOLISM. By EINAR LANGFELDT. Christiania, Norway. (From the Physiological Laboratory of Cornell University Medical College, New York City.) (Received for publication, June 1, 1921.) INTRODUCTION. The biological action of inorganic hydrosols has been exten- sively studied during the years which have elapsed since Credé? introduced the use of colloidal silver into medicine. From these investigations it seems very probable that the action of the inor- ganic hydrosols is much the same as the action of small doses of salts of the same metals (see the literature by Bechhold?). The colloidal metal dissociates slowly in water into metal ions, and the bactericidal action, the property which has been the most studied, is supposed to be due to these dissociated ions. After intravenous injection the colloids are distributed all over the body within 4 to 1 hour and, while still in the colloidal state, are deposited in almost every organ with subsequent slow dis- sociation of ions. Investigations on the influence of these substances upon the metabolism are very rare. It has been found that silver hydrosol increases the protein metabolism.® A special interest is connected with the negative iron oxide hydrosol. While the positive iron oxide hydrosol, according to 1Credé, B., Arch. klin. Chir., 1897, lv, 861. 2 Bechhold, H., Die Kolloide in Biologie und Medizin, Dresden and Leipsic, 2nd edition, 1919. | 3 Bechhold,? p. 402. 557 558 Animal Calorimetry Bechhold, unites with the negative blood colloids, forming an irreversible gel, the negative iron oxide hydrosol easily mixes with serum without producing any coagulation. This colloid solution is said to act as an oxygen carrier and to have properties similar to hemoglobin. The purpose of this investigation has been to examine the influence of such an intravenously injected colloidal iron solution on the basal metabolism. Methods. Two dogs, trained for calorimeter work, were used in the experi- ments. The dogs were fed once daily with the ‘‘standard diet.’”® The bladder was emptied and washed before and after each experiment. The hydrosol was injected into the jugular vein, and immediately thereafter the dog was placed in the calorimeter. In each experi- ment 5 cc.® were used. The movements of the dog in the calorimeter were recorded on a smoked drum. The efficiency of the calorimeter was controlled by frequent alcohol checks. ; Basal Metabolism. The standard diet was adininistered at 5 p. m. the day before the experiment. Thus, the basal metabolism was determined 18 to 20 hours after feeding. In all experiments the dog was perfectly quiet, and the temper- ature of the calorimeter was between 25 and 26°C. The basal metabolism of Dog 20 was found to average 14.92 calories per hour and the respiratory quotient 0.80 (Table I). . The basal metabolism of Dog 18 was found to be 16.18 calories - per hour and the respiratory quotient 0.80. The Influence of Colloidal Tron Intravenously. The results of intravenous injection of 5 ee. of colloidal iron are given in Table II. The average total heat production per hour ‘ Bechhold,? p. 417. * Lusk, G., J. Biol. Chem., 1912-13, xiii, 185. * Made by Laboratoires Clin, Paris. | E. Langfeldt 559 of Dog 20 was 16.00 calories and the average respiratory quotient 0.84. One of the experiments, No. 10-A, was performed imme- diately after the basal metabolism was determined, on the same day. After administering colloidal iron the heat production of Dog 18 was found to be 18.72 calories per hour and the respiratory quotient 0.82. TABLE I. Basal Metabolism of Dog 20. Date es | cee ee 1921 PATO SEM ste SIE SRS oo Cet ee 3 14.45 0.81 WIER TOs paact ocean ene bomen tone ¢ 5 14.77 0.79 CCS a ee a TE rf 14,94 0.81 BRED eo abc «5 oto acs 12 5 OR 10 15225 0.78 "TA eye ic ea RA ag SOP aR rea Nr a aL 11 NG 22 0.81 AN GGT er ahs STIS Re PRM re cio 6 OL aoe oe 14.92 0.80 TABLE II. Colloidal Iron Intravenously—Dog 20. Date ee || cee, |e 1921 PANINI Sees ce ay ese ee koi ie ee 4 5.63 0.85 UALS) 7S ea eI a 0 8 6 5.79 0.87 1-12 aE See RES PACE LO 10-A 16.40 0.81 PAFETA Oy oe Brac oc ae hh ee ee et. 16.00 0.84 The total heat production was thus increased about 7 per cent in Dog 20 and about 15 per cent in Dog 18. The average CO: output per hour of Dog 20 after the injection was 5.51 gm., but only 4.89 gm. in the basal metabolism. The average O2 consumption was 4.68 gm. per hour after injection and 4.46 gm. in the basal metabolism. In Dog 18 the CO, production was increased from 5.37 to 6.35 gm. per hour and the O. consumption from 4.87 to 5.63 gm. per hour after the injection. | “‘qamnb ‘Teseg Sod “3X O16 F4SI9M |F6 FT |Fe°ZI | 09°S |860°0 |ZI'II | 180 | 68°F | 26°F | 20'S -ZO ZT Zi 6. as e ‘yetnb S0q “AJSNOUDARI} ‘00 G ‘A[SNOWOAVIZUL UOT — -UI WOT [BPIOT[OD T8PIOTI9D “3H O16 FUFIOM |62°ST |66°ZI | 08'S |SOT'O |ZF' IT | 28°O | LFF | 89'S | 2G°T -2¢°TT 9 eS ‘yornb S0q] “B34 026 % MSP “1990 HO “Wose | TEseg . 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Si | OPS 07 ic V-OL €1 », ‘gernb Soq “SY 086 WSIOM “8990 “O “Y os [Tesvg “AOA “OT ABI YoY [oyoory |¢z'st |ce'er | 06'T |1Z0'0 [erst | 8Z°0 | 09°F | 16°F | OT'T Ola Or eT Avy Animal Calorimetry 562 ee ‘yomnb Soq = “3y “A[SNOUDARIY CF'6 WYBIOM *99 g ‘ATSNO -UI UOT [BpIO]]JOH ee rey uerr TSPlolleD | |eL Sr 120° St | So" |Ze OZ SL | 870 | €9°¢ | Geo" | 96°T —98° TT 98 ST » “qornb | [888g Bod “SY SFG SIEM |SI'9T eet | es'z |20T"0 |98°2 | 08°0 | 28°F | 2e°¢ | ez°% -Se'@r Seeks LT AvIN “ud “ub "ub “mb . 161 "pezel | 2 ais ve S66 "urazorg| "sy IBUIOY 18901, enti OF) POT i 20 709 ‘oun, ‘ON quourtrodxiy “07 “Sallo[BO Se a eee ‘unoy Jod sonyea oBvsi9Av ut possoidxo ore s}pnsor oy, “ABp snoraord oy} ‘urd ¢ ye uaAIs yoIp pre pueyg g] b0q “AT ATAVL K. Langfeldt 563 The respiratory quotient was slightly higher in both dogs during the injection experiments than during the determinations of the basal metabolism. There was no marked increase of the protein metabolism. The chief increase of the total heat production fell on the non-protein metabolism. The results are summarized in Tables III and TV. CONCLUSIONS. 1. Intravenous injections of iron hydrosol in dogs cause an increase of the O. consumption and the CO, production. The average increase of the heat production in one of these dogs was 7 per cent and in one experiment on the other the increase was 15 per cent. 2. The increased metabolism coincides with a slight increase of the respiratory quotient. 3. The chief increase of the total heat production falls on the non-protein metabolism. I wish to express my sincerest thanks to Professor Graham Lusk for advice and help in running the calorimeter, and to Mr. James Evenden for technical assistance. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVII, NO. 3 ee he pie Oe <8 ‘ae 5 roe rs ‘ Jy ' +¢ E> sat r a ¥ od a Rel ts, a | Perl! 1s. * '. (‘ye tae 5 ) -_. ag st . a4 ¢ ' ae ; 1 al CHEMICAL FACTORS IN FATIGUE. I. THE EFFECT OF MUSCULAR EXERCISE UPON CERTAIN COMMON BLOOD CONSTITUENTS.* By NORRIS W. RAKESTRAW. (From the Department of Chemistry of Stanford University, Stanford University.) (Received for publication, June 2, 1921.) The beginning of the truly chemical study of muscular exercise and fatigue may be traced back to Du Bois Raymond who, in 1859, showed that contracting muscles become acid as a result of their activity. Others, following closely, pointed out that not only is lactic acid formed in the working muscle but there is a simultaneous disappearance of carbohydrates, notably glycogen, the partial oxidation of which presumably gives rise to the acid. Space does not permit of an extended review of the work which followed from then till the present day, but a few of the outstand- ing features may be noted. Ranke, in 1865, first investigated the effect upon the contrac- tility of surviving, excised muscles produced by perfusion with solutions of various substances. His experiments were later extended and elaborated by Lee, Burridge, and others with the result that a number of substances were found whose influence upon surviving muscle produced to a certain extent the recognized phenomena of fatigue, as evidenced by a decrease in power of contraction. There have been reported from time to time among these so called ‘fatigue products’? (some or all of which may possibly be formed in muscles during their activity and produce the condition of fatigue) the following: lactic and oxy- butyric acids and their alkali salts, HCl, HeSO.:, COs, NaH2POs, KH2POs,, phenol, indole, skatole, and potassium ions. *A thesis presented tothe Department of Chemistry of Leland Stanford Junior University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 565 566 Chemical Factors in Fatigue. I Weichardt, in 1904, claimed to have isolated a specific fatigue toxin, similar to the bacterial toxins, which produced all the symptoms of fatigue when injected into the blood stream. He purported furthermore to have isolated an antitoxin, by regular immunological methods, but his results have never been confirmed. The excretion of substances in the urine during and following muscular work has received considerable attention. It seems to be generally conceded that no increase in nitrogen metabolism accompanies muscular work. Nevertheless, a number of workers have reported substantial increases in nitrogen elimi- nation for some time following a period of strenuous activity. The current opinion of the independence of nitrogen metab- olism is therefore not without question. The commonly reported increases in sulfur and phosphorus elimination may also have a bearing on this point. The question of changes in the excretion of nitrogenous extrac- tives is still unsettled. Creatine and creatinine are apparently not involved in any regular way, but uric acid and the purines have in general been found to increase after work. a The acidity of the urine is generally, but not invariably, found to increase, as might be expected from the formation of lactic acid in the muscles and the lowering of the alkaline reserve of the blood. The output of carbon dioxide is invariably increased in exercise, which is important when the demonstrated fatigue-producing action of carbon dioxide on the muscles is borne in mind. Sugar is practically the only constituent of the blood which has been quantitatively followed during muscular exercise, but there is no agreement between the results from different sources. A number of secondary physiological concomitants of fatigue have been reported, such as an increased number of red and of white corpuscles. | In summarizing the whole question we may lay down certain general principles: (1) Substances giving rise to hydrogen ions, such as lactic and oxybutyric acids, monopotassium phosphate, and carbon dioxide, are causal agents in fatigue. (2) Certain products of protein disintegration, indole, skatole, and phenol, are apparently capable of producing fatigue symptoms and may EE ———=— es — N. W. Rakestraw 567 be closely related to normal fatigue. (3) There is some evidence that certain negative ions, as the lactate and oxybutyrate, as well as certain positive ions, especially potassium, may be causal agents in fatigue. (4) There is no evidence substantiating Weichardt’s theory, still largely quoted, of specific fatigue toxins. (5) The formation and excretion of a number of materials such as uric acid, purines, urea, etc., may be influenced by muscular work and fatigue, without their standing in any causal relation to fatigue. EXPERIMENTAL. The present study was undertaken with the purpose of deter- mining, by means of improved biochemical methods of reasonable accuracy, the changes which occur in the more common con- stituents of the blood during fairly severe muscular exercise. The investigation will later be extended to other substances, and other conditions. A certain amount of preliminary work was done with dogs as subjects, but the results were so irregular and the conditions of the experiments so difficult to control that it was entirely abandoned in favor of human subjects, nearly all of whom were university students. A few sets of results were obtained with running as a mode of exercise. These data, as far as they go, are given in Table I. Later it was deemed advisable to lengthen the period of exercise and two subjects undertook a 65 mile bicyele ride, extending over about 12 hours and including a considerable amount of hill-climbing. The data covering this period are given in Table II. A consideration of the results from these widely differing types of activity, especially as regards the changes in blood sugar, suggested the possibility that the human organism differs in its reaction to short strenuous effort and to long tedious work. Accordingly it was decided to divide the muscular work into two kinds, a short, strenuous period of activity, and an easier, but longer period. For the first type rapid stair-climbing was chosen—a mode of exercise often made use of in this connection. A double flight of stairs in the labora- tory building, about 20 feet in height, was used, and the subjects ran up and down these stairs as rapidly as possible until nearly exhausted. The total time of the exercise did not exceed 15 568 Chemical Factors in Fatigue. I minutes in any case. For the other type of exercise bicycle riding was chosen. Two subjects generally rode simultaneously and at such speed that they were in a distinct state of fatigue at TABLE I.* : Non-protein Urea nitrogen Sugar Uric acid Total nitrogen. | nitrogen per 100 ce. per 100 ce. per 100 ce. per 100 ce. Subject. s|r|K| s |R|K|sS|R/K|S|R|K| st|R|K —— —_ ff Sf fj ——— | | | per per per cent | cent|cent | 7: | ™v-| mg. | mg.| mg. | mg. \mg.\mg.jmg. mg .\mg. I |3.37|3.55|3.35) 49.0 |39.6)45.3/19.2/14.8/20. 7/118) 86/110) < |2.3 II |3.49/3.56|3.44)(45.6)|40.6)/47, 1/19.7/13.6)18.7) 97) 86)108} = |3.7 III |3.57/3.62/3.40] 49.4 |44.9/46.7/20.0/15.0/18.9)127|131)131} > |5.1 BG 4 3.72\3.51 \46.4.50.0 18 .6|23 .2 92)111 7.6 *Of the horizontal rows numbered with Roman numerals I contains the results from the samples taken before the exercise; II,-after the subject had run 100 yards; III, after running one mile; and IV, after 24 hours subsequent rest. The vertical columns contain the results belonging to each of the three subjects, S, R, and K. 7 The results in this column are only qualitative. The samples were not compared in the colorimeter, but II showed distinctly more uric acid than I, and III distinctly more than IT. TABLE II.* Non-protein . : 5 3 = 2 U N U d iS} : Subject. Time. Total N. mer K Ae per 100 ce. pariT00 08. per 1008s per cent mg. mg. mg. mg. s Before. SEB} 39.7 eS 4.5 ilahs) ‘ After. 3.63 50.2 25.1 12.5 79 R Before. 3.40 39.6 2220 PG?) 106 After. 3.48 43.9 29.3 eral 100 * Showing results before and after a 65 mile bicycle ride, occupying 12 hours. A small amount of food was taken during that time, principally four or five pieces of bread and butter, but the second sample was taken 5 hours after eating. the end of from 2 to 3 hours. Most of the subjects were not accustomed to riding long distances. It was decided to confine the attention to the changes in sugar, uric acid, non-protein and urea nitrogen, preformed and total N. W. Rakestraw 569 creatinine, and cholesterol. These were determined in the plasma as well as in the whole blood, whence an insight was obtained into the changes in the distribution of these materials between plasma and corpuscles. Total nitrogen was determined in the preliminary experiments, but this was not extended into the major portion of the work. In addition to these chemical determinations it was considered important to note the changes in certain physical properties of the blood, especially such as would throw light upon any possible variations in the blood volume. A direct determination of the changes in blood volume was originally undertaken with dogs, but was necessarily given up when the work was carried over to human subjects. The experiments were generally carried out in the late after- noon, not closer than 3 to 4 hours from the noon meal. For collecting the blood samples a paraffined 50 ce. flask was used, fitted with a two-hole stopper carrying two right-angled glass tubes about 2 inches in length. One of these was connected with a piece of heavy rubber tubing through which the operator could exert suction at will; the other tube was paraffined internally and connected by a short piece of rubber tubing to a 20 gauge. needle. A small amount of powdered potassium oxalate was introduced into the flask and 40 to 50 cc. of blood were drawn from an arm vein. A preliminary, or control, sample was taken in this way and another one, similarly, after the exercise. Methods. Chemical Determinations—For the determination of sugar, uric acid, non-protein nitrogen, urea, and creatinine, both pre- formed and total, the general procedure of Folin and Wu (1) was adopted because of its simplicity, reasonable accuracy, and economy cf material. The proteins were precipitated both in plasma and whole blood in exactly the same manner, and 15 ce. of whole blood and 10 ce. of plasma yielded ample protein-free filtrate in each case to make determinations of sugar in duplicate or triplicate, urea and non-protein nitrogen in triplicate, total creatinine in duplicate, and preformed creatinine and uric acid in single determinations. Because of the uncertainty in pre- paring and keeping active urease solutions and because of the large number of samples urea was determined by the autoclave 570 Chemical Factors in Fatigue. I modification, 7.e. autoclaving at 150°C. for 10 minutes, in which case the total creatinine determinations could be started at the same time. One more or less important departure from the original Folin- Wu method was made in the case of non-protein nitrogen. Largely as a matter of personal preference, but also because of some difficulty with cloudy solutions and refractory precipitates after digestion with the phosphoric-sulfuric mixture, non-protein nitro- gen was determined as follows: To 5 cc. of the protein-free filtrate in a large Pyrex test-tube about 0.5 gm. of potassium sulfate was added, one or two small pieces of broken porcelain, and 1 ec. of a 1:1 solution of sulfuric acid containing 1 gm. of copper sulfate per 100 ce. The solution was then evaporated to fumes of H.SO,, the tube covered with a 2 em. watch-glass, and digested slowly for 5 minutes after the solution had become clear and bluish. It was then cooled and diluted to about 25 ec. After the addition of 5 cc. of 20 per cent NaOH, a drop of paraffin oil, and a little finely granulated zinc, the ammonia was distilled over into 5 ce. of twentieth normal HCl in a test-tube graduated at 50 ec. and Nesslerized as usual with 5 cc. of the reagent as prepared by Folin and Wu. For the distillation, the test-tube was fitted with a two-hole rubber stopper carrying in one hole an outlet tube to a 6 inch, three-bulb condenser, and in the other hole a 5 ce. pipette closed with a short piece of rubber. tubing and a pinch-cock, which served as a means of introducing the NaOH after the test-tube was in position. The solution in the tube was gradually brought to boiling with a microburner and the air slowly expelled from the apparatus. The distillation was carried on for 4 minutes after the condenser began to deliver. Toward the end the apparatus was allowed to cool slightly and the acid to suck back for a short distance. The pinch-cock, closing the pipette in the distillation tube, was then opened for a moment to draw back into the tube any traces of ammonia which might have been trapped within the pipette. Distillation was then continued for a few moments longer before stopping and washing. Although this method is somewhat longer than the direct Nesslerization, it is easily possible to distil ten to twelve samples © in 14 hours. With ordinary care there is no loss during distil- N. W. Rakestraw Gye lation; the method recovers ammonia quantitatively and is highly satisfactory. A blank determination of the reagents is of course necessary. The same distilling apparatus above described was used for urea. For receiving the distillate the Folin blood sugar tubes are excellently adapted, being graduated at 25 cc. and having a constriction in the neck at the proper place to prevent any loss of ammonia by too rapid bubbling during the early stages of the distillation. , Trouble with excessive frothing is generally traceable to the quality of paraffin oil used, and finely granulated zinc is preferable to quartz or porcelain to prevent bumping. Cholesterol was determined by the method of Myers and Wardell (2), this procedure being found most satisfactory after trying out several of the colorimetric methods now in use. The only important modification made was the use of a standard solution of pure cholesterol in chloroform for the colorimetric comparison, in place of naphthol green B recommended by the authors of the method. Specific Gravity—The Hammerschlag method was first tried but was abandoned in favor of the Westphal balance, since ample material was available. For this purpose a round glass plummet was made having a volume of about 2 cc. This was suspended on a silk thread from the beam of the analytical balance, and by determining its weight in air, water, and blood, the specific gravity was calculated in the usual manner. Viscosity—A capillary pipette was made which delivered its contents of blood in 40 to 60 seconds. A fairly rapid delivery was advisable to avoid the settling of the corpuscles. This pipette was carefully calibrated with water and its time-of- delivery curve determined over a 10° range of temperature. Conditions did not permit of a very accurate temperature control of the blood, but it was found sufficiently accurate to use the average of the blood temperatures before and after the determin- ation. It was ascertained that a change of 2 or 3° made practically no difference in the delivery time for blood, and the temperature regulation was well within these limits. For -the measurement of the plasma viscosity the ordinary type of capil- lary viscosimeter was used, immersed in a constant temperature bath. The delivery time for plasma was about 2 minutes. All the data for viscosity are relative to water. 572 Chemical Factors in Fatigue. I Hemoglobin.—The method of Cohen and Smith (8) was modi- fied by pipetting 1 ec. of blood into about 200 cc. of water in a 250 ec. graduated flask. Then 25 ec. of normal HCl were added and the contents made up to the mark with water. Since only the relative, and not the absolute, amounts of hemoglobin were of interest two samples of blood were treated in this way, one taken before and one after the exercise, and the resulting colors of the acid hematin simply compared against each other in the colorimeter to obtain the “‘ hemoglobin ratio,’’ which is caleulated on the assumption that the blood had a content of 100 per cent before the exercise. Relative Corpuscle Volume.—The relative volume of corpuscles was determined in the customary way by the hematocrit method. The suggestions of Sundstroem and Bloor (4) in regard to the construction of hematocrit tubes, were more or less closely followed. A number of capillary tubes were cut about 11 cm. in length and ground flat at one end with emery. These were all filled from the same sample of blood, centrifuged for the same time, and those which indicated concordant corpuscle volumes accepted for use. A more rigid method of checking the regularity of bore was used in some cases, as follows: A short column of mercury was drawn up into a tube and the length of this mercury column carefully measured as it was moved to different positions within the capillary. If little or no variation in the length of the mercury column was observed the bore of the tube was considered sufficiently regular. When it was possible the same capillary was used for the second blood sample as for the first and centrifuged in each case for the same time at the same speed (1 hour at 2,000 r.p.m.). In other cases two capillaries, cali- brated or checked against each other, were used to contain blood from the two samples, respectively, and centrifuged simulta- neously for 1 hour at 2,000 revolutions per minute, at the end of which time the length of column was constant. Therefore, the comparative nature of these results is practically certain. Plasma-Corpuscle Relations —The concentrations of the various substances in the corpuscles were calculated in the usual manner from the plasma and whole blood concentrations. It must be kept in mind that the determination of corpuscle concentrations . by difference in this way leaves considerable opportunity for the N. W. Rakestraw 573 accumulation of errors in the corpuscle results, in which there is consequently less certainty than in the directly determined values for plasma. DISCUSSION. A consideration of the data in Table I, which are more or less fragmentary, points out certain preliminary facts. . Total nitrogen exhibited:a slight tendency to rise during short exercise, which apparently continued for some time after. Non- protein and urea nitrogen underwent no regular change excepting a possible slight increase after the exercise was over. These changes are in accordance with the heretofore observed tendency for the nitrogen elimination to increase after work. Sugar was increased considerably by an amount of exercise represented by a mile run but returned to normal within 23 hours. Uric acid was noticeably increased by short exercise and continued to increase for some time after. This also agrees with the general finding in regard to uric acid eliminated in the urine. Table II, representing a much longer work period, presents a somewhat different picture. Total nitrogen and non-protein nitrogen, as well as urea, were increased. The concentration of uric acid rose considerably, but the longer exercise caused a drop in the blood sugar. The more exhaustive investigation, represented in Tables ITI and IV, substantiates these preliminary conclusions and goes somewhat further. In order to facilitate comparison of the effects of the two kinds of exercise the average results in the two cases are brought together in Table V. Let us consider each factor separately. Sugar. The shorter exercise period, which represented an expenditure, on the average, of about 58,000 foot pounds of energy in 10 minutes, resulted in an increase in whole blood sugar in seventeen cases out of eighteen. This increase averaged 0.036 per cent. The one instance of decrease was in the case of a subject who exhibited at another time an increase of about the same amount. It may possibly be significant that this subject, a boxer in training, was in the best physical condition of any who participated. 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It will immediately be observed that the distribution of sugar in all these experiments is different from the distribution relation now generally accepted as correct. The consistent fact that the sugar in the corpuscles proved to be greater in amount than that in the plasma was the subject of much concern. The recent work of Ege (5) and others seems to have shown conclusively that the concentration of sugar in the corpuscles is about three- fourths of that in the plasma. Wishart (6) insists that results showing as much sugar in the corpuscles as in the plasma are due to faulty technique. Falta and Richter-Quittner (7) have even presented results indicating that the corpuscles normally contain no sugar whatever, but their work does not seem to have been generally accepted as yet. Thus far, however, no distri- bution data appear to have been given on the basis of the Folin- Wu method, and the reason for the discrepancy in this respect between the latter and other methods is being further investi- gated in this laboratory. It may be pointed out that the normal, resting value for whole blood is no higher than the commonly accepted average, and there seems to be no explainable reason why the plasma values should be so low. That an incomplete removal of proteins in whole blood did not result in these cases from too low an acidity at least, has been demonstrated, since blood sugar values were no lower when a distinct excess of sulfuric acid had been added. Whatever the true explanation of the apparent discrepancy it will not affect the comparative nature of the values given in the tables. When we turn to the longer period we find only one case out of nine in which there was an inerease of more than 0.002 per cent, but on the contrary an average decrease of 0.005 per cent in the nine cases observed. The decrease was much more pro- nounced in the plasma, amounting to 0.015 per. cent on the average, as a result of which the calculated sugar in the corpuscles increased 0.008 per cent. In only one case did the corpuscle sugar drop as much as 0.005 per cent, although the decrease in the plasma was often many times this. In fact there seems to N. W. Rakestraw 585 have been a distinct tendency for the corpuscles to resist a decrease in sugar content, and this is also observed in the one ease of decrease in Table III. In other words, the longer exertion changed the distribution coefficient for sugar slightly, with the result that, on the average, there was relatively more sugar in the corpuscles after the exercise than before. This conclusion must be taken advisedly, how- ever, until the matter of normal distribution of sugar, using the Folin-Wu method, has been cleared up. Uric Acid. - Both the short and the long exercise caused an increase in uric acid which was greater, on the average, in the plasma than in the whole blood, so that in many cases the calculated uric acid in the corpuscles actually decreased. This is the most distinct effect upon distribution which was observed in any of the materials, there being no doubt that there was relatively less uric acid in the corpuscles after exercise than before. The further increase in uric acid after the completion of the exercise has been noted in the one case in Table III. Unfortu- nately it was impossible to follow this subsequent change in the rest of the subjects, but a more intensive investigation of this point will be made later. ; Urea and Non-Protein Nitrogen. The short period of exercise had no particular effect upon either urea or non-protein nitrogen, nor upon their distribution between corpuscles and plasma. Not only do the averages show close agreement but there are few wide variations in the individual results. These determinations were made in triplicate and each value generally represents the average of three results. The urea includes, as usual, the ammonia nitrogen. The longer period of exercise apparently had a very slight augmenting effect upon the urea and a somewhat greater one upon the non-protein nitrogen. There was no change in the distribution coefficient of either urea or non-protein nitrogen, however. 586 Chemical Factors in Fatigue. I Preformed and Total Creatinine. The values for ‘preformed creatinine remained remarkably constant in both periods of exercise. This is no more than would be expected from the repeatedly observed constancy of creatinine as a blood constituent. There seemed to be a very slight ten- dency to increase, however, since a number of cases showed a noticeable rise, but no instance of a fall was observed. This increase amounted to less than 0.1 mg. on the average and was equally distributed between corpuscles and plasma. Total creatinine was slightly increased, on the average, in both periods of exercise, although the distribution coefficient remained unchanged. The increase was not invariable, however; in several cases there was no observable change but in only two was there even a slight decrease. : The colorimetric comparison in the creatinine determinations is by far the most difficult of any, giving a certain element of uncertainty to small differences. The effect of exercise upon the preformed and total creatinine of the blood must therefore be considered more or less doubtful. Number of Red Corpuscles. Along with the other determinations red blood counts were made on the first thirteen subjects in the short period. The time consumed was so considerable and the results themselves so uncertain and variable in unskilled hands that, after establish- ing the fact that an actual increase occurred, the counts were not made beyond this point. Though the individual results are not given in the table the average of 13 sets of determinations showed an increase from 5.36 million to 5.55 million per c.mm. This increase, though small, is in fair agreement with those reported by Hawk (8) and by Schneider and Havens (9) over relatively long periods of exercise. ~ Specific Gravity, Corpuscle Volume, and Hemoglobin. Except in three cases in which no change was observed the specific gravity increased invariably in both periods of exercise. This increase amounted, on the average, to 0.004 and 0.002 in the two periods. EEL L——<$— - -§ ti. it nee ite nek ee a N. W. Rakestraw 587 With very few exceptions the relative volume of the corpuscles increased noticeably. The relative hemoglobin values, or “hemoglobin ratios,” showed a slight increase in most of the cases observed. This increase averaged 3 to 5 per cent. These changes in specific gravity, corpuscle volume, and hemoglobin content are undoubtedly to some extent attributable to increases in the number of corpuscles. An increase in the corpuscle volume has also been shown to result from the higher carbon dioxide content of the blood. Viscosity. With very few exceptions the viscosity of the whole blood increased considerably in both periods. The reason for this increase has been pointed out by Adam (10), who has shown that the blood viscosity is dependent not only upon the concentration of the blood and the number of corpuscles, but especially upon the carbon dioxide content. Since the latter fact was not fully appreciated until after this work was nearly completed no careful precautions were taken to make the viscosity measurements under similar conditions of carbon dioxide tension. In all cases the blood was mixed sufficiently to prevent settling of the cor- puscles, but not shaken enough to result in the loss of much carbon dioxide. The values are, therefore, purely qualitative. Adam has shown that the viscosity of the plasma is also affected by the carbon dioxide content and from his results it would seem that the slight increases in the plasma viscosity found in this work may be traced almost wholly to that source, since the plasma was not arterialized by shaking or passage of oxygen. Cholesterol. The data on cholesterol are collected separately in Table VI. The relative corpuscle volumes are not included, since most of the subjects are the same as those in Tables III and IV. The changes in cholesterol were generally small and not thoroughly consistent. On the whole, however, there was a general tendency to decrease, more noticeable in the short period than in the long, to which the corpuscles contributed more than the plasma. Not only did the average show a slight decrease but in only three cases, in each period, was there an increase. 588 Chemical Factors in Fatigue. TABLE VI. Cholesterol per 100 cc. Short period. Whole Corpus- blood. cles. Subject. Plasma. oWGB 11 | ABS JMWG 13 |*EDB 165 14 | 9GK 161 16 | 9IGM 17 | ¢FSE 18 | (CEH 19 | 7HCM 20 | SMJH 21 JRWS Averages.... 11 13 14 15 16 20 - Subject. NWR NWR J ELG o' ABS co EDB 9GK SWAC 2? BBR 29 IGM ? MEC 9 MJH I Long period. Whole Corpus- blood. ee Plasma. alas 136 N. W. Rakestraw 589 Considerations Bearing on Blood Volume. There are a number of considerations which substantiate the conclusion that the total blood volume underwent no considerable change during the course of these experiments: (1) The hemo- globin content did not increase much over 5 per cent in any case, which can to some extent at least be attributed to an increase in the number of red corpuscles which is known to have occurred. (2) Increases in specific gravity were slight and may also be explained as in (1). (8) The increase in the plasma viscosity, which would quickly reflect any considerable change in the blood concentration, was slight, and it is known that a consider- able portion of this increase was due to a higher content of carbon dioxide. (4) The values for preformed creatinine, recognized as an especially constant blood constituent, showed very little increasing tendency. It is very likely that any considerable change in the blood volume would be here apparent. The combined evidence from these different sources makes it practically conclusive that changes in blood volume cannot be regarded as important disturbing influences in the validity of the analytical results_of these experiments. SUMMARY. 1. An investigation was undertaken on twenty-one human subjects to determine the changes produced by severe muscular exercise upon the following constituents of blood and plasma: Non-protein nitrogen, urea, sugar, uric acid, preformed and total creatinine, cholesterol, and hemoglobin, as well as specific gravity, viscosity, and the number and relative volume of corpuscles. 2. Two types of exercise were employed, representing short, strenuous effort and longer, more tedious work. 3. Short. strenuous exercise was invariably found to increase the blood sugar concentration both in plasma and corpuscles, while a longer period of exercise was generally accompanied by a drop in blood sugar, which was greater in the plasma than in the whole blood. 4, Both kinds of exercise were accompanied by a small increase in uric acid, of about the same order, which was greater in the plasma than in the whole blood. 590 Chemical Factors in Fatigue. I 5. Short, strenuous exercise had no effect upon urea or non- protein nitrogen, but longer work increased both slightly, in whole blood as well as plasma. 6. In both types of exercise the total creatinine increased very little, while the preformed creatinine underwent almost no change. 7. It is shown conclusively that there were no considerable changes in the total blood volume during the muscular exercise and that variations in the concentration of the blood are not, therefore, disturbing factors in the above conclusions, 8. Cholesterol was found to decrease very slightly, although results were not thoroughly consistent. The decrease seemed to be somewhat more noticeable in the corpuscles than in the plasma. 9. The specific gravity, hemoglobin, and the number and relative volume of corpuscles were found to increase during the periods of exercise. The viscosity of the whole blood was found _ to increase considerably and that of the plasma slightly. 10. Some incomplete data are given suggesting that total nitrogen is increased in the blood by exercise and that urea, non- protein nitrogen, and uric acid continue to increase for some time after a work period, while the sugar concentration, on the other hand, returns to normal within 23 hours. The author wishes to express his thanks to Professor R. E. Swain, at whose suggestion this work was undertaken, for many helpful suggestions; to the several students and others who volunteered as subjects; and to Miss Dorothy E. Bernard, who was responsible for the analyses of cholesterol. BIBLIOGRAPHY. . Folin, O., and Wu, H., J. Biol. Chem-, 1919, xxxviii, 81. Myers, V. C., and Wardell, E. L., J. Biol. Chem., 1918, xxxvi, 147. . Cohen, B., and Smith, A. H., J. Biol. Chem., 1919, xxxix, 489. . Sundstroem, E. S., and Bloor, W. R., J. Biol. Chem., 1920-21, xlv, 157. . Ege, R., Biochem. Z., 1920, exi, 189. . Wishart, M. B., J. Biol. Chem., 1920, xliv, 563. . Falta, W., and Richter-Quittner, M., Biochem. Z., 1919, c, 148. . Hawk, P. B., Am. J. Physiol., 1903-04, x, 384. om wh ~, ° i | a SS ‘ ——E——————— ee eee ws N. W. Rakestraw 591 9. Schneider, E. C., and Havens, L. C., Am. J. Physiol., 1914-15, xxxvi, 239. 10. Adam, H., Z. klin. Med., 1909, Ixviii, 177. More or less complete bibliographies of the general subject of fatigue will be found in the following: Brésamlen, O., and Sterkel, H., Deutsch. Arch. klin. Med., 1919, exxx, 358. Burian, R., Z. physiol. Chem., 1904-05, xlili, 5382. ~ Grote, L., Ueber die Beziehungen der Muskelarbeit zum Blutzucker, Halle, 1918. Hastings, A. B., Bull. Hyg. Lab., U. S. P. H., 1919, xxxiv, 1682. Lee, F.S., J. Am. Med. Assn., 1906, xlvi, 1491; Am. J. Physiol., 1907, xviii, 267; 1907-08, xx, 170. Lichtwitz, L., Berl. klin. Woch., 1914, l, 1018. von Moraczewski, W., Biochem. Z., 1915, Ixxi, 268. Reach, F., Biochem. Z., 1911, xxxiii, 436. Seaffidi, V., Biochem. Z., 1911, xxx, 473. Scott, E. L., Bull. Hyg. Lab., U. S. P. H., 1918, xxxiii, 605. Scott, E. L., and Hastings, A. B., Bull. Hyg. Lab., U. S. P. H., 1920, xxxv, 2445. Sherman, H. C., J. Am. Chem. Soc., 1903, xxv, 1159. Spaeth, R. A., J. Ind. Hyg., 1919, i, 1. Weiland, W., Deutsch. Arch. klin. Med., 1908,. xcii, 223. ee Nero ee Sad cloran INDEX TO VOLUME XLVII. ABSORPTION of calcium salts i in man, note on, 3 Acid, arcealieviet a reagent for the estimation of sugar in nor- mal and diabetic urine, 5 ——, hydrochloric, ingestion, effect upon the composition of the urine in man, 315 ——, phosphoric, and calcium, dis- tribution in the blood of normal children, 321 , ——, distribution in the blood of normal infants, 53 Acids, dicarboxylic, hydrolysis of the esters of some, by the lipase of the liver, 495 ApotpeH, Epwarp F., and Ferry, Ronatp M. The oxygen dis- sociation of hemoglobin, and the effect of electrolytes upon it,547 Alkali, blood, and respiration during carbon monoxide asphyxia, 421 Analogy, in vitro, of antiketogenesis, 433 Analysis apparatus, improved Hal- dane gas, and a simple labora- tory gas meter, 489 ——, certain biochemical methods of substitution of turbidimetry for nephelometry, 27 ——., gas, sampling bottle for, 281 Antiketogenesis, 433, 449 Antiketogenetic balance in man, ketogenetic, 449 Antiscorbutic vitamine, effect of heat and oxidation upon, 483 Apparatus for reducing milk, fruit juices, and other fluids to a powder without destruction of vitamines, methods of extract- 5 93 ing and concentrating vitamines A, B, and C together with, 411 Apparatus, improved Haldane gas analysis, and a simple labora- tory gas meter, 489 —— used in determining the res- piratory exchange in man, notes on, 277, 281 Arginine and cystine as precursors of creatine, 33 Asphyxia, carbon monoxide, respi- ration and blood alkali during, 421 Autolysis of brain, 333 ——.,, studies of, 333 BAILEY, Cameron V. Notes on apparatus used in determining the respiratory exchange in man. I. An adaptation of the French gas mask for use in res- piratory work, 277. II. A sam- pling bottle for gas analysis, 281 Basal metabolism, influence of col- loidal iron on, 557 —— —— of normal women, 69 Beans, Chinese and Georgia velvet, relative digestibility of various preparations of the proteins from, 285 Benzidine method, determination of inorganic sulfate, total sulfate, and total sulfur in urine, 59 Biochemical methods of Scr. substitution of turbidimetry for nephelometry, 27 Blood alkali and respiration during carbon monoxide asphyxia, 421 594° Index Blood constituents, certain com- mon, effect of muscular exercise upon, 565 of normal children, distribu- tion of calcium and phosphoric acid in, 321 infants, distribution of phosphoric acid in, 53 —— serum, diffusible calcium of, a method for its determination, 529 Bioor, W. R. See McKeuurps, De Youna, and Buioor, 53 Buiunt, KaTHARINE, and Dye, Martz. Basal metabolism of normal women, 69 BovuTtweELL, P. W. See STEENBOCK, SELL, and BoutweE tu, 303 Brapuey, H. C. See GIBSON, UmpreiT, and Braptey, 333 Brain, autoloysis of, 333 BuELL, Mary V. See STEENBOCK, Sevx, and Buett, 89 (CALCIUM and magnesium in small amounts of serum, a simple technique for the de- termination, 475 —— —— phosphoric acid distribu- tion in the blood of normal children, 321 . —, diffusible, of the blood serum, a method for its determination, 529, 541 —— salts in man, note on the ab- sorption, 3 Calorimetry, animal, 557 Carbon dioxide, relation of migra- tion of ions between cells and . plasma to the transport of, 377 —— monoxide asphyxia, respiration and blood alkali during, 421 Catalase reaction, study of, 341 Cells and plasma, relation of migra- tion of ions between, to the transport of carbon dioxide, 377 Cereal and legume seeds, supple- mentary dietary relations be- tween animal tissues and, 139 — grains, proteins of, supple- mentary dietary relations be- tween the potato and, 175 ——-— supplementary relations of cereal grains with, with respect to improvement in the quality of their proteins, 207 —-- ; relations of legume seed with, with respect to im- provement in the quality of their proteins, 207 ——, supplementary relations of the proteins of milk for those of, and of milk for those of legume seeds, 235 CuHaAPIN, Ropert M. The deter- mination of cresol by the phenol reagent of Folin and Denis, 309 Chemical factors in fatigue, 565 Chinese and Georgia velvet beans, relative digestibility of various preparations of the proteins from, 285 CHRISTMAN, ADAM A., and Lewis, HowarpB. Lipasestudies. I. The hydrolysis of the esters of some dicarboxylic acids by the lipase of the liver, 495 Criark, E. P. Preparation of galae- tose, 1 Colloidal iron on basal metabolism, influence, 557 Colorimeter, nephelometer-, further improvements, 19 Constituents, certain common blood, effect of muscular exercise upon, 565 Creatine, arginine and cystine as precursors of, 33 Creatinuria, 33, 45 , effect of thyroid feeding upon, 45 Cresol, determination by the phenol reagent of Folin and Denis, 309 _ OEE EO EEE EEE ———_ Sh — ee - Cystine and arginine as precursors of creatine, 33 ENIS, W. On the substitution of turbidimetry for nephelo- metry in certain biochemical methods of analysis, 27 — and Folin’s phenol reagent, de- termination of cresol by, 309 Determination, gasometric, of ni- trogen, 11 ——, ——, of urea in urine, 13 — of calcium and magnesium in small amounts of serum, a simple technique for, 475 eresol by the phenol re- agent of Folin and Denis, 309 inorganic sulfate, total sul- fate, and total sulfur in urine by the benzidine method, 59 sugar in normal and dia- betic urine, dinitrosalicylic acid as a reagent, 5 the diffusible calcium of the blood serum, a method for, 529 respiratory exchange in man, notes on apparatus used in, 277, 281 DrYoune, I. M. See McKE tips, De Young, and Bioor, 53 Diabetic urine, dinitrosalicylic acid as a reagent for the estimation of sugar in, 5 Dicarboxylic acids, hydrolysis of esters of some, by the lipase of the liver, 495 Diet, deficient in vitamine A, failure of rats to develop rickets on, 395 Dietary relations between the pro- teins of the cereal grains and the potato, 175 —— ——, supplementary, between animal tissues and cereal and legume seeds, 139 Diets low in phosphorus and fat- soluble A, production of rickets by, 507 Index 595 Diffusible calcium of the blood se- rum, a method for its determi- nation, 529 Digestibility of proteins in vitro, studies on, 285 ——., relative, of various prepara- tions of the protein from the Chinese and Georgia velvet beans, 285 Dinitrosalicylic acid, a reagent for the estimation of sugar in nor- mal and diabetic urine, 5 Dotsy, Epwarp A., and Eaton, Emity P. The relation of the migration of ions between cells and plasma to the transport of carbon dioxide, 377 DutcueEr, R. Apams, Harsuaw, H. M., and Hatt, J. S. Vitamine studies. VIII. The effect of heat and oxidation upon the antiscorbutic vitamine, 483 Dye, Marie. See Biunt and Dye, 69 FATON, Emity P. See Dorsy and Eaton, 377 Eppy, Water H., Hert, Harrie L., StevENsSON, HELEN C., and Jounson, RurH. Studies in the vitamine content. II. The yeast test as a measure of vita- mine B, 249 Electrolytes upon the oxygen dis- sociation of hemoglobin, 547 Esters of some dicarboxylic acids, hydrolysis of, by the lipase of the liver, 495 Exercise, muscular, effect upon cer- tain common blood constitu- ents, 565 FATIGUE, chemical factors in, 565 Fats, animal, fat-soluble vitamine and yellow pigmentation in, with some observations on its stability to saponification, 89 596 Index Fat-soluble A and phosphorus, diets low in, production of rickets by, 507 — vitamine, 89, 303 —— —— and yellow pigmentation in animal fats with some obser- vations on its stability to sapon- ification, 89 content of peas in relation to their pigmentation, 303 Feeding, thyroid, effect upon crea- tinuria, 45 Ferry, Ronatp M. See ApDoLpH and Ferry, 547 Fiske, Crrus H. The determina- tion of inorganic sulfate, total sulfate, and total sulfur in urine by the benzidine method, 59 Folin and Denis’ phenol reagent, determination of cresol by, 309 Foods, supplementary protein val- ues in, 111, 139, 175, 207, 235 (GALACTOSE, preparation, 1 Gas analysis apparatus, Haldane, improved, and a simple labora- tory gas meter, 489 —— ——-, sampling bottle for, 281 — mask, French, for use in respi- ratory work, an adaptation, 277 meter, simple laboratory, and an improved Haldane gas analy- sis apparatus, 489 Gasometric determination of nitro- gen, ll —— —— —— urea in urine, 13 Georgia and Chinese velvet beans, relative digestibility of various preparations of the proteins from, 285 Gipson, CHarutes A., UMBREIT, Frepa, and Brapury, H. C. Studies of autolysis. VII. Au- tolysis of brain, 333 Grain, cereal, proteins of, supple- mentary dietary relations be- tween the potato and, 175 ——, ——, supplementary relations of cereal grain with, with respect to improvement in the quality of their proteins, 207 , , —— relations of legume seed with, with respect to im- provement in the quality of their proteins, 207 Gross, E. G., and Strensock, H. Creatinuria. II. Arginine and cystine asprecursors of creatine, 33. III. The effect of thyroid feeding upon creatinuria, 45 Hccarp, Howarp W., and Hen- DERSON, YANDELL. Hemato- respiratory functions. XII. Respiration and blood alkali during carbon monoxide as- phyxia, 421 Haldane gas analysis apparatus, im- proved, and a simple laboratory gas meter, 489 Hatz, J. S. See DurcHer, Har- sHAaw, and Hat, 483 Harsuaw, H. M. See Durcusr, Harsuaw, and Hatt, 483 Heat and oxidation, effect upon the antiscorbutic vitamine, 483 Hert, Harrie L. See Eppy, Hert, STEVENSON, and JoHNson, 249 Hemato-respiratory functions, 421 Hemoglobin, oxygen dissociation of, and the effect of electrolytes upon it, 547 HENDERSON, YANDELL. See Hac- GARD and HENDERSON, 421 Hess, A. F., McCann, G. F., and PAppENHEIMER, A.M. Experi- mental rickets in rats. II. The failure of rats to develop rickets on a diet deficient in vitamine A, 395 a <=. oe a a ' Index Hydrochloric acid ingestion, effect upon the composition of the urine in man, 315 Hydrolysis of the esters of some dicarboxylic acids by the lipase of the liver, 495 Hynes, Water A. See SHERWIN and Hynes, 297 INGESTION, hydrochloric acid, effect upon the composition of the urine in man, 315 Inorganic sulfate, total sulfate, and total sulfur in urine determina- tion, by the benzidine method, 59 In vitro analogy of antiketogenesis, 433 —— -——., digestibility of proteins, studies on, 285 Ions, relation of migration of, be- tween cells and plasma to the transport of carbon dioxide, 377 Iron, colloidal, influence on basal metabolism, 557 JOHNSON, Rurs. See Eppy, Hert, STEVENSON, and JoHN- SON, 249 Jones, D. BREESE. and JoNnss, 285 Jones, Martua R., and Nys, Lit- LIAN L. The distribution of calcium and phosphoric acid in the blood of normal children, 321 See WATERMAN JK. ETOGENETIC antiketogenetic balance in man, 449 Kuett, Ropert E. See Koper and Kerr, 19 Koper, Partie Apoupu, and Kier, Rosert E. Further improve- ments in the nephelometer- colorimeter, 19 597 Kramer, BENJAMIN, and TIsDALL, Freperick F. A simple tech- nique for the determination of calcium and magnesium in small amounts of serum, 475 J, ANGFELDT, Ervar. Animal calorimetry. Seventeenth pa- per. The influence of colloidal iron on the basal metabolism, 557 Legume seeds and cereal, supple- mentary dietary relations be- tween animal tissues and, 139 —— ——, supplementary relations of cereal grain with, with re- spect to improvement in the quality of their proteins, 207 ; relations of legume seed with, with respect to im- provement in the quality of their proteins, 207 relations of the pro- * teins of milk for those of, and of milk for those of cereals, 235 Lewis, Howarp B. See Curist- MAN and Lewis, 495 Lipase of the liver, hydrolysis of the esters of some dicarboxylic acids by, 495 — studies, 495 Liver, lipase of, hydrolysis of the esters of some dicarboxylic acids by, 495 MAGNESIUM and caleium in small amounts of serum, a simple technique for the deter- mination, 475 Mask, French gas, for use in respi- ratory work, an adaptation, 277 Mason, Epwarp H. A note on the absorption of calcium salts in man, 3 McCann, G.F. See Hess, McCann, and PAPPENHEIMER, 395 598 Index McCann, G.F. See von Mrysen- BuG and McCann, 541 McCarry, Artour C. See SrTexLe and McCarry, 315 McCuienpon, J. F. Methods of ex- tracting and concentrating vi- tamines A, B, and C, together with an apparatus for reducing milk, fruit juices, and other fluids to a powder without des- truction of vitamines, 411 McCo.tuivm, E. V., Srmmonps, NINA, and Parsons, H. T. Supple- mentary protein values in foods. I. The nutritive properties of animal tissues, 111. II. Sup- plementary dietary relations be- tween animal tissues and cereal and legume seeds, 139. III. The supplementary dietary re- lations between the proteins of cereal grains and the potato, 175. IV. The supplementary relations of cereal grain with cereal grain; legume seed with legume seed; and cereal grain with legume seed, with respect to improvement in the quality of their proteins, 207. V. Sup- plementary relations of the pro- teins of milk for those of cereals and of milk for those of legume seeds, 235 ——, —, Suiptey, P. G., and Park, E. A. Studies on experi- mental rickets. VIII. The pro- duction of rickets by diets low in phosphorus and fat-soluble A, 507 McKetuirs, G. M., De Youna, I. M., and Bioor, W. R. The distribution of phosphoric acid in the blood of normal infants, 53 Metabolism, basal, influence of col- loidal iron on, 557 Metabolism, basal, of normal wo- men, 69 —— of nitrobenzaldehydes and ni- trophenylacetaldehyde, 297 Meter, simple laboratory gas, and an improved Haldane gas anal- © ysis apparatus, 489 Method, benzidine, determination of inorganic sulfate, total sul- fate, and total sulfur in urine by, 59 for the determination of diffu- sible calcium of the blood serum, 529 Methods, biochemical, of analysis, substitution of turbidimetry for nephelometry in certain, 27 —— of extracting and concentrating vitamines A, B, and C together with an apparatus for reducing milk, fruit juices, and other fluids to a powder without des- truction of vitamines, 411 Milk, fruit juices, and other fluids, apparatus for reducing, to a powder without destruction of vitamines, methods of extract- ing and concentrating vitamines A, B, and C together with, 411 ——., supplementary relations of the proteins of, for those of cereals, and of milk for those of legume seeds, 235 ——, —— —— of the proteins of, for those of legume seeds, and those of milk for those of cereals, 235 Morevuuis, Serearus. A study of the catalase reaction, 341 Murray, Margortn F. See von MEYSENBUG, PAPPENHEIMER, Zucker, and Murray, 529 Muscular exercise, effect upon cer- tain common blood constitu-. ents, 565 | : | Index 599 N EPHELOMETER-C OLORIM- ETER, further improvements, 19 Nephelometry substituted by tur- bidimetry in certain biochemi- cal methods of analysis, 27 Newcomer, H. 8S. A simple labor- atory gas meter and an im- proved Haldane gas analysis apparatus, 489 Nitrobenzaldehydes and nitrophe- nylacetaldehyde, metabolism of, 297 Nitrogen, gasometric determina- tion, 11 Nitrophenylacetaldehyde and nitro- benzaldehyde, metabolism of, 297 Nutritive properties of animal tis- sues, 111 Nye, Linytran L. See Jones and Ny, 321 ()XIDATION and heat, effect up- on the antiscorbutic vitamine, 483 Oxygen dissociation of hemoglobin and the effect of electrolytes upon it, 547 PAPPENHEIMER, A. M. See Hess, McCann, and Pappren- HEIMER, 395 ——. See von MryYSENBUG, PAPPENHEIMER, ZUCKER; and Murray, 529 Park, E. A. See McCouivum, Siu- MONDS, SHIPLEY, and Park, 507 Parsons, H. T. See McCouivum, Srumonps, and Parsons, 111, 139, 175, 207, 235 Peas, fat-soluble vitamine content of, in relation to their pigmen- tation, 303 Phenol reagent of Folin and Denis, determination of cresol by, 309 Phosphoric acid and calcium, dis- tribution in the blood of normal children, 321 , distribution in the blood of normal infants, 53 Phosphorus and fat-soluble A, diets low in, production of rickets, by, 507 Pigmentation, fat-soluble content of peas in relation to, 303 —, yellow, and fat-soluble vi- tamine in animal fats with some observations on its stability to saponification, 89 Plasma and cells, relation of migra- tion of ions between, to the transport of carbon dioxide, 377 Potato, supplementary dietary re- lations between the proteins of the cereal grains and, 175 Precursors of creatine, arginine and cystine as, 33 Protein values in foods, supplemen- tary, 111, 139, 175, 207, 235 Proteins, digestibility of, in vitro, studies on, 285 — from the Chinese and Georgia velvet beans, the relative di- gestibility of various prepara- tions of, 285 — of milk, supplementary rela- tions of, for those of cereals and of milk for those of legume seeds, 235 —— —— the cereal grains, supple- mentary dietary relations be- tween the potato and, 175 RAKESTRAW, Norris W. Chemi- eal factors in fatigue. I. The effect of muscular exercise upon certain common blood constit- uents, 565 Reaction, catalase, study of, 341 Reagent, dinitrosalicylie acid, for the estimation of sugar in nor- mal and diabetic urine, 5 600 Reagent, phenol, of Folin and Denis, determination of cresol by, 309 Respiration and blood alkali during carbon monoxide asphyxia, 421 Respiratory exchange in man, notes on apparatus used in determin- ing, 277, 281 —— functions, hemato-, 421 —— work, an adaptation of the French gas mask for use in, 277 Rickets, experimental, in rats, 395 —, , studies on, 507 ——.,, failure of rats to develop, on a diet deficient in vitamine A, 395 ——, human, and experimental dog tetany, 541 —, production by diets low in phosphorus and fat-soluble A, 507 ALTS, calcium, in man, note on the absorption, 3 Sampling bottle for gas analysis, 281 Saponification, fat-soluble vitamine and yellow pigmentation in animal fats with some observa- tions on its stability to, 89 —, —— —— stability to, some observations on, 89 Seed, legume, and cereal, supple- mentary dietary relations be- tween animal tissues and, 139 ——, ——.,, supplementary relations of cereal grain with, with re- spect to improvement in the quality of their proteins, 207 — ' relations of legume seed with, with respect to im- provement in the quality of their proteins, 207 , ——, —— relations of the pro- teins of milk for those of, and of milk for those of cereals, 235 Sevt, Mariana T. See SreENBOCK, Setx, and Buett, 89 See Sreenpock, SELL, and 30UTWELL, 303 Index Serum, blood, diffusible calcium of, a method for its determination, 529 ——., determination of calcium and magnesium in small amounts of, a simple technique for, 475 Suarrer, Puirie A. Antiketo-gen- esis. I. An in vitro analogy, 433. II. The ketogenic antike- togenic balance in man, 449 SHerwin, Cart P., and Hyngs, Water A. The metabolism of nitrobenzaldehydes and nitro- phenylacetaldehyde, 297 SuipLrey, P. G. See McCouiium, StmmMonps, SHIPLEY, and Park, 507 Srmonps, Nina. See McCouiium, Srmmonps, and Parsons, 111, 139,175, 207, 235 See McCotitum, Srmmonps, Surpuey, and Park, 507 Sreensock, H., Sevy, Mariana T., and Buett, Mary V. Fat-sol- uble vitamine. VII. The fat- soluble vitamine and yellow pig- mentation in animal fats with some observations on its stabil- ity to saponification, 89. VIII. The fat-soluble vitamine con- tent of peas in relation to their pigmentation, 303 See Gross and STEENBOCK, 33, 45 SrreHLe, Raymonp L. The gaso- metric determination of urea in urine, 13 Note on the gasometric de- termination of nitrogen, 11 — and McCarry, Artuur C. The effect of hydrochloric acid in- gestion upon the composition of the urine in man, 315 Srpvenson, HeLten C. See Eppy, . Hert, Stevenson, and JoHN- soN, 249 Index Sugar in normal and diabetic urine, dinitrosalicylic acid as a reagent for the estimation, 5 Sulfate, inorganic and total, and total sulfur in urine, determina- tion by the benzidine method, 59 Sulfur, total, inorganic sulfate, and total sulfate in urine, determi- nation by the benzidine method, 59 Sumner, James’ B. Dinitrosali- cylic acid: A reagent for the estimation of sugar in normal and diabetic urine, 5 "TEST, yeast, as a measure of vi- tamine B, 249 Tetany, experimental dog, human rickets, 541 Thyroid feeding upon creatinuria, effect, 45 and TIsDALL, FrepERICK F. See Kra- MER and TIsDALL, 475 Tissues, animal, nutritive proper- ties, 111 ——, ——., supplementary dietary relations between cereal and legume seeds and, 139 Turbidimetry for nephelometry in certain biochemical methods of analysis, substitution, 27 UMBREIT, Frepa. See Gisson, Umprett, and BRraDLey, 333 Urea in urine, gasometric determi- nation, 13 Urine, composition in man, effect of hydrochlorie acid ingestion upon, 315 ——, determination of inorganic sul- fate, total sulfate, and total sul- fur in, by the benzidine method, 59 ——., diabetic and normal, dinitro- salicylic acid as a reagent for the estimation of sugar in, 5 601 Urine, gasometric determination of urea in, 13 ——, normal and diabetic, dinitro- salicylic acid as a reagent, for the estimation of sugar in, 5 VITAMINE A, failure of rats to develop rickets on a diet defi- cient in, 395 ——, antiscorbutic, effect of heat and oxidation upon, 483 —— B, yeast test as a measure of, 249 —— content, fat-soluble, of peas in relation to their pigmentation, 303 ———, studies in, 249, ——, fat-soluble, 89, 303 —., , and yellow pigmentation in animal fats with some obser- vations on its stability to sapon- ification, 89 —— studies, 483 Vitamines A, B, and C, methods of extracting and concentrating, together with an apparatus for reducing milk, fruit juices, and other fluids to a powder without destruction of vitamines, 411 ——., apparatus for reducing milk, fruit juices, and other fluids to a powder without destruction of, together with methods of extracting and concentrating vitamines A, B, and C, 411 von Mrysensua, L., and McCann, G. F. The diffusible calcium of the blood serum. II. Hu- man rickets and experimental dog tetany, 541 ——, ParpeNHEIMER, A. M., Zucker, T. F., and Murray, Marsorig F. The diffusible calcium of blood serum. I. A method for its determination, 529 602 - Index WATERMAN, Henry C., and ‘YEAST test asa measure of vita- Jones, D. Breese. Studies mine B, 249 ' on the digestibility of proteins in vitro. II. The relative di- gestibility of various prepara- tions of the proteins from the Chinese and Georgia velvet beans, 285 7 UCKER, T. F. See von Mery- SENBUG, PAPPENHEIMER, ZUCK- eR, and Murray, 529 THE JOURNAL OF BIOLOGICAL CHEMISTRY FOUNDED BY CHRISTIAN A. HERTER AND SUSTAINED IN PART BY THE CHRISTIAN A. HERTER MEMORIAL FUND OFFICIAL ORGAN OF THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS EDITED BY STANLEY R. BENEDICT, New York,N. Y. LAFAYETTE B. MENDEL, New Haven, Conn. HENRY D. DAKIN, Scarborough, N. Y. DONALD D. VAN SLYKE, New York, N. Y. WITH THE COOPERATION OF THE EDITORIAL COMMITTEE OTTO FOLIN, Boston, Mass. WALTER JONES, Baltimore, Md. L. J. HENDERSON, Cambridge, Mass. GRAHAM LUSK, New York, N.Y. ANDREW HUNTER, Toronto, Canada THOMAS B. OSBORNE, New Haven, Conn, WALTER W. PALMER, New York, N. Y. A. N. RICHARDS, Philadelphia, Pa. L. L. VAN SLYKE, Geneva, N. Y. VOLUME XLVIII BALTIMORE 1921 Copyrnriaut 1921 BY THE JOURNAL OF BIOLOGICAL CHEMISTRY PUBLISHED BY THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH FOR THE JOURNAL OF BIOLOGICAL CHEMISTRY, INC. WAVERLY PRESS Tue Witiiams & WiLKiIns Company Battimore, U.S. A. CONTENTS OF VOLUME XLVIII. No.1. September, 1921. TispALL, FREDERICK F., and Kramer, Bensamin. Methods for the direct quantitative determination of sodium, potassium, cal- cium, and magnesium in urine and stools..................... Kinessury, F. B., and Swanson, W. W. A rapid method for the determination of hippuricraeiauin urine... 25... 6eeeseooneee MILtER, C. W., and Sweet, J. E. Note on a possible source of error in Fesuine sOreasence-WOnResupLOLee cs <2 35s. psc. 252 ae oe ne anes AnvERSON, R. J. Acerin. The globulin of the maple seed (Acer sac- CHOTAILUNT) eae Pe ET oe Se OT AE EE Hart, E. B., Sreensock, H., and Hoprert, C. A. Dietary factors in- fluencing calcium assimilation. I. The comparative influ- ence of green and dried plant tissue, cabbage, orange juice, and cod liver oll om calcium assimilatione....5....+.5 0050. 0ees a. BENEDIcT, STANLEY R., and OsterBEeRG, Emiu. A method for the determination or-sucaran normaleunmes:. 42.2242. seeeso eee GREENE, CHARLES W. Chemical development of the ovaries of the king salmon during the spawning migration..... See eee es Ditt, D. B. A chemical study of certain Pacific Coast fishes........ . Myers, Victor C., and SHort, JAMEs J. The potassium content of normal and some pathological human bloods................... Ditt, D. B. A chemical study of the California sardine (Sardinia DOAROTNT BSE A ARIES Beto e dc G0 © 3.0 JRE AE aor Buavu, NatHan F. The estimation of creatinine in the presence of ace- bone said) diaicetic™ ae lle «eee oie esa sw ahot se ehend oles slo aia Levene, P. A. On the structure of thymus nucleic acid and on its pos- sible bearing on the structure of plant nucleic acid............. Hammett, FrepericK 8. Creatinine and creatine in muscle ex- tracts. I. A comparison of the picric acid and the tungstic acid, methods of deprotemizatiames. 3.5 ..n6.s ..sdacstac een see Hammett, FREDERICK S. Creatinine and creatine in muscle extracts. Il. The influence of the reaction of the medium on the crea- tinine-creatine balance in incubated extracts of muscle tissue of (avepenlll yield eh repaid ciao a\nicits 3.510 > RONIERC eR ROE RCI Boo b cio urkoig ur: Hammett, Freperick 8. Studies of the thyroid apparatus. IV. The influence of parathyroid and thyroid tissue on the creatinine- creatine balance in incubated extracts of muscle tissue of the 312) ona Og PERE ORE Sxo\ic3 Shs: coho 2): CeO ee ike RISE pee tcc Van Stryke, Donatp D. Studies of acidosis. XVII. The normal and abnormal variations in the acid-base balance of the blood... ili 105 119 127 133 143 153 lv Contents Levenn, P. A. Preparation and analysis of animal nucleic acid...... Wes LEvENE, P. A., and Simms, H.S. The liver lecithin................. 185 Levene, P. A. On the numerical values of the optical rotations in the SUPA ACIOSs.4: yn eee ie hen sense es) banana ee 197 Eacrertu, ARNotp H. The preparation and standardization of collo- CUOMINEMPEAN CH re ete eines sietsce. oie. s cole: s.0.0'o ateyeeiehe a ale bere eRe 203 Kramer, BensJAMIN, and TispauL, Freperick F. The direct quan- titative determination of sodium, potassium, calcium, and magnesium in small amounts of blood...............0--+fseeee 223 Levenp, P. A., and Meyer, G. M. Phosphoric esters of some sub- stituted glucoses and their rate of hydrolysis...............++. 233 No. 2. October, 1921. Hamitton, T. S., Nevens, W. B., and Grinp.tey, H..S. The quan- titative determination of amino-acids of feeds................ 249 Dakin, H. D. The synthesis of inactive para- and anti- hydroxy- aBPArLIC ACOs (Amminomalic acids)... ....... 232 sirsieyiaweutas FP ee 273 LeuMan, Epwin P. Studies in inorganic blood phosphate............ 293 Hart, E. B., and Humpurey, G. C. Can “home grown rations” supply proteins of adequate quality and quantity for high milk BREE UIGEION ow cis Si oa gs os aa nels eee het a 305 Fitz, Reainavp, and Bock, Artin V. Studies on blood sugar. The total amount of circulating sugar in the blood in diabetes mel- Litts anc sObHeriCOnGUIbIONS. . «os... «=. eo eee eee 313 Smita, Erma, and Mrepes, Grace. Effect of heating the antiscor- butic vitamine in the presence of invertase.............-....--. 323 | Mituer, Exvizasetu W. The effect of certain stimulating substances onthe invertase activity of yeast... .... 2. ca. acelae ae rae te ee 329 Jones, D. Breese, and Jouns, Cart O. Determination of the mono- amino-acids in the hydrolytic cleavage products of lactalbumin. 347 Bopansky, Meyer. The zine and copper content of the human brain. 361 GuTuRI£, CHARLES CLAuprE. A simplified form of apparatus for air CRAG M eS eRe ae yee. 2) nn Se SO eT At oa: 365 GUTHRIE, CHARLES CLAUDE. A gas receiver of convenient and prac- tical form for sampling expired air for analysis................. 373 Damon, Samurt R. Bacteria as a source of the water-soluble B VI GARIN recap Ph see dihs coon ie Pie's (o'o,e us cdc, oe o> = ORE eo eee 379 Frep, E. B., Pererson, W. H., and AnpEerRson, J. A. The charac- teristics of certain pentose-destroying bacteria, especially as concerns their action on arabinose and xylose. Plates 1 and 2. 385 Briacs, A. P., and SHarrer, Purtirp A. The excretion of acetone Prom) the uv Pe aat ee testa ate... c.0'-s -s.c-s-12 215 2 Eee ee 413 GREENE, CHARLES W. Carbohydrate content of the king salmon tissues during the spawninp mipration........... ccsseeeeie asso ae 429 I'unx, Casrmrr, and Dupin, Harry E. Vitamine requirements of Certain yeasis AnNGWDACTORIA SMe. rcs « oe-::s)s...c ee cee ee eee 437 Contents Vv WELKER, WILLIAM H., and Botuman, J. L. The effect of subcutaneous injections of solutions of potassium eyanide on the catalase eememinor the: blood.< Jagere done nk ©: cnn S350 Oot eee ey oe 445 Suppies, G. C., and Betis, B. Citric acid content of milk and milk RAMEE SS oo «co's. saree MR ec « ol cucrd oe varce ronty punk aya vere oe eee 453 Nasu, THomas P., Jr., and Benepict, STANLEY R. The ammonia content of the blood, and its bearing on the mechanism of acid Metin ization angheranimas | eOreaAMISINe- 4.) eee oie 463 ’ Baupiscu, Oskar. The mechanism of reduction of nitrates and MItLIGES 1 PTLOCESsesuOMmassimilation= |5. s6 4... eee eee eee 489 RINGER, MicHArEL, and UNDERHILL, FRANK P. Studies on the physio- logical action of some protein derivatives. VII. The in- fluence of various protein split products on the metabolism of LASUING? LOLS saat ee eam no 5 aint said’ =. nian dis atom nary 503 Rincer, MicHak&L, and UNDERHILL, FRANK P. Studies on the physio- logical action of some protein derivatives. VIII. The in- fluence of nucleic acids on the metabolism of fasting dogs..... 523 UNDERHILL, FRANK P., and RincrER, MicHaEL. Studies on the physio- logical action of some protein derivatives. IX. Alkali reserve AIG XP SLIME LAL SHOCK.) cele MoE eos csc 25 5.0. iis Sete ee 533 UNDERHILL, FRANK P., and Lonc, Mary Louisa. Studies on the physiological action of some protein derivatives. X. The influence of nucleic acid on the metabolism of the fasting UNDERHILL, FRANK P., GREENBERG, PHILip, and ALu, ANTHONY F. Studies on the physiological action of some protein deriva- tives. XI. The influence of some protein split products upon the metabolism of fasting rabbits........................ 549 UNDERHILL, FRANK P., and NELLANS, CHartes T. The influence of thyroparathyroidectomy upon blood sugar content and alkali MESCIAViG hls tacts ek SS Ree ss, = 0 Rqeisiens oie aavate herrea = 557 Roses, Witi1amM C. The influence of food ingestion upon endogenous PUTIN HIME EAD Ol Sia lente ONE RS ot... cares Stes earn sae 563 Rose, Wiuu1amM C. The influence of food ingestion upon endogenous Purine metabolismiy asleep rps ls... 6. crcysyses x sine PPT Cs 575 iedexto, Volumes Vili eo = ives oa s sarees eo sae empes 591 METHODS FOR THE DIRECT QUANTITATIVE DETER- MINATION OF SODIUM, POTASSIUM, CALCIUM, AND MAGNESIUM IN URINE AND STOOLS. By FREDERICK F. TISDALL anp BENJAMIN KRAMER. (From the Department of Pediatrics, the Johns Hopkins University, Baltimore.) (Received for publication, May 23, 1921.) In the study of the inorganic metabolism of children it is fre- quently necessary to perform a large number of determinations of the various inorganic elements in urine and stools. The ques- tion of the amount of material available and the time required for a given determination becomes an important consideration. We have elsewhere reported simple methods for the quantitative estimation of sodium, potassium, calcium, and magnesium in serum (1, 2, and 3). It has been found possible to modify the sodium, potassium, and calcium methods so as to make them appli- cable to the acid extract of the partly ashed residue of urine and stools. For the determination of magnesium in urine and stools we have used the principle of alkalimetric titration of ammonium magnesium phosphate first suggested by Stolba (4). This pro- cedure was subsequently modified by Mohr (5) and Kraus (6). Recently the same principle has been used by Bauzil (7), Angiolani (8), and Fiske (9) for the estimation of inorganic phosphates in urine. By the use of the methods described below a considerable saving in time and material is effected. The degree of accuracy of these methods is indicated in the tables. Preparation of Material. Stools —The fresh stool for a measured period is collected in a weighed porcelain dish. This is heated on the water bath until dry. 95 per cent alcohol is added and evaporated. This latter procedure is repeated, making two additions of alcohol in all. After thorough drying over the water bath the dish and contents 1 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIIJ, No. 1 2 Inorganic Elements in Urine and Stools are weighed and the weight of the air-dried stool is found. The fecal material is then ground to a fine powder and placed in a stop- pered container. 2 gm. of this material are weighed in a platinum crucible and ashed for 14 hours by the Stolte (10) method.!. An ash-free filter paper (No. 40 Whatman, 11 cm. in diameter) is then washed by allowing 20 to 30 cc. of 0.5 n HCl to run through it. The platinum crucible containing the partly ashed stool is placed on the water bath and 10 cc. of 0.5 N HCl are added. After this has become hot it is transferred with a 10 cc. pipette and filtered through the washed filter paper into a 100 cc. flask. The pro- cedure is repeated until the volume has been made up to 100 ce. The results obtained on this extract are identical with those found on a solution of completely ashed stool (Table VI). Urine-—A measured quantity of urine (50 or 100 cc.) is evapo- rated in a platinum dish, ashed, and extracted in a similar manner to the stool. The extract is then made up to the original volume of the urine. Sodium. The sodium is determined directly by precipitation as the pyro- antimonate. As the precipitation must be carried out in an alka- line medium, calcium and any other elements present, which would form insoluble compounds such as tertiary calcium phosphate, must be removed, otherwise they would interfere with the gravi- metric determination of the sodium pyroantimonate. This is accomplished by the following procedure. 15 to 20 cc. of the stool extract or 5 to 10 cc. of the urine extract are placed in a platinum dish and evaporated to dryness. The ash is transferred to a graduated centrifuge tube with 2.5 cc. of 0.6 Nn HCl. 3 ce. of a saturated solution of ammonium oxalate are added and the mixture is allowed to stand for 10 minutes. This precipitates prac- tically all the calcium. Concentrated NH,OH is then added to 7 ec., the contents are mixed and allowed to stand 45 minutes. 1 The platinum dish which contains the material to be ashed is placed in a quartz dish 10 em. in diameter and 6 cm. deep, in the bottom of which are placed several pieces of porcelain. The outer dish is gradually heated with a Meeker burner until no more fumes are given off when the flame is turned on full until the charred material is immobile. The large dish is then covered with a quartz plate and heating continued for 1} hours. F. F. Tisdall and B. Kramer 3 Magnesium is thereby precipitated as ammonium magnesium _phosphate. The sample is centrifuged for 5 minutes, affer which 5 ec. of the supernatant fluid are placed in a platinum dish and evaporated to dryness. The dish is placed in the oven at 100°C. for a few minutes to thoroughly dry the residue in order to avoid spattering during the subsequent ashing. The sample is then ashed by the Stolte method for 15 to 30 minutes. This volatilizes all the ammonium salts. A small amount of ash remains in the dish. This dissolves readily in 2 cc. of 0.1 Nn HCl. A drop of phenolsulfonephthalein is added and the solution made just alka- line by the addition of 2 or 3 drops of 10 per cent KOH. This solution which contains all the sodium present in the original sam- ple is now ready for the direct estimation of this element. TABLE I. Determination of Sodium in Known Solutions of Inorganic Salts.* NazH2Sb207+6H20 Na2H2Sb207+6H20 Amount of sample. ealcnlateee found. = Error. cc. mg. mg. per cent ? of 10 30.0 36.0 —4,2 pee 10 37.5 38.1 +1.7 Be 15 56.0 54.7 —2.3 me 15 56.0 56.2 +0.4 B90 75.0 12.5 —3.3 F “ 20 75.0 | 72.8 —3.0 * Composition of Solution Y [S175 I SE eS ree er | | 2) re are 0.241 gm Ti CSL Ree Ed ON A itn 6 cd a ae rh rr OF20i |. WIS) Oe yall s Ei OMT EARE AER Sara w ) 2 ey es OMS0RS Ca3(PO,4)2 PO ORIOO DOWER OOO O8IO A OO OS 0-5 0-050 GIO GID DIGRTOR ROI OREN CIS Oxs30N. ool CO RAR MR RR os, ile) 2 SE rr 0.500 “ (Cusirisel sl Cl RRO eee EU Oe ol 200 ce. To the sample, prepared as outlined above, are added 10 ce. of the potassium pyroantimonate reagent followed by 3 ce. of 95 per cent alcohol. The alcohol should be added, drop by drop, and the specimen stirred with a rubber-tipped rod. After standing 30 minutes, the precipitate is transferred to a weighed Gooch crucible and washed with 5 to 10 ce. of 30 per cent alcohol. The crucible is dried at 110°C. for 1 hour,? cooled in a desiccator for 30 minutes, 2 The temperature is gradually raised to 110°C. é 4 Inorganic Elements in Urine and Stools and weighed. The weight of the precipitate divided by 11.08 equals the number of mg. of sodium present in the sample. The method of preparation of the potassium pyroantimonate reagent has been fully described in a former paper on the deter- mination of sodium in serum (1). The details of the method of preparation of the Gooch crucibles, the precautions to be ob- served during the addition of the alcohol and the filtration and also the care of the platinum are fully outlined in the same paper. The results given in Table I show that as little as 3 or 4 mg. of sodium may be quantitatively recovered from solutions containing relatively large amounts of calcium phosphate. Potassium. The potassium method is identical with that reported a short time ago by the authors for the estimation of this element in serum (2). The optimum amount of stool extract for the potassium determination is generally 1 cc. The concentration of this element in urine, however, is so high that it is necessary to take only 0.2 to 0.5 ec. The sample to be analyzed is placed in a graduated centrifuge tube and diluted with distilled water to 2 cc. The centrifuge tube should be previously cleaned with the use of a brush, washed out with strong cleaning fluid (commercial H.SO; and potassium dichromate), and then thoroughly rinsed with distilled water. If the tubes are not cleaned in this manner the precipitate will adhere to the sides and low results will be obtained. 1 ce. of the sodium cobalti-nitrite reagent is then slowly added, drop by drop. The contents of the tube are mixed and allowed to stand for 3 hour. The volume is made up to 5 ce. with water and the contents again mixed and the tube centrifuged for 7 minutes at about 1,300 revolutions per minute. The precipitate will then be found at the bottom of the tube. All but 0.2 to 0.3 cc. of the supernatant fluid is removed. This is accomplished by means of the following apparatus. Through one opening of a two-holed cork is inserted a glass tube by means of which a positive pressure can be made in the centrifuge tube. Through the other hole is placed a tube which reaches to about 3 or 4 mm. above the precipi- tate. The lower end of this tube is drawn out to a bore of about 1 mm. and curved so that the opening is directed upward. By F. F. Tisdall and B. Kramer 5 fitting the cork to the centrifuge tube and blowing through the first opening the supernatant fluid can be readily removed without disturbing the precipitate. 5 cc. of water are allowed to run down the side of the tube which is then gently agitated so that the added water is mixed thoroughly with the residual reagent. Care should be taken that the precipitate itself is disturbed as little as possible. This may be accomplished by holding the tube vertically and gently hitting the lower end with a circular motion. The brown fluid may be seen to rise and mix with the supernatant fluid. The tube is then centrifuged for 5 minutes. The procedure is repeated three times so that the precipitate is washed four times in all. The supernatant fluid from the last washing should be perfectly clear. After the removal of the fluid from the final washing the precip- itate is ready to be titrated. Titration—An excess of 0.02 N potassium permanganate (gen- erally 2 to 5 cc.) is added to the precipitate followed by 1 cc. of 4n H.SO,. It is rather difficult to judge the amount of perman- ganate necessary to be added, but by carefully watching the tube while it is being heated, more can be added. The precipitate is thoroughly mixed with the permanganate and H.SO, by means of a glass rod and the tube is placed in the boiling water bath. At the end of 20 to 25 seconds the tube is examined and if the pink color of the permanganate has nearly disappeared, more perman- ganate is added from the micro-burette. In this way it is not diffi- cult to find out how much permanganate is necessary to constitute an excess. At the end of 1 minute from the time the heating is begun, the solution should be of a perfectly clear pink color. If all the precipitate is not oxidized, the contents will be cloudy and the color will be seen to fade. Heating should then be continued until the solution is clear and pink. When the heating is continued too long, the contents again become cloudy and of a brownish color. If this is allowed to happen, the sample must be discarded as high results will be obtained. 2 cc. of 0.01 N sodium oxalate are promptly added and the contents mixed. If this is not suffi- cient to decolorize the permanganate, another 2 cc. should be immediately added. The excess of oxalate is then titrated with 0.02 N potassium permanganate delivered from a micro-burette, graduated in 0.02 cc., until a definite pink color is obtained which lasts for 1 minute. 6 Inorganic Elements in Urine and Stools Calculation —1 ec. of 0.01 N potassium permanganate will oxi- dize a quantity of potassium cobalti-nitrite corresponding to 0.071 mg. of potassium. Thus, if 2 cc. of 0.02 N potassium per- manganate are originally added and 0.43 cc. of the same solution used in the final titration and 2 cc. of 0.01 N sodium oxalate are required to decolorize the sample after the first oxidation, then 2.43 — 0.03 (the amount of permanganate necessary to colorize the same quantity of water) < 2 (to convert 0.02 to 0.01 x) —2.00 (ce. of 0.01 N sodium oxalate added to decolorize the sample) < 0.071= 0.199 mg. of K in sample. The details of the preparation of the reagents have been given in a former paper on the determination of potassium in serum.’ TABLE II. Determination of Potassium in Known Solutions of Inorganic Salts Con- taining an Excess of Calcium Phosphate.* Amount of sample. K present. K found. Error. ce. mq. ind mg. per cent 0.4 0.215 0.218 +1.4 025 0.269 0.274 +1.8 O27 0.376 0.384 +2.1 1.0 0.5388 0.538 +0.0 * Composition of Solution C. O20 A) Ae. yt a a ee RL Ra 2.5820 gm. Gee Wee ee oS te Sat Bes 0:2052° = INSLGuens 2 eee Pe, AN eet CS ag eh eee 0.2063 “ huts Oh. sli O) 5 aes ea eS ae ee ies & ree 0.6100 “ ee EArt Oe cree, Pee Bh yt yo, oe bg ee epee eee 200 ec. The results of the estimation of potassium by this method in a known solution of inorganic salts are given in Table II. It should: be noted that even a large amount of phosphate does not affect the potassium determination. Calcium. The calcium method is identical with that reported a short time ago by the authors for the estimation of this element in serum (3). - * Kramer and Tisdall (2), p. 343. F. F. Tisdall and B. Kramer Z The concentration of calcium in the stool extract, however, is so high that dilution is necessary. 5 cc. of the extract are diluted to 50 ec. with distilled water. The optimum amount of this solution for the calcium determination is generally between 1 and 4 cc. The amount of the urine extract corresponding to 1 to 4 ce. of urine is also found to be quite satisfactory. The sample (generally 2 ec.) is measured into a graduated cen- trifuge tube previously ¢leaned with commercial H»SO; and dichromate and the volume made up to 3 or 4 ce. with distilled water. A drop of phenolsulfonephthalein is added and 10 per cent NH,OH (10 ce. concentrated NH,OH in 90 ce. of HO) until the solution is alkaline. Approximately N H.SO, is added until the solution is just acid and any phosphates that may have been precipitated are redissolved. 1 cc. of approximately N oxalic acid is added followed by 1 ce. of a filtered saturated solution of sodium acetate which should be added drop by drop. The con- tents are mixed and allowed to stand for ? hour when they are centrifuged for 10 minutes at about 1,300 revolutions per minute. This throws all the calcium oxalate precipitate to the bottom of the tube. All but 0.3 cc. of the supernatant fluid is removed by means of the apparatus described under the potassium method. The remaining fluid and the precipitate are mixed by tapping the the tube. Enough 2 per cent ammonia (2 ce. of concentrated ammonia diluted to 100 cc.) is then added to bring the volume to 4 ee., care being taken to wash the sides of the centrifuge tube free from adherent oxalic acid. The tube is then centrifuged for 5 minutes. This procedure is repeated twice, thus making three washings in all. After the third washing the supernatant fluid is removed, the tube is shaken to suspend the precipitate, 2 ce. of approximately Nn sulfuric acid are added, and the tube is warmed in the boiling water bath for a few minutes and titrated with 0.01 N potassium permanganate until a definite pink color per- sists for at least 1 minute when viewed under a good light against a white background. The strength of the permanganate solution is determined by titrating against 0.01 N sodium oxalate (Sérensen). Calculation—The number of cc. of 0.01 N potassium perman- ganate used (generally 0.5 to 2 cc.) — 0.02 ec. (the blank) xX 0.2 = mg. of calcium in the sample. - Preparation of Reagents.—0.01 N sodium oxalate (Sérensen) is 8 Inorganic Elements in Urine and Stools the only reagent that must be quantitatively accurate. An 0.1 N sodium oxalate (Sérensen) is prepared in the usual way. 6.7 gm. of sodium oxalate (Sérensen) are dissolved in water. Solu- tion is facilitated by the addition of 5 ec. of concentrated sulfuric acid and the volume made up to 1 liter. This is diluted ten times to make a 0.01 N solution. The former solution will keep indefin- itely while the latter has been found still unchanged after the lapse of 2 months. Approximately N Oxalic Acid—This is prepared by dissolving 63 gm. of oxalie acid (Kahlbaum or J. T. Baker, c. p., calecium- free) in a liter of water. The acid need be weighed only roughly. Approximately N Sulfuric Acid.—50 ec. of concentrated sulfuric acid (c. p.) are diluted with water to 1 liter. TABLE III. Comparison of Calcium Determination on Solutions of Ash of Infants’ Stools by McCrudden’s Method and the Authors’ Method.* McCrudden’s method. Authors’ method. Specimen. Ca per 0.5 gm. stool. Ca per 0.5 gm. stool. Difference. . mg. mg. per cent gf 31.17. 30.09 —3.0 II 39.89 40.05 +0.4 III sae lal 34.50 —1.7 IV DoLl6 51.18 —3.7 * We are indebted to Dr. 8. G. Ross for most of the calcium and mag- nesium determinations made by McCrudden’s method. Saturated Sodium Acetate Solution.—This solution is made by adding an excess of the salt to water and allowing it to stand over night. The supernatant fluid is then filtered. Sodium acetate (J. T. Baker, c. p.) does not contain calcium. A comparison of the results obtained by this method and by the MeCrudden method (11) on a solution of stool ash is given in Table III. Magnesium. The principle used for the determination of magnesium is that originally advanced by Stolba (4) in 1877. The calcium is precip- | itated as the oxalate. The magnesium is then precipitated as F. F. Tisdall and Bp: Kramer 9 ammonium magnesium phosphate. An excess of HClis added and the following reaction takes place: NH.MgPO, + 3 HCl =NH.Cl + MgCl, +H;PO, The H3POs, is then titrated with 0.1 NaOH to NaH2POu,, the pH of which is 4.4. The indicator used to detect the end-point is cochineal. This indicator changes from yellow to purple at pH 4.8. The error produced by titrating to this pH instead of pH 4.4 is small (9). 0.1 gm. molecules of NHiMg PO, when titrated with 0.3 gm. molecules of HCl yields 0.1 gm. molecules of H3PO,. When this is titrated back with 0.1 n NaOH to a pH of 4.8, one TABLE IV. Determination of Magnesium in Samples of Solution A.* Salution SA. Hie anne, Mesnesin pee Boe cc. cc. mg. mg. per cent 3 0.75 0.91 0.90 ares a 5 1.20 1.45 1.50 38 10 2.50 3.02 3.00 ey 20 5.10 6.17 6.00 +28 * Composition of Solution A. Mi), CONPAINING WIC. «i202 ca eee ae ss clas's 3 0) a' 24 0.030 gm (CANCION Moca se cater a oie Mantes of aioe o-Occich a a ciciicis Ee eRe 1.154 “ IN 6 W Oe Sal oI Gees ee Bie oi 3 dh cool cece iceeneae OXGIT) Concentrated iC] eee. non ee cite ee oes os 5 OG: equivalent of H has been replaced by sodium, leaving two equiv- alents still united to PO, as NaH,PO,. Therefore, two equiv- alents of H are equal to two equivalents of Mg; 7.e., 1 cc. of 0.1 Nn HCl = 1 ce. of 0.1 nN Mg solution = 1.21 mg. of Mg. The procedure is as follows: 25 to 50 ce. of the urine extract or 10 to 30 ce. of the stool extract are placed in a 100 cc. beaker. To this is added a drop of phenolsulfonephthalein and 10 per cent NH.OH (10 cc. of concentrated NH,OH in 90 ce. of H,O) until the solution is just alkaline. 4 N H.SO, is added until the solution is acid and all the phosphates are redissolved. 10 cc. of saturated ammonium oxalate are then added to the stool extract or 5 cc. to the urine extract, mixed, and allowed to stand for 15 minutes. 10 Inorganic Elements in Urine and Stools This precipitates the calcium as the oxalate. 1 cc. of 10 per cent (NH,)2 HPO; is added to insure an excess of phosphate followed by 5 ce. of concentrated NH,OH. The mixture is thoroughly mixed, allowed to stand 1 hour, and then filtered through 9 cm. of TABLE V. Comparison of Magnesium Determinations on Solutions of Ash of Infants’ Stools by MceCrudden’s Method and the Authors’ Method. McCrudden’s method. Authors’ method. Specimen. Mg. in 0.5 gm. stool. Mg. in 0.5 gm. stool. Difference. mg. mg. . per cent J 2.84 2.88 +1.4 II 2.29 2.16 —5.7 iil 2.78 2.70 —2.9 IV 3.55 3.54 —0.3 No. 40 Whatman filter paper. The precipitate is all transferred from the beaker by the use of a rubber-tipped rod and 10 per _ cent NH,OH. The ammonia is then all removed from the filter paper by washing it four times with 30 per cent alcohol. The filter paper with the precipitate which includes the calcium oxalate TABLE VI. Comparison of Calcium, Magnesium, Sodium, and Potassium Determinations ~ on the Solutions of Stool Residues Which Were Ashed 13 and 12 Hours. Specimen. Inorganic element. Ashed for 13 hours. Ashed for 12 hours. gm. per 24 hrs. mg. per 24 hrs. I Ca 1.728 1.692 Mg 0.115 0.115 Na 0.514 0.510 K 0.630 0.649 Il Ca 2.244 2.352 Mg 0.104 0.108 Na 0.035 0.037 K 0.243 0.249 Ill Ca 1.083 1.095 Mg 0.081 0.084 Na 0.146 0.145 K 0.338 0.336 F. F. Tisdall and B. Kramer 11 is transferred to a 100 ce. beaker, about 30 cc. of warm water are added, and the filter paper and precipitate mixed by the use of a glass rod. 3 drops of tincture of cochineal* are added and an excess of 0.1 N HCl (generally 5 ee.). After 5 minutes the mixture is titrated with 0.1 Nn NaOH delivered from a burette graduated in 0.05 cc. until the color changes from a light yellow to a purple. This end-point is very definite and a decided change in color is produced by 1 drop of 0.1.N NaOH. The presence of the filter paper and the large amount of calcium oxalate do not interfere with the interpretation of this end-point. The number of cc. of 0.1 N HCl added — the number of cc. of 0.1 N NaOH X 1.21 = the num- ber of mg. of magnesium in the sample. An analysis of the mag- nesium content of a known solution of stool and urine salts is given in Table IV. A comparison of the results obtained by the above method and by McCrudden’s method (11) is given in Table V. Table VI shows that practically identical results are obtained on the acid extracts of partly ashed stools as on the acid solution of the completely ashed stools. y CONCLUSIONS 1. Rapid methods for the determination of eae) potassium, calcium, and magnesium in urine and stools, including a direct method for the determination of sodium in the presence of laige quantities of other salts, particularly calcium phosphate, have been described. Aa, 2. The determination of potassium by means of sodium cobalti- . nitrite reagent has been used for the estimation of small amounts of this element in urine and stools. The error by this method is generally within 1 or 2 per cent. 3. The determination of calcium and magnesium has been com- pared with the results obtained by the standard McCrudden method. The deviation from the standard method has generally | been within 3 per cent. 4. By means of these methods a considerable saving of time is effected and all the so called fixed alkali elements may be deter- mined quantitatively in 50 ce. of urine or 2 gm. of dry stool. 4 The tincture is made by digesting 1 part of crushed cochineal with 10 parts of 25 per cent alcohol. 12 wo — Go Inorganic Elements in Urine and Stools BIBLIOGRAPHY. . Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 467. . Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 339. . Kramer, B., and Tisdall, F. F., Bull. Johns Hopkins Hosp., 1921, Xxxil, 44. . Stolba, F., Z. anal. Chem., 1877, xvi, 100. . Mohr, F., Z. anal. Chem., 1877, xvi, 326. . Kraus, F., Z. physiol. Chem., 1881, v, 422. . Bauzil, L., J. pharm. chim., 1917, xvi, series7, 321. . Angiolani, A., Gior. farm. chim., 1917, Ixvi, 251, abstracted in Chem. Abstr., 1919, xiii, 27. . Fiske, C. H., J. Biol. Chem., 1921, xlvi, 285. . Stolte, K., Biochem. Z., 1911, xxxv, 104. . MeCrudden, F. H., J. Biol. Chem., 1909-10, vii, 83. A RAPID METHOD FOR THE DETERMINATION OF HIPPURIC ACID IN URINE.* By F. B. KINGSBURY anp W. W. SWANSON. (From the Biochemical Laboratory, Department of Physiology, University of Minnesota, Minneapolis.) (Received for publication, June 20, 1921.) In making benzoate tests for renal efficiency we were confronted with the necessity for having a rapid and accurate method for the determination of hippuric acid in urine. The Folin-Flanders method which we have been using required more time than was thought necessary. By means of this method analyses could be made in 9 or 10 hours when necessary, but with the routine of teaching and other university work 24 hours were usually required. It was our object to devise a method which would conserve the accuracy of the Folin-Flanders (1) method, but one which could be completed within 2 or 3 hours and be as applicable for hospital routine work as are any of the other modern biochemical methods. A careful review of the more recent methods for the determina- tion of hippuric acid shows that at present there is only one method which fulfils the requirements of accuracy and simplicity. This is the method of Folin and Flanders. Two other methods which appeared at about the same time, Steenbock’s (2) and Hrynt- schak’s (3) methods meet the requirements of accuracy fairly well but are too tedious to compete with the Folin-Flanders method. Ito’s (4) method appearing 4 years later is more complicated than those mentioned above and does not represent an advance in this field. Steenbock’s and Hryntschak’s procedures depend upon the isolation and weighing of benzoic acid, which are accompanied by slight losses, more in the latter method than in the former, and are necessary only in those cases in which benzoic acid cannot be directly titrated, as for instance, in the presence of other titratable *Acknowledgment is made to the Graduate School of the University of Minnesota for the purchase of a portion of the chemicals used in this work. 13 14 Determination of Hippuric Acid acids. Since there are no other acids present in the final extrac- tion and ‘titration stages of the Folin-Flanders method, titration in this case is not only easier to accomplish but more accurate. Folin and Flanders proved that their method gave quantitative results with pure hippuric acid solutions which we have confirmed many times in the last few years. They did not compare their method, as applied to urine with any other procedure of analysis, nor as far as we can find, has any other investigator. They have assumed, however, that their method gives the most accurate re- sults of any method devised up to that time. We have proved in the experiments which are directly to follow that the Folin- Flanders method does correctly estimate the amount of hippuric acid that can be extracted directly from urine by means of ethyl acetate. Experiment 1.— 0.561 gm. of pure sodium hippurate was dissolved in 100 ec. of water, 1 cc. of concentrated nitric acid added, and the mixture then extracted with ten 50 cc. portions of ethyl acetate, shaking exactly 2 minutes each time. The aqueous mixture left was then filtered, the filtrate evap- orated to dryness over night on the steam bath with 10 ec. more of 5 per cent sodium hydroxide than that required for neutralization of the nitric acid present. The residue was then analyzed for any remaining hippuric acid by the Folin-Flanders method. The titration value was 0.07 cc., which is the ordinary blank of the method. The hippuric acid was com- pletely extracted by this procedure. 100 ce. of urine, the hippuric acid titration value of which was 13.58 ce. of one-tenth normal sodium ethylate were acidified with 2 cc. of concen- trated nitric acid and extracted with ten 50 cc. portions of ethyl acetate, shaking 2 minutes each time. The combined extracts were washed with two 200 ce. portions of the Folin-Flanders sodium chloride solution and then steam distilled until all of the ethyl acetate and approximately 300 ec. of water had passed over. The aqueous solution of hippuric acid remain- ing in the distilling flask was quantitatively transferred to a casserole and analyzed according to the Folin-Flanders method. The titration value was 13.43 ec. of one-tenth normal sodium ethylate, agreeing with the value obtained directly as well as duplicates can usually be obtained by this method. Experimental Methods of Analysis. Our problem resolved itself into increasing the speed of the hydrolysis of hippuric acid either by acids or alkalies and the effec- tive oxidation of urinary pigments and other disturbing sub- stances. Without going into the details of many experiments F. B. Kingsbury and W. W. Swanson 15 carried out it may be stated that by using 15 gm. of solid sodium hydroxide in hydrolyzing the hippuric acid of 100 cc. of urine at the boiling point for 30 minutes and subsequently acidi- fying, extracting, and titrating, results were obtained that were, in one experiment, 22 per cent higher than the known titration value for this specimen of urine. It was also found that values from 10 to 33 per cent higher than those obtained by the Folin- Flanders method resulted when urine was boiled with an equal volume of a mixture of concentrated nitric and sulfuric acids for 30 minutes ina process that gave 100 per cent recovery when applied to solutions of pure hippuric acid. Oxidation of the urine with alkaline potassium permanganate after the plan of Hryntschak was tried and yielded such promising results that the details of one typical experiment are given below: Experiment 2.— 50 cc. of urine were boiled with 7.5 gm. of solid sodium hydroxide and 1.5 gm. of potassium permanganate for 30 minutes in a Kjeldahl flask with a rather closely fitting test-tube condenser in the neck. The flask was cooled and 50 cc. of concentrated nitric acid slowly poured down the side of the condenser. The brown mixture cleared up after boil- ing a few minutes, but this was continued for 30 minutes, then cooled and extracted as in the Folin-Flanders procedure using comparative amounts of the various materials; The titration value was 16.72 cc. of one-tenth normal sodium ethylate; by the regular Folin-Flanders method, 16.95 ce. In a series of 12 analyses made in this way it was found that values from 97 to 99 per cent of the Folin-Flanders figures could always be obtained when these were as large as 15 ce., but with lower values the error was sometimes as large as 25 per cent. This was believed to be due to the action of the potassium permanganate on the benzoic acid present as it was always most pronounced in the urines which were the most dilute and therefore con- taining less of the other substances to combine with the permanganate. It was difficult to estimate the correct amount of potassium permanganate to be added in each case and it frequently happened that 1.5 gm. were a greater amount than could be reduced beyond the manganate stage and 0.5 gm. portions of sodium bisulfite had to be added to complete the reduction. It was also found that if this method were applied toa pure solution of hip- puric acid, allowing the potassium permanganate to act only 2 or 3 minutes before reducing it with sodium bisulfite that it was impossible to obtain more than 95 per cent of the theoretical amount. In Hryntschak’s method the urine was boiled with 10 gm. of sodium hydroxide for 2.5 hours then 10 gm. of potassium permanganate were added and the boiling was continued for6or7 minutes. The excess of permanganate was removed by adding about 15 gm. of sodium bisulfite prior to acidification and extraction. He subjected benzoic acid to the same conditions and was able to recover 16 Determination of Hippurie Acid 98.24 and 98.17 per cent in two experiments and concluded from this that potassium permanganate did not destroy any benzoic acid. This is con- trary to our findings using the more sensistive titration method. We were reluctant about giving up the use of potassium permanganate because the subsequent chloroform extracts were always practically color- less and remained so until the definite pink end-point of titration was reached. No decidedly yellow extracts such as are rather frequent in the Folin-Flanders method were ever encountered. It was found by one of us that if a small quantity of magnesium oxide were present the effect of the permanganate in decreasing the titration value was prevented. The de- tails of the method as we have adopted it follow: Description of the Method. 50 ee. of urine are treated with 7.5 gm. of sodium hydroxide and 0.5 gm. of magnesium oxide in a 500 or 800 ec. Kjeldahl flask. This mixture is boiled at such a rate as to bring its volume down to approximately 25 ec. in the course of half an hour. At the end of this time, while still at the boiling temperature, 1.0 cc. of a 7 per cent solution of potassium permanganate (a solution approxi- _ mately saturated at room temperature) is added, care being taken to rinse down any that may remain on the neck of the flask with the smallest possible amount of water since no unchanged per- manganate must be present when the acid is subsequently added. The flask with its brown contents is twirled gently for a minute or two, cooled under the tap, a fairly closely fitting test-tube con- denser placed in the neck and 30 ce. of concentrated nitric acid slowly poured in down the side of the condenser. The mixture, which rapidly clears up on the addition of the acid, is now gently boiled for 45 minutes (30 minutes are sufficient for accurate results, but a less colored, more easily titratable extract is obtained by boiling it 45 minutes) with a good current of water flowing through the condenser, cooled under the tap, and the extraction with chloroform carried out approximately according to the Folin- Flanders method. The condenser is rinsed down with 25 cc. of water to remove any benzoic acid sublimed on the bottom of the condenser, the contents of the flask are transferred to a 500 cc. separatory funnel containing 25 gm. of solid ammonium sulfate. The flask is rinsed with 20 cc. of water which is poured into the separatory funnel. After dissolving the ammonium sulfate the benzoic acid is extracted successively with one 50 ec., one 35 ce., F. B. Kingsbury and W. W. Swanson if and two 25 ec. portions of neutral, well washed chloroform. The first 2 portions of chloroform are used to rinse the Kjeldahl flask. The combined extracts in a second separatory funnel are washed once with 100 ce. of the Folin- Flanders salt solution (containing 1.0 ce. of concentrated HCl in 2 liters of saturated NaCl solution) and drawn off through a dry filter paper into a dry Erlenmeyer flask. The separatory funnel from which the extract was drawn is rinsed with 20 ce. of chloroform. This is drawn off into a small beaker to which the wet filter paper had been transferred.. The paper is rinsed with the chloroform and the latter is poured through a dry filter into the main bulk of extract in the Erlenmeyer flask. 4 drops of 1 per cent phenolphthalein in absolute alcohol are added and the benzoic acid solution titrated to a faint, but definite pink with tenth normal sodium ethylate. The preparation and stand- ardization of this alkali solution are adequately described in the original paper of Folin and Flanders. We have found that the following treatment of the chloroform used in this method insures a product that is reliable as far as its neutrality is concerned: New chloroform, which is of the v. s. P. grade and contains about 0.75 per cent of ethyl alcohol, should be washed with an equal vol- ume of distilled water twice before being used for the extraction of benzoic acid. Chloroform which has been used in analysis and therefore contains sodium benzoate and alcohol is first filtered through a dry filter paper which removes a considerable part of the sodium benzoate in those determinations in which the titra- tion figure was fairly large. It is now washed successively with: equal volumes of tap water, once; tap water containing 5 to 10 ce. of a saturated solution of NaOH, twice; tap water, twice; and distilled water, once; six washings in all. Since the accuracy of this method depends primarily upon the use of a sample of chloro- form which not only reacts neutral when tested, but which must remain neutral after being shaken with nitric acid, we have used the test which follows to determine this point: 155 ec. of chloroform, the amount used in an analysis, washed as described above, are shaken with dilute nitric acid, washed with 100 cc. of the Folin-Flanders salt solution, filtered through a dry paper, and titrated. The titration of this amount of chloroform suitable for use should not exceed 0.10 cc. of tenth normal sodium ethylate. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 1 18 Determination of Hippurie Acid The application of this method or that of Folin and Flanders requires the removal of protein from the urine when this is present, asin nephritic urines. Directions for doing this have already been published, but perhaps may be repeated here. The albuminous urine is collected in 2 per cent nitric acid which was found by Raiziss and Dubin (5) to be effective in preventing the hydrolysis of hippuric acid. 15 cc. of this dilute nitric acid are sufficient for a 3 hour nephritic urine. 50 ce. of this urine, treated with 3 or 4 drops of 0.1 per cent methyl red solution in alcohol, are brought to the first definite yellow by the addition of approximately normal NaOH. The solution is then boiled, and during the boiling sufficient one-tenth normal HCl is added to pro- duce the first definite red color. This procedure removes the albumin nearly quantitatively so that there is no increase in the resulting titration, as has already been shown (6). The coagulum of albumin on the filter paper is washed twice with 50 cc. of boiling water. The combined washings and main bulk of filtrate are evaporated rapidly over a free flame in an 800 cc. Kjeldahl flask after being made slightly alkaline to methyl red by the addition of a small amount of dilute alkali. Bumping and frothing, should they occur, are checked by adding a glass pearl and a drop of caprylic alcohol. By supporting the funnel in the neck of the flask by means of a slice of a large cork stopper the filtration and evaporation are continued simultaneously. When the contents of the flask have been evaporated to approximately 50 ec., 7.5 gm. of NaOH and 0.5 gm. of MgO are added and the analysis made according to the directions already given. In Table I are given the comparative results with various spec- imens of urine, normal and pathological, obtained by the new method and by that of Folinand Flanders. In a series of approxi- mately half of the determinations one of us used one method and the other, the other method. The results of neither of us were known tothe other until all the determinations of this series had been made, when they were compared. No.19 in TableI is a comparison of the two methods with 50 ce. aliquots of a pure sodium hippurate solution. The only modification in this case was the reduction of the permanganate with 0.5 gm. of sodium bisulfite as a substitute for the urinary constituents which ordinarily function in this man- ner, prior to the acid treatment. It is noted that the agreement F. B. Kingsbury and W. W. Swanson 19 between the two methods is good, as close in general as duplicates can be made by the older method, and that duplicates by the new method, where they have been made show a very close agreement. TABLE I. ee 0.1N Na ethylate. Tine oO. ee New method. Folin-Flanders method. CC. cc. 1 4.50 4.70 4.35 4.50 4.80 4.50 2 29.40 28.95 29.50 29.60 3 13.95 13.55 13.70 4 33.65 33.10 33.50 33.80 5 1.05 0.95 6 P.* 24.55 24.80 Tepes 26.60 26.75 8 P.* 24.50 24.50 9 14.20 14.20 10 20.10 19.80 11 8.50 8.65 12 5.25 5.35 13 8.65 8.70 14 17.55 16.95 15 8.55 8.80 16 8.95 8.80 17 12.56 12.35 18 13.75 14.00 13.55 19+ 14.25 14.25 13.80 14.15 *Urines designated by “‘P.’’are pathological specimens. All others are normal. 750 ce. of a sodium hippurate solution were used. A few duplicate determinations have been made several days apart with no evidence of loss of hippuric acid in acid urines at room temperature preserved with a small amount of a 10 per cent solution of thymol in chloroform. 20 Determination of Hippuric Acid CONCLUSION. An accurate, rapid method for the determination of hippuric acid in urine is described which requires about 2 hours for comple- tion with normal urine and about 3 hours with urine containing albumin. BIBLIOGRAPHY. . Folin, O., and Flanders, F. F., J. Biol. Chem., 1912, xi, 257. . Steenbock, H., J. Biol. Chem., 1912, xi, 201. . Hryntschak, T., Biochem. Z., 1912, xliii, 315. . Ito, H., J. Am. Chem. Soc., 1916, xxxviii, 2188. . Raiziss, G. W., and Dubin, H., J. Biol. Chem., 1915, xxi, 331. 6. Kingsbury, F. B., and Swanson, W. W., Arch. Int. Med., 1921, xxviii, 220. corm © te NOTE ON A POSSIBLE SOURCE OF ERROR IN TESTING FOR BENCE-JONES PROTEIN. By C. W. MILLER anv J. E. SWEET. (From the Departments of Physiological Chemistry and Research Surgery, University of Pennsylvania, Philadelphia.) (Received for publication, June 30, 1921.) In a long series of attempts to produce Bence-Jones proteinuria in dogs by administration of agents known to cause lesions of the bone marrow, we found a possible source of error in making quali- tative tests for this protein to which it may be well to call atten- tion. If urine, especially of dogs, containing a small amount of serum protein is allowed to stand at room temperature for from 8 to 24 hours after voiding, it will occasionally be found that the heat coagulation test, at first clear, will after standing become less marked or even disappear entirely. On the other hand a con- siderable cloud will still be produced by potassium ferrocyanide and acetic acid; and the addition of an equal volume of a _ satu- rated solution of ammonium sulfate will also give a precipitate. This change occurs even when the urine is preserved with toluene, and is apparently due to the proteolytic enzyme of the urine. Now since it is well known that certain digestion products give the same heat precipitation and resolution reactions as Bence- Jones protein, the desirability of using fresh urine when testing for this substance is obvious. In working with dogs it is also necessary to collect the urine by catheter, or at least to watch the dog carefully so as to be certain of obtaining urine free from con- tamination (vomitus, feces). Even slightly contaminated urine from the cage was several times found to contain enough diges- tion products to give Bence-Jones reactions, which could not be confirmed in specially collected urine. The use of toluene as a preservative for urine may temporarily give rise to confusion. If a couple of drops of toluene are emulsi- fied by shaking in a test-tube with urine or water an opalescent 21 22 Bence-Jones Protein fluid results. If this be heated it becomes clear, and when cooled the cloudiness immediately returns, and this can be repeated, thus simulating the behavior'of Bence-Jones protein. Hither the emul- sion becomes invisible when hot on account of the change of the refractive index of the toluene, or the latter goes into solution at boiling heat and is reprecipitated upon cooling. Very little agita- tion, e. g. filtration, of urine preserved with toluene can effect sufficient emulsification to cause this phenomenon; however, by confirmatory tests it is easily differentiated. a oe ACERIN. THE GLOBULIN OF THE MAPLE SEED (ACER SACCHARINUM).* By R. J. ANDERSON. WITH THE COLLABORATION OF W. L. KULp. (From the Biochemical Laboratory, New York Agricultural Experiment Station, Geneva.) (Received for publication, June 27, 1921.) INTRODUCTION. In an earlier paper from this laboratory! it was indicated that the cotyledons of maple seed contained a high percentage of ni- trogen, and preliminary experiments showed that a large amount of this nitrogen was present in the form of a globulin which could be extracted with water or with dilute solutions of sodium chloride. In the pure state the globulin is insoluble in water but owing to the presence of soluble salts in the seed it is extracted by small quantities of distilled water just as readily as by dilute solutions of sodium chloride. On diluting such aqueous extracts a large part of the globulin is precipitated. The amount of total and soluble nitrogen in the cotyledons is shown in Table I. The nitrogen in the 70 per cent alcoholic extract was non-protein in character, but the nature of the nitrogen compounds insoluble in dilute sodium chloride solution was not determined. The amount of pure globulin obtained from 100 gm. of powdered seed was only about 6.5 gm. This corresponds to about only one-half of the amount of nitrogen soluble in a dilute solution of sodium chloride. However, no effort was made to secure @ quantitative yield. * Read at the Chicago meeting of the American Association of Biological Chemists, December, 1929. 1 Anderson, R. J., J. Biol. Chem., 1918, xxxiv, 509. 93 24. Acerin The globulin separates on dialysis into small uniform globular particles whieh show no crystalline structure and it is precipitated completely from saline solutions by 0.6 saturation with ammonium sulfate. It has been isolated and purified by alternately precipi- tating it with ammonium sulfate and by dialyzing its dilute salt solutions. After dehydrating with alcohol and drying in vacuum it was obtained as a compact light gray or nearly white powder which was non-hygroscopic. Since this is the first pro- tein obtained from maple seed we propose, provisionally, to call this globulin acerin. TABLE I. Nitrogen in Maple Seed. per cent JURE ETAT LY 2 Ge eho oA bos REE BR RES Oops Suce aca bc0 4.40 Nitrogen soluble in 70 per cent alcohol....................6 0.39 ‘ s coy S -“.. sodium chloride.s..,.0.¢ shee 2.06 < Kernisiniie an. SClU TESIGUE. ”.....). Seicwis +.) saae eee eee 1.93 TABLE II. Composition of Acerin and Arachin. Constituents. Acerin. Arachin. per cent per cent Reese eatin Pe Wied Lia iis © » i srdiatg. 51.44 52.15 Ee Nea ers ci oo s cbs dio cid 6.80 6.93 I ei eos oa.) 2x = 8 wine's 18.34 18.29 “5 Pe Ee SSRI, Pes (ree a a 0.55 0.40 RAE: Sey: Sa 2 See eae 22.87 22.23 A number of different preparations were made in different ways and on analysis, all gave practically identical results. These various preparations were so nearly ash-free that a non-weighable residue remained after combustion. ° The percentage composition of acerin is very similar to that of legumin, amandin, or the globulin from cottonseed described by Osborne and Campbell? and Osborne and Voorhees.? However, 2 Osborne, T. B., and Campbell, G. F., J. Am. Chem. Soc., 1896, xviii, 609; 1898, xx, 348. + Osborne, T. B., and Voorhees, C. G., J. Am. Chem. Soc., 1894, xvi, 778. R. J. Anderson 25 the nitrogen distribution and particularly the percentage of basic amino-acids vary widely from results obtained by the above authors, indicating a decided difference of the molecular constitu- tion of these proteins. Arachin, the globulin from peanut, recently described by Johns and Jones! is very similar in compo- sition and nitrogen distribution to acerin, but the percentage of the basic amino-acids differs in the two globulins. These relations are indicated in Tables IT and III. TABLE III. Nitrogen Distribution in Acerin and Arachin. Form of nitrogen. Acerin. Arachin. per cent per cent (Annade mitrogen 62213). hie 2h seas 2.53 2.03 Humin See 2, mbes fo) Si cae 0.15 0.22 Basic EE MEN, Sch Se Res HEE 4.86 4.96 BION =DASI Ciesla ta tmetacuntes 10.63 11.07 ECS AE eae oe LS eS, 0.55 0.85 LS GUTTA hee ae | 1.43 1.88 AT AAMINIC RA ES eee idols: ken ee 10.07 13-51 LTE Se Be 5 ie ieee en ee cele 6.07 4.98 EXPERIMENTAL. The air-dried cotyledons, freed from the testa or outer brown membrane, were powdered and extracted with ether at room tem- perature. After the ether had evaporated, the seed residue was used for the isolation of the globulin. A solution of the globulin is obtained on digesting this powdered material in a small quantity of water or in a dilute solution of sodium chloride. On filtering the extract through a layer of paper pulp a clear brownish yellow solution is obtained. After extracting the powdered maple seed with 5 per cent sodium chloride solution and filtering as indicated above, the filtrate gave the following reactions: (1) It was slightly acid to litmus. (2) The globulin was precipitated on the addition of a Johns, C. O., and Jones, D. B., J. Biol. Chem., 1916-17, xxviii, 77. 26 Acerin dilute acids, but saturating the solution with carbon dioxide did not produce’ any precipitate. (3) The solution turned slightly cloudy when gradually heated to 50°C. The cloudiness increased with a rise in temperature and at 75°C. a flocculent precipitate began to form and at 100°C. the amount of the precipitate in- creased. (4) Ammonium sulfate added to the clear solution caused only a very faint cloudiness up to 0.2 saturation, but with 0.3 and 0.4 saturation, a heavy precipitate is produced. When this precipitate is filtered off further addition of ammonium sulfate up to complete saturation produces only a very slight cloudiness. Isolation of Acerin. Of the powdered ether-extracted seed, 200 gm. were digested in 900 ec. of 10 per cent sodium chloride solution to which were added 40 ce. of a saturated solution of barium hydrate. The amount of barium hydrate necessary to maintain a neutral re- action in the extract was determined by titration. The mixture was stirred for about 15 minutes and it was then filtered through a layer of paper pulp. A perfectly clear brownish yellow filtrate was obtained. It was saturated with ammonium sulfate and the precipitate which formed was separated from the mother liquor as thoroughly as possible by centrifuging. The precipitate was dissolved by adding about 100 ec. of water and the solution was filtered through paper pulp. The globulin was again precipitated by saturating the filtrate with ammonium sulfate. The mixture was centrifuged; the precipitate dissolved in 100 cc. of water, filtered through paper pulp, and reprecipitated a third time with ammonium sulfate. After centrifuging, dissolving in water, and filtering through a layer of paper pulp, toluene was added and the solution dialyzed in a collodion bag suspended in distilled water. The water was frequently changed and the dialysis was continued until the solution was free from sulfates. The protein separated in the form of small uniform globular particles which showed no crystalline structure. The precipitated globulin was removed from the dialyzer and collected on a Buchner funnel and washed with water. It was — then suspended in 95 per cent alcohol, again filtered, and washed successively with 95 per cent alcohol, absolute alcohol, and ether, R. J. Anderson 27 and finally dried in vacuum over sulfuric acid. The dry substance was a heavy, nearly white powder, and it weighed 16.3 gm. The substance was moistened with a little saturated solution of ammonium sulfate and dissolved by adding about 100 cc. of water. After filtering off a small amount of insoluble material through a layer of paper pulp the filtrate was again dialyzed. The precipitated globulin was dehydrated by treating it succes- sively with 25, 50, 75, and 95 per cent alcohol and finally with absolute alcohol. It was filtered, washed with absolute alcohol and ether, and dried in vacuum over sulfuric acid. The dry substance weighed 13.5 gm. The substance was analyzed after drying at 110° in vacuum over phosphorus pentoxide. 0.1252 gm. substance lost 0.0067 gm. = 5.25 per cent H.O. Outsaac “gave 0.0708 “ H,O and 0.2231 gm. COs. 0.7932 “ . “0.0351 “ BaSO.. 0.1420 “ 7 required 18.5 cc. 0.1 N H2SO, (Kjeldahl). Found: C, 51.22; H, 6.68; S, 0.60; N, 18.24 per cent. Second Preparation of Acerin. Extraction with Water. 50 gm. of the powdered, ether-extracted maple seed were digested in 200 cc. of distilled water for about 5 minutes. The extract was then filtered through paper pulp and washed with water until 200 cc. of filtrate were obtained. Ammonium sul- fate was added nearly to saturation; the precipitate was centri- fuged, dissolved by adding 100 cc. of water, and the solution was filtered through paper pulp and the filtrate dialyzed under toluene until free from sulfate. The precipitated globulin was dehydrated and washed with alcohol and ether as before and dried in vacuum over sulfuric acid. The yield was 3.6 gm. There is a sufficient amount of soluble salts in the maple seed to permit practically all of the globulin to be extracted with dis- tilled water. The seed residue in the above preparation was extracted with 200 cc. of 10 per cent sodium chloride solution, but the extract contained an exceedingly small quantity of protein. The globulin obtained above was identical in appearance and properties with the first preparation. It was analyzed without further purification after drying at 110° in vacuum over phos- phorus pentoxide. 28 Acerim 0.1652 gm.substance lost 0.0097 gm. = 5.87 per cent H,0. 0.1555 “ « gave 0.0944 “ H,O and 0.2947 gm. COs. 0.8454 “ as “ 0.0326 “ BaSQ,. 0.1412 “ - required 18.5 ec. 0.1 N H,SO, (Kjeldahl). Found: C, 51.68; H, 6.79; S, 0.56; N, 18.34 per cent. Third Preparation of Acerin. 200 gm. of the powdered maple seed were digested in 800 cc. of distilled water for 15 minutes, filtered, and washed with water until 800 ce. were obtained. The filtrate was saturated with ammonium sulfate and the precipitate centrifuged and then trans- ferred to a Buchner funnel and washed with saturated ammonium sulfate solution. The precipitated globulin was redissolved by adding 250 ce. of water, the solution filtered and dialyzed until free from sulfates. The contents of the dialyzer were transferred to a beaker and allowed to settle. The supernatant liquid was poured off and the globulin brought on a Buchner funnel and washed with water. It was then suspended in about 200 ec. of water and dis- - solved by adding about 5 per cent of sodium chloride. The solution was filtered and dialyzed until free from chlorides. The precipitated globulin was removed from the dialyzer, dehydrated with alcohol, washed in alcohol and ether, and dried in vacuum over phosphorus pentoxide. The dry substance weighed 12.6 gm. This preparation was analyzed after drying at 110° in vacuum over phosphorus pentoxide. 0.1285 gm. substance lost 0.0077 gm.= 5.99 per cent H.O. 0.1208 ¥ gave 0.0751 “ H,O and 0.2279 gm. CO,. 0.7884 “ : “0.0300 “ BaSQ,. 0.1411 “ Es required 18.8 ec. 0.1 N H,SO, (Kjeldahl). Found: C, 51.45; H, 6.95; 8, 0.52; N, 18.65 per cent. Fourth Preparation of Acerin. 50 gm. of the powdered maple seed were digested in 200 ee. of 70 per cent alcohol for 2 hours with frequent shaking. It was then filtered on a Buchner funnel and washed with 70 per cent alcohol until the filtrate came through colorless. The seed residue was dried in vacuum over sulfuric acid and then digested in 200 ce. of 5 per cent sodium chloride solution to which sufficient barium hydrate had been added to maintain a neutral reaction eS al R. J. Anderson 29 in the extract. It was filtered through paper pulp and washed with water until 300 cc. of extract were obtained. The extract was precipitated by adding ammonium sulfate _ to 0.6 saturation. The mixture was then centrifuged and the globulin transferred to a Buchner funnel and washed with ammo- nium sulfate solution of the same strength. The precipitate was brought into solution by adding 100 cc. of water. It was again filtered through paper pulp and dialyzed until free from sulfate. The precipitated globulin was suspended in 100 cc. of water and dissolved by adding a little ammonium sulfate. The solution was filtered and again dialyzed until free from sulfates. The globulin which separated was dehydrated with 30, 50, 70, and 95 per cent alcohol and finally with absolute alcohol and ether and dried in vacuum over phosphorus pentoxide. The nearly white powder weighed 3.3 gm. The extraction with 70 per cent alcohol removed about 16 per cent of solid matter from the maple seed but this material contained only 0.3 per cent of non-protein nitrogen. After precipitating the globulin from the extract obtained on digesting the seed residue in 5 per cent sodium chloride with ammonium sulfate to 0.6 saturation, further addition of ammon- ium sulfate to the filtered extract gave no additional precipitate, showing that all of the protein was precipitated with the above concentration of ammonium sulfate. The globulin was analyzed after drying at 105° in vacuum over phosphorus pentoxide. 0.1553 gm. substance lost 0.0097 gm. = 6.24 per cent H,0. 0.1456 “ : gave 0.0883 “ H.O and 0.2748 gm. COsz. 0.7749 “ a “ 0.0314 “ BaSOx,. 0.1419 <“ 2 required 18.5 cc. 0.1 N H.SO, (Kjeldahl). Found: C, 51.46; H, 6.77; 8, 0.55; N, 18.35 per cent. Fifth Preparation of Acerin. The seed residue, after extracting 300 gm. of maple seed with 70 per cent alcohol, was digested in 1,500 cc. of 5 per cent sodium chloride solution containing 60 cc. of Baryta water. The extract was filtered and washed with 5 per cent sodium chloride solution until about 1,600 ce. of filtrate were obtained. The clear filtrate was precipitated by adding ammonium sulfate to 0.6 saturation. After centrifuging, filtering, and washing with ammonium sulfate 30 Acerin solution, the globulin was dissolved by adding about 300 ce. of water. This solution was filtered and dialyzed. The globulin was removed from the dialyzer and suspended in water and dis- solved by adding a small amount of ammonium sulfate. After filtering the solution it was precipitated with ammonium sulfate to 0.6 saturation, filtered, washed with ammonium sulfate solu- tion, dissolved in about 300 cc. of water, and dialyzed until free from sulfate. After dehydrating with alcohol and ether as be- fore it was dried in vacuum over sulfuric acid. The yield was 19.5 gm. The substance was analyzed after drying at 105° in vacuum over phosphorus pentoxide. 0.1578 gm. substance lost 0.0097 gm. = 5.89 per cent H,0. 0.1485 ‘“ 43 gave 0.0913 “ H.O and 0.2801 gm. COy,. 0.8011 “ rf “0.0309 “ BaSQ,. 0.1412 “ x required 18.4 cc. 0.1 N H2SO, (Kjeldahl). Found: C, 51.44; H, 6.87; S, 0.53; N, 18.24 per cent. Sixth Preparation of Acerin. This was prepared from 300 gm. of powdered maple seed exactly as described for the fifth preparation. The dry product weighed 20 gm. It was analyzed after drying at 105° in vacuum over phosphorus pentoxide. 0.2738 gm. substance lost 0.0160 gm. = 5. 84 per cent H.O. 0.2578 “ es gave 0.1556 “ H.O and 0.4861 gm. CO,. 0.7552.“ 2 * 0.0319 “ Ba SOx,. 0.1413 “ ‘ required 18.4 ce. 0.1 N H,SO, (Kjeldahl). Found: C, 51.41; H, 6.72; S, 0.58; N, 18.23 per cent. TABLE IV. Summary of Analyses of Acerin. Preparation:. 3220.) ..c0cck 1 2 3 4 5 6 ee tion. per cent | per cent | per cent | per cent per cent | per cent | per cent Cota SA cosh OL 51.22 | 51.68 | 51.45 | 51.46 | 51.44 51.41 | 51.44 H CAO Mee otter 27 6.68 6.79 6.95 | 6.77 6.87 6.72 6.80 Devreeeeseeceseees es} 0.60] 0.56] 0.52] 0.55] 0.53] 0.581 0.55 N....... MT RR TE, & 18.24 | 18.34 | 18.65 | 18.35 | 18.924 18.23 | 18.34 O (by difference)...| 23.26 | 22.63 | 22.43 | 22.87 22.92 | 23.01 | 22.87 R. J. Anderson oa A composite sample containing all of the above preparations gave 0.55 per cent of sulfur and 18.32 per cent of nitrogen (Table IV), which values are identical with the above averages. After hydrolysis according to the method of Van Slyke® the nitrogen constituents were determined as shown in Table V. The basic amino-acids were determined in the phosphotungstic acid precipitate by the micro method of Van Slyke.* The results calculated to the basis of the original globulin are given in Table WT: TABLE V. Nitrogen Distribution in Acerin. Form of nitrogen. Amount. per cent Amide nitrogen....... Bic ost atone G Hb 6 Oo OCIA 2.53 Humin CER aac IN! &. PEGA (Mt edie Lt a ac 0.15 Basic Fie RA Ok TA Se es ee 4.86 ISGins OFT fon Ca ey ree ee ee eee 10.63 Notalnitrogen recovereds. -- ese eee eee 18.17 TABLE VI. Basic Amino-Acids in Acerin. per cent TATE LETC a NR oP he et 0.55 PRR OEREH EY oto yc 20. ay2.dea cores « athe er rt ee RR ee 10.07 |S STAY o Lay st a aa it MOON ren hoyle Lip. “bed VES SCN 1.43 l LASTER Oe Be Oca SOR MOE Rane baa de esan a toe 6.07 SUMMARY. The principal protein of the seed of the silver maple (Acer Saccharinum) has been isolated and purified. This protein, for which we propose the name acerin, is a globulin. It could not be obtained in crystalline form but separated on dialysis in small uniform globular particles. The purified acerin is a nearly white heavy powder which on combustion leaves no weighable ash. A number of different preparations were made and all of these preparations showed close agreement on analysis. 5 Van Slyke, D. D., J. Biol. Chem., 1912, xii, 275. 6 Van Slyke, D. D., J. Biol. Chem., 1915, xxiii, 407. i ri wy Pf ; Ty ae — i" A y A\X 1, pass ‘i i Pt Myf i od nt ere rh TS oahu il rae, Anant Tae Et} eo oie ae ~— ae ie a A ll ce Tat The average composition of acerin is as follows: aif ‘ a C, 51.44; H, 6.80; N, 18.34; S, 0.55; O, 22.87 per cent. pie: . When analyzed by the Van Slyke method it was found that a considerable percentage of the basic nitrogen was present as lysine. DIETARY FACTORS INFLUENCING CALCIUM ASSIMILATION. I. THE COMPARATIVE INFLUENCE OF GREEN AND DRIED PLANT TISSUE, CABBAGE, ORANGE JUICE, AND COD LIVER OIL ON CALCIUM ASSIMILATION.* By E. B. HART, H. STEENBOCK, anp C. A. HOPPERT. (From the Department of Agricultural Chemistry, University of Wisconsin, Madison.) (Received for publication, July 11, 1921.) The problem of the dietary factors influencing calcium assimila- tion in domestic animals is not new. In 1913 we drew attention to an observation (1), which, when worked out in its details will, no doubt, have a very important bearing on the question of cal- cium assimilation. In this early publication data were presented which showed that there were marked differences in the amount of calcium eliminated in the feces of a milking goat when that animal was changed from old dried roughage to green pasture, and after a period of fresh green grass intake placed in the metabolism cage and returned to the dried feed ration for a calcium balance experi- ment. After the period of green pasture feeding, the fecal calcium elimination was so reduced as to give a positive calcium balance as compared with a high fecal calcium output and negative calcium balance on the old dry roughage. Evidently something had been ingested with the green material that allowed a more perfect skele- tal storage of calcium or a more complete assimilation of this element from the intestine. In an earlier piece of work (2) we had observed that a nega- tive calcium balance could prevail with a lactating cow for a very long time with a slow shrinkage in milk flow but no observant physiological disturbances. This animal was under actual quanti- * Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 33 34 Calcium Assimilation. I tative observation for 110 days and on a ration consisting of ordinary oat straw, wheat bran (natural or washed), rice meal, and wheat gluten. The fecal output alone of calcium was nearly equal to the daily intake of this element. Her daily output of calcium oxide was approximately 50 gm. per day with an intake of but 25 em. The constancy in maintaining the percentage of calcium oxide in the milk was remarkable. We would not want to leave the impression that there were not deep seated and seriously abnor- mal conditions developing in this individual and that ultimately nutritional failure and milk production must cease, but we did not observe this in a period of 110 days. We have observed (3) disas- ter in reproduction with cows where probably negative calcium balances are long continued. In this same direction Forbes and his associates (4) have made important contributions showing that high milking cows receiving rations that are supposed to provide an ample intake of calcium, nevertheless may eliminate a larger amount of calcium than is ingested. Even the addition of calcium salts to a ration of grains, dry alfalfa hay, and corn silage did not lead in their experience to the establishment of positive calcium balances. In a similar direction Meigs, Blatherwick, and Cary (5) have contributed interesting data showing that a dry but pregnant cow is probably not assimilating sufficient calcium from a calcium-rich ration suchas dry alfalfa hay, corn silage, and a grain mixture for a positive balance, but is actually transferring calcium salts from her, skeleton for fetal skeleton building. Meigs and his associates are inclined to interpret these observed negative calcium balances as only temporary and merely incident to the collection of the excreta and due to nervous disturbance of the animal. While we recognize the possibility of such a factor as operative with some individuals, yet we believe that the main factor of influence in this connection is of dietary origin. While the problem of calcium assimilation and metabolism is of very great importance in relation to growth, milk production, and egg production of our farm animals, it is of equal importance in human nutrition, and no doubt likewise related to dietary factors other than mere calcium supply. Ina recent short note (6) where we briefly discussed this subject we said: PR rere ew Hart, Steenbock, and Hoppert 35 Ss In the case of nursing women the relation of diet to a posi- tive or negative calcium balance and to dental conditions will assume new aspects. “The supposition that we are dealing with something influencing calcium assimilation and which is more abundant in green than in dried plant tissue and consequently variable with the season’s milk, would explain the varia- tions in the seasonal frequency of rickets, as observed and commented upon by Hess (7).”’ In continuation of this line of reasoning we are assuming that it is entirely probable that the factor or factors shown to be opera- tive in optimum calcium assimilation in any one of our farm ani- mals can be translated as applicable not only to other types of ani- mals but to human nutrition as well. No doubt there will be species differences. One species, under adverse conditions will assimilate calcium more completely than another, but these dif- ferences will be quantitative and not qualitative. Just as the guinea pig is more sensitive to a lower supply of the antiscorbutic vitamine than the rat, cow, or pig, so the human infant and puppy are probably more sensitive than the rat or pig to a low supply of the dietary factors affecting calcium assimilation, but it is ‘‘a dif- ference of degree and not of kind.” The statement that faulty calcium assimilation or poor bone formation is due to lack of a balance of dietary factors is too indefi- nite to satisfy students of nutrition. Of course, it is self-evident that a low calcium- or a low phosphorus-containing ration would be a primary factor in poor skeletal development with a rapidly growing species, but the real problem before us is the dis- closure of the nature of that dietary factor whose absence leads to faulty calcium assimilation even in the presence of an ample supply of this element. Faulty calcium assimilation extending over a comparatively long time can occur in cows and mature swine without an exhibi- tion of the quick and complete collapse shown by a growing puppy suffering from rickets, and yet we have no doubt that some of the nutritive failures exhibited by growing swine (8) and even ma- ture swine and mature cattle on certain restricted diets will ulti- mately be classed as in the main a condition simulating rickets. In this paper there will be presented only what preliminary data we have accumulated on the influence of dietary factors on cal- cium assimilation by the dry and milking goat. Work with other 36 Calcium Assimilation. I types of animals and with food materials other than those used in these experiments is now in progress and will be reported on when the accumulated data warrant it. EXPERIMENTAL. The goats used were common American grades with no distinct breeding. They were confined in our metabolism cages with quan- titative collection of the excreta. When milking animals were under observation they were milked twice daily. Analyses were applied to the weekly collection of feces and to the weekly com- posites of aliquots of urine and milk taken daily. Calcium deter- minations were made on the feeds, milk, and feces by the MeCrud- den method, after ashing. In the urine the calcium estimation was made directly and without ashing as further suggested by MeCrudden (9). Record of Animal 1. Animal 1 was a milch goat producing 700 to 800 ce. of milk per _ day. She, as well as the others, was fed a grain mixture consisting of 30 parts of yellow corn, 15 of oil meal, 30 of whole oats, 24 of wheat bran, and 1 of common salt. The roughages were varied in the successive periods of observation being, respectively, alfalfa hay, oat straw, green oats, and dried green oats. In view of the fact that Forbes, Meigs, and their associates, as already stated, observed a negative calcium balance in feeding cows with alfalfa hay—a roughage distinctly high in its calcium content—we used alfalfa hay in the first period. This gave us data for comparing the behavior of the cow with the goat. Alfalfa Hay Period.—After 2 weeks preliminary feeding, collec- tions were made for a period of 5 weeks on the alfalfa ration. During this time 3,500 gm. of alfalfa and 3,500 gm. of grain mix- ture were consumed weekly. Contrary to expectations the animal did not go into negative calcium balance as can be seen in Table I where the record of this as well as of the other trials are tabulated. As this animal was purchased locally and had been receiving a varied and unknown ration before her purchase there is a possi- bility that previous storage of the factor influencing calcium assim- ilation had occurred which later during the alfalfa feeding period aided in maintaining normal calcium assimilation. We are in- ee, ee ee ee Se ote Hart, Steenbock, and Hoppert ot clined to doubt the validity of this assumption, in the first place, because the record was started in April—a time of the year when the animal kept on ordinary rations would be expected to be deple- ted of this dietary factor—and in the second place, because on the oat straw ration, immediately following, a negative calcium bal- ance was readily established. It seems plausible to assume that TABLE I. Record of the Calcium Balance of Animal 1. pete, | asst [a0] es | Use| AB: [Fash | tg mame Alfalfa hay period. gm. per cent gm. gm. gm. gm. gm. PAT TS ecoyecss tacks 2,330) 2.74 | 63.84) 0.32 | 12.31) 86.45) 76.47|+ 9.98 “ 25-May 2.....; 2,240) 3.01 | 67.42) 0.18 | 18.72) 86.45) 81.32/4- 5.13 NB YEO wie. soe 2,341) 3.22 | 75.49) 0.29 | 12.50) 86.45/ 88.28)— 1.83 eee O= 168 oe. 2,248] 3.07 | 69.09) 0.17 | 15.61} 86.45) 84.87/4+ 1.57 P20) 15 ee a 2,375| 2.87 | 68.16} 0.09 | 11.70} 86.45} 79.95|+ 6.50 Oat straw period. June 6-18.........| 1,363] 1.84 | 25.18) 0.02 | 10.48 12.92| 35.68| 22.76 Se SO ccccicte-s Seen 1,222} 1.70 | 20.74; 0.10 | 7.39) 8.69] 28.23)—19.54 DOD ino ane | Le 28] Lesh mom OLO2 8.13] 8.71] 27.89|—19.18 eet —Nitlhyere. ok Oao| 1e29) | oneal OnO2 7.38} 8.50) 20.77|—12.27 Green oats period. dub to: 888] 0.68 | 6.06) 0.02 | 6.20) 8.04) 12.28)/— 4.24 “8 IS Sa a a 871| 0.65 | 5.66) 0:10 | 6.63) 8.00) 12.39/— 4.39 1 |. 761| 1.07 | 8.15} 0.07 | 6.21) 7.78) 14.43/— 6.65 « 25-Aug.1....| 811) 1.20 | 9.66] 0.10 | 5.51) 8.62) 15.27|— 6.65 Oat hay period. ‘a ES 893} 1.10 | 9.90) 0.10 | 5.05] 8.23) 15.05)— 6.82 “Te tS |S rae comes 945] 1.15 | 10.86] 0.08 | 4.93] 8.23) 15.87/— 7.64 Be 2d et Be One| 8213) 0:06"|) 3.35) - 7.06)" 544748 the alfalfa hay is considerably richer than oat straw in its content of the unknown factor influencing calcium assimilation and that the goat is less sensitive than the cow to its scarcity. In this connection it is well recognized that the goat is an animal of extraordinary persistent milking tendencies under most adverse conditions. 38 Caleium Assimilation. I Oat Straw Period.—When feeding oat straw in place of alfalfa, casein was added to bring up the protein. As an example of a week’s ration there were consumed during the second week 2,750 gm. of grain, 1,550 gm. of oat straw, and 186 gm. of casein. During this period the calcium balance was always decidedly negative, but the decided deficit of the first week must be accepted with some reservations, as with a 6 to 8 fold decrease in calcium intake the lag of calcium excretion from the alfalfa period is decid- edly contributory to the negative balance. As the pronounced negative balance persisted, there need, however, be no question as to the character of the change produced. In the table, to bring out these facts, there are given in addition to other data both the weights of the feces and the percentage of calcium contained therein. These are important data and must be used in the interpretation of the results. A depression in the mass of feces with a rise in the percentage of calcium oxide with change of diet means nothing, but either a constancy in the fecal mass accompanied by a lowered percentage of calcium oxide or a depression in both means quite as much on a low calcium intake as does the actual establishment of a positive calcium balance. This latter will depend not only upon the kind of diet but also upon the amount of calcium in the ration. Green Oats Period.—During this period, which ran for 4 weeks, fresh green oats (entire plant) were cut daily, sampled daily, and fed fresh, in amounts so that the calcium intake was approximately equal to that of the oat straw period; vz.,8 to 9 gm. On this uniform intake, though a positive balance was not established, probably because the intake was too low, the loss of calcium was reduced to approximately one-third of its former value. This has especial significance taking into consideration what was said in the preceding paragraph in regard to the importance of the relations of fecal mass to percentage of calcium contained therein, for while the fecal mass was reduced by the feeding of this succulent material, its percentage content of calcium was also reduced. Such a situation leaves no doubt as to the corrective effects brought about by the unknown factors of the fresh green roughage. Mere difference in solubility of the lime content was not the factor, as in harmony with our previous results where all the cal- » cium in oat straw was found soluble in 0.05 n HCl, 95 per cent of Hart, Steenbock, and Hoppert 39 the calcium was extracted by digestion for 24 hours at 37°C. with a 0.2 per cent hydrochloric acid-pepsin solution. Oat Hay Period—During this period the green oats were substituted by the same material in the dried form as a hay, anticipating from what we expect occurs in farm practice, that this material would give about the same results as those secured with oat straw. For this purpose the oats were cut and dried inthe diffused light of an attic hghted by skylights. Leaching by dew and rains was thus prevented but in addition changes induced by the fermentation of the curing process in the cock or mow were also eliminated so that the material was not strictly comparable to what is most commonly fed in general farm practice. Probably our expectations in regard to results obtained were also unwar- ranted, for feeding experiments with rats have shown that while oat straw contains little fat-soluble vitamine, this oat hay con- tained an abundance of it. So in certain relations at least, dis- tinct differences in the effect of feeding these materials were to be expected. 3 weeks of record in the metabolism cage gave results unlike those obtained with oat straw but comparable to those secured with the green oats as seen in Table I. There was no rise in the fecal calcium elimination and no distinct difference in the calcium balance on practically the same intake. Possibly the factor influ- encing assimilation had not been greatly reduced in the oat hay as we dried it. Possibly our period of observation was too short. This phase will receive further study. Record of Animal 2. In the case of this animal our plan was to duplicate the proce- dure used with Animal 1. We did this in every respect but with the exception of omitting the dry alfalfa hay period. This animal was likewise milking and received the same materials as used in the first experiment. These materials consisted of the grain mix- ture and casein, with ordinary oat straw, green oats, or the attic dried oat hay. In all these experiments the amount of green oat hay allowed was made equivalent in dry matter to the dry matter of the oat straw. The records are shown in Table II. These results duplicate in principle those secured with Animal 1. On the oat straw ration there was a high fecal calcium output and 40 Calcium Awivaivten: I a very pronounced negative calcium balance. On the green oats the dry weight of feces was practically the same as during the oat straw period but the percentage of calcium was reduced, making the total calcium elimination much less. Here a positive calctum balance was not established due to a low intake of calcium, but the degree of negative balance was greatly reduced. The effect with the oat hay was similar to our experience with Animal 1 and did TABLE II. Record of the Calcium Balance of Animal 2. Duis | Rest | See] He | Base | 20. [Tate Pate atom Oat straw period. gm. per cent qm. gm. gm. gm. gm. June 17-24.........| 1,547} 1.50 | 23.26) 0.03 | 10.86} 13.34) 34.15|—20.81 “« 24-July 1....| 1,578] 1.44 | 22.84) 0.03 | 8.48} 13.57] 31.35)—17.78 RG PEW cess se 1,681) 0.89 | 14.99) 0.08 | 7.70) 18.89) 22.77|— 8.88 6 OS ADL........| | 968) 0.96 | .9.35] 0.04 | 4.48) 7.91) 13.87/— 5.96 Green oats period. POLY: FOTO) 6) 1,368] 0.80 | 10.99} 0.16 | 7.07} 12.47| 18.22)— 5.75 “ 19-26.........| 1,109] 0.78 | 8.75] 0.05 | 7.30] 12.59) 16.10|— 3.51 “« 26-Aug. 2....| 1,263) 0.77 | 9.73] 0.09 | 6.86] 14.07) 16.68)/— 2.61 it ee . 1,037} 0.75 | 7.80) 0.04 | 5.58) 11.47) 13.42);— 1.95 Oat hay period. Ang. 8-15... Fes... 1,509} 0.59 | 8.90) 0.05 | 5.75] 18.83] 14.70|— 0.87 “ 15-21 1,261) 0.65 | 8.24) 0.04 | 4.69} 11.86} 12.97)— 1.11 not give the results expected; we expected an increased fecal cal- cium elimination and an increased negative calcium balance, but: this did not result during the time of observations. Increased water intake was not a factor in these experiments since quite as much water was consumed on the oat hay ration as on the green oat ration making due allowance for its water content. For ex- ample, in a selected week on the oat hay ration the water consump- tion was 6,680 cc., on the green oats ration it was 5,940 ce. Ee ee —— ——E Hart, Steenbock, and Hoppert 4] Record of Animal 3. Goat 3 was not a heavy producing animal, giving approximately only 200 gm. of milk per day when put on the basalration of grain, TABLE III. Record of the Calcium Balance of Animal 3. Dried |CaOin| Feces | Urine | Milk | Intake |Outpu EES feces. | feces. | CaO. | CaO. | CaO. | CaO. | CaO. Balance. Oat straw period. gm. |percent| gm. gm. gm. | gm. gm. Ore it 18h. 1,284| 1.44 | 18.59) 3.31 | 0.39 | 16.01) 22.29] —6.28 22 a kc Sse 1, 736} 0.92 | 15.97} 3.24 | 0.26 | 16.01] 19.47] —3.46 “ 25-Nov. 1 ....| 1,647] 0.99 | 16.35] 3.28 | 0.25 | 16.01] 19.88] —3.87 Butter fat period. Nov. 1-8..........| 1,411] 1.00 | 14.16] 2.32 | 0:22 | 14.86] 16.70) —1.84 ie a) a ey 1, 283] 1.09 | 14.05} 1.81 | 0.17 | 9.42} 16.03) —6.61 Orange juice period (60 cc. per day). Now. 15-22)... ....'.: 1,551] 0.79 | 12.25) 2.36 | 0.26 | 12.49) 14.87) —2.38 See Oana ssc 1,291} 0.75 9.74] 1.74 | 0.29 | 14.32] 11.77) +2.55 “ 29-Dec. 6....| 1,463} 0.81 | 11.89] 1.29 | 0.26 | 14.52) 13.44) +1.06 Orange juice period (120 ce. per day). 0.29 | 14.71] 14.32} +0.39 0.16 | 14.71] 14.49] +0.22 11.98 12.60 2.05 1.73 See 1, 408| 0.85 pa) a ae 1,581| 0.79 Dried cabbage period (30 gm. per day). Rane ae 1, 467| 0.81 | 11.92] 1.63 | 0.24 | 14.64] 13.79} +0.85 “« 97-Jan. 3. ...| 1,229) 0.98 | 12.06] 2.32 | 0.26 | 16.30] 14.64) +1.66 Neat 3102.0. | 1,359] 0.94 | 12.77) 1.50 | 0.24 | 15.99] 14.51) +1.48 “ 10-17.........| 1,371] 1.01 | 13.94] 1.65 | 0.22 | 15.99] 15.81] +0.18 “ 17-24.........| 1,350] 0.99 | 12.19] 2.15 | 0.10 | 10.33] 14.44] —4.11 et 3 ts re 1,119] 1.11 | 12.43] 1.39 | 0.12 | 14.56] 13.94) —0.62 Fin. (eee 1,221] 1.18 | 14.42] 1.44 | 0.19 | 15.75] 16.05} —0.30 eee t4 | 1,325] 1.02 | 13.62] 2.64 | 0.09 | 15.39] 16.35} —0.96 Raw cabbage period (300 gm. per day). Feb. 14-21.........| 1,383] 0.94 | 18.12} 2.22 | 0.07 | 14.71) 15.41) —0.70 <-21-28.........| 1,328] 0.97 | 13.01} 2.12 | 0.06 | 14.70] 15.20) —0.50 «< =6©°28-Mar. 7. ..| 1,357| 0.99 | 18.52) 3.26 | 0.05 | 15.39) 16.84) —1.45 Wier (1455, 2 2,5 - 1, 447| 0.93 | 18.48] 2.53 | 0.05 | 14.34) 16.07; —1.73 42 Calcium Assimilation. I TABLE I1Il—Conlinued. | ied | CaO i ie s J ae: 3 il is Date. tet ena! Gao. | Cao Cao. oi cate Balance. Raw cabbage period (1,000 gm. per day). wl BERS, Pee nt| om. gm. gm. | gm. qm. Mar. 14-21.........| 1,019] 1.12 | 11.47, 2.19 | 0.08 14.01) 18.74) +0.27 ME DIDS ted bt 829) 1.47 | 12.23) 3.36 0.08 | 13.65) 15.67) —2.02 “ 98-Apr. 4...) 928) 1.30 | 12.12] 2.77 | 0.07 | 14.87] 14.96) —-0.09 Apr. 4-11........./ 870] 1.51 | 13.21) 2.96 | 0.05 | 13.73) 16.22, —2.49 Cod liver oil period (5 cc. per day). a a | 87| 1.68 | 14.96| 3.97 | 0.06 | 17.15] 19.00] —1.85 2 a | 892} 1.18 | 10.52| 3.35 | 0.06 | 13.88] 13.94] —0.06 Cod liver oil period (10 ce. per day). Apr. 25-May 2.....| _883| 0.98 | 8.72| 2.96 | 0.05 | 12.67| 11.73| +0.94 May: 229. 2s3e: Ss | 670] 0.95 | 6.39] 3.38 | 0.05 9.83! 9.52) —0.31 pple! vai Seana Discontinued; not eating. casein, and oat straw. Her daily consumption was 600 gm. of grain, 40 gm. of casein, and 200 gm. of straw. On the ration, she rapidly went into negative calcium balance (see Table III). Our results with green material, in these trials with green oats and in former work with mixed grasses, were so consistent that we believed that we were warranted at this stage of our experimenta- tion to attempt to determine what factor in the green materials was operative in facilitating calcium retention. For this purpose. our basal ration was supplemented successively with butter fat, orange juice, dried cabbage, fresh cabbage, and cod liver oil, all of which are materials which are well known to be rich in the fat-sol- uble vitamine or the antiscorbutic vitamine. These were selected not because we were inclined to the opinion that the factor we were dealing with was necessarily either one of these, but because we believed that this selection gave us a sufficient range of variables, including two well known factors with the possibility of the inclu- sion of others, so that the effect of the green oats might be dupli- cated. The relation of the water-soluble vitamine to this prob- lem of calcium assimilation we felt free to disregard as our basal ration was, we believe, amply supplied with this factor. * ee Hart, Steenbock, and Hoppert 43 Butter Fat Period.—In this period we gave 45 to 60 gm. daily of a clear filtered butter fat reducing the grain allowance by an amount equivalent in energy to the butter fat added. Butter fat feeding was continued 2 weeks. With this animal its ingestion affected the appetite adversely and milk production fell off very perceptibly, decreasing to approximately 50 cc. per day. In the short time of observation, the butter fat had no effect on the per- centage of calcium eliminated by the feces, and the negative cal- cium balance was quite as large as on the basal ration. However, our data with butter fat are entirely too limited to give us an opinion as to whether or not it possessed any specific therapeutic value in influencing calcium assimilation. Orange Juice Period.—Observations were next made with orange juice plus the basal ration. 60 cc. of orange juice were given daily for 3 weeks and then the dosage was increased to 120 ec. per day for 2 weeks. We had no difficulty in getting this ani- mal to consume the orange juice with a complete recovery of appe- tite from the depressed condition experienced in the butter fat period. Milk flow was not completely restored although the daily volume now reached about 100 cc. as compared with but 50 ce. in the butter fat period. The calcium percentage in the feces was slightly but perceptibly decreased, and with the lowered milk flow the net result was a positive calcium balance. From these data alone one would be inclined to ascribe to the antiscorbutic vita- mine some influence on calcium assimilation, but when, as will be shown later, no such positive calcium balances followed with the daily consumption of 1,000 gm. of fresh cabbage, also a very po- tent antiscorbutic material, and that with two other animals orange juice was not effective, there is left slight support for the assumption as made by Robb (10) that we were dealing with the antiscorbutic vitamine in green material as the factor influencing calcium assimilation. Possibly the orange juice we used at this time contained some of the food accessory which influences cal- cium assimilation. Dried Cabbage Period.—Following the orange juice feeding came a long period where 30 gm. daily of dried cabbage (equivalent to about 300 gm. of fresh cabbage) were fed, plus the usual basal ration of grain mixture, casein,and oat straw. This dried cabbage was prepared by autoclaving at 15 pounds for 13 hours and then 44 Calcium Assimilation. I drying at 65-75° C. In this period there was a slow increase in the fecal caleium elimination, finally resulting in a negative cal- cium balance, although this did not take place until a lapse of 4 weeks of dried cabbage feeding. Since the behavior of this ani- mal was indicating that the antiscorbutic vitamine might have some influence on calcium assimilation, in the next period of feeding we used fresh cabbage, feeding 300 gm. daily for 4 weeks and increasing this amount to 1,000 gm. daily for 4 more weeks with constant quantitative observations. Fresh Cabbage Period.—During the period where we fed 300 gm. daily of raw fresh cabbage no reduction in the other constituents of the ration was made. The mass of feces remained practically the same as in the previous period and there was no appreciable change in the percentage of calcium in the feces. Negative calcium balance continued. When the fresh cabbage was in- creased to 1,000 gm. per day we reduced the straw intake from 170 to 25 gm. per day with the intention of keeping the fiber content of the ration approximately the same as in previous periods, and thereby holding the fecal mass constant. We did not succeed in this and consequently added ordinary filter paper, 60 gm. per day, for the purpose of increasing the fecal residue. With this goat the added paper did not raise the fecal residue. The result of a lowered fecal residue during the 1,000 gm. of raw cabbage feeding was a marked increase in the percentage of cal- cium in the feces with the net result that just as much calcium continued to be excreted in the feces as in the previous feeding period, and a negative calcium balance continued. These results left no question as to the negative relation of the antiscorbutic vitamine to this phenomenon of calcium assimilation. While fresh cabbage or dried cabbage contains some fat-soluble vitamine (11), yet it is not particularly rich in this food factor; should it be established later that this vitamine is the one related to cal- cium assimilation it would be necessary to assume that cabbage was not sufficiently rich in this factor to bring about such an influence. The failure to induce a positive calcium balance with such large amounts of cabbage is interesting when it is compared with the one positive effect of the orange juice administration. Both are rich in the antiscorbutie vitamine, and weight for weight they are Eo » » Hart, Steenbock, and Hoppert 45 probably of approximately equal value as sources of the fat- soluble vitamine as shown by our feeding experiment with rats. Yet before attempting to make extensive analytical use of these discrepancies it is well to bear in mind that negative results must not be given too absolute a valuation as the recuperative elasticity of an animal in trials of this nature has decided limitations. Cod Liver Oil Period.—The general recognition of cod liver oil as a successful therapeutic agent in rickets led us to use it in these experiments. It was emulsified with acacia gum and water and for 2 weeks this emulsion was administered in amounts equivalent to 5 ec. daily of the original oil. In the 3rd and 4th week of this period an equivalent of 10 cc. of oil was given daily. There was no influence on calcium assimilation in the Ist week, but in the 2nd week the fecal calcium oxide dropped from 1.68 to 1.18 per cent and in the 3rd week to 0.98 per cent with the output of feces practically the same as in the cabbage period. This influence of the oil resulted in establishing a positive calcium balance in the 3rd week, with a decrease in the calcium oxide of the feces from 14 to 8.7 gm. There was soon developed a dis- tinct dislike for the oil and in the 4th week there occurred a loss of appetite with the result that the food intake was reduced in this week and a slight negative calcium balance resulted. It is safe, however, to conclude that cod liver oil was an effective agent in assisting calcium assimilation in this species. Record of Animal 4. Basal Ration Period.—While a milking goat receiving our ration of grain mixture, casein, and dry oat straw, responded readily with a negative calcium balance, we were not sure that such a reaction would follow with a dry animal. To determine this, Animal 4 was started on our basal ration October 11, 1920. She remained in positive calcium balance or equilibrium until the 2nd week in December, when a distinct negative calcium balance was assured. Feeding of this ration was continued until January 31, 1921, at which time the animal’s appetite was becoming poor and her general condition apparently somewhat impaired. Her feces were hard and dry. She showed a negative calcium balance of 3.04 gm. of calcium oxide for the week. See Table IV for the record of results. 46 Calcium Assimilation. I TABLE IV. « Record of the Calcium Balance of Animal 4. et Dri ‘a0 i | Urine | Intake | Output Date, palo | eos oeee: |" Urine | Tatese amar Oat straw period. ree : | gm per cent gm i: qm. gm. gm. Oot.? T1138 YS, ae 1, 486 0.81 | 12.06 | 0.17 | 13508) 122238 rake MLS. ee 1, 5038 0.84 | 12.70 | 0.33 | 15.08 | 18.03 ss ~625-Now: 1 3/9526 0.83 | 12.65 0.84 | 14.66 | 13.49 INOW. S185 oe ones, f ei, 1,590 | 0.81 | 12.91 1.00 | 18.91 | 18.91 i SLO Lae icon 1,394 | 0.75 | 10.56 0.73) |) LealGn | Pie2S Dee: 6-13... 7. 3 1,338 | 0.84 | 11.27 | 0.40 | 13.07 | 11.67 See S-20 se sete: 1,483 | 0.85 | 12.58 | 0.29 | 11.68 | 12.97 ANON 24aF os) nee 1,381 0.98 | 13.54 0:45 | 12.-73)) 13.99 a2 ee 657 0.90 | 5.95 0.13 | 3.03 6.08 Orange juice period (120 ce. per day). Jan. 31-—Feb: 7 .... 741 1307 || 7295) |) OSs) 182525 eSel0 Renee fal45 ne 1,192 1.00 } 11.98 0.12 | 10.59 | 12.10 Aiea: 794| 1.06| 8.48| 0.11| 7.42] 8.59 MOL DOr es. : 1,054 | 1.00 | 10.57 | 0.08 | 10.29 | 10.65 — ~“26-Mar. 7,...| 1,044 | 1/00 | 10-77 |” 0.067) ~9390")) 10283 Mar 71424 55 022 1,015 ANON ee e7, 0505) 9.02>| 11222 Orange juice period (240 cc. per day). Mar. 44-91). 42. 963 LeU) LOS75 5) (OLO7a ese Ole | LOrS2 SD eee ae eed 1,093 1.13 | 12.38 |} 0.04 | 9.09 | 12.42 “< 28-Apr. 4 | 851 | 1.22 | 10.55 0.05 7.42: | 10260 Cod liver oil period (5 ec. per day). ADDY: AqVios heb a 578 Dee GESOniG 04 5.36 | 6.84 yoy pLI=18) ae ee 643 1.16 7.46 0.03 | 6.24 7.49 ciate | (20713 nner py 513 | 0.95 | 4.90] 0.03 5.39 | 4.93 Cod liver oil period (10 ec. per day). Apr. 25-May 2..... 409 | 0.97 | 4.00! 0.02| 2.60! 4.02 May ds | 290] 0.93] 2.71 | 0.03] 3.45| 2.74 (a cays ae Discontinued; not eating. Balance. 41.85 42.05 lea —0.00 41.87 <1) 430 —1.26 —3.05 +0.22 —i ea =a. —0.36 —0.93 —2.20 Ion —3.33 —2.88 —1.48 —{ 28 +0.46 —1.42 +0.71 Hart, Steenbock, and Hoppert 47 Orange Juice Period.—On January 31, 1921, administration of 120 cc. of orange juice daily was begun. This resulted in some stimulation to appetite and increased food consumption, but there was no change in the amount of calcium assimilated and a nega- tive calcium balance continued. On February 14, 1921, the amount of orange juice allowed was increased to 240 cc. per day, but even this amount did not decrease the fecal calcium elimina- tion and the animal remained at negative calcium balance. Cod Liver Oil Period.—On April 4, 1921, 5 ce. of cod liver oil per day as an emulsion with acacia gum and water were given. For the first 2 weeks of oil administration there was little influence on the percentage of calcium in the feces, but with decreasing appetite and decreasing fecal output the net result was a lowered calcium elimination. This situation continued into the 3rd week with a decreasing percentage of calcium in the feces, and an actual change to a positive calcium balance in this last period. After 3 weeks on a daily allowance of 5 cc. of cod liver oil, we increased the amount to 10 cc. but the appetite of the animal had become so poor and the intake of food so low that the results of the last 2 weeks mean little. The balance experiments for this animal do show, however, that a negative calcium balance can be established with a dry goat receiving our basal ration; that orange juice had no influence on calcium assimilation; and that cod liver oil in as low amounts as 5 cc. per day did effect a better retention of the calcium of the feed. Record of Animal 5. Basal Ration Period.—The record of Animal 5 was substantially a duplication of that of No. 4, both being non-producers. The results are recorded in Table V.. After prolonged feeding (2 to 3 months) a negative calcium balance was established on our basal ration. This animal as well as No. 4 was not in the metabo- lism cage continuously as seen in the protocols, but received the basal ration through the entire period of observation whether in the cage or not. Orange Juice Period.—On February 7, 1921, after we were positive that the animal was in negative calcium balance, 120 ec. of orange juice were given daily. This was continued for 6 weeks but without any appreciable change in calcium assimilation. 48 Caleium Assimilation. I The mass of feces and percentage of calcium therein remained practically like that of the preceding period. TABLE V. Record of the Calcium Balance of Animal 6. : Dried | CaOin | Feces Urine Intake | Output Date. feces. feces. CaO. CaO. CaO. Cao. Oat straw ration. i gm. per cent gm. gm. gm. gm. Oot. td 18:6: dateve 1,190 | 0.87 | 10.37} 0.12) 11.80 | 10.49 ey locos earns eh 1, 068 PSs) 12.28 0.38 | 11°30) 12266 ‘e95-Nov..1....| 1,074] 1256,/ 16.70 | 0.74.) Th 30) shies INO Wie fl Sio aie csencies 1, 056 0.85 9.01 0.59 | 11.30 | 9.60 “ roize! ho IL yar 1, 121 0.74 8.32 0.59 | 11.30 8.91 Heer G13 4a. 1,029 1.09 | 11.28 0.06 | 11.22 | 11.34 RLS HAO Fclcaets ts 1,316 0.96 | 12.72 OF07% | A228 e209 Jane (24.5 s ee 1,297 0.92 | 12.238 0.08 | 10.92 | 12.31 Soe Oa acters 1, O86 1.00 | 10.91 0.08 | 10.92 | 10.99 oy vol Webs dane 1, 082 1.05 | 11.44 0.07 | 10.89 | 11.51 Rebs (14.22... 2 34,3 1,229 |} 0.97 | 11.92 0.07 | 10.28 | 11.99 allie! (| Gk 1,159 | 0.91 | 10.64 | 0.07 | 11.06 | 10.71 See ISIS ea ei 1,071 0.92 9.93 0.05 | 11.06 9.98 «6. 28-—Mar. 7....| 1,140 | 0.95 | 10.90 | 0.03 } 11.06 | 10.93 1 Ee 2: OR ear 1, 241 0.94 | 11.69 0.04 | 11.06 | 11.73 cae Eee 1,128 1.01 |} 11.39 0.03 | 11.06 | 11.42 Cod liver oil (20 cc. per day) for 2 days. Off feed. Oat straw period. feet ene | 1,007 | 0.90 | 9.11 0.07 | 9.64 | 9.18 | Cod liver oil period (5 ce. per day). Ars 11-1835 fe5e 894} 0.84] 7.52 | 0.06| 9.64] 7.58 Ce AER DD. en te 846 | 0.75 | 6.42] 0.06| 8.65] 6.48 awe 2o—Mava 2) ee 804 0.65 5.22 0.04 5.99 | 5.26 Balance. +0.81 —1.36 —67 14 +1.70 +2.39 =O512 Lar —1.39 —0.07 —0.62 —A aie +0.35 +1.08 +0.13 —0.67 —0.36 +0.46 +2.06 =e +0.73 Cod Liver Oil Period.—On March 21, 1921, we began the admin- istration of 20 cc. of cod liver oil per day. for 2 days and so completely upset the appetite of the animal . that further administration of it was discontinued. At this This was continued Hart, Steenbock, and Hoppert 49 point, we again reverted to the basal ration, only beginning quan- titative collection of the excreta from the basal ration on April 4to11l. This week showed a positive calcium balance which may be interpreted as the residual effect of the cod liver oil given earlier, but the decrease in fecal calcium was not marked. After reestablishing the animal on the basal ration, we continued to give 5 cc. of cod liver oil daily as an emulsion. The effect of this in reference to calcium assimilation was gradual but positive. The fecal residue decreased somewhat, while the percentage of calcium in the feces was decreased from 0.90 to 0.65 per cent, giving a distinct positive calcium balance. However, the long cod liver oil administration gradually impaired the food intake of this animal and the experiment was discontinued. The records of this animal are in accord with those of No. 4. With the basal ration a negative calcium balance was established; the added orange juice did not consistently influence calcium assimilation; the cod liver oil did decrease the calcium assimilation in the feces with the production of a distinct and continued positive calcium balance. SUMMARY. 1. Experiments with goats, milking and dry, show that there is something in fresh green oats as compared with a dry oat straw which increases the amount of calcium assimilated. The oat hay, dried out of direct sunlight, but in a fairly well lighted attic, seemed to retain the properties of the fresh green oats that we were studying. 2. Orange juice administered in generous quantities (120 to 240 cc. per day) had no consistent effect on calcium assimilation. 3. Raw cabbage (1,000 gm. per day) or dried cabbage, had no influence on calcium assimilation. These data eliminate the antiscorbutic vitamine as a factor in calcium assimilation and conform with clinical experience in rickets. 4, Cod liver oil (5 to 10 ce. per day) consistently changed nega- tive calcium balances to positive balances. 5. Our limited data show that the same factor affecting cal- cium assimilation and resident in green oats and grasses is present in cod liver oil. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 1 50 bo Calcium Assimilation. I BIBLIOGRAPHY. . Steenbock, H., and Hart, E. B., J. Biol. Chem., 1918, xiv, 59. . Hart, E. B., McCollum, E. V., and Humphrey, G. C., Research Bull. 5, Wisconsin Exp. Station, 1908. . Hart, E. B., Steenbock, H., and Humphrey, G. C., Research Bull. 49, Wisconsin Exp. Station, 1920. . Forbes, E. B., and associates, Bull. 295, Ohio Exp. Station, 1916; Bull. 308, Ohio Exp. Station, 1917; Bull. 330, Ohio Exp. Station, 1918. . Meigs, E. B., Blatherwick, N. R., and Cary, C. A., J. Biol. Chem., 1919, xl, 469. . Hart, E. B., Steenbock, H., and Hoppert, C. iA Science, 1920, lii, 318. . Hess, A. F., J. Am. Med. Assn., 1921, Ixxvi, 693: . Hart, E. B., Miller, W. S., and McCollum, E. V., J. Biol. Chem., 1916, xxv, 239. Hart, E. B., and Steenbock, H., J. Biol. Chem., 1919, Xxxix, 209. McCrudden, F. H., J. Biol. Chem., 1911-12, x, 199. . Robb, E. F., Science, 1920, lii, 510. . McCollum, E. V., Simmonds, N., and Pitz, W., J. Biol. Chem., 1917, xxx, 13. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 1919, © Xxxvii, 187. A METHOD FOR THE DETERMINATION OF SUGAR IN NORMAL URINE. By STANLEY R. BENEDICT anp EMIL OSTERBERG. (From the W. A. Clark Special Research Fund and the Department of Chem- istry, Cornell University Medical College, New York.) (Received for publication, June 27, 1921.) In a previous paper! we described a procedure for the determi- nation of sugar in normal urines based upon preliminary removal of the nitrogenous urinary constituents by means of mercury nitrate in the presence of sodium bicarbonate. The sugar was then determined in the filtrate by the use of picric acid and alkali under definite conditions. This method has been of considerable service. Frequent checkings by comparative de- terminations made upon the same filtrates by the Allihn gravi- metric method which we have carried out have convinced us that the method is accurate for both unfermented and fermented urines. Duplicates by the colorimetric and the Allihn method practically always agree with 0.02 per cent on the sugar content of the urines. The mercuric nitrate method has, however, a serious drawback in the laborious technique involved, which has interfered with the general usefulness of the method. We have, therefore, constantly kept in mind the development of a procedure which should permit of the determination of small quantities of sugar in urine without the use of so troublesome a technique. The method described in the present paper meets this requirement. It has been in constant use in our laboratories and elsewhere for about 2 years, and we now feel quite sure of its accuracy and reliability under widely varying. conditions. While the present method retains the use of picric acid, it seems probable, as will be pointed out later, that the actual reaction takes place between the sugar and an unidentified derivative of picric acid. 1 Benedict, S. R., and Osterberg, E., J. Biol. Chem., 1918, xxxiv, 195. ol 52 Sugar in Normal Urine The chief interfering substances in urine as regards the deter- mination of sugar by picric acid (or indeed by other methods as well) are creatinine and creatine. In constructing the present method we have, therefore, had in mind primarily the elimination of interference by these compounds. The procedure developed has accomplished this end. It appears that interference by minute traces of other substances has been eliminated as well. It has been noted in the literature that acetone causes a partial fading of the color resulting from the interaction of creatinine and picric acid in the presence of sodium hydroxide. Preliminary experiments convinced us that if the reaction between picric acid and sugar could be made to go quantitatively in the presence of hydroxide instead of carbonate, which we have had to employ heretofore, it would be possible to utilize the acetone effect on the creatinine-picriec acid product, so that sugar could be deter- mined in the presence of large amounts of creatinine or creatine. We have found that if a low concentration of picric acid is used the reaction with sugar proceeds quantitatively in the presence of sodium hydroxide. It has further been found that by addition of suitable quantities of acetone to such a mixture it is possible to destroy completely the color due to creatinine or creatine, without a serious effect on the color developed through the action of the sugar. Before describing the exact technique of our method the following points may be mentioned as of some general interest. The impression which quite commonly prevails that picramic acid is the colored product resulting from the interaction of creatinine and picrate in alkaline solution is incorrect. We have isolated many grams of the product of the action of picrate and creatinine, and in physical properties and stability it is quite different from picramic acid. The product of the creatinine reaction is a bright, carmine powder, which is unstable even in the dry form when exposed at all to light: When so exposed it rapidly becomes lemon yellow in color, as though reoxidized to picric acid. Analyses of the product indicate that its empirical formula is quite close to, or identical with, that of picramic acid. Nevertheless, the products are, as noted, entirely different. The procedure proposed in the present paper serves also to demon- strate the difference between the reaction products of sugar and EE Te — S. R. Benedict and E. Osterberg 53 of creatinine with picric acid. In our process we add picric acid, alkali, and acetone to the solution, and heat. A color develops due to the acetone alone, which color fades rapidly so that in a minute or two the mixture again assumes the color of picric acid. If creatinine or creatine be present the color developed during the early stage of the heating is much intensified, so that in such a case the solution may become very intensely colored during the first minute or two of heating. Within about a minute and half, however, the color begins to fade just as does the color due to acetone alone, so that after 5 minutes of heating such solutions can scarcely be differentiated from a blank. Upon subsequent dilution these solutions have only a light yellow color as found in a blank. If sugar is present the color due to this develops more slowly than the color due to acetone or to creatinine, and does not fade or change with continued heating for at least 45 minutes. It is interesting to note that the reaction is apparently not between the sugar and the picric acid. This is indicated by the fact that during the first part of the heating in the presence of acetone, and before the sugar apparently begins to react with picric acid, it can be shown that this latter substance has been completely destroyed by the acetone. If sugar (or creatinine) be added after the heating with acetone has been carried on for a short time no color whatever develops, showing that the solution no longer contains any picric acid. If the solu- tion after heating with the acetone until the color has faded to light yellow be acidified with hydrochloric acid and warmed, the mixture turns deep red-brown in color, and a dark precipitate forms. We have been unable to identify this product. It would seem probable that in our method the sugar reacts with an intermediate reaction product between acetone and picric acid. Creatinine will not react with this product, but reacts quickly with the original picric acid to form a compound which is not stable in the presence of acetone. It is interesting that by this adjust- ment of conditions it is possible to determine accurately sugar in the presence of three or more times its weight of creatinine, when under ordinary conditions creatinine yields with picric acid about three to five times as much color as does an equal weight of glucose. Indeed the reaction now proposed appears to be perhaps the most specific reduction test available for 54 Sugar in Normal Urine sugar. Certainly when applied directly to urine the reaction vives more accurate results for sugar than does any other test. One may have three or four times as much creatinine or creatine present as of sugar without affecting the results. Larger amounts of creatinine may cause a slight lowering of the figure for the sugar. Hydrogen sulfide, which readily reduces picrie acid in alkaline solution under ordinary conditions may be present in relatively very large amounts (1 cc. or more of a saturated solu- tion) without affecting the results. We had hoped that the reaction could be applied directly to urine with satisfactory results. The figures obtained in this way are, however, slightly too high, as will be evident from an inspection of Table II. We have therefore adopted a procedure of preliminary shaking of the urine with purified bone-black, which we have found removes the trace of unknown interfering substance. The bone-black used is prepared as follows. 250 gm. of commercial bone- black? are treated with about 1.5 liters of dilute hydrochloric acid (1 volume of concentrated acid diluted with 4 volumes of water) and the mixture is boiled for about 30 minutes. The bone- black is now filtered off on a large Buchner funnel and washed with water (preferably hot) until the washings are neutral to litmus. The product is then dried and powdered. The highly absorbent animal charcoals on the market should be avoided in this connection. Commercial bone-black should be used, and the final product should be tested by shaking a portion (15 cc.) of a glucose solution containing 1 mg. of the sugar in 2 ce. of water with 1 gm. of the bone-black and determining the sugar in the filtrate. There should be no detectable absorption of the sugar. Following is the procedure for the determination of sugar in urine. The urine should be diluted so that the specific gravity does not exceed 10.25 to 10.30. 15 ec. of the urine are treated with about 1 gm. of bone-black (smaller quantities of both may be used if desired) and the mixture is shaken vigorously occasion- ally for a period of 5 to 10 minutes. The mixture is then filtered through a small dry filter into a dry flask or beaker. The volume 2We have employed commercial bone-black supplied by Eimer and Amend. Different samples supplied over a period of 2 years have all: yielded satisfactory results. The crude bone-black must not be used without purification. S. R. Benedict and E. Osterberg 55 of this filtrate to be used in the determination will depend upon its sugar content, but should never exceed 3 cc. Such a volume should be used as will contain about 1 mg. of sugar. Usually 1 to 2 ce.is the right amount. The proper volume of the urine filtrate is measured into a large test-tube which is graduated at 25 cc., and if the volume used was less than 3 cc. enough water is added to make the volume exactly 3 cc. Now add exactly 1 ce. of 0.6 per cent picric acid solution (best prepared from dry picric acid) and 0.5 ec. of 5 per cent sodium hydroxide solution. Just before the tube is ready to be placed in boiling water add 5 drops of 50 per cent acetone (this should be prepared fresh every day or two by diluting some pure acetone with an equal volume of water) taking care that the drops fall into the solution and not on the sides of the tube. Shake the tube gently to mix the contents, and place immediately in boiling water and leave for 12 to 15 minutes. The standard solution should be simulta- neously prepared by treating 3 cc. of pure glucose solution (con- taining 1 mg. of the sugar) exactly as described for the unknown solution and heating simultaneously. The pure glucose solution containing 1 mg. of the sugar in 3 cc. of solution will keep in- definitely if preserved with a little toluene. We have not been able to find a colored solution which matches the colored product of the reaction and which is permanent. In connection with the use of the method attention may be called to the following points. The quantity of the picric acid solution used must be measured with exactness, just as are the un- known and standard sugar solutions. Slight variations in the alkali are not so important. Adding the same number of drops (about 10) to each of the tubes from the same pipette is sufficient. The acetone solution should be added last, and the tubes placed in the water bath within about a minute afterwards. The diluted acetone undergoes some peculiar change on standing which makes old solutions yield somewhat irregular results. It is therefore best to prepare the acetone solution fresh every day or two. Each solution should be so added that it falls into the bottom of the tube, and does not hit the sides. Standard and unknown must correspond in sugar content within reasonable limits. For a 1 mg. standard satisfactory results can be obtained for an un- known solution containing between 0.75 and 1.75 mg. of sugar. 56 Sugar in Normal Urine With wider variations between unknown and standard results are not so good, particularly when the quantity of sugar is low. If less than 0.7 mg. of sugar is present in the unknown it is better to have a standard solution containing 0.5 mg. of sugar in 3 ce., and to dilute both unknown and standard to 12.5 instead of to 25 cc. TABLE I. Comparative Results for Sugar in Normal Urine by the Mercuric Nitrate- Picric Acid Method and by the New Procedure. Mercurie nitrate-picric acid method. New method. Sample No. SS a | Before After Before After | fermentation. fermentation. fermentation. fermentation. Dog urine. per cent ‘per cent per cent per cent 1 0.073 0.022 0.062 0.024 2 9.095 0.055 0.087 0.042 3 0.065 0.041 0.063 0.042 4 0.056 0.635 0.042 0.017 5 0.111 0.057 0.092 0.059 6 0.107 0.034 0.094 0.034 7 0.079 0.036 0.081 0.037 8 0.084 0.034 0.079 0.027 Human urine. 1 0.147 0.083 0.140 0.075 2 0.077 0.033 ~ 0.069 0.033 3 0.111 0.065 0.091 0.056 4 0.086 0.043 0.077 0.043 5 0.065 0.048 0.068 0.046 6 0.064 0.034 0.068 0.046 i 0.109 0.050 0.119 0.062 8 0.211 0.068 0.220 0.060 Where the method is intelligently carried out it is very simple, and yields results of a high degree of accuracy. We have studied the procedure in detail as regards recovery of added sugar, and the determination of sugar in urine with and without creatinine and creatine addition. The results have been wholly satisfactory. About fifty determinations have been made comparing results obtained by the new method with those given by the mercuric S. R. Benedict and E. Osterberg Be nitrate procedure. A few of these results are recorded in Table I. It will be noted that the new procedure gives consistently slightly lower figures than does the old, but upon the whole the. agreement is excellent between the two methods. The new method applied to the mercuric nitrate -filtrates gives no lower figures than when the bone-black is employed, showing that there is no nitrogenous constituent of the urine which interferes with the method. TABLE Il. Comparison of the Figures Obtained for the Sugar Content of Urine with and without Preliminary Treatment with Bone-Black. _ | Sugar. Sample No. | With bone-black. Without bone-black. Dog urine. per cent per cent 1 0.078 0.100 2 0.074 0.096 3 0.072 0.096 4 0.096 0.133 5 0.110 0.144 6 0.060 0.077 7 0.119 0.168 Human urine. 1 0.083 0.110 2 0.066 0.110 3 0.181 0.195 4 0.037 0.053 For clinical purposes the use of bone-black might be omitted if desired. Under such conditions figures will be obtained which are about 0.03 to 0.04 per cent too high. Table II shows some comparative figures with and without the use of bone-black. We are studying the question of the adaptation of the new procedure to the determination of sugar in tissues and in blood. On account of its high degree of specificity for sugar the new procedure may prove to be of advantage in these determinations. MARE ere SW he o' J it Aiea eto. bt ¥ ‘ | Tha* Mi “4 © @ae f ? ‘tol pi oil. . 44 . : air a) rer eg ir 7 Neu js ; t Mi i =. } * 4 ‘ht rs bi » omy ea, a wis 1 ae) Pig 3 Vicetia es CHEMICAL DEVELOPMENT OF THE OVARIES OF THE KING SALMON DURING THE SPAWNING MIGRATION. By CHARLES W. GREENE. (From the Department of Physiology and Pharmacology, Laboratory of Physiology, Unaversity of Missouri, Columbia.) (Received for publication, May 24, 1921.) Analytical data are presented here of the chemical develop- ment of the ovaries of the king salmon during the prolonged stage of its migration. Active development of the sex gonads takes place chiefly after the entrance of the salmon into fresh water. The gonad, growth, therefore, occurs in the absence of an intake of food; 7.e., at the expense of stored materials on hand at the beginning of the migration. The migration and the time on the spawning beds preliminary to the act of spawning takes 2 to 4 months or even more. The ovaries increase in weight, during the migration of the spring run on the Columbia River, from 200 to 300 gm. at the beginning to as much as 2,500 gm. at the end. Coincident with the growth of the ovary there is the expendi- ture of much dynamic energy in the migration. This dynamic energy is derived primarily from the potential energy of the excess of proteins and fats stored in the muscles in large amounts and to some extent in other organs and tissues. There is an absolute loss of mass of the muscle tissue during the migration amounting to some 45 per cent of the total. The muscle tissue left is much poorer in both proteins and fats. The fats alone drop from 20 per cent per unit mass at the beginning of the journey to less than 2 per cent at the spawning. The percentage of protein calculated on a protoplasmic basis, decreases from 20 per cent at the close of feeding to 14 per cent at the spawning. In other words, of 100 per cent of protein of muscle per unit mass of proto- plasm at the beginning of migration, 30 per cent has disappeared at the spawning. Computing the loss of protein and of fat in 59 60 Ovaries of Salmon during Spawning the tissue remaining at the spawning time and deducting that from the 45 per cent total loss during the migration, it appears that some 25 per cent or more of the muscular tissue as such has totally disappeared. These losses provide a source for a large amount of potential energy as well as for the materials that re- appear in the ovaries. The foundation for these facts has been set forth in a previous publication.!. They bear directly on the problem of food storage in the developing ovaries, a process that takes place coincident with the retrogressive changes in the muscle and other tissues of the salmon. The problems undertaken in this paper are: first, the mass change in the total growth of the ovary; second, the percentage composition of the ovarian protoplasm; third, the proportionate amounts of typical stored food materials found in the eggs of the salmonoid fishes at different stages of the spawning journey; and finally, the history of the phospholipins present in such rich quantities in all eggs. Historical. Few references have been found in the literature giving the complete analysis of the constituents of fish eggs and ovaries, and none of the ovaries of American fishes except that of Atwood’s analyses of food fishes in which he gives the composition of shad roe.2. Earlier preliminary reports of the work detailed in this paper represent the only data available on the chemical development of the ovaries of American fishes. In Europe, Miescher’s classic studies of the Rhine salmon‘ and the studies of Paton of the Scottish salmon® give data of the composition of the salmon roe as regards the store of simple and compound fats which they regard as the chief food source in the yolk. Buttenberg® investigated the chemistry of different caviars. He also gives two analyses of fresh sturgeon roe. The averages for fresh sturgeon roe are: water 62.3, ash 1.71, nitrogenous substances (proteins) 22.8, and fat 9.9 per cent. The ash is higher than in salmon roe, the proteins are lower, and the fat content is comparable to that of the eggs of fasting salmon obtained at the spawning beds. 1Greene, C. W., J. Biol. Chem., 1919, xxxix, 435. * Atwater, W. O., Rep. U.S. Com. Fish and Fisheries, 1888, 679. ¢Greene, C. W., J. Biol. Chem., 1918, xxxiii, p. xiii. ‘Miescher, F., Schweizerischer Fischerei-Ausstellung in Berlin, 1880. . ‘Paton, D. N., Rep. Fishery Bd. Scotland, 1898, iv, 63. 'Buttenberg, P., Z. Untersuch. Nahrungs- u. Genussmittel., 1904, vii, 233. a e i C. W. Greene 61 Rimini’ also analyzed preserved fish roe, including different types of sturgeon caviar. The sodium chloride ran from 1.2 to 11 per cent, corre- spondingly raising the mineral content and lowering the percentages of the organic fractions. The fats of caviar ran from 14 to 28 per cent, the latter in a sample containing only 16.5 per cent of water. There is in fact no common basis of comparison between the composition of caviar as against fresh roe. Tangl and Farkas studied the chemical changes in the developing trout eggs. They make the following comparisons: eee Developing eggs. \ VEY HS Oe ecg RE HE et Bae : 66.67) 66.08 65.6 SOMMS hy aeb et eee MeO SRSo|" 130592 34.94 PRE See 8 te ani. 5 ka BR ee eet ORS) 21758 7.98 Saponified fats. These analyses indicate that the eggs lost weight, water, and energy (cal- ories), but gained fat during incubation. The author compares the loss in trout, chicken, and spider eggs, showing the proportionate loss in each during the incubation. The trout loses the least in total weight, 5.6 per cent; the chicken egg, 17 per cent; and the spider egg, 26 per cent. Solberg? analyzed ‘‘ Dorsch’? roe (Norway), finding water 66.03 per cent, proteins 29.92 per cent, amino-acids 4.67 per cent, fat 2.26 per cent, and ash 2.16 per cent. Kojo?® gives the following analyses of the white and the yolk of the chicken’s egg. White. Yolk. VAUD STO 8 a aR A eg as ai Ae es a ME 87.71 49.73 STU Eye Ea Cn A 1 Re 12.29 50.27 ANS) Th oR ee Dee IE EIST RE Say cE fe eg A a Ye 0.4 1.44 INCOSE 585.62 Stadia FE ne AILS ee a ren dhelone 0.55 0.27 ISS EAE a IRE! 8 nd UN Seed nS ieio 2.49 The comparison between the salmon egg and the hen’s egg is in the yolk. The percentage of water and total solids checks closely but the yolk of the hen’s egg contains only a trifle more than half as much protein, three times as much lecithin, and some- what more neutral fat than is contained in the salmon egg. The 7Rimini, E., Z. Untersuch. Nahrungs- u. Genussmittel., 1904, vii, 232. °Tangl, F., and Farkas, K., Arch. ges. Physiol., 1904, civ, 624. 9Solberg, E., Z. Untersuch. Nahrungs- u. Genussmittel., 1908, xvi, 364. 10 Kojo, K., Z. physiol. Chem., 1911, Ixxv, 1. 62 Ovaries of Salmon during Spawning important point made in Kojo’s analyses is the demonstration of glucose; namely, 0.27 per cent in the yolk and 0.55 per cent in the white. Glucose is also constantly present in the salmon egg, but only to the extent of 0.09 per cent." Collecting Stations. The king salmon readily available for a study of this nature are the schools which migrate up the Columbia River to spawning beds in the cold waters of the Cascades and the Rocky Mountain rivers, and those which make similar migrations in the Sacramento River in California to spawning beds in the head waters of streams arising on the slopes of Mt. Shasta. Both series have been studied but the present report is based on a collection of salmon from the Columbia River basin made in the summer of 1908. In this series, seventeen salmon chosen from five different stations of the Columbia River and its tributaries have been analyzed in detail. The stations chosen are in order, Ilwaco at the mouth of the Columbia River; Warrendale in the Cascades, 135 miles up the Columbia; Seufert’s fishery on The Dalles of the Columbia, 210 miles up; and at Ontario on the Snake River, 700 miles above the mouth of the Columbia. The spawning fish were obtained from Cazadero, some 30 miles above Portland on the San Lorenzo River. The spawners were undoubtedly of the spring migration which entered the San Lorenzo River through the Willamette River. The other samples are from spring and summer migrants. Methods of Sampling and of Analysis. The method of selecting salmon types and of taking and pre- serving samples followed in this series of fish has already been described. The analytical procedures are the same as used on muscles and are presented in a previous paper.! Samples of ovaries and of free eggs are rather difficult to pulverize and require greater precaution in extractions, especially of the lecithins. The method in brief was as follows. The sample preserved in alcohol was transferred to a Gooch crucible and extracted in a Soxhlet modified to insure extraction at the boiling point of the’ 41 Greene, C. W., in press. C. W. Greene 63 solvent.’2 The sample was then extracted first in alcohol, then ether, then alcohol, and finally in ether, 6 to 8 hours each. The residue was finely pulverized after the first ether extraction. The insoluble residue was dried to constant weight at 105°C. and given six extractions in distilled water. The water-soluble was evapo- rated and dried to constant weight, then ashed and weighed. The alcohol-ether-water-insoluble was obtained by difference and the organic extractives also by difference. The alcohol and ether were driven off the alcohol-ether-soluble fraction and the lecithins thrown down with acid chloroform-water by the method of Koch. The emulsion of lecithins and fats was oxidized, and the phospho- rus determined by the official gravimetric method. The phospho- lipins were computed by the lecithin-phosphorus factor. Several analyses of phospholipins of critical samples were lost during the oxidations and the determinations do not reach our ideal of accuracy for the purposes. The organic extractives in the alcohol- ether-soluble fraction and from the alcohol-ether-insoluble frac- tions and the corresponding ashes were determined separately, but are combined in Table I. The waters were determined on a separate sample, and the neutral fats computed by difference, Growth of the Ovary. The growth of the ovaries in the salmon takes place chiefly during the migration. This fact is shown from the weights of the ovaries of fishes from the different stations in this series and in data obtained in the Sacramento River basin of California. The contrasts would be much more striking if sea-run fish could have been secured for the Columbia River series comparable to feeding salmon from Monterey Bay or Bolinus Bay, California. The weights of the ovaries of fish taken in July and August from the lower Columbia River fishing stations vary from 501 to 747 gm. which compare with similar weights at tide-water on the Sacramento. The weights from the spawning beds are from 775 to 2,243 gm. for ovaries, 2,596 for ripe ova and ovaries. This represents a maximum increase of 400 to 500 per cent and is an enormous storage of food substances. Fish of the size indicative of approaching maturity, 8 to 10 kilos body weight, 2Greene, C. W., J. Biol. 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The smallest ovaries collected at the mouth of the Columbia and at tide-water on the Sacramento River have reached 500 gm. and more in weight. The development of the ovaries of salmon at the time of begin- ning the migration varies with the season. The August salmon of the mouth of the Columbia at Ilwaco are more mature as regards the sex gonads than are those from the same station that begin the migration in the spring. It is the spring run that spawn in the San Lorenzo River and apparently that migrate up the Snake River. The August fish spawn in the White Salmon River and other streams of the Cascade Mountains. The observations seem to justify the conclusion that from 90 to 95 per cent of the total weight of the mature ovaries of the king salmon is acquired during the spawning migration; 7e., while the salmon is in fresh water and not taking food. Itis a unique case of synthesis and growth of the tissues of the gonads while all other organs are decreasing by a process analogous to tissue starvation. The Chemistry of the Salmon Ovaries. The analytical data presented in this series follows the quantita- tive distribution of the proteins, lipins, phospholipins, extractives, inorganic salts, and water in the ovaries and in ripe ova. The distribution of the nitrogen and of the. various phosphorus frac- tions has been followed, but the data $e‘not now presented. The data have been paleniared on the three bases used in pre- senting results of the analyses of muscle. These are: first, per cent of the wet sample as collected; second, per cent of the dry or water-free sample; and third, per cent of the fat-free sample. The stored food material in the egg is out of all proportion to the active protoplasm of the germ cell or cells. This is obvious when one remembers that the protoplasm of the ovum itself is micro- scopic whereas the total egg is 6 to 7mm. indiameter. Neither of the above classifications therefore can be considered as repre- sentative of the composition of the ovarian protoplasm. The data represent only the composition of stored food material, however computed. C. W. Greene 67 The Mature Ova. Two of the four female fish sampled at the spawning grounds contained mature ova separated from the ovarian tissue and float- ing free in the ovarian fluid in the body cavity. The composition of these two samples of mature eggs is as follows in terms of per cent of the total sample: water 57.68 and 58.19 per cent; total solids 42.32 and 41.91 per cent; protein 26.56 and 27.01 per cent; phospholipins 1.9 (probably low) and 3.6 per cent; neutral fats 11.70 and 9.15 per cent; total organic extractives 1.43 and 1.41 per cent; and inorganic ash 0.68 and 0.62 per cent. This composition is characterized by its low content of salts and extractives and high content of protein. The protein is at least. 70 per cent higher than in the yolk of the hen’s egg. On the other hand the lecithins and neutral fats are less than half the amount stored in the yolk of the hen’s egg. In the preincuba- tion stage the organic extractives are low, much lower than in the muscle of the same fish. The Developing Ovaries. The ovaries contain ovarian tissue and developing ovules, the ovules with their stored food yolk forming an ever increasing pro- portion of the mass. This fact doubtless accounts for the slighter variations of composition as development progresses. The changes during development are presented under subtopics describing the various group constituents. The Water of the Ovaries. The water of the developing ovary is greatest in the mature ovary. The averages for each station are 54.08, 54.21, 52.05, 54.36, and 58.27 per cent. The increase at the spawning grounds coincides with the decreasing concentration of the blood observed in the king salmon of the Sacramento River.“ It cannot be accounted for on the ground of loss of fat since the percentage of water on a fat-free basis is higher in spawning salmon. 13Greene, C. W., Bull. U. S. Bureau Fisheries, 1904, xxiv. 445. 68 Ovaries of Salmon during Spawning The Inorganic Salts. The total inorganic salts vary slightly but are a trifle higher in mature ovaries, averaging 0.57 per cent in relatively young ovaries in salmon from the mouth of the Columbia in comparison with 0.72 per cent from mature ovaries and in ripe ova. This variation is of little significance except that it gives one more confirmation of the fact that the saline content of salmon tissues is independent of the saline content of the water in which the salmon lives. The Proteins. The proteins of the developing ovaries differ little in amount from the content of ripe eggs. The station averages are: 25.62, 25.99, 25.71, 27.08, 26.10 per cent for the five stations, a variation of only 1.46 per cent. The highest protein observed was at the Ontario station and the lowest on the spawning grounds. The average for ripe eggs is 26.7 per cent which is nearly 11 per cent greater than the 16 per cent of protein present in the yolk of the hen’s egg. The proteins form no inconsiderable part of the stored food of the salmon eggs. The Organic Extractives. The organic extractives of the ovaries are never great in amount. The averages are 1.55, 1.62, 1.63, 1.39, and 1.45 per cent from the five stations. The carbohydrates constitute 0.09 per cent of the extractives and the remainder is assumed to be nitrogenous extractives. The average amount decreases slightly but not greater than the variation in duplicate determination. On the whole the ovarian extractives seem to obey the law of constant level of saturation as given for the nitrogenous extractives of muscle.! The maintenance of the high level of protein during the growth in mass of the ovaries can only occur by the synthesis of proteins from the amino-acids of the blood and ovarian waters. The degree of saturation of the muscle waters by organic extractives amounts to 3.8 per cent, the ovarian waters are saturated only ‘Greene, C. W., The glucose of salmon tissues, in press. '6Greene, C. H., J. Biol. Chem., 1919, xxxix, 457. C. W. Greene 69 to 2.5 per cent. While the muscle protein is being hydrolyzed ‘ protein is in process of synthesis in the developing ovaries. The loss of muscle proteins during the migration is more than enough to account for the gain in the proteins stored in the ovaries, a point supported by Paton in 1898. However, my view of the process is that the ovarian proteins are resynthesized from the hydrolytic products arising from the muscle proteins. The Neutral Fats. The neutral fats represent the chief store of energy in the sal- mon’s egg, just as the‘‘egg oils’”’ serve that function in the yolk of the hen’s egg. The youngest ovaries carry the greatest amount of fats. The amounts at the three lower river fishing stations are 14.2, 18.5, and 17.4 per cent. The individual extremes are 13.3 and 18.2 per cent. Ontario salmon average 13.6 per cent with 10.3 and 15.1 per cent for the extremes. The aver- age from the spawning grounds is 10.6 per cent. The lowest fat found was 9.1 per cent in the ripe eggs of Salmon 1,299. The series shows a great variation in stored fats of the ovaries, just as was found for the muscles. But the spawning fish have a decidedly lower content of fat. This fact argues for the depend- ence of the ovarian fats on the general fat stores and on the hpoly- tic processes of the body of the salmon. The ovaries are not de- pleted of their fats to the low level of the fats of muscle, but do yield from 30 to 40 per cent of the store of fats present in the younger stages. . As with the proteins so with the egg oils, the storage takes place from the lipins liberated from supplies in the muscles, liver, connective tissues, etc. The reversible action of the lipases is adequate to account both for the storage of ovarian fats and for their percentage decrease as the fats are depleted from the body by oxidation during the migration. The Phospholipins. The phospholipins represent the most complex food product stored in the developing ovaries. In the yolk of the hen’s egg the phospholipins are present to the extent of 11 per cent and more along with about twice that amount of neutral fats. But in the 70 Ovaries of Salmon during Spawning salmon ovary the amount is far less, from 2 to 3.6 per cent of the mature eggs. The phospholipins of the ovaries from the three stations on the lower Columbia amount to 4.1, 4, and 3.4 per cent. At Ontario the average has dropped to 2.9 per cent, although one ovary contained asmuch as 4 per cent of phospholipins. At the spawning grounds the average phospholipin content is 2.6 per cent, with 1.9 and 3.6 per cent as the extremes. The determinations given for the phospholipins represent the greatest variations in the data. The variations bear no direct relation to the neutral fats present in individual ovaries. How- ever, like the neutral fats, the phospholipins decrease in amount with approaching maturity of the ova. The 4 per cent average for an ovary of 500 gm. weight drops to an average of 2.6 per cent for mature ovaries weighing 700 to 2,500 gm. The significance of this decrease and variability is somewhat a matter of conjecture. It was argued by Paton! that the lecithins of the muscle are not adequate to provide the store found in the mature ovaries of the Scottish salmon, but loss of muscle phos- phorus was equal to gain of ovarian lecithin phosphorus. The muscles of the king salmon have an average of 1.18 per cent of phospholipins when they begin the migration.! This drops to 0.44 per cent for spawning salmon. This loss, considering the large mass of the muscle tissue, is much greater than the gain to the developing ovaries. However, in view of the more recent advances in knowledge of lecithin metabolism" it is not to be assumed that the muscle lecithins are transported unchanged from muscle to ovary. It is more plausible to assume that these phospholipins are resynthesized in the ovary from available fats and phosphorus rests coming to the organs in the blood. It would be indeed difficult to prove this contention by direct tests on the salmon itself, a fact the writer keenly realizes. On the hole it seems evident that the phospholipins play a less dominant rdle, at least they form a far less proportionate” amount of the stored food materials, in the salmon ovaries and eggs than they play in the case of the hen’s egg. The fats, too, ‘6Paton, D. N., Rep. Fishery Bd. Scotland, 1898, iv, 143. ‘7Bloor, W. R., J. Biol. Chem., 1916, xxv, 577. This paper contains a full reference list. C. W. Greene (js form a smaller percentage of the food store of the egg. The dependence of the salmon on fats and fatty bodies for energy during the migration is reflected in the partial depletion of both neutral fats and phospholipins in the mature salmon eggs. The proteins are present in unusual amount and doubtless play a leading part in the nutrition of the developing embryo. A CHEMICAL STUDY OF CERTAIN PACIFIC COAST FISHES.* By D. B. DILL. (From the Pacific Coast Fish Investigations, Food Research Laboratory, Bureau of Chemistry, San Diego.) (Received for publication, July 5, 1921.) INTRODUCTION. In reviewing the literature on the chemical composition of fish, it is noticed that very few species have been the subject of extended investigations. The first extensive record of analyses of fish is that of Atwater published in 1888 (1). His notable in- vestigations covered fifty-two American species, chiefly of Atlan- tic source but including some from the Pacific. Of his analyses, twenty-seven were based on one sample, thirteen on two samples, and twelve on from three to seven samples. Varying numbers of individuals entered into the composition of each sample. He examined nine species at different seasons of the year, the results in the case of three of these indicating seasonal variation. His figures on salmon also clearly indicate a loss in fat content during the spawning migration. A second noteworthy investigation was conducted by Clark and Almy (2). They made a series of analyses of Atlantic Coast fishes during 1915 in order to obtain additional data on the sea- sonal variation both in the proximate composition and in the physical and chemical fat constants. Most of their samples were of a composite nature based on an average of three or four fish. '* The work presented here was begun in May, 1918, under the direction of Dr. E. D. Clark and continued under Mr. A. W. Hansen until July, 1919. Since that time until June, 1920, it was carried on under the supervision of Dr. C. L. Alsberg, Chief of the Bureau. The analyses were begun at Stan- ford University, continued in the laboratory of the National Canners Asso- ciation at San Pedro, California, and completed at the San Diego branch of the Food Research Laboratory under the supervision of Dr. L. H. Almy. 73 74 Chemical Study of Pacifie Coast Fishes One series was completed in the spring of 1915 and the other in the fall of the same year. They analyzed the shad before and after spawning. They also analyzed four composite samples of three fish each from the same school of weakfish. To summarize their findings briefly, they found considerable variation in many species (bluefish, butterfish, carp sucker, and weakfish) from spring to fall; the shad loses greatly in fat content during spawn- ing; weakfish of the same school, caught at the same time, may show a wide variation in fat content (four composite samples of three fish each showed fat percentages of 1.35, 2.47, 4.88, and 8.03). Many studies have been made of the changing composition of the king salmon during their fasting migration to the spawn- ing grounds. In a recent publication, Greene (3) shows that spawning king salmon which have been in fresh water without eating at an estimated time of from 4 to 5 months have a fat content of 2.63 per cent and a protein content of 13.71 per cent contrasted with 16.43 per cent fat and 16.97 per cent protein in tide-water fish. Greene concludes that the king salmon stores up both fat and protein for its spawning migration. It has frequently been pointed out that there is a relation between sea temperature and fat content of fishes. Murray and Hjort (4) stated that the fat contents of the sprat which abounds off the coast of Norway increase during summer when there is a rise in sea temperature, while both decrease toward the end of the year; it is concluded that the growth of the fish must be in- fluenced by the prevailing temperatures in different waters. Perhaps the most detailed investigation of the proximate composition of a selected species has been made by Johnstone (5). He selected the herring as a subject and his analyses were made of fish caught during the years 1914, 1916, and 1917. His samples were always composite ones usually based on ten fish, © five males and five females. Most of the fish he analyzed were mature although he does not usually make mention of the size in connection with the anaylsis. However, he has made a care- ful record of the degree of development of the gonads in every case and hence has obtained an exact record of the relation of the chemical composition of both sexes to the sexual cycle. In this report Johnstone remarks: DB. Dall 75 “Tn all races of herrings the maturation of the gonads is accompanied by an increase of fat in the flesh. For some time before the fish spawns (but after the major part of increase in the mass of the gonads has taken place) the fat contents decrease, and after spawning this decrease becomes very rapid. Between the time of spawning and the time at which matu- ration of the gonads begins again, the fat content of the flesh is at its minimum value.’ Procedure. The primary objects of our investigation were to study the seasonal variation in several species of fish and to obtain data from which food values can be ascertained. Many of the earlier analyses, based on but a few fish and made at only two or three widely separated dates, have been omitted and this report has been confined to two subjects; the variation in composition of individual fish, and the seasonal variation in the composition of the mackerel and mackerel-like fishes. The fish used for analysis were obtained from the wholesale fish markets, from boats, or from canneries. From five to ten fish were usually selected for analysis. When the fish were small, all of the flesh was removed from one side of each, scales and bones were separated, and the remaining edible portion ground three times in a meat chopper. A sufficient quantity of the sam- ple thus obtained was kept in a stoppered flask until the analysis was completed. In the case of larger-fish, sections 1 inch or more in thickness were taken from one side of each fish and pre- pared for analysis as above. Ether extract and ash were determined by the official meth- ods.!. Total solids were found by weighing out 10 gm. of the sample into a lead dish which contained a small quantity of ignited sand. After drying to constant weight in a water bath oven at 98-99°, the same sample was used for the determination of ether extract. Total nitrogen was determined by the Kjeldahl and Gunning method. The results in all cases represent the average of two closely agreeing determinations. Variation in the Composition of Individual Fish. Evidence that analyses based on but a few fish may lead to incorrect conclusions has been found in studies of several species. 1 Bull. 107 (revised), Bureau of Chemistry. 76 Chemical Study of Pacific Coast Fishes Those which have been investigated and which show marked and often erratic difference in composition are yellow fin tuna, blue fin tuna, sable-fish, barracuda, mackerel, and sardine. Data on the last named species will be presented in another paper. In September, 1920, six yellow fin tuna (Germo macropterus) each weighing between 25 and 30 pounds were found to have fat percentages of 5.84, 5.71, 4.88, 2.65, 1.82, and 0.20. These six fish were selected at random from a large catch. The average fat content is 3.52 per cent although the first three fish average 5.48 per cent and the last three 1.56 per cent, a striking variation. In September, 1918, five individual blue fin tuna showed fat percentages of 7.95, 8.71, 9.39, 10.04, and 10.76. These fish were of about the same size and were from the same boat-load. Al- though the variation in fat content is not striking yet the figures are valuable in showing a certain individual range in composition. It would seem to be a natural inference that immature fish should show a lower fat content than mature fish of the same species. Some data in agreement with this inference has been found. Thus an immature sable-fish (Anaplopoma affinis) caught April 4, 1918, had a fat content of 0.07 per cent while a mature sable-fish, caught 2 months later, had 14.87 per cent fat. These results are in accordance with the expectation and may be characteristic of this species. That this condition does not hold for all species was found when large, medium, and small barracuda were analyzed. Three analyses were made of this species (Sphyrena argentea). The first sample, consisting of ten fish caught off the coast of Lower California in December, 1918, weighing about 5 pounds apiece, had 1.85 per cent fat. The second sample, from ten fish caught at the same time and place and weighing about 2 pounds apiece, contained 6.45 per cent fat. The third, based on fish caught off San Pedro a month later, averaging 0.5 pound apiece had a fat content of 1.51 per cent. On several occasions a number of individual mackerel from the same catch have been analyzed. These analyses, as shown in Table I, are arranged in several chronological series and the fish in each series are in an increasing order as regards weight. In the series of October 25, 1918, variation from a minimum of 0.85 . | iD: Be Dall 77 per cent fat to a maximum of 7.88 per cent was found although the fish were. .of nearly the same size. About a month later a sample from six males had about the same composition as TABLE I, Variation in the Composition of Individual Mackerel (Scomber japonicus). v4 ° ODN OA rR WN | No. analyzed. eo ee ee ee el oon a Descrip Males. Females. tion. Male spent. Female spent. Male. Female. “ce Male. Average weight. Date. Composition of the edible portion. Solids. per cent 1918/25.92 1918|26.20 1918)31.71 1918)24.35) 1918 24.89 1919)28.16 1919|26.83 1919 28.03 1919 28.18 1919)32.65 1919 30.36 1919/30.69 1919/28.57| : 1919|32.57 1919 32.31 1919)34.21| 1919 32.96 1919|29.33 1919/37.67)15.45 1919)37.25)1: 1919)39.84/18.93 1919)41.05 20.32 1919)30.65| 8.41 1919/33.00 10.32! 1919 33.99 12.35) 1919137.48 117.78 1919 30.31) 7.77 Ash. 3 a sa} cent a ee ee ee ee ae 20 Ww or He bo 41 OT 45 47 47 44 .32 (NX6.25). gen. Total nitro- Protein per cent 3.76|23.50 3.89 24.31 3.66 22.88 3.69 23.06 3.74/23 .37 3.77|23.56 3.65)22.81 3.69 23.06 3.49 21.81 3.43/21 .44 40 ol 56 43 23 00 OT 33 28 .20 04 04 .23 ol 30 34 .O2 23.06 .50)21.87 22.38 21.88 5\22.19 22.88 22.06 21.68 20.81 20.44 5\20.31 3.27|20.44 one based on six females, both having less than 1 per cent Five mackerel analyzed August 7, 1919, had fat contents increasing in much the same order as their increasing weights. fat. 78 Chemical Study of Pacific Coast Fishes The same relation was found November 18, 1919, with the ex- ception of Simple 18 which had a proportionately low fat content. On December 10, 1919, Nos. 23 to 26 showed close relationships between size and fat contents, while No. 27, the largest of the series, had the lowest fat content. . The data given in Table I indicate that fat content may vary widely in mackerel of the same catch and of about the same size (Nos. 1 to 3); sex appears to bear little if any relation to proxi- mate chemical composition; although the percentage of fat generally increases with the size, Nos. 3, 18, and 27 are marked exceptions to this rule; finally, mackerel of the same size may have different fat contents at corresponding times of consecu- tive seasons (Nos. 1 to 5 contrasted with Nos. 13 to 17). As some of the mackerel analyzed in August, 1919, were spent (Nos. 6 to 9) and some were full (Nos. 10 to 12), it is evident that the spawning time of this species is midsummer. Seasonal Variation in the Composition of the Mackerel-Like Fishes. Of the various mackerel-like fishes, the species which is avail- able throughout the year and most easily secured in the whole- sale markets is the California mackerel (Scomber japonicus). Accordingly, most of the analyses of mackerel-like fishes have been confined to this species. As has been shown, there is con- siderable variation in individual mackerel and hence the samples analyzed, with the exception of a few of the earlier ones, are based on not less than five fish. It will be seen by referring to Table II that in many cases five small and five large fish of the same catch were segregated and the two composite samples analyzed separately. This was not always possible because the larger sized fish often could not be obtained. With the excep- tion of Samples 1, 4, 5, and 6, obtained from Monterey Bay, all of the samples were taken off southern California and brought in to San Pedro. Samples 4, 5, and 6 were analyzed by Mr. A. W. Hansen of the Bureau of Chemistry. It will be seen that of the samples averaging more than 1 kilo in weight, the two highest in fat content, Nos. 12 and 17, were in October, 1919, and February, 1920; the next highest was in August, 1919 (No. 9); while the lowest, No. 21, was in May, 1920. It has already been pointed out from the data shown in i i mi te! le 4 ee ee a. ee a — + haces Dabs Dil 79 Table I that small mackerel analyzed individually in the fall of 1918 at San Pedro were much lower in fat content than those of the succeeding fall. Samples 2 and 3 contrasted with Nos. 10 and 11 (Table II) further illustrate this fact. Considering mackerel caught during 1918-20 of from 375 to 680 gm. in weight, it is noticed that the sample of lowest fat content, No. 7, was TABLE II. Analyses of the California Mackerel (Scomber japonicus). | Composition of the edible portion. 3 | | ) No. 2 Description. ee Date < 3 s 5 | aie nen a ail 225! aX 2 = |25/ ¢ | $8] Be Z 2) iS) < BR AY | per per per per per gm. | cent cent cent cent cent dhe 400 |June 3, 1918|27.17| 3.62] 1.27] 3.50/21.87 By | 93) 523 |Oct. 25, 1918/27.94) 3.33] 1.38] 3.77/23.57 3 || 12 511 |Nov. 17, 1918)24.62) 0.63} 1 39] 3.71/23.19 A 2 Ai aelinat 1, 1918/28.61| 4.64) 1.46) 3.58/22.37 5| 4 503 | « 25, 1918/25.60) 1.51) 1.41] 3.56/22.25 6) 5 348 |Dec. 19, 1918|28.08|] 4.69] 1.25} 3.53 22.06 7| 10 | Filling. | 500 |May 12, 1919/23.38] 0.28) 1.47| 3.55/22.19 8 5 | 1 full; 4 spent.| 644 |Aug. 7, 1919/28.77| 4.91) 1.48) 3.61|22.56 9{ 9) All full. 22h la 11, 1919/30.69| 7.50) 1.31) 3.50/21.87 10 | 10 | Spent. 680 Oct. 9, 1919)33.8310.16) 1.35) 3.68/23.00 tel) 5 ee | 450 | « 20, 1919/31.69} 8.49) 1.37) 3.61/22.56 12) 5 cs 11,350 | “ 20, 1919/38.80/18.12) 1.31) 3.27/20.44 13 | 10 | Virgin. | 894 |Nov. 18, 1919|34.58|12.41) 1.28) 3.44/21.50 14 5 | Filling. 724 |Dec. 10, 1919/33.09)11.32] 1.35} 3.45|21.56 15 | 10 ss 375 |Jan. 19, 1920|27.59| 4.70] 1.41] 3.57|22.31 16) 5 SS 551 |Feb. 12, 1920|26.52] 3.65) 1.50) 3.61/22.56 LN eet) es 1,290 | “ 12, 1920/38.63/18.08] 1.24) 3.16)19.75 LSS g 510 |Apr. 7, 1920/27.46] 4.38] 1.41] 3.60)22.50 19; 5 en 760 | “ 7, 1920/30.53| 7.49) 1.42) 3.52/22.00 20) 5 Hy 666 |May 29, 1920/25.39) 1.02} 1.43) 3.72/23.25 PALA gs a 1,370 | “ 29, 1920/26.97| 3.45] 1.38) 3.59|22.44 caught in May, 1919, while No. 3, caught in November, 1918, and No. 20, caught in May, 1920 were nearly as low in percentage of fat. The two of maximum fat content are Nos. 10 and 11, caught in October, 1919. Nos. 15, 16, and 18, caught in January, February, and April, 1920, respectively, had an intermediate fat content. 80 Chemical Study of Pacific Coast Fishes We are therefore justified in concluding that the mackerel undergoes a seasonal variation in composition; that large mackerel are generally fatter than small mackerel of the same school, and that the variation in ‘one season may not be paralleled by the next season’s variation. The period of maximum fat content during 1919 followed the spawning season, while the period of minimum fat content preceded the spawning season. In the previous season the percentage of fat appeared to be at a minimum in the late fall after the spawning season. Several analyses have been made of the other common mack- erel-like fishes of southern California. With the exception of the bonita, none of the species whose analyses are shown are commonly taken in a spawning condition on this coast. It is generally considered that the blue fin tuna and albacore of the size usually taken, from 16 to 25 pounds in weight, are immature fish. For this reason the only analyses of large tuna or albacore, Nos. 4 and 5, should be considered in a class by themselves. They are quite likely mature spent fish. Such fish are not commonly taken off southern California until late in the fall. Whether such species migrate great distances, or whether they seek deep water for spawning, is as yet a mystery. The analyses of the fish shown in Table III are too limited in number to give satisfactory information as to seasonal varia- tion. It is apparent that considerable fat is stored in the flesh of these species during June, July, August, and September. In considering these analyses, it should be borne in mind that they are based on flesh only. The skin and heads frequently have a relatively high fat content when the amount of fat in the flesh is small. Thus, on one occasion it was found that a sample of skins, heads, and bones of albacore, even after the loss of some of their fat by cooking, had a fat content of 11.16 per cent while the per cent of fat in the flesh of the same cooked fish was 4.91. Before cooking the flesh had a fat content of 5.22 per cent (Sample 1). CONCLUSIONS. Large variations in the composition of individual fish of several species (yellow fin tuna, blue fin tuna, sable fish, barracuda, and mackerel) have been found. 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Tks 6I16T ‘03, “49O G OF ay.4) a10, 0) 6, 0 € 6 el ele oe 5 a 3 6F' 8e 616T *Q “ydagq G GZ-0Z Gursiere 16) 0 8.61.6) 6) onenate . 5 5 3 os’ Te 6161 VAG Ane G GZ-0Z eee eee eee eee ee . 5 a % GO'ZS |616T ‘61 eung G (O50 eee NS Se eee O Deir ** (pBunjpojp OWA) BLODVATV quao wad “41 “SPrI[Og ‘V0 Tak albeeuiers Bernd yun gy a1ayon PY ay) fo saysrg snoriw A fo sashjouy ‘TIl ATAVL ‘ON THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 1 82 Chemical Study of Pacifie Coast Fishes The variation in the composition of the mackerel during one season was. not paralleled by the next season’s variation. The spawning season of the mackerel was found to be mid- summer. No evidence of a connection between decreasing fat content and the approach of the spawning season was found in the mackerel. On the contrary, the spawning season in 1919 appeared to come during a time of increasing fat content. No evidence of a connection between the sex and the prox- imate chemical composition of the mackerel was found. With some exceptions, the mackerel and the mackerel-like fishes were found to have an increasing fat content through the summer and early fall. BIBLIOGRAPHY. 1. Atwater, W. O., The chemical composition and nutritive values of food fishes and aquatic invertebrates, Rep. Com. Fish and Fisheries, 1888, 679 ff. . Clark, E. D., and Almy, L. H., A chemical study of food fishes. The analysis of twenty common food fishes with especial reference to a seasonal variation in composition, J. Biol. Chem., 1918, xxxili, 483. 3. Greene, C. W., Biochemical changes in the muscle ti ssue of king salmon during the fast of spawning migration, J. Biol. Chem., 1919, xxxix, 435. 4. Murray, J., and Hjort, J., The depths of the ocean. A general account of the modern science of oceanography based largely on the scientific researches of the Norwegian steamer Michael Sars in the north Atlan- tic, London, 1912, 761-762. 5. Johnstone, J., Rep. Lancashire Sea-Fisheries Lab., 1917. bo —-e THE POTASSIUM CONTENT OF NORMAL AND SOME PATHOLOGICAL HUMAN BLOODS.* By VICTOR C. MYERS anp JAMES J. SHORT. (From the Laboratory of Pathological Chemistry, New York Post-Graduate Medical School and Hospital, New York.) (Received for publication, June 21, 1921.) Our interest in the potassium content of human blood was aroused some time ago by the observation of Smillie! that poisoning may result from the administration of potassium salts to certain nephritic patients. This was noted in a case of nephritis and later confirmed in experimental uranium nephritis. Smillie states: “In human beings, potassium chlorid, in doses which have no effect on normal individuals, will cause acute poisoning in individuals with chronic nephritis. “This acute poisoning occurs because the salt, whichis normally readily absorbed and very rapidly excreted, in nephritis is readily absorbed and not excreted, thus reaching a concentration in the blood whichis injurious.”’ Owing to the fact that in the human species potassium is an important constituent of the corpuscles and present in them ina much higher concentration than in the plasma, it is quite necessary that this factor should always be taken into account in any estima- tion of the potassium content of human blood. The composition of the blood of different species of animals was carefully considered by Abderhalden,? and Table I is recalculated from his data. As will be noted the potassium content of whole blood in the horse, pig, and rabbit, and also man (Tables IT and ITI) stand in marked *A preliminary report of these observations was made before the Society for Experimental Biology and Medicine, November 17, 1920, Myers, V. C., and Short, J. J., Proc. Soc. Exp. Biol. and Med., 1920-21, XVlli, 72. 1 Smillie, W. G., Arch. Int. Med., 1915, xvi, 330. 2 Abderhalden, E., Z. physiol. Chem., 1898, xxv, 106. 83 . TABLE I. S4 ~ Potassium Content of Blood 1 | Potassium Content of the Blood of Different Species of Animals.* Potassium. Species. pn nESe T ire | Whole blood. Serum. | mg. per 100 ce, : | ROTO los 0, echnical eee 227 22 By De ofbin ator oe cies eee ee eeeee Ee 123 21 12 | MAME ME ce eas cite oe 192 23 Rabbit. sass a: oe eis orn eee 175 22 Peer etcidi c's “is ese aie yore eee 34 22 12110 | eRe Seer cere Ae rus coer 34 PAL BIBRE Lec onk te ee ek rei ie Sco 34 22 BPA RE eae te SERN AT BRAG & 34 Dil Goats 2 i255 ee SRP ae 33 21 Catrasacicatioethine sR eR eee betas 22 22 1 Tot ne [eet eee aati eRe gk a eee 22 22 ae UO OOHRS Aan OORT ae 21 19 * Observations recalculated from Abderhalden.? TABLE II. Potassium Content of Human Blood.* Potassium. Case. Diagnosis. Age. Sex. ca ; Mole | Serum | mg. per 100 cc. 1 Normal. 25 rot 174 31 2 § 30 fo) 161 33 3 Cholera. 26 9 185 4 “ 55 fof 225 5 ss 20 fo) 166 43 6 oe 71 fof 203 ie 7 as ZS rofl 194 62 8 | Diabetes. 34 rot 170 oe 9 | Chronic edema with albu- 39 rofl 116 21. minuria. 10 | Anasareca without albumi- 42 fot 190 63 nuria. 11 | Normal dog. 37 29 * Observations recalculated from Schmidt. V. C. Myers and J. J. Short 85 contrast with that found in such carnivorous animals as the cat and dog, where the findings for whole blood and serum are almost identical. We have long possessed data on the potassium content of human blood as the result of the analyses carried out by Schmidt? in 1850. The data in Table II are recalculated from his analyses. The rather high findings for potassium in the whole blood of the cholera cases can probably be explained on the basis of the con- centration of the blood found in this condition, but this would scarcely explain the high figures for the serum in two of these cases and one of the cases of nephritis. As has been pointed out by Macallum,* Schmidt’s figures for the potassium content of the normal blood are likewise rather high. Despite the fact that figures for the potassium of human blood were given by Schmidt in 1850, comparatively few data have since been recorded in the literature. A few analyses were reported by Macallum in 1917. He gives the normal potassium content of human blood plasma as 19 to 21 mg. per 100 ce., which, as will be noted, is about 60 per cent lower than the figures given by Schmidt for normal individuals. Regarding pathological cases Macallum states that his results obtained for the plasma in Bright’s disease are quite incomplete but those for puerperal eclampsia are far enough advanced to furnish some points of interest. His po- tassium figures (four cases) are not given in absolute amount, but in relation to the sodium, taking the latter’as 100. In these cases the ratio of the potassium to the sodium was increased two to four times. Since severe eclamptics generally suffer from quite pronounced acidosis, and sometimes from salt retention, it would seem logical to expect greater fluctuation in the sodium than in the potassium. On this account it is difficult to draw con- clusions from changes in the ratio between the elements. A few figures for the potassium content of blood have also been given by Drushel,® Greenwald,* Clausen,’ Kramer,’ and Kramer 3 Schmidt, C., Charakteristik der epidemischen Cholera gegeniiber verwandten Transsudationsanamalien, Leipsic and Mitau, 1850. 4Macallum, A. B., Tr. College Phys. Philadelphia, 1917, xxxix, series 3, 286. 5 Drushel, W. A., Am. J. Sc., 1908, xxvi, 555. § Greenwald, I., J. Pharmacol. and Exp.-Therap., 1918, xi, 281; J. Biol. Chem., 1919, xxxvili, 439. 7 Clausen, S. W., J. Biol. Chem., 1918, xxxvi, 479. 8 Kramer, B., J. Biol. Chem., 1920, xli, 263. 86 Potassium Content of Blood and Tisdall.2 Drushel obtained 166 mg. of potassium per 100 cc. in defibrinated pig’s blood, 50 mg. in sheep’s blood, 20 mg. in the serum of dog’s blood, and 16 mg. in dog’s lymph. Green- wald found the potassium content of dog’s blood (serum and whole blood) to vary from 14 to 27 mg. per 100 cc., while Clausen found the potassium content of human whole blood (pathological cases) to vary from 143 to 290 mg. per 100 ce. and in plasma from 53 to 90 mg. In his first paper, Kramer gives the normal potassium content of human serum as varying between 16 to 22 mg., while in a more recent paper Kramer and Tisdall state that the potassium content of the serum of both normal children and adults is singu- larly constant, the maximum variation being from 18 to 21 mg. per 100 cc. Ina series of fifteen miscellaneous pathological condi- tions in children they report figures ranging from 23 to 70 mg. per 100 cc. of serum. The analyses reported by Kramer and Tisdall were made by a direct precipitation method without ashing. It seems rather difficult to understand why miscellaneous pathological conditions should show such a marked difference from the normal as is the case with the results of Clausen, and Kramer and Tisdall. In 1909 Myers" carried out a study of the potassium content of the spinal fluid of insane patients. The interesting observa- tion was made that the potassium content of spinal fluid in- creased very rapidly after death, as high figures being obtained one-half hour post mortem as at any later time. It was stated at that time that: Soe the potassium content of the cerebrospinal fluid during life corresponds very closely to the amount of potassium in the blood serum, while after death the quantity of potassium in the cerebrospinal fluid agrees more nearly with that of the whole blood.” The figures obtained for fifteen living cases varied from 14 to 28 mg. and averaged 22 mg. potassium per 100 cc. of spinal fluid. For the twenty-two specimens of spinal fluid obtained after death the findings ranging from 57 to 105 mg. with an average of 83 mg. per 100 ce. ® Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 339. 10 Myers, V. C., J. Biol. Chem., 1909, vi, 115. V. C. Myers and J. J. Short 87 The estimations of Abderhalden and Schmidt previously re- ferred to were carried out with the chloroplatinate method, but in most recent observations, the cobalti-nitrite method has been used. Drushel, Clausen, Kramer, and Kramer and Tisdall have des- eribed adaptations of this method to blood analysis. In the last mentioned method the potassium is precipitated directly from the serum without ashing. We have used the cobalti-nitrite method of Drushel,®!! essentially as it was employed by one of us for the spinal fluid more than 12 years ago. Our experience would lead us to believe that more satisfactory results are obtained on serum than on plasma, owing to the fact that hemolysis is more likely to take place when sodium citrate or ammonium oxalate have been added. Since human whole blood contains about ten times as much potassium as the serum it is essential to guard against the passage of any potassium from the cells. In most of our analyses the corpuscles have been separated from the serum by a double centrifuging about 2 hours after the specimens have been taken. Furthermore, when there has been any question about hemolysis, the serum has been subjected to spectroscopic examination for absorption bands of oxyhemoglobin. Slight hemolysis, however, does not necessarily greatly increase the potassium content of the serum. The potassium estimations on whole blood have been made on blood to which (potassium-free) sodium citrate was added as the anticoagulant. The analytical procedures we have employed are described below. Method. 5 ee. of blood serum or 1 cc. of whole blood are treated in a 125 cc. plati- num evaporating dish with 5 cc. of a 1 to 10 sulfuric-nitric acid mixture and evaporated down rapidly in a hood over a low Bunsen burner flame. When the mixture reaches a small volume and begins to char, nitric acid is added, a few drops at a time, and the heating continued until it foams up. At this point the dish is covered with small ashless filter paper to prevent loss of material from spattering during the final steps in the oxi- dation. After the material has become nearly dry from the low heat, the flame is turned on full until all the organic material, including the filter paper, is burned up, and the substance is completely ashed. The ash is now dissolved in 8 to 5 ce. of hot water, 1 to 2 cc. of glacial acetic acid added, and then transferred to a 50 cc. beaker with the aid of 11 Drushel, W. A., Am. J. Sc., 1907, xxiv, 433. Potassium Content of Blood 88 — a we 5 ih) a ‘ustprordyqioddAy ‘sty -uydeu [eyysieyur oruoryD Fee Os] O87] 12 Se Ieee abe 2°80 *pelp ‘stqyydou osnytp ommo1yD | Ze |8ze'0 | O'Sz | FIZ | FST | GOF | LZ'L | O'ST *porp ‘suo1s -[nauoo ‘suydou oTuoryy €91'0 | O'9L | SIT COpeu lac ae InGePl ‘poaorduir ‘sis -ov1x0} ‘sty11ydou poouvapy | T¢ |OST'O | 9's |_E¢ L0¢ | 0'F | 9°ST “perp > » 3 9% |09T'0 | T'S1 | SOT | 2°6 | FHF | OF | 21T ‘SIpUYdou [BIZQSIOJUT OIUOIYYD | 6% |ZFT'0 | 2°S | 86 G9 Le. ee Sr *BTULOIN ‘sqyuydou osnytp —,, OG JOST 0: 0O°ST"| COT. |-e°6- |-F6G [eore “ere ‘stpuydou [erp y4si0zut OTUOIYD | EF |6ST'0 | 8 | Zor | O'L | Teg | te | 2°¢T “perp ‘sryyuydou asnyip otuoryD | 2g |1Z1'0 | ¢'¢ | SF Ge levee v's »” ” PZ » 9°12 *[BULLON € 61 “99 |qua0 sad 99 001 | °29 OOI | *92 OT | ‘92 GOT suoyjiu|j;uaa sad Jad ‘fit | sad fiw \ sad *Bu| sad “bu - ‘oulu "'N ‘poe 09 “19801 | BOI ong, | ‘[OBN “SISOUDBIP [BOTUT[D SU “OLY “BING *S[[99 poy [@10], *sosA[VUB POO[g 1044 “SpI[OS | PTOUM I SFI | +82 69 +36 ¥6 0G 96 91 ¥6 6I ST 88 L zo | (OT HL | 81 G6 | SI 69% | SI scr | #1 sad Dre | aad Du a p 00 I q “UIn ag “UINISSBIO dT rey ioe oyapoy le) “XOQ G& 0g 20 G LP 9G 9G 0G 9G c& ‘any ‘spoojg ununyy yooibojoyjvg awmog pup powion fo JUa}UoY WnIssp}og 2Y,], HT WTA vib 89 . V. C. Myers and J. J. Short ‘poze roidde AT[NJ sea 4OT2 oy} WoIJ UNAES Jo UOTZeIedS OYVIPOUMUA! YSOUTTL BY} IOJ APISS9D0M 9Y} OLOJoq WOYL} suowuedg | ‘YJwop 1o4Je SoyNUIUA GF JNO JAvoY WOLJ UOYV} POOT| , “(%) etueue snororusd ‘siyrtydAg “BTULOUB SNOTOIUIIg ‘porp ‘eurod oTyeqvIq, “BISdULe OW *yU90SOT -eauood ‘eruoumnoud rieqoT ‘snsvydose Jo vulouloies) ” ” “uoIsue}IOdAy [BI}UeSsa "BOIS -vue ‘uolesuaduodap OBIPIBD ‘OSVOSIP IB[NOSBAOTPIBD ‘eruoue AIEpuodeS ‘ 3.20 20.00 ATER es cc sttet 85.5 2 20.65 Onda sioilil 19.09 IVES) S =< Caecum 93.7 2 21.31 0.60 3.11 19.09 DEC uate tr 86.5 2 32.25 12.06 3.03 18.93 1920 TaN ns ers 100.0 3 31.68 10.74 3357 19.81 Reb! Menccttes 104.0 5 32.12 11.66 3.09 19.31 MATS Lao 104.0 10 30.17 9.49 3.12 19.15 AT. Ai ies oH ord 105.0 8 26.20 4.50 Bieri 20.06 WMiaive- ce. ashe 113.0 5) 26.20 3.69 3.40 2125 UME se aoe 100.0 3 27.67 5.07 3.45 21.56 * All June analyses were made June 3, as no sardines of this size could be obtained later in the month. TABLE III. Variation in the Composition of Individual Sardines. Composition of the edible portion. ae the Average ay Solids pee Ash. eae RY gm. per cent per cent per cent per cent per cent 1 Male. 162 20.92 | 0.52 1.59 3.08 18.94 2 ef 170 20.89 |} 0.24 1.63 3.09 19.31 3 oe 165 19.07 | 0.13 1.80 2.87 17.94 4 Female. 184 25.92 4.66 1.46 3.23 20.19 5 - 172 21.43 |} 0.39 1.68 3.07 19.19 6 f 162 22.09 0.80 1.47 3.01 19.44 7 160 PANRTA! 0.09 1.59 Bye | 20.06 8 ee 162, | 24.14] 1.80 | 1.57 | 73.36% |eeateue 9 re 152 20.12 0.10 1.66 3.07 19.19 10 re 162 | 20:31] 0.13 i o9 2.99 18.69 ANCTA&O ooo earn 165 21.66 | 0.89 1.60 3.10 19.37 Dr By Ait a6 occasions ten relatively mature and ten relatively immature fish of each sex were segregated and analyzed. These results are shown in Tables VI, VII, and VIII, respectively. : TABLE IV. Monthly Average Composition of ‘‘Large Ovals.” Average Composition of the edible portion. Month. weight per No. of fish. analyses. Total | Protein nitrogen. | (NX 6.25). Ether extract. Solids. | Average weight 140 to 260 gm. 1919 | gm. per cent per cent per cent per cent ACN Sa) Pea eae 229 4 37.71 19.20 2.79 17.44 IRs Oe aeons 186 2 33.34 14.02 2.88 18.00 WVilarsertx efits ae 225 3 35.41 15.83 2.89 18.06 ANDI. Gis x obese 188 2 21.08 0.75 3.03 18.93 Met Seyeerac, Ste. « 191 5 23.27 2.74 3.11 19.07 SUIT e een eo 552 meee! 188 4 40.30 | 21.38 288i) 18%00 1920 Ue Senate ae 170 4 37.91 17.89 3.04 19.00 elie s.f 6.2%: 186 il7/ 38.34 18.88 2.89 18.06 IN Detter’ a.s.2 6% 186 20 36.53 17.04 2.89 18.06 /A\ Oe ee ee 169 20 21202 6.67 3.08 19.25 Wityerces 2 2} 180 10 20.0 4.00 3.32 20.75 [Wise eee 170 + 25.22 2.75 3.38 21713 * All June analyses were made June 3, as no sardines of this size could be obtained later in the month. DISCUSSION. There may be a striking variation in the composition of indi- vidual sardines as willbe seenfromastudy of Table HI. Although the average fat content of the ten sardines analyzed was 0.89 per cent, eight of the ten fish had less than this amount while one, No. 8, had twice the average amount and another, No. 4, had over five times the average. Even when a composite sample is based on as many as ten fish, it has been found that erratic results may be obtained. There is usually a fairly regular increase in fat content with increasing size of the sardine. This increase is illustrated in the analyses of March 29 as shown in Table V which are typical 98 Chemical Study of Sardinia cerulea of fourteen similar series made from January to June, 1920. How- ever, the analyses of April 8, shown in the same table, are quite variable due, no doubt, to the fact that there was great variation in individuals at this time just as there was during the same period of the previous season, as indicated in Table III. It is apparent from the analyses of small sardines as presented in Table I that there is considerable although sometimes inconsis- tent, seasonal variation in fat content. The minimum fat per- centages in “quarters”? was found in October, 1918, and January and February, 1919. Yet the period of maximum fat content extended from July, 1919 to September, 1919. Unfortunately, sardines of these two groups were not available later in the year and so it is impossible to say whether there is always to be expected a sudden decrease in the fat content of small sardines in the fall. It does seem certain that their maximum fat content is reached during the late summer. It has been shown in a previous paper that mackerel were fatter in the fall of 1919 than in the previous fall. It was also true, as indicated in Tables I and IV, that large sardines were fatter in the season of 1919-20 than in the previous season. ‘This is true month for month in the case of ‘‘small ovals”’ and with the appar- ent exception of January, 1919, is true of ‘large ovals” as well. This one exception may be explained by the high average weight of the January, 1919, samples, 225 gm. contrasted with 170 gm., the average weight of the January, 1920, samples. Fairly consistent variations in the fat contents of large sardines are revealed by a study of Tables I and IV. The fat contents were at a maximum from December to February or March and appeared to drop. off rapidly in April of both 1919 and 1920. Evidence was found that this sudden dropping off may have a different explanation from the obvious one. Thus in Table V are shown the analyses of sardines of three different dates during the transition period from high to low fat content. The fish analyzed . on March 29 and April 12 were relatively fat while those ana- lyzed at an intermediate date, April 8, were thin. This strongly indicates that there is much difference in schools at this season of the year and suggests that the fish of March 29 and April 12 were from the same or similar schools while those of April 8 were from a school of thin fish. SR — iy DYE; Dill 99 The opinion has been expressed that the decrease in fat content of the large sardine is closely related to the development of the gonads and the approach of the spawning season. The fact that the time of low fat content and the spawning period are nearly coincident lends support to this view. Considerable attention has TABLE V. Variation in the Composition of Sardines from Different Schools. Description of fish analyzed. Composition of the edible portion. Weight of Date. weight per| divided | 4zeaz° | souds. | Ether | Total, | Protein, weight. 1920 gm. cm. per cent per cent per cent per cent Miaire 298 52h. 80 0.037 19.2 25.56 3.20 | 3.00 21.00 os DL ee 100 0.036 | 21.0 25.92 4.20 | 3.24 20.25 ee OS) acs 120 0.031 21.8 29.93 9.11 BEI 19.81 |) ere 140 0.058 | 22.4 | 34.038 14.09 | 2.96 18.50 cemee Der 33. 160 02052, || "23.7 34.57 14.78 | 2.93 18.31 OEE eae 180 0.054 | 24.0 35.07 | 15.89 | 2.938 18.31 i =, ae 200 0.044 | 25.1 39.27 | 15.06] 2.98 18.63 4, ic) late 220 0.048 | 25.9 35-25 et: 2.94 18.37 CPL) ae ee 240 0.048 | 26.9 33.43 | 13.39 | 2.99 18.69 ANOi3 “oj5 ee 100 0.028 | 21.1 23.30 2.13 | 3.16 19.75 a eae 120 0.025 | 22.0 23.63 3.220) | o&.08 19.25 he Cee 140 0.033 | 23.1 24.04 3.38 | 3.11 19.44 SON Ree 160 0.043 | 24.0 23-009 2.65 | - 3.14 19.63 ean ORE ,* 180 0.027 | 25.4 21.66 0.88 | 3.11 19.44 U2 ants wee 200 0.038 | 26.2 23.31 3.42 | 3.00 18.75 HS 0 Dey 220 0.042 | 27.0 27.09 6.25 | 3.10 19.37 1S 0) Aaa 100 0.049 2085 29.19 7.89 SHLS 19.69 PAL Es: 160 0.082 | 23.7 31.84 10.95 | 3.09 19.31 Average of 100 and 160 gm. samples from each group. Mian 29))../. 130 0.044 | 22.3 30.24 9.49 | 3.08 19.25 Apr Ost s. 130 0.035, ||- 22.5 23 .42 2.39 3.15 19.69 seen BUDE. 130 0.065 | 22.1 30.51 9:42 | 3.12 19.50 been devoted to this question of the relation of percentage of fat to degree of sexual development. In Table VI are shown the analyses of ovaries and testes of the sardine in March, April, and June, 1920. It is evident that there is no great change in the composition of the reproductive organs as spawning season 100 Chemical Study of Sardinia cerulea approaches. The only consistent change is a decreasing fat percentage both in the ovaries and testes. Table VII shows the changing weight of the gonads as the season advances. The maximum was reached during April or May at which time spent fish began to appear; at this point the relative TABLE VI. Composition of the Gonads of Sardines. No. | Sample. Dates? | Solids. | Péter | pie ee 1920 per cent per cent per cent per cent per cent 1 Testes. Mar. 25 20.84 | 2.59 1.28 2.80 17.50 2 Ovaries. BB 27.66 | 4.54 1.08 3.24 PY. PAs 3 Testes. Apr.) 12 PAV AT eek) IPA 74 Ao)L 18.19 4 Ovaries. Se a2 29.59 | 3.76 le l7/ 3.66 22.88 5 Testes. June 3 20.52 1.07 25 2.97 18.56 6 Ovaries. a 3 PS tee) ||, Pp iP? 0.98 3.00 18.75 | TABLE VII. Ratio of Weight of Gonads to Total Body Weight. No. Date. Ratio for ‘‘small ovals.’’| Ratio for “‘large ovals.”’ 1920 1 Jan. 20 0.015 0.018 2 Feb. 2 0.017 0.023 3 By Pb 0.022 0.029 + Mar. 3 0.025 0.028 5 aay 119) 0.036 0.035 6 25 Palle) 0.047 0.045 7 ee!) 0.035 0.051 8 Apr. 8 0.029 0.037 9 ae 1 0.049 0.082 10 se mi) 0.047 0.067 11 i 2h 0.026 0.057 12 May 5 0.046 0.072 13 seal, 0.023 0.046 14 June 3 0.009 0.052 weight of gonads began to decrease as the proportion of spent fish increased. It was found that “small ovals” tend to spawn before the larger sardines. This tendency is shown by the data of June 3 in Table VII. Gonads composed only 0.009 of the. total weight of ‘‘small ovals” and 0.052 of the weight of “large | 24 —a- oA ave Gp Re 2 DB Dil 101 ovals.” Of the fish analyzed on June 3, 28 out of 30 “small ovals”? were spent while only 12 out of 40 “large ovals” were spent. The maximum weight of gonads found was on April 12 (Table VII, No. 9) and was 0.082 of the body weight. The analy- sis of this particular sample is shown in Table V (160 gm. sample of April 12)—the fat content was relatively high, 10.95 per cent, showing that a considerable growth of the reproductive organs can take place without drawing to any great extent on the reserve store of fat. TABLE VIII. Composition of Sardines of Different Degrees of Sexual Development. Desecration of fish analyzed. Composition of the edible portion. Weight of Date. freight per) divided, | HYG | sotias. | AMS. | astigun, ASU. weight. 1920 gm. cm. per cent per cent per cent per cent Feb. 23 (a).. 154 0.040 | 23.3 38.89 | .18.18 | 2.92 18.25 areca (0)... 151 0.017 | 23.0 37.86 | 18.13 | 2.93 18.31 “2 (oe 149 0.025 | 23.2 37.42 | 17.21 | 2.96 18.50 = 28 (d):. 151 0.016 | 23.3 36.73 | 16.27} 3.03 18.94 Apr. 2*(a).. 130 0.082 34.22 | 14.35 | 2.88 18.00 paren (D) 1. 124 0.052 32073: lI 358')) 202k 18225 ae aca). . 136 0.044 33.70 | 14.07 |. 2.92 | 18.25 peeeen a). 131 0.029 Sl52-), Ul 46 |) 290) 18.13 | (a) represents the values found with relatively mature males; (6), relatively immature males; (c), relatively mature females; and (d), rela- tively immature females. This same question was approached from another angle. Ad- vantage was taken of the fact that the reproductive organs are of different degrees of maturity in sardines of the same size from the same school. On two occasions fifty or more fish of approxi- mately the same weight were selected and from these ten relatively mature and ten relatively immature fish of each sex were segre- gated and analyzed. The results, shown in Table VIII, are remarkable for their close similarity. The only consistent vari- ation is that the samples of highest fat content are from the more mature fish. This strongly indicates that in sardines the develop- 102 Chemical Study of Sardinia cerulea ment of the reproductive organs is not closely related to the decreasing fat content although both take place at about the same time. In some cases the relation between the fat content of fish and the sea temperature has been established. It has been shown, for example, that sea temperature is a factor in the variable fat content of the herring of European waters. It has been found that the minimum sea temperature at the surface off southern California is reached in January or February while the maximum is reached in July or August with a range of 6 or 7°C.! Since the fat content of large sardines is near the maximum in January or February, the possibility of more than a remote relationship between these factors in the case of the sardine must be slight. Aside from the determination of constituents shown in the tables, several determinations of glycogen in sardines have been made. Three of these five determinations yielded 0.50, 0.17, and 0.22 per cent of glycogen in the edible portion. The other two showed no trace of glycogen but, as these two samples were from fish which had been out of the water for several hours while the other three were based on live fish, the absence of glycogen may have been due to hydrolysis. On one occasion several hundred grams of flesh from live sardines were rapidly ground and mixed with a strong potassium hydroxide solution. Instead of hydrolysis of the glycogen and determination of the sugar in the usual way, the glycogen was separated and purified by repeated reprecipitation with alcohol. In this way several decigrams of an amorphous brown powder were obtained which gave an opales- cent aqueous solution which produced a red color with iodine. CONCLUSIONS. Considerable variation in the composition of individual sardines of the same size and from the same school may occur. Small sardines were found to have a maximum fat content in the summer months. With some exceptions, other factors being the same, the fat content of sardines increases with the increasing size of the fish. ‘McEwen, G. F., Summary and interpretation of the hydrographic observations, made by the Scripps Institution for Biological Research of the University of California, 1908 to 1915, Berkeley, 1916. De Bs, bull 103 Marked variations in the fat content of a seasonal nature were found in large sardines, the percentage of fat dropping from a maximum in December or earlier to a minimum in May. This variation was more extreme in 1918-19 than in the following season. Great difference in the fat content of sardines of the same size from different schools was observed. The migration of schools may be related to the sudden decrease in fat content that takes place in April of each season. No evidence that the growth of the reproductive organs draws to any great extent on the reserve store of fat was derived. The relation between the percentage of fat in the sardine and the sea temperature, if any, is remote. There are appreciable percentages of glycogen in the flesh of the sardine. THE ESTIMATION OF CREATININE IN THE PRESENCE OF ACETONE AND DIACETIC ACID. By NATHAN F. BLAU. (From the Department of Chemistry, Cornell University Medical College, New York, and the Russell Sage Institute of Pathology, in affiliation with The Second Medical Division of Bellevue Hospital, New York.) (Received for publication, June 27, 1921.) Since the adaptation by Folin of the Jaffé reaction to the quanti- tative estimation of creatinine in urine, the procedure has been made the basis of much important research bearing on the prob- lem of the metabolism of creatine and creatinine. The accuracy and reliability of the method have only been questioned in the ease of pathological urines, more particularly in specimens con- taining acetone and diacetic acid. Many of the experiments dealing with this question that were described in the literature offer inconclusive and contradictory evidence as to the exact mode and extent of interferences of the acetone bodies with the deter- mination of creatinine by the Folin method. Klercker! stated that large amounts of acetone cause a rapid fading of the creati- nine color, while van Hoogenhuyze and Verploegh? claimed that the color is at first too dark, but soon fades to a point where it gives correct creatinine reading. Similar results were reported by Rose’ with regard to the effect of diacetic acid on the color of creatinine and alkaline picrate. His attention was arrested by the assertion of Krause! that diacetic acid causes a measurable increase in color, leading ultimately to too low creatine values. From his own investigation, Rose draws the conclusion that diacetic acid, if present in amounts not exceeding 0.25 per cent, 1 af Klercker, K. O., Biochem. Z., 1907, ii, 45. ? van Hoogenhuyze, C. J. C., and Verploegh, H., Z. physiol. Chem., 1908, Ivii, 161. 3 Rose, W. C., J. Biol. Chem., 1912, xii, 73. 4Krause, R. A., Quart. J. Exp. Physiol., 1910, iii, 289. 105 106 Creatinine in Acetone and Diacetie Acid gives an increase in color, which, however, soon fades and offers therefore no serious obstacle to the correct estimation of creatinine. In concentrations larger than 0.25 per cent, the color due to diace- tic acid is more persistent and hence objectionable. He further- more states that acetone in all concentrations is without influence on the reading. The conclusions with regard to the effect of diacetic acid are far from incontestable, for the reason that Rose used the ethyl ester of diacetic acid and not the acid itself in his experiments. As was shown by Graham and Poulton,® and we can confirm their find- ings, these two substances do not behave in an analogous manner with respect to their effect on the color reaction of creatinine. The same criticism may be applied to the work of Wolf and Osterberg,® who found that 1.0 per cent of ethyl acetoacetate caused no marked change in the creatinine reading. In contrast to the results of Rose stand the reported findings of Greenwald’ that acetone in amounts greater than 0.5 per cent gives at first an undue increase in color, which soon drops below its normal value on account of fading. In the presence of diacetic acid the creatinine color is always too light. Greenwald esti- mated the amount of diacetic acid added to urine by the intensity of the ferric chloride reaction and made no attempt to determine the minimum amount of diacetic acid which may possibly interfere. Graham and Poulton® detail careful experiments in which they have added graded quantities of acetone, ethyl acetoacetate, and sodium acetoacetate to urine and studied the effect of these sub- stances on the creatinine color. They concluded that acetone, if less than 0.2 per cent, causes no error; with larger amounts the color is lighter than normal. Ethyl acetoacetate in small amounts: (0.1 to 0.7 per cent) gives a lighter color; larger amounts give a darker color which increases on standing. Sodium acetoacetate, even in small amounts, causes a lighter color; with larger amounts the color is still lighter and fades rapidly on standing. Conse- quent upon these findings, they have investigated the question of alleged creatinuria in carbohydrate starvation. As a result of ® Graham, G., and Poulton, E. P., Proc. Roy. Soc. London, Series B, 1913-14, Ixxxvii, 205. ® Wolf, C. G. L., and Osterberg, E., Am. J. Physiol., 1911, xxviii, 71. 7 Greenwald, I., J. Biol. Chem., 1913, xiv, 87. Nathan F. Blau AOZ their study, they have come to the conclusion that the figures for urine creatine in carbohydrate starvation reported by previous observers merely represented discrepancies between correct and incorrect creatinine determinations, due to the respective absence and presence of diacetic acid in the urine after and before heating of the specimen for the conversion of creatine to creatinine. In view of these conflicting statements with regard to the effect of acetone and diacetic acid on the estimation of creatinine and their bearing on the problem of creatine metabolism, as pointed out by Graham and Poulton, it was deemed worth while to subject the matter to a critical experimental examination. We have studied the effect of added acetone, diacetic acid, and ethyl acetoacetate on the color reaction of creatinine in pure solution and in urine. Effect of Acetone on the Color Reaction of Creatinine in Pure Solution.—A stock solution of pure creatinine was made by dis- solving 1.0 gm. in 1,000 ce. of 0.1 N HCl (1.0 cc. — 1.0 mg. of creati- nine), 2 and 5 ce. portions, respectively, were placed in a 500 ce. volumetric flask, acetone and water were added to a volume of 10 cc. The solution was then treated with 15 cc. of saturated picric acid and 5 ce. of 10 per cent sodium hydroxide solution, allowed to stand for 8 to 10 minutes, and after diluting to the mark, compared with a standard creatinine solution similarly treated with picric acid and alkali. The results are given in Table I. Effect of Added Acetone on the Determination of Creatinine in Urine-—A quantity of urine was boiled in an open flask for about 10 minutes to remove all traces of acetone and diacetic acid. It was then cooled to room temperature and its creatinine content estimated, using a 0.5 N KeCr.0; solution as astandard. Different portions of the sample were mixed with varying amounts of acetone and each analyzed for creatinine (Table II). Tables III and IV give the results of experiments with ethyl acetoacetate. Experiments with Diacetic Acid and Acetone.—A pure solution of diacetic acid was prepared by the hydrolysis of its ethyl ester according to the directions of Ceresole.2 Just before using, the solution was placed in a tall cylinder and a current of acetone-free § Ceresole, M., Ber. chem. Ges., 1882, xv, 1871. 108 Creatinine in Acetone and Diacetie Acid air slowly drawn through it for about 45 minutes to remove traces of acetone. that have been formed from the spontaneous decompo- sition of the acid. A measured volume of it was then transferred to a Kjeldahl flask, diluted with several volumes of water, acidi- fied with H.SO,, and distilled into water. The acetone caught in the TABLE I. Effect of Acetone on the Color Reaction of Creatinine in Alkaline Picrate. Creatinine taken. Acetone added to 10 ce. Color. Oranuene Error. mg. mq. per cent mm. mg. per cent 2.0 0.0 0.0 10.0 2.0 0.0 2.0 20.0 0.20 10.0 2.0 0.0 2.0 40.0 0.40 10.1 1.98 — 1.0 2.0 50.0 0.50 10.1 1.98 — 1.0 2.0 60.0 0.60 10.3 1.94 — 3.0 2.0 70.0 0.70 10.6 1.88 — 6.0 2.0 80.0 0.80 10.8 1.85 — 7.5 2.0 100.0 1.00 11-5 1.74 —13.0 2.0 150.0 1.50 128 1.55 —22.0 7 Ug 200.0 2.0 Loe 1.27 36.5 5.0 0.0 0.0 10.0 5.0 0.0 5.0 40.0 0.40 10.0 5:0 0.0 5.0 50.0 0.50 10.0 5.0 0.0 5.0 60.0 0.60 10.1 4.95 — 1.0 5.0 70.0 0.70 10.3 4.85 — 3.0 5.0 90.0 0.90 10.5 4.7 — 4.8 5.0 120.0 1.20 11.0 4.54 — 9.2 5.0 180.0 1.8 IE ¢/ 4.27 —14.6 5.0 500.0 5.0 14.0 3.57 —28.6 5.07 800.0 8.0 33.0 1.51 —65.8 * Standard, 2.0 mg. of pure creatinine set at 10 mm. 7 Standard, 5.0 mg. of pure creatinine set at 10 mm. receiver was then determined by the Messinger titration. From the figures obtained, the percentage of diacetic acid in the solution was calculated. Definite quantities of this solution were added to known amounts of creatinine in pure solution, and in urine, and their effect noted. The results are summarized in Tables V and VI. ee ee ee ee ee cee AE vit TLS ee oe — Nathan F. Blau 109 DISCUSSION. The data in the tables show consistently that, barring a few minor exceptions, our results are in substantial agreement with those obtained by Graham and Poulton. Acetone in large amounts undoubtedly fades the creatinine color from the very out- set. We are inclined to set the upper limit of allowable acetone con- centration at 0.50 per cent. The figures presented by Graham and TABLE II. Effect of Acetone on the Determination of Creatinine in Urine.* Acetone added to 10 ce. of urine. Color. Sreatinine ia 200 Error. mg. per cent mm. mg. per cent 0.0 0.0 Wall 105.19 0.0 40.0 0.4 Uadh 105.19 0.0 50.0 0.5 Cath 105.19 0.0 60.0 0.6 7.8 103.87 — 1.19 70.0 i 7.9 102.53 — 2.52 90.0 0.9 8.4 96.42 — 8.35 120.0 Wes 8.8 92.04 —12.46 150.0 5) Del 89.01 —15.38 200.0 2.0 9.8 §2.65 —21.42 0.0 0.0 8.1 100.00 0.0 30.0 0.3 So 100.00 0.0 50.0 0.5 ona 98.78 — 1.22 60.0 0.6 8.1 100.00 0.0 70.0 0.7 8.4 97.62 — 2.38 80.0 0.8 8.6 94.18 — 5.82 90.0 0.9 8.7 93.0 — 7.00 100.0 10 9.0 90.0 —10.00 150.0 15) 3355) 60.0 —40.00 300.0 3.0 16.0 50.62 —49.38 * Standard, 0.5 N potassium dichromate. All tests, giving higher read- ing, fade on standing. Poulton show that 0.17 per cent acetone had no effect on the reading, but 1.0 per cent gave a markedly lighter color. The gap between 0.17 and 1.0 per cent is obviously a wide one. We can say that the fading effect of acetone does not begin markedly to show itself until a concentration of 0.70 per cent has been reached. With no amount of acetone did we observe an increase in color (Tables I and II). 110 Creatinine in Acetone and Diacetie Acid Creatinine taken. or or 0 .0 5.0 5.0 5.0 .0 0 or on TABLE III. Effect of Ethyl Acetoacetate on Color of Pure Creatinine.* Ester added to 10 ce. mg. 0.0 5.0 10.0 20.0 50.0 60.0 100.¢ * Standard, 5 mg. of pure creatinine set at 10 mm. per cent 0.0 0.05 0.10 0.20 0.50 0.60 1.00 Color. soiree © mm. mg. 10.0 5.0 10.0 5.0 10.1 4.95 10.3 4.85 9.8 5.10 9.0 IS 535) 3.0" GrZo { Did not fade after standing for 10 minutes. Ester added to 10 ce. mg. 0.0 10.0 20.0 50.0 60.0 100.0 200.0 per cent 0.0 TABLE IV. Effect of Ethyl Acetoacetate on Determination of Creatinine in Urine.* Color. mm. mg. 66.39 64.80 64.28 65.32 67.57 90.07 * Standard, 0.5 N potassium dichromate. + No fading, but slight increase of color after 10 minutes. Creatinine taken. on ar ae toreeee or or or Or or or TABLE V. Effect of Diacetic Acid on Color of Pure Creatinine in Alkaline Picrate.* Diacetic acid : 10 ce. wre 3 conorae * oo — Ww p~ per cent Creatinine in 100 ec. of urine. 73.63 Error. per cent 0.00 0.00 — 1.00 — 3.00 + 2.00 +11.00T +25. 00T Error. per cent 0.00 — 2.39 on = I2GE + Cem +10.907 +35.56T Error. per cent 0.00 0.00 = 3.00 — 6.60' —10.80 —18.80 Golor, | Creatine: mm. | omg. | per cent 10.0 5.00 10.04 5.00 10.3 4.85 10.7 4.67 11.2 4.46 12.3 4.06 13.5 3.70 —26.00 * Standard, 5 mg. of pure creatinine treated the same as the unknown. 7 Fades after about 5 minutes to 10.2. rapidly. All other tests fade even more ef: Nathan F. Blau eli Ethyl acetoacetate in small amounts causes a fading in the creatinine color, while in larger amounts it causes an increase in color. The line of demarkation is difficult to establish, as the two TABLE VI. Effect. of Diacetic Acid on the Determination of Creatinine in Urine. Acid added to 10 ce. Color. Creatinine in Error. mg. per cent mm. mg. per cent 0.0 0.0 3.0 270.00 0.00 Pad 0.021 3.1 261.29 — 3.22 3.0 0.030 3.2 253.12 — 6.25 4.5 0.045 3.4 238.23 —12.138 6.0 0.06 3.8 213.16 —21.05 9.0 0.09 4.1 197.56 —26.82 15.0 0.150 5.5 147.27 —45.45 0.0 0.0 5.1 158.82 0.00 Za 0.021 5.3 152.83 — 3.51 3.0 0.0350 5.5 147.27 = oZii 4.5 0.045 B50 142.10 —10.52 6.0 0.060 6.1 132.78 —16.39 9.0 0.090 6.9 117.39 —26.08 15.0 0.150 8.2 98.78 —37.80 0.0 0.0 6.8 119.11 0.00 1.107 0.011 ZW 115.71 — 2.85 2.214 0.022 7.3 110.16 — 7.51 3.321 0.033 Tol 105.19 —11.68 5.535 0.55 8.4 96.42 —19.05 0.0 0.0 8.3 98.79 0.00 2.07 0.027 9.8 82.55 —16.42 5.535 0.055 10.8 75.00 —24.08 11.07 0.110 13.0 62.30 —36.93 0.0 0.0 12.5 64.80 0.00 1.107 : 0.011 13.0 62.30 — 3.83 2.214 0.022 13.3 60.90 — 6.01 4.428 0.044 15.0 54.00 —16.66 5.535 0.055 NG e/ 48.50 —25.15 limits seem to merge into each other by an almost insensible grada- tion (Tables III and IV). This is, of course, of no great importance, since diacetic acid does not occur in urine in the form of its ethyl ester. LE Creatinine in Acetone and Diacetic Acid Our experiments with diacetic acid bear out the conclusion of Graham and Poulton that this substance, even in small amounts, has a great influence on the creatinine reading. 0.015 or 0.02 per cent causes a perceptible fading, and the color becomes pro- gressively lighter, as the concentration of the acid or the time for which the test is allowed to stand is increased (Tables V and VI). The tables show uniformly that the influence of a given amount of acetone or diacetic acid on the determination of creatinine is more marked in dilute solutions than in more concentrated ones, and also that the effect is the same, whether the creatinine is In a pure solution or in urine. The above findings point clearly to the necessity of removing diacetic acid from urine, preparatory to the determination of creatinine in the sample. Of the efforts that have been made towards the development of a suitable technique to serve this pur- pose, worthy of note are those of Greenwald and of Graham and Poulton.’ Following the suggestion of Rona, Greenwald has attempted to drive off diacetic acid from urine by boiling, but, after a few trials with urines from cases of muscular dystrophy, abandoned the method because it invariably resulted in higher creatinine values. Whether this was due to the expulsion of diacetic acid, and was therefore the desired result, to the conver- sion of creatine to creatinine or to the undue concentration of the urine leading to the formation of pigments and chromogenic con- densation products; whether any or all of these factors were opera- tive in the production of a greater intensity of color, Greenwald did not ascertain. He adopted the procedure of extracting the urines with ether in a continuous extractor for 2 hours, blowing off the ether, diluting the cool solution to twice its original volume, and developing the creatinine color by the addition of 30 ee. of picrie acid and 10 ec. of NaOH. Needless to say that this method is impractical where a great many determinations have to be made. It is tedious and time consuming, and is further limited in its usefulness by the fact that it requires expensive apparatus. The ~ same objections apply, though perhaps with not such great force, to the method described, by Graham and Poulton. They direct adding 1.0 ce. of a 10 per cent solution of H;PO,; to a measured volume of urine and removing the diacetic acid by distillation at 70°C, and 210 mm. for 45 minutes. Then they neutralize with OH | 0.) == 20s 2 H aN | »yP—0—CH; CH CH(CHOH); C—CsH.ON; OH | @ eG H oN | : Va »»P—0—CH; CHOHCH(CHOH), C—CaHON, OH | 4 Levene, P. A., and Jacobs, W. A., J. Biol. Chem., 1912, xii, 411. 122 Thymus Nucleic Acid Incidentally the barium salt of the hexothymidindiphosphoric acid was obtained in crystalline form. For the final solution of the problem of the structure of thymus nucleic acid larger quantities of the material are needed. Since the manufacture of this nucleic acid in Europe has been discon- tinued, it will have to be prepared with our laboratory facilities. Because of this, the progress of the work will be delayed, but work is now in progress. EXPERIMENTAL. Commercial animal nucleic acid (Merck) was hydrolyzed in 100 gm. lots. This amount of the acid was heated on flame with reflux condenser for 2 hours with 1,000 ce. of 2 per cent sulfuric acid. The sulfuric acid and the free phosphoric acid are re- moved by a slight excess of barium hydroxide solution. The excess of this reagent was then removed quantitatively and the solution concentrated under diminished pressure at room tem- perature to a volume of 300 cc. The nucleotides were then pre- cipitated with a 25 per cent solution of neutral lead acetate. The precipitate was washed repeatedly with cold water and then suspended in water, decomposed by means of hydrogen sulfide gas. The filtrate from the lead sulfide was again concentrated under diminished pressure at room temperature to a volume of 300 ce. This was again neutralized with barium hydroxide, filtered from the slight trace of barium phosphate, and brought to a boil over a free flame. A flocculent precipitate soon appears, which on prolonged boiling assumes a granular character. The filtrate from this precipitate was then concentrated to small volume and again heated as before; generally a second precipitate formed. The material which formed on boiling (Precipitate I) had the composition of the diphosphonucleotides. The mother liquor from the diphosphonucleotides was precipi- tated by alcohol (Precipitate II). This precipitate had the elementary composition of monophosphonucleotides. However, when freed from barium and purified through conversion into lead salt and reconversion with barium salt again, there is formed a barium salt insoluble in boiling water. Thus finally, practi- cally all is converted into the diphosphonucleotides. P. A. Levene: 123 There seems to be a discrepancy between the present result and that obtained by Levene and Jacobs. Two alternative explanations may be given to the discrepancy; either the mono- phosphonucleotides found previously by Levene and Mandel and by Levene and Jacobs are products of further decomposition of the diphosphonucleotides formed in the course of further manipulation, or the monophosphonucleotides are missed in the present procedure. Composition of Crude Barium Salts. No. of sample. P N 358 20/21 8.82 5.36 359 20 /21 8.27 5.14 361 20 /21 7.74 5.89 366 20/21 7.39 5.62 370 20 /21 8.27 5.0L 362 20 /21 5.66 5.96 474 20/21 5.15 6.11 Samples 362 and 474 were combined, freed from barium, converted into lead salt, and this reconverted into barium salt. The greater part settled out on boiling and had the following composition. No. of sample. iP N 408 20 /21 askin 5.50 For further purification and for the separation of individual diphosphonucleotides the older procedure was modified. The barium salts were converted into lead salts and these into brucine salts. The brucine salts were fractionated by recrystallization from 35 per cent alcohol until the more insoluble fraction had the elementary composition of the hexothymidindiphosphoric brucine salt. The combined mother liquors were then concentrated and allowed to stand until a crystalline deposit formed. This was again refractionated. After two refractionations the more soluble brucine salt on conversion into the barium salt analyzed for the hexocytidindiphosphoric acid barium salt. 124 Thymus Nucleic Acid Analysis of the Brucine Salts. The most insoluble fraction of the brucine salt analyzed as follows: 0.1001 gm. of the substance on combustion gave 0.1999 gm. of COz and 0.2000 gm. of the substance gave 11.4 cc. of nitrogen gas at T = 21°C. and P = 751 mm. 0.3000 gm. of the substance gave on fusion 0.288 gm. of Mg2P.0O7. Cy. sNeoP2O13C 92H ogN sO16 Calculated. (6, 54.29, H 6.64, N 6.15, 12 2.73. +14H,0. Found. C 54.45, H 6.77, N 6.54, P 2.67. The most soluble fraction analyzed as follows: 0.1004 gm. of the substance gave on combustion 0.1990 gm. of COz and 0.0591 gm. of H,O. 0.2000 gm. of the substance gave 12.0 cc. of nitrogen gas at T = 23°C. and P = 752mm. 0.3000 gm. of the substance gave 0.0287 gm. of MgeP2O;. Cy 0Hi7N3P2012.C 921 o4N sOr6 Calculated. C 54.10, H 6.64, N 6.81, P 2.75. +14H,0. Found. C 54.05, H 6.58, N 6.85, P 2.67. This material was then converted into the barium salt. It must be remarked however, that the elementary compositions of the brucine salts of the above two nucleotides do not differ sufficiently one from another to permit identification of the nucleo- tide on the basis of the analysis of the brucine salts. Often, samples, which on the basis of the analysis of the brucine salt seemed to be the cytidin nucleotide, proved on conversion into the barium salt to be the thymidin nucleotide. Conversion of Brucine Salts into Barium Salts. For this purpose the brucine salts were dissolved in 35 per cent alcohol, an excess of ammonia water added, and the pro- duct allowed to stand in the refrigerator. The brucine was then removed by filtration, the filtrate was again concentrated and allowed to stand to permit further crystallization of brucine. The operation was repeated as long as brucine crystallized out. From the final solution the nucleotides were precipitated by a 25 per cent solution of neutral lead acetate. The lead salt was washed repeatedly with water, filtered, suspended in water, and P. A. Levene 125 freed from lead by means of hydrogen sulfide. The filtrate from lead acetate is concentrated under diminished pressure at room temperature and then converted into the barium salt. h®The hexothymidin salt was then converted into the crystalline form. Preparation of Crystalline Hexothymidindiphosphoric Barium Salt. 9 gm. of the barium salt obtained from the brucine salt were taken up in 500 cc. of water and shaken for 1 hour. Part re- mained insoluble. All was allowed to stand over night when a crystalline deposit was found covering the undissolved amorphous material. The mixed deposit was then taken in 1.5 liters of water at 30°C. and shaken for 1 hour in a shaking machine. The insoluble part was removed by filtration and the filtrate concen- trated under diminished pressure at- room temperature to a volume of 350 cc. After several hours of standing there appeared a crystalline deposit consisting of long needles grouped into star-shaped aggregates. The composition of the substance was the following: 0.1050 gm. of the substance gave 0.0700 gm. of CO, and 0.0182 gm. of H,0. 0.1719 gm. of the substance used for Kjeldahl nitrogen estimation required for neutralization 4.97 cc. of 0.1 N acid. 0.2579 gm. of the substance gave on fusion 0.0830 gm. of Mg.P207. CyoHisN3P2012Baz. Calculated. Cc 18.37, H OTE N 3.89, 12 8.62. Found. C 18.11, H 1.93, N 4.04, P 8.97. Barium Salt of Hexocytidindiphosphoric Acid. This was prepared from the brucine salt in the same manner as the thymidin salt. As yet it has not been converted into the crystalline form. It analyzed as follows: 0.1052 gm. of the substance gave on combustion 0.0658 gm. of CO. and 0.0260 gm. of HO. 0.1870 gm. of the substance employed for Kjeldahl nitrogen estimation required for neutralization 8.21 ce. of 0.1 N acid. 0.2805 gm. of the substance gave 0.0946 gm. of Mg,P.07. CioH13N3P2012Baz. Calculated. C 17.05, H 1.86, N 5.97, P 8.81. Found. © 17.05, HL 2:12, N 6.17, P 9.40. CREATININE AND CREATINE IN MUSCLE EXTRACTS. I. A COMPARISON OF THE PICRIC ACID AND THE TUNGSTIC ACID METHODS OF DEPROTEINIZATION. By FREDERICK 8. HAMMETT. (From The Wistar Institute of Anatomy and Biology, Philadelphia.) (Received for publication, June 29, 1921.) Since the publication by Folin (1) in 1914 of the determination of creatinine and creatine in the filtrates from the deproteinization of blood, milk, and tissues by saturated aqueous picric acid solu- tion plus the addition of solid picric acid, criticisms of the method have been made by Benedict (2), McCrudden and Sargent (3), Hunter and Campbell (4), and others. The most serious objection to the method is that when the picric acid filtrate is heated for the transformation of the creatine to creatinine, certain changes apparently take place which yield results that are higher than those obtained when filtrates from other methods of precipitation are similarly treated. Although Folin and Wu (5) in their system of blood analysis, for obvious reasons of convenience, do not use the picric acid deproteinization, they, nevertheless, still feel that the original process can be utilized when the proper precautions are taken. Because of this controversy and because of the fact that the direct picric acid precipitation of tissue extracts, when creatinine and creatine alone are to be determined, has its elements of convenience in the reduction of dilution and manipulation, a comparison was made of this method and the method of deproteinization with sodium tungstate and 2/3 n sulfuric acid. In this as in the studies to follow, the standards were made from purified creatinine zine chloride prepared according to Benedict (6). The stock solution was standardized against 0.5 n potassium bichromate and the requisite dilutions made therefrom according to Folin (7). For each series of determinations adequate standard solutions were prepared so that there would be at hand 127 128 Creatinine-Creatine in Muscle Extracts. I one with which the unknown solutions would closely correspond. This was considered necessary since the curves of Hunter and Campbell (8), while valid for the conditions under which they worked, may or may not be suitable for use in these studies. The picric acid was purified according to the method of Folin and Doisy (9) and satisfied the requirements stated in their report. Freshly saturated picric acid solutions were always'used and were made up in the ratio of 2.5 gm. of the acid to every 100 cc. of distilled water. They were well shaken and the portions that were used for the reaction with the standards were always fil- tered clear through cotton. The muscle extracts were prepared from fresh cleaned tissue obtained from the posterior limbs of albino rats killed by ether. The tissue was first ground in a meat chopper, then macerated with an equal weight of fine sand in a mortar, and mixed with an equal volume of Tyrode’s solution and 5 cc. of toluene. The suspension was put into a small press and the expressed extract was measured and diluted with an equal volume of Tyrode’s solution. 5cc. portions were used in all the tests. After several trials the following procedures were developed for the determination of the preformed and total creatinine in the muscle extracts. In the picrie acid deproteinization 5 cc. of the diluted extract are measured into a test-tube or centrifuge tube previously marked at the 15 ec. level, and 10 ce. of a saturated picric acid solution in distilled water are added. After the addition of a small amount of solid picric acid the whole is thoroughly shaken and centrifuged for 3 or 4 minutes. It was found that a better sedi- mentation is obtained if the contents of the tube are vigorously mixed with a small stirring rod immediately before centrifuging. 10 ce. of the filtrate, obtained by pouring the supernatant fluid in the tube through a bit of cotton in a funnel, are measured into a small flask or vial. 1 ec. of distilled water is added and 1 ce. of 20 per cent sodium hydroxide accurately measured. This ratio between the picric acid and the sodium hydroxide is that used by Folin and Wu (5). The standards for comparison are each made up of 1 ec. of stock creatinine solution containing the appropriate amount of creatinine (0.05 and 0.10 mg. per ce. for the extracts used in these studies), 10 ec. of saturated picric acid, and 1 ce. of F. 8S. Hammett 129 20 per cent sodium hydroxide. Both the unknown and the stand- ard solutions are allowed to stand for 10 minutes and are then compared in the colorimeter. Sometimes it is necessary to filter off through cotton a light flocculent precipitate from the unknown solutions. The addition of the sodium hydroxide should be made with the same pipette and with the same procedure in all cases. It should be noted that in all circumstances the standard solutions are identical with the unknown solutions with respect to the amount of picric acid, and the amount of sodium hydroxide, and are closely similar in colorimetric value. For the determination of the total creatinine, that is to say, the creatine as creatinine plus the preformed creatinine, 10 cc. of the filtrate from a second 5 cc. sample of extract precipitated as de- scribed are. put in a small Erlenmeyer flask, diluted with 10 ce. of distilled water, and heated at the boiling point for 2 hours on an electric hot-plate. Partial evaporation is allowable. Complete evaporation is disastrous and is prevented by the addition of small amounts of water from time to time as the occasion demands. During the last half hour of heating the solution may be allowed to concentrate to about 3 or 5 cc. although a 10 cc. final volume does not affect the end-result. The flask is then removed from the hot-plate, cooled to room temperature, and the contents are made to about 16 cc. with distilled water. 1 cc. of 20 per cent sodium hydroxide is added and the mixture is allowed to stand for 10 minutes when it is transferred to a 100 cc. flask and diluted to the mark. The solution so made up is compared with the appropriate standard. It has been found that for extracts prepared as described a standard consisting of 1.5 mg. of creatinine plus 10 cc. of saturated picric acid solution and 1 cc. of 20 per cent sodium hydroxide diluted to 50 ce. after 10 minutes standing is satisfactory. The final procedure developed for the determination of creatinine and creatine in the tungstic acid deproteinization method is as follows. 5 ec. of the diluted muscle extract are put into a test- tube or centrifuge tube marked at the 15 cc. level, and 5 cc. of distilled water are added. Then 2 cc. each of a 10 per cent solu- tion of sodium tungstate and 2/3 n sulfuric acid are added, the whole is made to 15:cc. with water, shaken thoroughly, and centri- fuged. For the creatinine determination the supernatant solution 130 Creatinine-Creatine in Muscle Extracts. I is filtered through a bit of cotton and 10 ce. of the filtrate are trans- ferred to a small vial. 10 cc. of saturated picric acid are added and 1 ce. of 20 per cent sodium hydroxide. The standards for these unknown solutions are made by taking 1 cc. of the appropri- ate original creatinine concentrations, adding 10 cc. saturated picric acid, 9 ce. of distilled water, and 1 cc. of sodium hydroxide. Both standard solutions and unknown solutions are allowed to stand for 10 minutes and are then compared in the colorimeter as previously described. For the determination of the total creati- nine, 10 cc. of the filtrate from the tungstic acid precipitation are put into a small Erlenmeyer flask, diluted with 10 ce. of distilled TABLE I. The Amounts of Preformed and Total Creatinine in Muscle Extracts after Deproteinization by Picric Acid and Tungstic Acid. Preformed creatinine. Total creatinine. Method...Picric acid. Tungstic acid. Picrie acid. Tungstie acid. aaa Pato mg. mg. mg. 0.125 08125 Lae. (By 0.288 0.300 9.89 10.25 0.148 0.112 5.14 4.95 0.120 0.117 5.49 5.40 0.108 0.114 5.49 5.54 0.111 0.114 5.45 5.40 0.071 0.073 5a22 5.22 water, 1 ce. of n hydrochloric acid is added, and the whole is heated for 2 hours as described for the picrie acid filtrates, save that in this case the final solution should not exceed 2 or 3 ec. in volume. The flask is removed from the hot-plate, cooled, and 10 cc. of picric acid solution are added and 1 ee. of 20 per cent sodium hydroxide. The mixture is allowed to stand for 10 min- utes, is then transferred to a 100 ce. graduated flask, and diluted to the mark. The standard for this determination is exactly the same as that for the analysis of the picric acid filtrate. When the methods as outlined are carried out on one and the same extract concordant results are obtained as shown in Table I. Parallel determinations were made in all cases. moO DD ee COONAN F. S. Hammett fat BIBLIOGRAPHY. . Folin, O., J. Biol. Chem., 1914, xvii, 475. . Benedict, 8S. R., J. Biol. Chem., 1914, xviii, 191. . McCrudden, F. H., and Sargent, C. S., J. Biol. Chem., 1916, xxiv, 423. Hunter, A., and Campbell, W. R., J. Biol. Chem., 1917, xxxii, 195. Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. Benedict, S. R., J. Biol. Chem., 1914, xviii, 183. Folin, O., J. Biol. Chem., 1914, xvii, 463. . Hunter, A., and Campbell, W. R., J. Biol. Chem., 1916-17, xxviii, 335. . Folin, O., and Doisy, E. A., J. Biol. Chem., 1916-17, xxviii, 349. CREATININE AND CREATINE IN MUSCLE EXTRACTS. Il. THE INFLUENCE OF THE REACTION OF THE MEDIUM ON THE CREATININE-CREATINE BALANCE IN INCUBATED EXTRACTS OF MUSCLE TISSUE OF THE ALBINO RAT. By FREDERICK S. HAMMETT. (From The Wistar Institute of Anatomy and Biology, Philadelphia.) (Received for publication, June 29, 1921.) The object of this investigation was the determination of the changes that take place in the creatinine and creatine content of extracts of muscle tissue of the albino rat when incubated at body temperature for 24 hours when the reaction of the extract is buffered to neutrality or alkalinity, and when the extract is unbuffered save by the Tyrode’s solution used as diluent, and is allowed to develop its own reaction which is slightly acid to rosolic acid. The study is to serve as a foundation for an inquiry into the factors concerned in the creatinine-creatine balance in such tissue extracts with the hope that some light may be thrown on the problems of the metabolism of these compounds. There are those who have doubted that the demon- stration by Stangassinger (1), Gottlieb and Stangassinger (2, 3), Rothmann (4), Mellanby (5), and Myers and Fine (6) of an increase in creatinine accompanied by a decrease in creatine content of muscle tissue or muscle extracts in in vitro experiments is a valid indication that such a process occurs in the living organism. The objections raised to such an application of the findings have been based on evidence which has its contradictory phases, while on the other hand, the main facts of the strictly laboratory tests consistently point in one direction. The uni- formity of the results of these latter methods of attack can be attributed to the elimination of the interfering factors of digestion, assimilation, utilization, bacterial action, and the probable influ- ence of organs other than the muscles on the reaction being studied. It is not necessary to go into the literature dealing with 133 134 Creatinine-Creatine in Muscle Extracts. II this controversy, for to those interested in the problem its main features are well known. The results of the studies of autolyzed muscle tissue or extracts have shown that the increase of the creatinine content in such preparations occurs whether the reaction of the medium is acid, neutral, or alkaline. But the data are conflicting with respect to the relative influence of the reaction on the amount of creatine formation. The recent report of Hahn and Barkan (7) on the effects of sodium hydroxide and hydrochloric acid on this change in aqueous solutions of creatine while of interest is hardly directly comparable with the studies in which tissue extracts were used. For the purposes of this study extracts were prepared from the voluntary muscles of the hind limbs of albino rats as described in the preceding paper (8). Rats of the same sex and age were used within each series, although the sex and age differed for the different series. For each series a set of sixteen centrifuge tubes was used and into each tube 5 cc. of muscle extract were measured, using the same pipette throughout. To the first group of four tubes there were added 4 drops of distilled water; to the second group 4 drops of Henderson’s (9) phosphate mixture; and to the third 4 drops of a saturated solution of NasZHPO,. The remaining four lots were used for the estimation of the preformed and total creatinine of the fresh extract. 0.5 cc. of toluene was added to the above mixtures and they were tho- roughly mixed by means of a fine stream of air blown through a glass capillary dipped to the bottom of the tubes. When the reaction of the various groups was tested with rosolic acid, they were found to be slightly acid, neutral, and alkaline, respectively, both before and after incubation. After the contents of the tubes had been prepared as described they were incubated for 24 hours usually at a temperature of 38°, although some lots were kept at 36 and 40°. After incubation the preformed and total creatinine were determined according to the picric acid deproteinization method previously described (8). Parallel determinations were made and the reported values represent their averages. The statistical values of the parallel determina- tions are given in Table I in terms of 0.1 mm. and demonstrate that a considerable degree of reliance can be placed upon the findings. ee ee F. S. Hammett fs5 Turning now to a consideration of the results of these experi- ments the figures in Table II are given. They represent in mg. the amounts of total and preformed creatinine found in the fresh extracts and in the extracts after incubation under the con- ditions described. The percentage increase is also tabulated as are the statistical values for the series as a whole. No figures are given for creatine since they are obtained by difference and cal- culation and would add nothing to the argument. It is evident that here, as with other workers, there has been an increase in the creatinine content of the extracts on incubation TABLE I. Statistical Values of the Colorimetric Readings of the Parallel Determinations. _Mean Froese Standard pe ae difference. of wenn: deviation. sa 0.1 mm. 0.1 mm. 0.1 mm. 0.1 mm. Creatinine before incubation ...... ee 0825 1.76 0.18 Creatinine after incubation. Acid & tae Ce he ee» 2.4 0.31 1.98 0.22 INetitiral 25. 254: 2.4 0.31 1.84 0.22 Alkaline-3..2) 5. 3.0 0.38 2.24 0.27 Creatinine; all determinations..... 2.5 0.15 1.97 0.11 Total creatinine before incubation. Pall 0.25 1.80. 0.18 Total creatinine after incubation. INCIdets Soccer eee 2,9: 0.39 2.03 0.28 Neutralia 3s": ane, eo sO 0.49 1.94 0.35 AMikaliney. a4. 2.6 0.46 1.91 0.32 Total creatinine; all determinations.| 2.4 0.18 1.94 0.13 whether the reaction of the medium was acid, neutral, or alkaline. This increase is statistically valid as measured by the usual criterion that the probable error of the mean must be contained in the difference between the means at least twice, and three times for definitely satisfactory differences. No changes in the amounts of total creatinine occur on incubation when the same standard of validity is applied. Such being the case and since there has been neither gain nor loss of total creatinine under these conditions, the increase in the creatinine must perforce have been at the expense of the creatine. Since the muscle extracts exhibiting this phenomenon were extracts made with - II Muscle Extracts. ine in -Creat inine 136 Creat Z°0 80°0 |2°0 80°0 |F°0 80°0 | 80°0 |2°8 = |z00°0 |8°IT €°0 cI 0 |€°0 II‘0 |$°0 ILO | 22°00 |9 TL |200°0 i a | 6S°0 |Z'T 8¢°0 |9°% 1¢°0 | 09°0 |9°6S |Z10°0 9°0— | 88°F |F 0O— | 88°F |S I— | &8°P | 16°F |T PZT |69T 0 9°0— | 08% |0'0 €8°F |9 O— | O8 F | &8'F |9'6E |Z9T'O O'SZ— | 86 F 19 S— | 86°F |S I— | 66 P | 90°92 |F' 82. 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S. Hammett 137 Tyrode’s solution which simulates to a considerable degree the medium in which the reactions of the living organism take place, I am of the opinion that we are justified in assuming, until it has been disproved by critical experiment, that there occurs in the muscles of the living organism a formation of creatinine from the muscle creatine and that the endogenous source of the urinary creatinine is the muscle creatine. This lack of destruction of total creatinine just discussed confirms the findings of Mellanby (5) and Myers and Fine (6) and fails to substantiate the results of Gottlieb and Stangassinger (2). Experiments where putrefaction was allowed to occur, and which will be presented presently, tell another story. Now when the percentage increase in creatinine is considered it is seen that this increase is regulated in part by the reaction of the medium, for it is least in the acid solutions, greatest in the neutral solutions, and between the two in the alkaline solutions. This relationship is consistently constant in all of the twelve experiments reported and is substantiated by the statistical calculations. It is not in agreement with the result of Rothmann (4), Myers and Fine (6), or Hahn and Barkan (7). For the two former found an apparent acceleration of the reaction by acid and the latter that alkali retarded the change of creatinine to creatine as compared with acid. The studies of Hahn and Barkan (7), however, are hardly comparable with the studies made with tissue extracts. When one looks at the results of Myers and Fine (6) given in Table VII of their paper, it is seen that when the autolyzing mixtures were buffered to neutrality by phosphate mixture a somewhat greater creatinine formation took place than when the tissue was treated with water alone. My results confirm this finding in principle. Nevertheless the ’ studies of Myers and Fine (6) are not strictly comparable with mine inasmuch as they used whole muscle tissue, their periods of autolysis were extended over a longer period and they used an acid not normally found in muscle tissue. Such being the case it is evident that a slightly acid or an alka- line reaction retards the transformation of creatine to creatinine in muscle extracts when incubated for 24 hours at body tempera- ture. This transformation occurs at a maximum when the reaction of the digesting mixtures is buffered to neutrality by a 138 Creatinine-Creatine in Muscle Extracts. II phosphate mixture. These facts serve as a partial explanation of the observations of Underhill (10) that creatinuria is frequently an accompaniment of induced acidosis and of Underhill and Baumann (11) that a marked increased creatine excretion may be found in experimental alkalosis, and other apparently anomalous results of the studies of creatinuria, if we admit that the urinary creatinine is largely derived from the muscle creatine, and in spite of the opinions of Denis and Minot (12) and Gamble and Goldschmidt (13) that the acid-base equilibrium has nothing to do with the condition. For since it is shown that both a slight acidity and an alkalinity retard the transformation of creatine to creatinine in muscle extracts it is possible to consider that if similar tendencies are present in the living organism, even though fleeting, they may give rise to similar effects, and if that phase of muscle metabolism which results in creatine formation continues at the same or even a diminished rate, there is produced a relatively greater concentration of creatine in the circulation, part of which at least finds its way to the kidneys and is excreted. That such an increase in blood creatine can occur and continue for days under changed conditions of muscular activity I have already demonstrated (14), though this phase of the problem is not necessa- rily at present connected with the question of creatinuria and acid-base equilibrium. Since these experiments demonstrate conclusively that there is no loss of total creatinine on incubation under sterile conditions the figures given in Table III are particularly interesting, from the point of view of the contention of Gottlieb and Stangassinger (3) of the presence in muscle tissue of creatinine—and creatine— destroying enzymes. ‘The results in this table were obtained from extracts which had been allowed to undergo putrefaction during the incubation. It will be seen that there has occurred a marked loss of total creatinine that is statistically valid. This supports the findings of Mellanby (5) that only when bacterial decomposi- tion occurs does there take place a destruction of creatine or creatinine, and plainly shows the cause of the results reported by the proponents of the ‘‘creatinase’”’ and ‘‘crea- tase” theory. However, it is quite probable that the transforma- tion of creatine to creatinine in muscle extracts is brought about by an enzyme, in view of the fact that the change occurs in the ee eh oe 139 F. S. Hammett oF | 21:0 \¢-2 | FO |8'z | 9t'0 | OL’ \e'sT |9T0°0 |9°2F \OP0'0 |8°6e (9200 |900°0 | BOF -vIAop piepueys jo Lo119 a[qQeqoig Lg ¥o'0 |9 OL | 89°0 |8'€ 1z'0 | #1°0 [0°92 \ez0'0 |¢°29 |Z90°0 |7°9¢ |280°0 |200°0 | uUBoUT jo LO119 a[qBqold L291 | 2:0 |p'Te | TZ'T |Z'IT | $9'0 | Th'0 |4°924 |890°O |T°00G |69T'O |Z 29T |IIT O |120°0 “MOTPIAOP PLEPUBZS L°GL—| €F'T |8 0F— ST‘ |¢°zl—| OFT | LI°S |8"LTS |1Z9°0- |O'SFF S890 |0°96E \E8h°0 |S0T'O | “<> SUBOTN: 6'I8—| 96°0 |z°ZLZ—| STI |8°SL—| ZIT | 08'S |T'OLF [0690 |€°ZTS |TFZ'O |0'98E |88S°0 | TZI°0 9°0S—| s9°s |0 0 9¢°¢ |6°6S—| ST's | 98°S |G'°GEPF |Z89'0 |P TST \€22'0 |Z PST |O0E'O |S8IT 0 G’18—| €8°0 €°148—| 29°0 | SF'F |6'LzE |€S9°0 |S°S9F |889°O |6 Scr |PFS°O | FOTO 6'9L—| 82° |6'SF—| 00'S |IT'F9—| G6 | GE'S |2°2E9 |60S°0 |8°Z89 |OFS°0 |6°FZ9 |00S'0 |690°0 seovous] py enesonsl pug |oenesrea| ow | writes that the presence of a true nucleic acid in the spleen is still in need of proof. His method of proving it is not more rigorous than the one employed by the previous writer. The substances now prepared had the following composition: * Levene, P. A., Z. physiol. Chem., 1905, xlv, 370. 4 Levene, P. A., and Mandel, J. A., Biochem. Z., 1908, x, 215. * Steudel, H., Z. physiol. Chem., 192i, cxiv, 255. P. A. Levene 179 Cc H N Vz Spleensnucleic acids... .....s00- sa. Sor oN 407 15.52 9.71 Pancreas “ Roth orth ore eee 36.20 | 4.76 15.26 9.58 $ is Bi Ak (ais er ee 35.07 | "8.43 14.95 9.59 Liver < BP anole Sea ee 36.75 | 4.65 11765" | 10°60 ef i eT oc do aden aes 37.08 | 4.96 115r | 10k Theory for a hexosetetranucleotide....| 36.30 | 4.19 14.79 8.73 None of the samples showed the presence of pentose and all, even the samples from the liver, contained the two purine bases. The yield of the bases was smaller, however, from the pancreas and liver nucleic acids. They were as follows: From: Adenin picrate.| Crude guanin. gm. gm ‘shoe miele evel soe caten dbo phe eeeianoene Tt 16.0 10.0 Pancreas “ SI) WARNE FLD 2 SecA ee een Ae oe 9.0 Teo Liver y MB aes FE BLE BAG ESD iu eg ha EOI 8.0 TAG It is obvious that the elementary composition of amorphous substances of the nature of nucleic acids, which are never free from impurities, is of comparatively little service for the purpose of forming theories of molecular structure. However, great deviations from the theory, such as are observed on liver nucleic acid, require an explanation. Also, the fact that the pancreas nucleic acid, which in its elementary composition does not differ much from the spleen nucleic acid and yet furnishes on hydrolysis less purine bases than the other, needs further explanation. Work on these problems is now in progress. EXPERIMENTAL. Preparation of Nucleic Acids.—This is uniform for all three of the tissues, and the details of the procedure are as follows: 2,500 gm. of minced fresh tissue (previously freed from fat) are taken up in 3,000 cc. of water. 300 gm. of sodium chloride are added and all is kept boiling (with a steam coil) for 4 hours. Then 80 gm. of sodium acetate and 60 ce. of a 33 per cent solution of sodium hydroxide are added, and the mixture is allowed to 180 Animal Nucleic Acid stand over night. The mixture is then neutralized with acetic acid and treated with picric as long as a precipitate forms. To the filtrate of this mixture hydrochloric acid is added until it turns slightly opalescent and the nucleic acid is precipitated with a 10 per cent solution of copper chloride. The copper salt of the nucleic acid is filtered and converted into the free acid by treat- ment with a 5 per cent solution of hydrochloric acid. The treatment is repeated once. The resulting free nucleic acid is redissolved in a 5 per cent solution of sodium hydroxide, the solu- tion is made acid with acetic acid and precipitated with 95 per cent alcohol, containing 4 per cent of hydrochloric acid. The precipitate is then washed with 95 per cent alcohol until the washing no longer shows the presence of chlorine ions. The nucleic acid is then washed with absolute aleohol and ether, and dried. The substance so obtained does not show the presence of even traces of biuret-giving substances, and gives a negative test with orcin. Alcoholysis. 10 gm. of nucleic acid are suspended in 200 ce. of dry methyl alcohol and hydrochloric acid gas is passed in a lively stream. After 2 hours the reaction is interrupted and the flask is allowed to stand over night. Guanin and adenin are separated in the usual way. Guanin was purified by conversion into the sulfate, which again was converted into the free base. Adenin picrate for purification was twice recrystallized out of 10 per cent acetic acid and then dissolved in water by the addition of the required quantity of ammonia and precipitated by means of acetic acid. Analysis of Nucleic Acids. Spleen Nucleic Acid. 0.9880 gm. of the substance gave on combustion 0.1310 gm. of CO2 and 0.0360 gm. of H.O. 0.0884 gm. of the substance employed for Kjeldahl nitrogen estimation required for neutralization 9.8 ec. of 0.1 N acid. 0.1768 gm. of the substance gave 0.0616 gm. of Mg2P2O7. EE a PenAe Levene 181 percent ‘ per cert Ol nee aoe A eo en nee cae See ee 36.30 36.15 130... bg! Sak hr renee ee 4.19 4.07 had, = fe I a ee 14.79 15.52 Perret: 35-522 2 ccatqatebat dently bate 8.83 9.71 Bases.—10.0 gm. of this material yield 1.6 gm. of adenin picrate and 1.0 gm. of guanin. The guanin was analyzed as the free base. 0.2000 gm. of the substance employed for Kjeldahl nitrogen estimation required 66.30 cc. of 0.1 N acid for neutralization. Calculated F ound for CsHsN:0O. per cent per cent IN do CHO RO OE DOE EIE.G NO OOT OLIOE G Stet nied Sac aays 46.53 46.41 The adenin picrate analyzed as follows: 0.0946 gm. of the dry substance employed for Kjeldahl nitrogen estima- tion (reduced with zine metal) required for neutralization 21 cc. of 0.1 N acid. Calculated for CsHsNs.CeH2(NO2)30H. Found. per cent per cent ee hg r ee AE tec 30.71 31.06 Pancreas Nucleic Acid. A. 0.1056 gm. of the dry substance on combustion gave 0.1358 gm. of CO, and 0.0572 gm. of H,0O. B. 0.1080 gm. of the dry subtsance of a second sample gave 0.1434 gm. of CO» and 0.0460 gm. of H.O. A. 0.1818 gm. of the substance employed for Kjeldahl nitrogen estima- tion required for neutralization 19.40 cc. of 0.1 N acid. B. 0.1780 gm. of another sample required for neutralization 19.40 ee. of 0.1 N acid. A. 0.2726 gm. of the substance gave 0 0988 gm. of Mg2P.207. B. 0.2670 gm. of another sample gave 0.0918 gm. of Mg»P2O7. ’ Calculated for CusHesNiPsOz. Found. per cent per cent A B Occ c SR Oe One aE tn abies ab 36.30 30.07 36.02 pes f..63 cc ober teeene hee 4.19 5.43 4.76 INP d dioraicts Mea PS Asia 14.79 14.95 15.26 8.73 9.59 9.58 182 Animal Nucleic Acid Bases.—The substance was alcoholyzed in 10.0 gm. lots. The average yield was 0.900 gm. of adenin picrate and 0. 750 gm. of crude guanin. Guanin was analyzed as the free base and gave the following values. 0.1066 gm. of the substance gave 0.1566 gm. of CO, and 0.0310 gm. of H.0. 0.0996 gm. of the substance gave 39.4 gm. nitrogen gas at 7 = 26° and P = 756mm. for CstleNeO, Found. per cent per cent 5s TE, RE Ras cle LS Ck ia PRET ns Ok Reape ae ; 39.738 39.05 |S DE ee cen er ASE IE es MRM RLA SS Butt 0 3.30 3.26 Uy Re Oe e505 cick NOR Reon KEG on CREE IO Cot 46.60 46.62 Adenin picrate analyzed as follows: 0.0935 gm. of substance employed for nitrogen estimation (modified Kjeldahl) required for neutralization 20.25 cc. of 0.1 N acid. Calculated for CsHsN. CcH2(NO:);0H, Found. per cent per cent Lia Or a 2 eee eS A 30271 30.32 Liver Nucleic Acid. A. 0.9720 gm. of the dry substance gave 0.1330 gm. of CO». and 0.0434 em. of H,0. B. 0.1090 gm. of another sample gave 0.1390 gm. of CO, and 0.0566 gm. of H.0. A. 0.0900 gm. of the dry substance employed for Kjeldahl nitrogen estimation required for neutralization 7.40 cc. of 0.1 N acid. B. 0.1816 gm. of another sample required for neutralization 15.60 ee. of 0.1 N acid. A. 0.1800 gm. of the dry substance gave 0.0666 gm. of Mg2P.20;. B. 0.2724 gm. of another sample gave 0.1108 gm. of Mg2P207. for Go e.On. Found. per cent per cent A B | Cae Sin cry cero a2 omer 36.30 37.08 34.79 | & RS oA ek riser cnn oo ol ae 4.19 4.96 5.81 Lk BPRS cs Ota ae Se oo ene 14.79 11.51 10.48 | SE Te ear a ae cpa. Sater 8.73 ~ 10.31 11.33 Bases.—One lot of 50.0 gm. was treated in methyl alcohol and hydrochloric acid gas as described above. This yield was 3.8 gm. of crude guanin and 4.0 gm. of crude adenin picrate. P. A. Levene 183 Guanin was identified as the free base and analyzed as follows: 0.0997 gm. of the base gave on combustion 0.1566 gm. of CO2 and 0.0338 gm. of H,0. Calculated Round: for CsH;NsO. per cent per cent (Giese eee BAERS DORE Bid CACO CCIE Oe 39.73 39.55 Ee i ke Ee UD 1 ot a cin yn ea ae 3.30 3.47 Adenin was identified as picrate and analyzed as follows: 0.0940 gm. of the substance employed for nitrogen estimation (modified Kjeldahl) required for neutralization 20.50 cc. of 0.1 N acid. Calculated for CsHsNs.Cs.H2(NOz);0K, Found. per cent per cent UN Ges etait fe os sh ot A DANG oF epee 30.71 30.53 THE LIVER LECITHIN. By P. A. LEVENE anp H. S. SIMMS. (From the Laboratories of The Rockefeller Institute for Medical Research.) (Received for publication, July 7, 1921.) The older work on the unsaturated lipoids of the liver has been reviewed in the paper of Levene and Ingvaldsen. In the same paper a new analysis of the liver lecithin was reported. The new facts reported in that paper related principally to the nature of the fatty acids. In the course of the work reported by Levene and Ingvaldsen! two fatty acids were found, one saturated, and the other unsaturated which analyzed for a polyunsaturated arachidic acid. Since an acid of that structure has never before been described in connection with lecithin, further corroboration of the finding seemed desirable. Furthermore, in the course of the present year it was shown by Levene and Rolf? that the egg lecithin and that of the brain contained two saturated fatty acids. This result was obtained by means of fractional distilla- tion of the esters of the combined saturated acids. In light of this observation it became necessary to reinvestigate all lecithins in regard to the number of fatty acid radicles, saturated and un- saturated, present in their molecule. In regard to the liver lecithin it was now found that it contained two saturated and two unsaturated acids.* The saturated acids are palmitic and stearic. They were isolated and identified by the same procedure as described by Levene and Rolf. The unsaturated acids are: one, unsaturated stearic, and the other, unsaturated arachidic. On reduction one is converted into stearic and the other into arachidic. The exact degree of 1 Levene, P. A., and Ingvaldsen, T., J. Biol. Chem., 1920, xliii, 359. 2 Levene, P. A., and Rolf, Ida P., J. Biol. Chem., 1921, xlvi, 193, 353. 3 Evidence has recently been obtained in this laboratory that egg lecithin also contains the two unsaturated acids. 185 186 Liver Lecithin unsaturation of either one of the two acids is as yet not known. There are however, indications that one (arachidic) may be tetra- unsaturated. On the addition of bromine a substance was obtained which analyzed for an octobromide of arachidic acid. However, it will require a larger quantity of material to establish the degree of unsaturation of each of the two acids with certainty. The presence of several acids in the liver lecithin again empha- sizes the question of the existence of more than one lecithin. It was attempted to answer this question by the molecular weight estimation of the hydrolecithin. The hydrolecithin from the liver lecithin has been prepared essentially according to Paal’s procedure. The molecular weight of the substance was found 810 and 700 (in two estimations). The theory of a monophosphatide re- quires 809, that of a diphosphatide 1600. Consequently, liver lecithin consists of a mixture of monolecithins. In the course of the present work the process of preparation of pure free lecithin from its cadmium chloride salt has been im- proved so that analytically pure substance is prepared in good yield; namely, about 50 gm. of free lecithin from 100 gm. of the cadmium chloride salt. The procedure in the main is as follows: The salt of lecithin is dissolved in chloroform and this solution is transferred into a solution of dry ammonia gas in dry methyl alcohol. The re- sulting lecithin is purified from the slight quantity of impurities by the acetic acid process developed by Levene and Ingvaldsen. The details of the procedure are given in the experimental part. EXPERIMENTAL. I. Preparation of Pure Lecithin. Various attempts to produce pure liver lecithin without con- verting it into the cadmium chloride salt met with little success. The following method proved to be the easiest and most efficient. The liver, in 100 pound lots, is minced, dried, and extracted, first with acetone, second with ether, and last with alcohol. These extracts are treated separately as follows: Acetone Extract.—This is allowed to stand at 0°C. over night. A precipitate of fat is deposited, which isremoved by filtration. eee ee ae ee ap P. A. Levene and H. 8. Simms 187 The filtrate is concentrated (if necessary) and the lecithin pre- cipitated by adding a saturated solution of cadmium chloride in alcohol until no further precipitate is formed. The residue from the above filtration is suspended in alcohol and warmed until the fat is melted. The mixture is then cooled over night. The fat precipitated on standing is again filtered from the alcoholic solution and again treated with alcohol as before. This extraction is repeated until the mother liquor no longer gives with cadmium chloride a precipitate of lecithin cad- mium salt. The latter is recognized by the fact that on dissolving in a small amount of moist ether it is again precipitated by the addition of an excess of acetone. From three to seven extrac- tions may be required. The alcoholic mother liquors are then precipitated with cadmium chloride. Ether Extract.—This is concentrated to a small volume and allowed to stand at 0°C., when a precipitate consisting of fat and cerebrosides is formed. The precipitate is extracted with ether. The ethereal extract is added to the original filtrate and cooled once more to permit the separation of the cerebrosides which the solution may still contain. After filtering, the com- bined mother liquors are concentrated and treated with alcohol to separate the lecithin from cephalin. The alcoholic liquors are then treated with cadmium chloride. Alcoholic Extract.—This is likewise concentrated and cooled to remove cerebrosides, the mother liquor being decanted if possible, otherwise filtered, or centrifuged if necessary. The cerebrosides are again extracted with warm ether. The extract is cooled and centrifuged. The alcoholic and ethereal liquors are then treated with cadmium chloride. Treatment of Cadmium Chloride Salts. It is necessary to allow the cadmium chloride precipitate of lecithin to stand at least half an hour until it is sufficiently coagu- lated to permit filtration. The filtered material, which is not quite dry, is transferred to a large beaker or precipitating Jar and stirred up with a large volume of cold acetone. If the acetone liquor turns dark from dissolved material the suspen- sion is allowed to settle, the liquor decanted off, and more cold acetone is added. Finally the material is filtered by suction. 188 Liver Lecithin This material is purified in two steps: the one, is the ‘‘ether crystallization,’’ the other is the ‘‘toluene-ether’ process.’ It is a matter of judgment as to which shall be used first and the number of times which each should be repeated. The aim is to obtain a white granular material which filters quickly. The ether crystallization consists in dissolving the cadmium chloride salt in warm ether, water being added, a few drops at a time, until the suspended material goes into solution. An excess of water hinders the solution of the larger particles. The solu- tion is allowed to stand over night, or longer, at O°C. The substance should separate in a granular form, easily filterable by suction. If it forms a pasty solution not easily filtered, time and material will be wasted in attempting a filtration. Another precipitation with acetone should remove impurities which interfere with the process. This purification removes not only the fats and oils but also takes out most of the cephalin present. Since the cadmium chloride salt is itself slightly soluble in cold ether, some of the material may be lost in purification. Hence the following pre- cautions are necessary. 1. Excess of ether is to be avoided. With very impure material it.is more advisable to repeat the purifi- cation several times with small quantities of solvent than to use a large excess at one time. The amount of ether filtered off should not be more than twice the volume of the residue. 2. The filtered material should not be washed with ether, but should be filtered as quickly as possible until the solvent runs very slowly. 3. In case the material fails to filter properly, it should be transferred to a beaker, warmed slightly until dis- solved, precipitated with acetone, purified by the toluene- ether method, and subsequently passed through the ether crystalli- zation process. 4. The filtration should be carried out in the cold. The toluene-ether process consists in dissolving the cadmium chloride salt in a minimum volume of toluene (adding a slight amount of water if necessary). If the toluene fails to dissolve all the material the residue should be centrifuged off. The solution is then treated with 4 volumes of ether containing 1 per cent water. The solution is cooled to 0°C. over night and filtered. P. A. Levene and H. S. Simms 189 The latter method gives larger yields but removes less of the cephalin and other impurities. It probably removes impuri- ties not taken out by the former method, hence the cadmium chloride compound should be purified by both methods. Experience shows that in the case of liver lecithin the toluene- ether method should precede the ether crystallization method of purification of the cadmium chloride salt in order to obviate difficulty in filtering from the ether. One purification by each method should be sufficient to give almost white dry material with an amino content of less than 3 per cent of the total nitrogen present. Such a product may be converted into free lecithin. Conversion of the Cadmium Chloride Compound into Free Lecithin. The cadmium chloride salt is dissolved in chloroform and is converted into free lecithin by means of a solution of ammonia in methyl alcohol. 100 gm. of the cadmium chloride salt are dissolved in 300 cc. of warm chloroform and poured into 400 ce. of methyl alcohol containing 20 gm. of ammonia gas. This is added slowly with rapid stirring. The product of reaction is allowed to stand a short time before filtering. The precipitate may be filtered off through a folded filter paper. The chloroform methyl alcohol solution of lecithin is then concentrated under diminished pressure. Near the end of the concentration the material foams considerably for a short time and then the foam- ing subsides. The vacuum concentration should be carried out at a low temperature. If during the operation a precipitate of fat settles out this should be filtered off. The remaining lecithin is practically free from solvent. It is dissolved in a minimum (5 to 10 cc.) of glacial acetic acid. This is poured into 800 ce. of boiling hot acetone, stirred, and allowed to cool to room tem- perature. A very small dark precipitate (1 to 2 gm.) settles out. The supernatant liquid is decanted or filtered. The precipitate is slightly soluble in ether and insoluble in acetone but somewhat soluble in ethyl! alcohol and more soluble in methy] alcohol. No. 126. 0.0154 gm. of substance gave on combustion 0.0954 gm. of H2O, 0.2315 gm. of COs, and 0.0114 gm. of ash. 190 Liver Lecithin 0.1910 gm. of substance used for Kjeldahl nitrogen determination required 3.90. cc. of 0.1 N acid corresponding to 0.00546 gm. of N. 0.2865 gm. of substance gave 0.0390 gm. of Mg2P207;. CysHs7OoNP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. Found. C 61.16, H 10.34, N 2.92, P 3.88. It contains 10 per cent amino nitrogen. No. 124. 1.5 gm. of substance were hydrolyzed with HCl, neutralized, concen- trated, and made up to 15 ce. 5 ec. of this solution required for Kjeldahl nitrogen determination 5.60 ec. of 0.1 N HCl. 2 ec. of this solution for Van Slyke determination gave 0.57 ec. of Ne tel, — on aa oeee mn Amino N 10 Total N 100 The liquors are then cooled in a freezing mixture to —5°C. Frequently at this phase a second small precipitate settles out. A sample of this material analyzed as follows: No. 122. 0.1024 gm. of substance gave on combustion 0.1098 gm. of H,O, 0.2216 gm. of COz, and 0.0118 gm. of ash. 0.1832 gm. of substance for Kjeldahl nitrogen determination required 3.10 cc. of 0.1 N acid corresponding to 0.00434 gm. of N. 0.2748 gm. of substance gave 0.0464 gm. of Mg2P20 7. CysHs702NP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. Found. C 59.50, H 12.10, N 2.87, P 4.75. The mother liquors are concentrated under diminished pres- sure until all the ether and most of the acetic acid are removed. Water is added a little at a time and the material is shaken or stirred until a thick emulsion of a light brown color is formed. This is poured into 800 to 1,000 ec. of acetone, chilled down to —5°C. It is carefully stirred and allowed to stand at 0 to—5°C. over night, when it is transferred to a crystallizing dish and washed free from excess water by stirring with cold dry acetone. The acetone is decanted off and the lecithin dried in a vacuum desiccator. From 40 to 45 gm. of pure material may be obtained from 100 gm. of cadmium chloride salt. (Theoretical yield, 81 to 82 gm.) Several samples have been analyzed. They differed little one from another in their elementary composition. The analysis of one of them is as follows: i ee O————<—<« — see ee P. A. Levene and H. S. Simms 191 No. 119. 0.0996 gm. of substance gave on combustion 0.0994 gm. of H20, 0.2768 gm. of COz, and 0.0090 gm. of ash. 0.1798 gm. of substance required 2.40 cc. of 0.1 N acid, corresponding to 0.00336 gm. of N. 0.2697 gm. of substance gave 0.0390 gm. of Mg2P.O7. CyuHs70oNP.* Calculated. C 65.59, H 10.89, N 1.74, P 3.86. ‘Found. C 64.83, H 11.16, N 1.87, P 4.03. * This formula represents material consisting of equal parts of two lecithins, each one containing two of the four fatty acids. II. The Fatty Acids of Lecithin. For the preparation of fatty acids from lecithin, the material was hydrolyzed 8 to 15 hours with 10 parts of 10 per cent HCL. The fatty acids on cooling appeared as a semisolid cake. They were dissolved in methyl alcohol and precipitated in the pres- ence of ammonium hydroxide with a nearly equal weight of lead acetate dissolved in a minimum quantity of water. After freezing, the mother liquors were filtered off. The lead salts which con- tained both the saturated and unsaturated fatty acids were extracted repeatedly with boiling ether until further extraction produced only slight precipitate with hydrochloric acid. The ether solution then contained the lead salts of the un- saturated acids while the ether-insoluble material consisted of the lead salts of the saturated acids. Both fractions were de- composed with HCl, dissolved in ether, washed with water, dried, and the solvent evaporated off. A lot of 528 gm. of the lecithin cadmium chloride free from amino nitrogen was hydrolyzed with 10 per cent solution of hydrochloric acid. The yield of fatty acids was 223 gm. Unsaturated Fatty Acids. These were obtained by extracting the lead salt first by means of acetone and then by means of ether. Each extract was worked over separately. The acetone extract was concentrated and the residue thus. obtained extracted with ether. From both of these fractions the acids were liberated and reconverted into the lead salts. These were again purified and again converted into the free acids. A sample of the acids gave the following iodine and hydrogen values. 192 Liver Lecithin 0.2907 gm. of substance absorbed 0.435 gm. of iodine by the Wijs method. 0.5141 gm. of substance reduced by Paal’s method absorbed 67 cc. of H, in 3 hours at 17°C., 759 mm. pressure, or 1.103 gm. of H» per 100 gm. of substance. CysH;,02. Caleulated. Iodine value 91, Hydrogen number 0.721. CisH3202. Calculated. ‘ ae le, a +; 1.447. Found. ou bs: :% § 1.103. It was later found that this material consisted of two fatty acids, hence it is possible that one was a singly unsaturated, and that the other contained two or more double bonds. The free fatty acids were finally reduced by Paal’s method. The samples of reduced acids obtained from each fraction analyzed as follows: No. 84 (material obtained from the acetone extract of the lead salts). 0.1020 gm. of substance gave on combustion 0.1198 gm. of H,O and 0.2830 gm. of COr. No. 85 (material obtained from the ether extract of the lead salts). 0.1012 gm. of substance gave on combustion 0.1190 gm. of H.O and 0.2828 gm. of COs. No. 84. C 75.96, H 13.19. f -8b., C7621, aos Since the two fractions proved practically of identical elemen- tary composition they were combined and converted into the methyl esters. These were freed from adhering sulfuric acid by washing with water and finally by recrystallization from methyl alcohol. They were then fractionated by distilling at a pres- sure of 1 to 2mm. The following fractions were obtained. ara Dre Pa brs ks Oe RE ARS es haa NTE AR cae 182-185°C. Do cdos esc aee ot aera vinte ste: + 2:3 Beles Salty pe eats cytes 175-185°C. 208 CA Are eee te Bo che nies 0 ies SRNR nee eae ete 182-195°C. DT Sie, SARE SR cee eet sacs Sibge tea he teases eres 185-203°C. Fractions A and D were redistilled and the following fractions were obtained. From A I SRE A, SR Carts ins silos 5 TARE 158-165°C. Drak Ce eae ad ss Ais dy St Re RR Ty ey Ste 170-182°C. From D Dy. PR I te. 22 ee ae 182-192°C, Dias'.ot Sie site ses aagetonpece iets ke Ps eis eee 187-197°C, P.-A. Levene and H. 8. Simms 193 For identification, the esters were saponified with an alcoholic solution of sodium hydroxide. The acids were liberated and con- verted into the lead salts. The acids were again liberated from the lead salts and analyzed. Fraction A; corresponded apparently to pure stearic acid. Analysis 101. 0.1024 gm. of substance gave on combustion 0.1186 gm. of H:O and 0.2860 gm. of CO». 0.8950 gm. of substance in a molecular weight determination required 6.50 ce. of 0.6 Nn NaOH. CysH36Oo. Calculated. Cc 75.93, H 12.76. Found. C 76.16, H 12.96. Molecular weight was 275, that of stearic acid is 284. The substance melted at 70.5-71°C., stearic acid melts at 70-71°C. When this was mixed with some very pure stearic acid melting at 74°C., the mixture melted at 74°C. Fraction D. apparently corresponded to pure arachidic acid. Analysis 100. 0.1000 gm. of substance gave on combustion 0.1166 gm. of HO and 0.2822 gm. of COs. 0.9760 gm. of substance neutralized 6.75 cc. of 0.6 N NaOH. CooHaqOo. Calculated. C; 76.95, H 12.91. Found. C 76.97, H 13.24. Molecular weight was 314, that of arachidic acid is 313. The substance melted at 75.5-76°C., arachidic acid melts at 75-77°C. When this was mixed with some pure arachidic acid melting at 75°C., the mixture melted at 75°C. Saturated Fatty Acids. The lead salts which were insoluble in acetone and ether - were converted into free acids. These were twice esterified with methyl alcohol. The mixture of methyl esters thus obtained was distilled at a pressure of 1 to 2 mm. into the following fractions: Dos oe RM eg 2 Oe 160-163°C. EY 2, 2/08 SV Rt ie Als | a seal 159-167°C. 251 OR Bo ec sane A OSE 158-172°C.. FN). -. SON ain 5.0.0 a age 170-180°C. and residue. Fractions a and d were redistilled as follows. 194 Liver Lecithin From a Sis; 2 e's ee rae ec ee ie eee 156-162°C. Dake & incistoh ee ee aren ini ree oie Residue. From. di 4. di27) eee or orien oon se eee 180-183°C. Oa Sete Ee in canoe Sa ee ee 182-188°C. Fraction a; apparently corresponded to pure palmitic acid. Analysis 92. 0.1009 gm. of substance gave on combustion 0.1220 gm. of H.O and 0.2802 gm. of CO». 0.8168 gm. of substance neutralized 6.14 gm. of 0.5 n NaOH. CisH3202. Calculated. C 74.92, H 12.58. . Found. C 75.09, H 12.98. Molecular weight was found to be 266, palmitic acid had a molecular weight of 256. The melting point was 62°C., palmitic acid melts at 63-64°C. When this was mixed with some pure palmitic acid melting at 64°C.. the mixture melted at 63°C. Fraction ds apparently corresponded to pure stearic acid. Analysis 94. 0.1009 gm. of substance gave on combustion 0.1220 gm. of H2O and 0.6686 gm. of substance neutralized 4.82 ec. of 0.5 n NaOH. CisH 3602. Calculated. Cc 75.93, Jel IBArs. Found. C 75.72, H. 13.53. Molecular weight was 278, stearic acid had a molecular weight of 284. The substance melted at 71°C., stearic acid melts at 70-71°C. When this was mixed with a sample of very pure stearic acid melting at 74°C. the mixture melted at 74°C. III. Bromine Addition Products of the Unsaturated Acids. An attempt was made to separate and to characterize the individual unsaturated acids by preparing the bromine addition products. 40 gm. of pure lecithin which had been prepared from the cadmium chloride salt as described above, were used. No. 119. 0.0996 gm. of substance gave on combustion 0.0994 gm. of H.O, 0.2768 gm. of COs, and 0.0090 gm. of ash. 0.1798 gm. of substance required 2.40 cc. of 0.1 N acid, corresponding to 0.00336 gm. of N. 0.2697 gm. of substance gave 0.0390 gm. of Mg2P207. CysHg7Os5NP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. Found. C 64.83, H 11.16, N 1.87, P 4.03. P. A. Levene and H. 8. Simms 195 This was hydrolyzed with a 10 per cent solution of hydrochloric acid, the acids were dissolved in ether, washed with water, dried, and the ether evaporated off. The iodine number of the mixed acids was 91. 0.2457 gm. of substance absorbed 0.232 gm. of iodine by the Wijs method. Average molecular weight of 280. Calculated. Iodine value 91. Found. . So OFF The acids were converted into the lead salts, the unsaturated acids extracted with ether and converted into the free acids. These were dissolved in 18-30° petrolic ether and brominated at 0°C. with 3 cc. of bromine dissolved in petrolic ether. On freezing to —10° a precipitate was obtained. The mother liquor was concentrated and again cooled to —10°. The com- bined precipitate was recrystallized from petrolic ether and then recrystallized from ethyl ether. This gives three fractions: A, the petrolic ether-soluble fraction; B, the fraction insoluble in petrolic ether but soluble in ethyl ether; and C, the fraction insoluble in both solvents. This last fraction contains the material having most bromine (namely, the hexabromides and octobromides, if present). The first fraction should be largely dibromides while the tetrabromides should predominate in the fraction insoluble in petrolic ether but soluble in ethyl ether. Fraction C (the material insoluble in both solvents) was re- crystallized from ethyl ether; the yield was 1 gm. In an open tube melting point determination it darkened, turning black at 200°C. It contracted at 240°C. and decomposed at 248°C. In a closed tube it contracted at 239°C. and melted without decomposition at 243°C. This analyzed as follows: No. 129. 0.2012 gm. of substance gave 0.2936 gm. of AgBr. This would indicate a hexabromide. CooH3,02Brs. Calculated for hexabromarachidic acid. 61.0. CisH;,02Bre Calculated for hexabromstearic acid. 63.2. Found. 62.11. The material was recrystallized from ether. On heating in an open tube it darkened at 180-200°C:, contracted at 240°C., and decomposed at 244°C. 196 Liver Lecithin It analyzed as follows: Analysis 130. 0.1068 gm. of substance gave 0.1670 gm. of AgBr. This corresponds more closely to an octobromide. CooHg202Brs. Calculated for octobromarachidie acid. 67.80. Found. 66.55. There was not sufficient material for further treatment. IV. Hydrolecithin from Liver Lecithin. For the preparation of hydrolecithin 10 gm. of pure liver lecithin free from amino nitrogen were used (Analysis 119 given above). This was reduced by Paal’s method. The hydrolecithin pro- duced was recrystallized twice from acetone and once from methyl ethyl ketone. This analyzed as follows: No. 128. 0.1074 gm. of substance gave on combustion 0.1082 gm. of H.O, 0.2554 gm. of COs, and 0.0098 gm. of ash. 0.1926 gm. of substance for Kjeldahl nitrogen determination required 2.40 cc. of 0.1 N acid, corresponding to 0.00336 gm. of N. 0.2839 gm. of substance gave 0.0400 gm. of MgeP2O;. CuHoOsNP. Calculated. C 65.30, H 11.33, N 1.73, P 3.84. Found. C 65.03, H 11.29, N 1.74, P 3.86. A molecular weight determination was made as follows: 1.036 gm. of substance raised the boiling point of 16 gm. methyl alcohol 0.071°C. 0.964 gm. of substance raised the boiling point of 16 gm. methyl! alcohol 0.077°C. CysHoOoNP. Calculated. Molecular weight 809. Found. First determination 810. Second a 700. eS ee a ee ee ee eee ON THE NUMERICAL VALUES OF THE OPTICAL ROTA- TIONS IN THE SUGAR ACIDS. By P. A. LEVENE. (From the Laboratories of the Rockefeller Institute for Medical Research.) (Received for publication, July 7, 1921.) In recent years the van’t Hoff theory of optical superposition has been on several occasions the subject of discussion. Some writers, Rosanoff, and independently of him Patterson and Taylor have challenged the theory, whereas in a series of very important publications Hudson! has demonstrated the validity of the theory and made it the foundation of many important contributions to the chemistry of carbohydrates. The present writer also has made use of the theory for the purpose of establishing relationships between the configuration of the carbon atom 2 and the optical rotation of epimeric sugar acids. It was found that in a pair of epimeric sugar acids as in gluconic and mannonic acids the molecular structure may be regarded as consisting of two parts, one (A) consisting of carbon atoms 1 and 2, and the other (B) of carbon atoms 3, 4, 5, and 6 as seen from the following. (’: “COOH COOH + Aj ay. vl | HCOH OHCH | (OHCH (OHCH HCOH HCOH +B | +B | HCOH ee CH,OH CH,OH 1 Hudson, C.S., J. Am. Chem. Soc., 1909, xxxi, 66; 1917, xxxix, 462; 1918, xl, 813. 197 198 Optical Rotations in Sugar Acids It is evident that such pairs of epimers may be represented as +A —A | and ‘ +B +B. 2—hexosaminic acids the superposition theory holds to the follow- ing extent; the direction of the rotation of the carbon atom 2 is in agreement with the theory in all of the four pairs of hexonic and all of the four pairs of hexosaminic acids. The numerical value of the rotation of carbon atom 2 should according to the theory of van’t Hoff be identical for all hexonic and for all hexos- aminic acids. This expection was not realized. In three of the epimers of each group the values are identical, but in the fourth it is markedly different as seen from the following table. Tt was found that in sugar acids and in Phenylhy- D drazides of f| : La]o0 [a] ; atom 2. [ a], of [m]> earbon D atom 2. Epichitosaminie . .|+12.5 |+24.37 (102)} Gluconic. ...|+14.25)+42.18 (102) Chitosaminic...... \—12.5 |—24.37 (102) Mannonic:. .|—14.25]—42.18 (102) Dextro-xylohexos- | PHPANVO, = s 5605006 +12.5 +24.37 (102)| Gulonic...../+14.25)+42.18 (10?) Levo-xvlohexos- | ALAUDIC. Sp, e125 | 24.37 (102)|= Ldoniceereee —14.25)—42.18 (10?) Epichondrosaminie)/+12.5 4-24.37 (102)| Galaetonic ..'+ 8.25|/4+24.42 (10?) Chondrosaminic. ..|;—12.5 |—24.37 (10?)} Talonic...... — 8.25)—24.42 (10?) Dextro-ribohexos- AAT Ce eee +19.12)+-37.28 (10?)} Allonic...... +20.8 |+61.56 (102) Levo-ribohexos- | Sea. 5 aehadade [= 19.12|—37.28 (107)| Altronic ..... —20.8 |—61.56 (10?) Hudson was stimulated by our findings on the hexonie and hexosaminiec acids and developed a more general conclusion, that the direction of the rotation of the carbon atom 2 determines the direction of the optical rotation of the acid. This is seen by mere comparison of our tables without resorting to calculation. Hudson arrived at his conclusion in a very ingenious way. He assumed that the superposition theory holds literally in every detail and he concluded that the magnitude and the direction of the rotation of any one of the four asymmetric carbon atoms ty os P, A. Levene 199 in hexonic acid can be calculated from four equations each re- presenting the algebraic sum of the four asymmetric carbon atoms of one acid and of the value of its optical rotation in the following way. 1. d-Gluconie acid + a — Bty+s = (+12.0) (286) = +34.3 (102) 2. d-Gulonie “ + a—6— y+ 6 = (413.7) (286) = +39.2 (10?) 3. d-Idonic “ -—-a+t+Bp—7y+6 = (—12.4) (286) = —35.5 (107) 4. d-Galactonic“ + a— 6 — y+ 6 = (+11.0) (286) = +31.5 (10?) Solving these four equations, the following values are obtained: a = +87.3 (102); 6 = +3.9 (102); y = +1.4 (102); 6 = — 0.6 (102). Weerman? later working on the amides of sugar acids, corrob- orated the conclusions of Levene* +> and of Hudson. Hudson alone, and later with Komatsu® repeated the observations on a series of amides. They found the following values. a-carbon = +47.25 (102); @-carbon = — 14.65 (10?) ; y-carbon = +0.95 (102) ; é-carbon = —2.05 (102). The authors then remark: ‘‘It will be noticed that the numerical values decrease as the carbon atom is further removed from the amide end. . . The alternation in the sign of the rotation of the successive carbon atoms is also noteworthy suggesting the alternation in positive and negative affinity that is often ascribed to the carbons in a chain.’”’ Inasmuch as we have found that the value of the rotation of carbon atom 2 (a, according to Hud- son’s nomenclature) is not a constant for all acids, it was sug- gested that also the magnitudes of rotation of the other carbon atoms are influenced by the configuration of the adjacent carbon atoms. If that were so then the system adopted by Hudson for his calculations may be incorrect and may lead to erroneous conclusions. We therefore calculated the magnitudes of rotation of each of the four carbon atoms in hexonie and 2-aminohexonic acids from several combinations of four equations. If the magnitudes * Weerman, Dissertation, Amsterdam, 1916. 3 Levene, P. A., and Meyer, G. M., J. Biol. Chem., 1917, xxxi, 623. 4 Levene, P. A., J. Biol. Chem., 1916, xxvi, 367. 5 Levene, P. A., and Clark, E. P., J. Biol. Chem., 1921, xlvi, 19. § Hudson, C. 8., and Komatsu, S., J. Am: Chem. Soc., 1919, xli, 1141. 200 Optical Rotations in Sugar Acids of rotation of each carbon atom were constant the same values should be obtained from all combinations. It will be seen from the following table that the results ob- tained by Hudson and his coworkers are due to special conditions and that some combinations of four equations lead to values of rotation of the a-carbon atom (Hudson’s nomenclature) which are lower than some one other carbon atom. In Combinations V, VI, and VII the values for the rotation of every carbon atom remain constant. These combinations are com- posed of members in which the a-carbon atom is constant and equal to +12.5. On the other hand, in all combinations in which at least one member had the a-carbon atom of the value 19.5 the values for the other carbon atoms were variable. It seemed therefore that the values for 8-, y-, and 6-carbon atoms may possess constant and equal values when derived from the values of Part B of each acid, and not from that of the entire acid. Calculations again showed that the values for B-, y-, and 6-carbon atoms remained equal and constant when derived from values of Part B of acids having the rotation of a-carbon atom = 12.5, and not otherwise. This observation naturally leads to the conelusions that the superposition theory holds only within certain limits. In certain substances, as in hexonie acids, the vicinity of a certain group (carboxyl) accentuates the rotation of the a-carbon atom to such an extent that the direction of its rotation determines the direction of the rotation of the entire molecule. The correct- ness of this conclusion with certainty can be demonstrated only on a comparison of the rotation of pairs of epimers. It is, however, interesting to note that for the series of gluconic and mannonic, galactonic and talonic, gulonic and idonic, the superposition theory holds completely. Hexosaminie acid. [a], laly [Mp btytbB+a = +125 a = +11.625 +22.65 (102) 5—y—-Bt+ta=+ 80 B=+ 2.125 + 4.14 (102) é6+y—B—a= —15.0 y= +0125 + 2.44 (102) 5 = ys: p — 6 = — 1875 = 26:825(109) Phenylhydrazides of hexonic acids. Seiya — & Mae ya Pt @ oy 6 —-a iyo pb —-@ Oyo Ba NSS 9% == (8) == ay Oa. y =p aa OS Warp sr & OF 97 se Bap @& OA yg ea si erated diay pl) Oy aie ee iY = Bir a SS 97 = (= @: oy — B+ @ fe Na 8 a, @ Diya iat 6-y-B-2a be wy. — 6 —.a QO= W558 = @ te Ve Par & Ory = bee raya ee 0 iB ae (ites e's ca se at) ey — Bb ie Dieteia = 2. PSG cal eee P. A. Levene + 10.0 —lo.0 +14.0 + 8.0 [aly +25.8 +1222 —10.5 —15.0 Ut %2DWR Ill 2 WR IV O22 DR o2DWR Wal %2DWR VII m2 WA WATT oO 2 DR I ll +16.875 1 Sy — 3.375 — 4.875 Stele ap od = 4575 — 9.29 +13.15 (10?) = 1t-22 (102) = 9:23, (0?) — 18.04 (10?) +32.95 (10?) — 2.68 (10?) = oem) — 9.50 (10?) +38.02 (10?) +13.65 (10?) — 9.23 (10?) — 18.05 (10?) +24.38 (10?) + 5.85 (10?) +19.50 (10?) — 0:98 10?) +24.38 (10?) + 5.85 (10?) +19.50 (10?) — 0.98 (10?) +24.38 (10?) + 5.85 (10?) +19.50 (10?) — 0.98 (10?) (M], +27.95 (10?) + 23.93 (10?) +30.38 (10?) — 8.50 (10?) 201 202 Optical Rotations in Sugar Acids 16.4 : 6+%+B8B—a= —15.8 a = +10.95 +31.32 (10?) 6—y—-Bt+a= 412.2 B = — 2.65 — 7.58 (10?) 6+y7—-—B-—a= —10.5 y = — 0.40 — 1.15 (10?) 6—y+t+B-—a= —15.0 6 = — 1.80 + 5.16 (10?) x 6+y+t+B+a= +25.8 a = +20.8 +59.48 (10?) 6+y+B-—a= —-15.8 p= -- 7.15 +20.44 (10?) 6—y-—Bt+e = +12.2 y = — 0.35 — 1.00 (10?) 6—y+t+B-—a@= —15.1 6 = — 1.80 — 5.14 (10?) XI as Values of Part B. [Q]p IM], le], [M)p 64+7 +8 +a = +125, +2437 (108 6+7+8 — a = —26.5 —51.67 (102) AIT Poy SB ie 7 0 = 3815 (107) 6+7+8 = —6.75 —1346 (10?) 6—y—B=-45 —8.58 (10) XII b—y+e+e= 4140 427.3 (10) ,_ ‘ : Satya sareiil.0. =2145 00). > Uo ° XIV 6+y—B+t+a=+10.0 +19.5 (10?) 43 : yg — ee 15.0. 29.25.00) “Oi, Solving for XII, XIII, and XIV, the values obtained are 8 = +3.0, y = +1.0, and 6 = —0.5. Solving for XI, XII, and XIII, the values obtained are 8 = +3.0, y = —4.125, and 6 = —5.625. THE PREPARATION AND STANDARDIZATION OF COLLODION MEMBRANES. By ARNOLD H. EGGERTH. (From the Hoagland Laboratory, New York.) (Received for publication, July 6, 1921.) The use of collodion membranes in the study of diffusion phenomena was begun by Fick (1855); the closed collodion sac‘ was devised 5 years later by Schumacher (1860). Metchnikoff, Roux, and Taurelli-Salimbeni (1896), in their researches on the vibrio of Asiatic cholera introduced these sacs into biological science. Because of their ease of preparation, availability, thinness, and ready permeability to ordinary crystalloids, they have largely replaced the older parchment paper membranes in the study of dialysis and diffusion. For a complete bibliog- raphy and literature review of this development of their use, the reader is referred to the paper of Bigelow and Gemberling (1907). Mention should be made, however, of the, work of Gorsline (1903), who showed that erystalloids were not the only substances that could diffuse through a collodion membrane, for at 35°C., peptone, albumose, albumin, starch, dextrin, and certain enzymes all dialyzed through in less than 24 hours in recognizable quantities. The first successful attempt to produce a raged series of membranes of increasing permeability was made by Bechhold (1907, 1908). By impregnating filter papers with varying percentage strengths of gelatin or glacial acetic acid collodion, he produced membranes whose permeability varied inversely with the concentration of the impregnating solution. These membranes were used as filters. With the use of pressure, often many atmospheres, substances like hemoglobin, albumin, and various inorganic colloids were filtered through. A mechanical stirring device was employed to prevent the precipitation of the colloid on the surface of the membrane; such precipitated colloid 203 — 204 Collodion Membranes otherwise forming a very impervious film. The higher the pres- sure the more prone is this superimposed film to form. In a former investigation, De Kruif and the author (1919) were unable to prevent the formation of this film in the filtration of anaphy- latoxin, the membranes becoming rapidly less permeable as filtration proceeded. Another method of increasing and regulating the permeability of collodion membranes is that of Schoep (1911). Increased permeability is secured by adding 2 to 10 per cent of glycerol to an alcohol-ether solution of Schering’s celloidin; 4 per cent of castor oil is added to give the membranes elasticity. A wide range of permeability is secured by varying the amount of glycerol added. These membranes are far more permeable than those of Bechhold; very low pressures are sufficient for the fil- tration of substances like albumin or hemoglobin. There is little tendency to form the objectionable colloidal film on the filtering surface. De Kruif and Eggerth (1919) used them with success In filtering the greater part of the serum proteins from anaphylatoxin. Brown (1915) developed another method of varying permeabil- itv. Collodion sacs are air-dried and then immersed in mixtures of ethyl alcohol and water; the greater the percentage of alcohol, the more they swell, and the more permeable is the resulting membrane. This method gives a valuable series, but even the most permeable members of it (those treated with 90 to 96 per cent alcohol) do not allow much diffusion of sustances such as Congo red or hemoglobin; dialysis for 2 to 7 days allows only small quantities to pass through. The Brown series, excellent in its scope, needs to be extended in the direction of greater permeability. The Schoep membranes seem to answer that need. But the Schoep membranes are made from Schering’s celloidin, which cannot be purchased in the United States now. Several brands of American pyroxylins were tried in the Schoep formulas with poor success. If little glycerol was used, the membranes were very impermeable; if the amount of glycerol was increased, they were so fragile as to be useless. Considerable improvement was effected by substituting 10 per cent of acetic or lactic acid for the castor oil used by Schoep; a stronger membrane with a A. H. Eggerth 205 considerable range of permeability was obtained. While attempting to adapt the Schoep formulas to the available pyroxylin, it was observed that merely altering the proportion of alcohol to ether in the solvent mixture sufficed to alter the permeability of the resulting membranes through a wide and easily controllable range. ‘These membranes have the advantage of being free from castor oil, they are transparent, and stronger than those of Schoep. A similar observation has been made by Malfitano, who states (1908) that the higher the proportion of alcohol in the solvent, the more permeable the membrane, and in 1910 he stated that Michel and Lazarus in his laboratory had demonstrated that alcohol-rich solutions give more permeable membranes than those that are ether-rich. No further details are given. The pyroxylin used in this investigation is that manufactured by Du Pont De Nemours and Company under the name of parlodion. All of the solutions referred to in this paper contained 6 gm. of the dry collodion in 100 gm. of alcohol-ether solvent. The parts of alcohol and ether were all taken by weight. To designate the different solutions and membranes, the following nomenclature was adopted: a solution is named according to the parts by weight of alcohol it contains; one containing 50 parts of alcohol and 50 of ether is a ‘‘50 alcohol solution;”’ a membrane made from this solution is designated a ‘‘50 alcohol membrane.” The ethyl alcohol first used in making up the solvent was distilled over anhydrous copper sulfate; later in the work it was distilled over calcium oxide and redistilled over metallic sodium. The ether was distilled over sodium. Alcohol-ether mixtures containing from 20 to 80 parts of alcohol by weight readily dissolve 6 per cent of parlodion; but to obtain solutions containing 10, 15, 85, and 90 parts of alcohol, it is necessary first to make a thick solution of the collodion in a portion of the solvents and later add the remainder of the alcohol or ether, as the case may be. On adding the last part of the ether to the 10 alcohol solution of collodion, the solution has on two occasions set to a gel, which could be redissolved by adding another per cent of alcohol. On two other occasions, a white flocculent precipitate formed, which failed to redissolve. This was allowed to settle out, and the supernatant solution was used in making the membranes. 206 Collodion Membranes’ The different collodion solutions differ considerably in their viscosity, as can be seen by referring to Table I. Viscosities were determined rather roughly by noting the time in seconds required for the solutions to flow out of the same 10 ce. pipette. The 20 alcohol solution was found to be the least viscous, viscosity increasing regularly in both directions in the series. The changes in viscosity of these solutions after a small amount of water has been added are noteworthy. The alcohol-rich TABLE I. Relative Viscosities at 20°C. 5 per cent Solution. Anhydrous.| of water Remarks. added. 10 alcohol. 35 18 Transparent emulsion. 15 as 26 20" ** 20 15 Granular precipitate. Redis- solves readily. S01" 23 AD as 29 26 Granular precipitate. Redis- solves readily. 50 cs 34 aoe” 40 41 Gelatinous precipitate. Redis- solves with difficulty. 70 “3 45 | 53 85* Sets to gel in 24 hours. a at 64 00 85 —* Sets-to gel. *2 per cent of water added. solutions are made more viscous (only 2 per cent of moisture setting the 90 alcohol solution to a firm gel) while the ether-rich solutions are made more fluid by the same agent. Several batches of 90 alcohol solutions, made up with supposedly anhy- drous solvents, slowly increased in viscosity, and after several days, set|to a semisolid gel; not until the last traces of water were removed from the alcohol by redistillation over sodium was a 90 alcohol solution obtained that could be kept for weeks with- out marked change in viscosity. A. H. Eggerth 207 Method of Making the Sacs. The collodion sacs were made by the method devised by Novy,! with a few slight modifications. A glass tube, melted down at one end to leave a small hole, is the mold. A small fragment of cigarette paper is slightly moistened and placed over the hole, where it dries quickly; a layer of collodion is painted over the paper and the end of the tube; this is allowed to dry for 20 to 30 seconds. A few cubic centimeters of collodion solution are poured into a test-tube; this is held nearly horizontal while the end of the mold tube is immersed in the collodion. The mold tube is rotated slowly, and slowly withdrawn; then it, with its covering of collodion, is thrust horizontally into a large test-tube that lies on the table before the operator, the mold tube being rotated rapidly all the time. In these experiments the membranes were always dried for 1 minute, and then immersed in water. Rotat- ing the membrane within the large test-tube in the manner de- scribed makes the drying slower and more uniform, and cuts off air currents. It was found to be good practice never to return any unused collodion to the stock bottle. By filling the mold tube with water and applying air pressure to the open end with the mouth, it is usually not difficult to force water between the glass and the membrane, and so easily remove the latter. The tube and the membrane should be immersed during this process. Sometimes the membranes adhere to the glass at the edge of the hole, and no amount of gentle manipula- tion with the fingers can free them. Increased pressure finally breaks through, but often tears the membrane. If a_ short rubber tube (not too thin-walled) is slipped over the end of the mold tube and filled with water, and a small cork stopper is pushed slowly down the rubber tube, the membrane can be ‘‘started’”’ with ease and without tearing, this expedient allow- ing the application of a great deal of force with a minimum of displacement. By pinching on the rubber tube as the cork is withdrawn, too much negative pressure is avoided. After loosening the membrane in this way, it can be easily removed by blowing with the mouth. The ether-rich membranes (the 14 description of this method is given in the paper by Bigelow and Gemberling (1907). 208 Collodion Membranes 20, 39, and 40 alcohol solutions), adhere very tightly to the glass and the percentage of spoiled membranes is very high unless the rubber tube and cork are used. The 20 and 30 alcohol membranes require in addition a little manipulation with the fingers at the very end to free them. The 10 and 15 aleohol membranes adhere so tightly to the glass that they cannot be made by this method at all, nor can they be made on the inside of a test-tube. A solid glass rod was ground down so that it tapered from a diameter of 11.0 mm. at a point 8 em. from the end to 10.6 mm. at the end itself. The end of the rod is dipped into the collodion solution to a depth of about 1 cm.; this coat is allowed to dry for 15 to 20 seconds, and then the entire membrane is made as described above. Two coats are necessary at the extreme end, otherwise this portion is so thin that it is certain to tear. After immersing in waier, the mem- brane is removed by peeling it down from the top. For other membranes of the series, this method does not work so well as the one first described. Method of Conducting Diffusion Experiments. The sacs, usually prepared on the preceding day, were each attached by means of a rubber band to a glass tube about 8 cm. long; this glass tube passed through a cork which had a longi- tudinal groove cut in the side. The corks fitted into the necks of test-tubes of 4 inch diameter. Sacs made by the first method described had a diameter of 8.3 mm. in all experiments, 3 ce. of test substance were placed in each sac, which filled it to a depth of 6cm. The volume of the dialysate was always 10 cc., this kept constant throughout the experiment. The sacs were always immersed to a depth of 6 em. The membranes made by the second method were larger, with an average diameter of 10.8 mm. 5 ec. of test substance were used, and they were immersed to a depth of 8 em. All of the membranes used in the diffusion of NaCl, KH».PO,, saccharose, and indigo carmine (Fig. 3) were made by the second method. In the diffusion experiments performed, four or five and sometimes more sacs made from the same solution were tested each time. Thus the experiment plotted in Fig. 1 required thirty sacs, five sacs of each of six grades of permeability. -- = ———— Es So. er ee “———s A. H. Eggerth 209 Wherever possible, quantitative determinations of the amount of test substance that had passed through the membranes were made at different time intervals and the results plotted as in Figs. 1 and 2. Test Substances and Method of Their Estimation in the Dialysate. Two Per Cent Aqueous Sodium Chloride.—Estimated by titration with a standard silver nitrate solution, using potassium di- chromate as an indicator. 0.2 M Potassium Dihydrogen Phosphate.—Estimated by titra- tion with 0.1 Nn sodium hydroxide using phenolphthalein as an indicator and titrating to a full red color. Saccharose, 20 Per Cent Solution—FEstimated with the polari- meter. Raffinose, 8 Per Cent Solution—Estimated with the polari- meter. Indigo Carmine (Sodium Sulfindigotate), 0.2 Per Cent Aqueous Solution.—Estimated with the colorimeter. Safranine, 1 Per Cent Aqueous Solution.—FKstimated with the colorimeter. Primary Proteose, 1 Per Cent Aqueous Solution at pH = 7.0.— This was prepared from Difco peptone by precipitating with (NH,4)2SO,, and purified by reprecipitating three times with (NH4)2SO, to half saturation, The proteose was estimated by adding 6 cc. of saturated (NH,)2SO, to 4 cc. of the dialysate and determining the precipitate nephelometrically. Dialyzed Serum Protein.—No effort was made to separate the albumin from the pseudoglobulin, but the euglobulin was removed by centrifugation. To 10 ce. of sheep serum 1.8 cc. of 0.2 N HCl were added; this brings the serum approximately to the optimum reaction for the flocculation of euglobulin. This was then dialyzed in a 40 alcohol membrane against distilled water. The volume was then made up to three times the original volume of the serum. In one experiment, the protein in the dialysate was precipitated with sulfosalicylic acid and determined nephe- lometrically; in another, the nitrogen was determined by the micro-Kjeldahl method of Folin and Wu (1919), and the protein computed. . oe THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 1 210 Collodion Membranes Oxyhemoglobin.—Sheep cells were washed three times with isotonic salt solution. An equal volume of distilled water was added to the packed cells and cooled; then a half volume of cold ether was added and shaken. After standing in the ice box over night, the lower layer was drawn off and diluted to a volume ten times as great as that of the original packed cells. It was estimated with the colorimeter. Carboxyhemoglobin.—Carbon monoxide, prepared from oxalic acid and sulfuric acid, was bubbled through a solution of oxyhemo- globin prepared as above. Methemoglobin.—To the concentrated oxyhemoglobin solution was added a half volume of 10 per cent potassium ferricyanide; this was now dialyzed in a large 40 alcohol sac until the dialysate was free from ferricyanide. It was estimated colorimetrically. Congo Red (Grubler’s).—A 1 per cent aqueous solution was used. This has a pH of about 8.6. It was estimated colorimetrically. ‘In the diffusion experiments the proteose, serum protein, and hemoglobin solutions were dialyzed against a buffered phosphate solution set at pH 7.0, made by adding 29.6 cc. of 0.2 n NaOH to 50 ce. of 0.2 mM KH»PO, and diluting to 300 ee. The other test substances were dialyzed against distilled water. An _ effort was made to stabilize the reaction of the Congo red solution by using a buffered dialysate, but the. presence of electrolytes greatly reduces the solubility of Congo red, so this was abandoned. Fig. 1 shows the passage of Congo red through membranes of six grades of permeability. The 85 alcohol membranes allow very rapid diffusion of this typical ‘‘non-dialyzable colloid,” while the 40 aleohol membranes allow only traces to pass through in the first 2 or 3 hours. The 30 alcohol membranes, not shown on the figure, allow no diffusion in 24 hours. In 5 hours, the 85 alcohol membranes have passed twelve times as much Congo red as the 50 alcohol ones. sv referring to Table II it will be seen that the thickness. of these membranes increases towards the alcohol-rich end of the the series. The higher alcohol membranes cannot be made any thinner without impairing their strength; any attempt to secure uniformity of thickness in the entire series would involve thickening the ether-rich membranes to an unnecessary and undesirable extent. It is obvious that if the membranes had Ss = ee eee eS Ss A. H. Eggerth ZAA all been of the same thickness, the differences between the curves shown would, if anything, be greater; certainly not less, since the alcohol-rich membranes are the thickest and at the same time the most permeable. A 1 per cent solution of Congo red exerts an osmotic pressure which causes an increase in the volume of the dialysee. This increase in volume is greatest with the least permeable mem- So BE Rest aay ge ; j Sa es | a : 2, plead om ee : me. = — Dee ay © ea Jae ee ae TIME IN HouRS Fig. 1. Diffusion of 1 per cent aqueous Congo red at 35°C. through membranes of six grades of permeability. The numbers at the margin are the alcohol numbers of the membranes. branes, and becomes less as permeability increases. Thus, the 40 alcohol membranes raised a column averaging 18.0 cm. in 8 hours; the 85 alcohol membranes raised a maximum column of 3.0 cm. in 3 hours, after which it began to fall. With the aid of a ruler, it was possible to place the thirty membranes used in this experiment in their correct order of permeability. Other test substances, such as hemoglobin and serum protein, Membrane. 10 alcohol. 30 40 60 a0 C= we bo 19.8 r Empty space Thickness (meas TABLE I. =D; mount filtered lated) 0.82 22.20.83 23.50.83 Fee '22.7|0.84 23.00.84 oe 0.89 25.6|0.89 25.6/0.90 2/0.92 .7|0.90 50.91 0.0.93 .2|0.92 410.92 23.00.93 mm. 0.017 0.015 20.015 0.015 0.011 0.011 0.015 0.016 0.014 0.020 0.017 0.015 10.035 0.025 inane 0.040 0.030 0.035 0.050 0.040 0.045 0.065 0.060 0.060 10.055 | Thickness (caleu mm. 0.014 0.011 0.011 0.011 0.011 0.010 0.009 0.013 0.013 0.011 0.021 0.019 0.015 0.034 0.028 0.032 0.043 0.035 0.043 0.050 0.038 10.040 10. 074 0.070 25.6/0.93,0.052)0. 063 21.40.93,0.050/0.055 |22.7 0.93 24.80.93 (24.00.93 .0.080,0. 080! 0.090 0.083 0.060) 0.051). 380 380 380 (0.011 0.010 200 3,0.012)0. 012/200 20.011 (200 187 215 187 212 min. Or i F RL 0.00033 2 0.8.0. 00017 6 0.00011 5/0. 00045 .10.00039 0,0. 00033 0.00210 0.00130 0.00075 bo oO — 0.00890 0.00580 0.00490 sI_ bt 0.02500 4 i] Wet weight. Dry weight. gm. 0.13)0.0380)0.0220)1.78 0.11)0.0290}0.0151)1.92 0.10)0.0290'0.0148)1.95 0.15)0.0250\0. 0099 2.5 0.14)0.0350)0.0164/2.1 0.13)0.0330)0.0165|2.0 0.21 0.020010: Cae 0.19,0.0190,0.0070/2.7 0.17|0.0170.0.0067|2.5 0.31 0.0240/0. 0072 3.3 0.28 0.0230 0.00703.3 0.26,0.0200 0.0064)3.1 0.40,0.0370 H> O> Sor ior) DON dO co Or &> 0 0 Or Or Or oO or 0.02000)/0.37,0.0340 0.01600\0.36)0.0270 ie may 0.02800|0.41,0.0560 0.02500/0.40,0.0480 0.02500/0.40'0. 0560 oe uo 0.04900)0.47|0.0730 0.03500/0.43)/0.0600 0.03300|0.43)0.0730 0.08000/0.53)/0.0840 0.06600)0.51/0.0650 0.05700)0.49/0. 0680 or) 0.10300)0.57|0. 1230 0.09100)0.55}0. 1000 0.07700/0.53/0.0860 4,0. 17000\0.65/0.1170 0,0. 16000'0.63/0. 1050 .0)}0. 14000)0.61)0.0920 (0.185000. 66/0. 1500 0.0095\3.9 0.0083/4.1 0.0066/4.1 0.0115/5.0 0.0095/5.0 0.01145.0 0.0116,6.3 0.0099)6.1 0.0106\6.9 0.0104'8.0 0.00956.9 0.0095 )7.2 0.0133)9.2 0.0120)8.3 0.0107)8.0 0.0124'9.4 0.0117,9.0 0.0102)9.0 0.01659.1 ot 0.15000 0.63/0. 1380 0.1 2600 0.60)0. 1340 0.0147/9.3 0.0146 9.3 . - ———— —— et Pe) A. H. Eggerth 213 which were dialyzed against a buffered phosphate solution, showed the same phenomenon, though here the columns raised did not exceed 1 to 2 cm. during the time of the experiment. Fig. 2 shows in detail the diffusion of methemoglobin through the same six grades of membranes. It will be observed that diffusion through the 40, 50, 60, and 70 alcohol membranes is more rapid than with Congo red; for the 85 alcohol membranes, 50 BO 70 4 a = 60 Le) = =r, SS 2 50 O82 od tr aw * TIME IN HOURS Fig 2. Diffusion of methemoglobin at 35°C. The numbers at the margin are the alcohol numbers of the membranes. The 85 alcohol series is omitted as the points have the same distribution as for the 80 alcohol series. it is slower. Also, there is no essential difference in the diffusion of this substance through the 70, 80, and 85 membranes. It is probable that the methemoglobin molecule is smaller than that of Congo red, hence it passes more easily through the less per- meable membranes. Further increase in permeability beyond the 70 alcohol membrane can no longer hasten the diffusion, which is limited by the diffusion constant of methemoglobin in water. 214 Collodion Membranes Congo red, with a larger aggregate, is more retarded by the lower membranes of the series; but being highly ionized relative to the methemoglobin, its diffusion constant in water is greater and it passes the 85 alcohol membranes more rapidly than the latter substance. The curves for oxyhemoglobin and carboxyhemoglobin are not given, as they so closely resemble those of methemoglobin. Ww ™® > a PER CENT DIFFUSED eine! ace OF MEMBRANES ~~ Fia. 3. A, sodium chloride, 10 minutes at 20°C. B, potassium dihy- drogen phosphate, 10 minutes at 20°C. C, saccharose, 30 ee at 20°C. D, indigo carmine, 20 minutes at 20°C. E, primary proteose, 2} hours at 20°C. F, methemoglobin, 3 hours at 35°C. G, Congo red, 3 hours at 35°C. H, dialyzed serum, 3 hours at 35°C. The behavior of indigo carmine and safranine was also studied in detail. Indigo carmine diffuses very slowly through the 10 alco- hol membranes; the speed of diffusion increases with the alcohol numbers up to the 40 alcohol membranes, after which there is no further appreciable increase. The behavior of this substance is given in abbreviated form in Fig. 3. Safranine is somewhat less diffusible than indigo carmine; otherwise their curves are very similar. A. H. Eggerth 215 Test substances, not colored, were tested for one time interval only. The behavior of these substances is shown in Fig. 3. Here the alcohol numbers of the membranes are plotted against the percentage of test substance diffused; the times and tempera- tures are indicated. Congo red and methemoglobin are in- cluded for comparison. The points on these two curves are omitted for the sake of simplifying the figure, as they may be found in Figs. 1 and 2. The curves of oxyhemoglobin and carboxy- hemoglobin, safranine, and raffinose are also omitted for the same reason. With all the test substances, it is apparent that the permeability of the membranes increases with their alcohol numbers. It is not altogether certain that the rise in the sodium chloride curve represents a real rise in the permeability of the membrane series to this test substance. If we consider a membrane as made ~ up of a solid meshwork containing water-filled pores or spaces, then the true diffusing area is not the area of the membrane, but the sum of the areas of the pores. The proportion of water- filled spaces is not the same throughout the membrane series, as may be seen by referring to Column 3 of Table II. The 20 alcohol membranes have, on this theory, one-sixth more water-filled space than the 10 aleohol membranes of the same area. This is sufficient to account for the rise of the sodium chloride curve. This line of reasoning will not, however, explain away the rise in the curves of the other test substances studied as over 50 per cent more of phosphate, and 100 per cent more of saccharose diffused through the 20 aleohol membrane than through the 10, in the same length of time. The 70 alcohol membranes have about one-twelfth more water-filled space than the 50 alcohol membranes, yet twenty times as much serum protein diffused through them in the same length of time. Fig. 4 shows the effect of different temperatures on the rate of diffusion of methemoglobin through 70 alcohol membranes. At the lower temperatures it will be noticed that the rise in the curve is preceded by an appreciable flat portion, representing a period during which little or no test substance diffuses through. The membrane, apparently, must be ‘‘saturated’”’ with the sub- stance before any of it can appear in the dialysate. If a methe- moglobin solution is filtered under low pressure, through a 60 216 Collodion Membranes or 70 alcohol membrane, the first few cubic centimeters are colorless; only after the membrane is saturated with the test substance will any of it come through. If a very dilute solution, such as 1 to 1,000 Congo red, is used in diffusion experiments, little or none may appear in the dialysate even when the mem- brane is very permeable to that substance. To obtain consistent results with Congo red, it was found necessary to use a more ts wr = ta, Ce, 23 10@, fo — Cx) ro) 2 GJ a. TIME IN Hours Fic. 4. Diffusion of methemoglobin through 70 alcohol membranes at different temperatures. concentrated solution, so that the amount taken up by the mem- brane is small in comparison to the total amount. The velocity of the filtration of water through membranes was used by Bechhold (1908), not only for demonstrating differ- ences of permeability but also for calculating the diameters of the pores. This method is based upon the application to mem- branes of the law of Poiseuille for the passage of fluid through a capillary. The membrane is considered as a number of capillaries whose length is the thickness of the membrane. A. H. Eggerth 217 4 : PD : : This law states that Q = k Te where Q is the quantity of fluid passed through unit area in unit time, P the pressure, D the diameter of the capillary (pore), L the length of the capillary (thickness of membrane), and k a constant depending on the nature of the fluid, the temperature, and the units chosen. jg From this equation, it follows that D = \~ . Hence, with Se See 2a e se eeeee ate eee See “ALCOHOL NUMBERS “OF MEMBRANES Fie. 5. Values of VOL for the geist series. Pressure, 60 mm. of Hg; temperature, 20°C. 0.2 constant pressure and temperature, D will vary as 4/QL. The value of QL can be readily calculated from the experimental data, This has been done in Table II. Only three membranes of each series are given in this table, selected to show maximum, mean, and minimum values of QL. The values of 4/QL are plotted in Fig. 5. No attempt was made to determine the value of the constant k, without which D cannot be computed. Method of Conducting Water Filtration Experiments. Bulbs of about 10 ce. capacity were blown from 8 mm. tubing; the two ends were cut down to 2 cm. in length. Each membrane 218 Collodion Membranes was attached to one of the ends of a bulb with a strong rubber band; the joint was dried with a towel and painted over with a thick 30 alcohol solution of collodion. When dry, the joint was tested for air-tightness. The sac was emptied and 12 cc. of distilled water were measured into it. A rubber tube over the other end of the bulb, led to a pressure tank and manometer. The filled sac was completely immersed in a test-tube of water, which stood in a constant temperature bath. A pressure of 60 mm. of Hg and a temperature of 20°C. were used in all experi- ments. Filtration was continued until the bulb, but not the sac, was empty. After filtration, the sac was emptied and tested for leaks; the volume of the residual water was measured. The thickness of the membranes was measured with a micro- _ meter, taking the mean of several readings at different places. The thickened bottom of the sac was removed; a piece 6 cm. long was cut off, rapidly blotted between filter papers, and weighed in a weighing bottle. This gave the wet weight. Each membrane was then air-dried and weighed again. The specific gravity of dry parlodion was found to be 1.608 at 20°C. The volume in cubic centimeters occupied by the membrane is then equal to dry weight Wet weight — dry weight + 1.608 all weights being expressed in grams. Dividing this value by the area of the membrane gives its thickness (Column 5, Table II). As the thickness obtained in this manner gave more consistent results than those obtained by measuring with the micrometer, this was used in calculating the value of QL. In making this calculation, the unit for L was taken as 0.01 mm. The ratio of water-filled space to total volume was calculated as follows: Pe wet weight — dry weight volume Walpole (1915) and Brown (1915, 1917) showed that the ratio of the dry to the wet weight of a membrane was an index of its permeability. Their conclusion is fully supported by this in- vestigation, as may be seen in the last column of Table II. A. H. Eggerth 219 In the course of this investigation, a number of other substances were added to the alcohol-ether solvents, in the hope of improving the series. The most promising were certain of the organic acids. If acetic acid, for instance, is added to the solvent, it becomes much easier to make the membrane; instead of shrinking to the glass on immersing in water, the collodion film loosens from it readily and slips off with ease. 10 to 20 per cent of acetic acid seems also to give added durability and elasticity to the resulting membrane, and it increases the amount of non- solvent, such as glycerol or water, that may be added to the solution. The addition of this amount of acetic acid makes the membranes less permeable (only the 50 and 70 alcohol mem- branes were tried). Oxalic and citric acids and phenol made 70 alcohol membranes slightly more permeable when added in 5 per cent amounts. Lactic acid, however, added to the solutions in amounts varying from 10 to 30 per cent, greatly increases the permeability of the resulting membranes. Glycerol and water both increase permeability, though neither are as effective as lactic acid. Since the work of Metchnikoff, collodion sacs have been exten- sively used in bacteriology and serology. For this work, it is desirable to sterilize the membranes by heat. Heating is accom- panied by two undesirable changes, marked shrinkage and marked decrease in permeability. This matter has recently been investigated by Gates (1921), who gels his membranes in 95 per cent alcohol before immersing them in water; on heat sterilization, his membranes shrink about 33 per cent in volume and are still readily permeable to simple salts and glucose, though quite impermeable to protein and hemoglobin. It would seem desirable to prepare membranes that would allow the passage of proteins after heat sterilization; with this in view, the following experiments were conducted: Sacs were prepared from the 80 alcohol solution. Some were immersed in 95 per cent alcohol, after the method of Gates. Others were made from the 80 alcohol solution plus 10 per cent of lactic acid, and immersed directly into water and washed free from acid. When autoclaved at 20 pounds pressure for 30 minutes, both sets of membranes shrank over 50 per cent in volume; when sterilized by three steamings in the Arnold 220 Collodion Membranes sterilizer, the shrinkage was very nearly 33 per cent for both sets of membranes. When tested with methemoglobin solution at 35°C., both sets of membranes were found to transmit it readily, the lactic acid membranes being more permeable. The latter had the permeability of unheated 60 alcohol membranes. Sacs made by both methods are serviceable and strong; they will stand over 25 em. Hg of pressure without bursting. Staphy- lococcus aureus was grown in such sterile membranes for 2 months without contaminating the surrounding broth. Bacillus influenze was grown in pure culture in plain broth without hemoglobin by growing it on the inside of a sterilized sac, with a living culture of staphylococcus, streptococcus, or pneumococcus growing on the outside; controls without the symbiotic organisms showed no growth of Bacillus influenze. By this means it is possible to study the symbiosis of organisms while keeping each of them in pure culture. The value of a method depends, among other things, on whether the results with it can regularly be reproduced. Most of the experiments described above were repeated many times, in whole or in part; fresh collodion solutions were made up several times from different lots of materials. Results ob- tained with membranes in the series between the alcohol numbers of 20 and 85 could be reproduced without any trouble. Such variations as occurred between different batches are believed to have been due to the presence of small amounts of water in solution. With the extremes of the series, more difficulty was encountered. The 90 alcohol membranes gave such irregular results that they have not been included in the series; they were several times found to be less permeable than the 85 or even the 80 alcohol membranes. This may in part have been due to their great variations in thickness, as the viscosity of the 90 alcohol solutions was found to be very variable. By referring to Table I, it will be seen that very small quantities of moisture suffice to cause great changes in viscosity. Variations in permeability were also found in the 10 and 15 aleohol membranes of different batches. One batch gave an almost horizontal line for the diffusion of sodium chloride and potassium dihy- drogen phosphate, when plotted out as in Fig. 3. Three other batches gave more constant results. A. H. Eggerth 221 SUMMARY. A simple method of preparing a graded series of collodion membranes of a wide range of permeability is presented, with quantitative data on the diffusion of various test substances through the different grades of the series. BIBLIOGRAPHY. Bechhold, H., Z. physik. Chem., 1907, lx, 257; 1908, Ixiv, 328. Bigelow, 8. L., and Gemberling, A., J. Am. Chem. Soc., 1907, xxix, 1576. Brown, W., Biochem. J., 1915, ix, 594; 1917, xi, 40. DeKruif, P. H., and Eggerth, A. H., J. Infect. Dis., 1919, xxiv, 505. Fick, A., Ann. Physik. uw. Chem., 1855, xciv, 59. Folin, O., and Wu, H., J. Biol. Chem., 1919, xxxviii, 81. Gates, F. L., J. Exp. Med., 1921, xxxiii, 25. Gorsline, C. 8., Contributions to medical research, Ann Arbor, 1903, 390. Malfitano, G., Rev. gén. Sci. pures et applig., 1908, xix, 617; Z. physik. Chem., 1910, Ixvili, 243. Metchnikoff, E., Roux, E., and Taurelli-Salimbeni, Ann. Inst. Pasteur 1896, x, 261. Schoep, A., Kolloid. Z., 1911, viii, 80. Schumacher, W., Ann. Physik. u. Chem., 1860, ex, 337. Walpole, G. 8., Biochem. J., 1915, ix, 287. ~ THE DIRECT QUANTITATIVE DETERMINATION OF SODIUM, POTASSIUM, CALCIUM, AND MAGNE- SIUM IN SMALL AMOUNTS OF BLOOD. By BENJAMIN KRAMER anp FREDERICK F. TISDALL. (From the Department of Pediatrics, The Johns Hopkins University, Baltimore.) (Received for publication, June 8, 1921.) The concentration of sodium, potassium, calcium, and magnes- ium in the blood of animals has been determined by Bunge (1), Abderhalden (2), and more recently by Greenwald (3). These investigators used from 25 to 100 cc. of blood for their deter- minations. Such quantities of blood cannot conveniently be used in studies with patients, particularly children. We have therefore devised a method by means of which the concentration of all these elements may be quantitatively determined on 7 cc, of blood. ’ Principle of the Method. Deproteinization is carried out by means of the trichloroacetic acid method recommended by Greenwald (4). The determina- tions of the individual elements, except that of magnesium, are then made directly on separate aliquots of the deproteinized fluid by modifications of methods previously decribed by us for serum (5, 6, 7). The inorganic or acid-soluble phosphorus may also be determined by any of the well known micro methods (8) on a portion of the supernatant fluid corresponding to 0.5 or 1.00 cc. of blood. Methods. Collection of Material and Deproteinization—25 cc. of distilled water are placed in a 50 ee. volumetric flask which is then weighed. From 7 to 83 ec. of blood are obtained by means of a 10 ce. grad- uated syringe and slowly added to the water in the flask. The flask should be continuously rotated during this procedure which 223 224 Inorganic Elements of Blood completely hemolyzes the blood. The flask and contents are again weighed and the exact amount of blood added thereby determined. 1 or 2 drops of octyl alcohol are added followed by 12 to 13 ce. of 12 per cent trichloroacetic acid which are added slowly while the flask is rotated. The contents are thoroughly mixed and allowed to stand 10 minutes. Water is added to 50 cc., the contents are again mixed, transferred to a large centrifuged tube and centrifuged for 5 to 10 minutes at about 1,000 revolutions per minute. The supernatant fluid is poured off and an aliquot (gener- ally 35 ce.) is placed in a beaker and evaporated. The presence of of a few particles of the precipitate which sometimes float on the surface of the supernatant fluid does not interfere with the subsequent determinations. If the particles are very numerous, the fluid may be allowed to stand in the ice chest for a few hours. They will then have settled to the bottom. The supernatant fluid may be kept at this stage for at least 2 weeks before com- pleting the determination. After the aliquot has been evapo- rated to dryness the residue is dissolved in 0.1 nN HCl and is transferred to a volumetric flask and the volume made up to 10 ec. This fluid has the appearance of serum. Should it be cloudy it may be centrifuged for a few minutes when a clear, straw- colored supernatant liquid will be obtained. The sodium, potassium, and calcium determinations are done directly on this material while the magnesium determination is done on the first supernatant fluid obtained from the calcium determination. Sodium Method. 4 ec. of the. material prepared as outlined above are placed in a platinum dish and evaporated to about 2 ec. A drop of phenolsulfonephthalein is added and the contents are made just alkaline with 10 per cent KOH (generally about 10 to 12 drops will suffice). 10 cc. of the potassium pyroantimonate reagent are added followed by 3 cc. of 95 per cent alcohol. The alcohol should be added drop by drop and the specimen stirred with a rubber-tipped rod. After standing 30 minutes the precipitate . is transferred to a weighed Gooch crucible and washed with 5 tol0 ec. of 30 per cent alcohol. The crucible is dried at 110° C. for 1 hour,! cooled in a desiccator for 30 minutes, and weighed. 1 The temperature should be gradually raised to 110°C. ——- | | . | B. Kramer and F. F. Tisdall 225 The weight of the precipitate divided by 11.08 equals the number of mg. of sodium present in the sample. The method of preparation of the potassium pyroantimonate ‘reagent has been fully described in a former paper on the deter- mination of sodium in serum (5). The details of the method of preparation of the Gooch crucibles and the precautions to be observed during the addition of the alcohol and the filtration, and also the care of the platinum are fully outlined in the same paper. For the determination of sodium in solutions of blood ash we used the same procedure as described for the determination of this element in solutions of the ash of urine and stools (9). Potassium Method. 0.2 ec. of the material prepared as previously outlined is placed in a graduated centrifuge tube. 0.5 cc. of water is added followed by 0.5 ce. of a solution of sodium nitrite prepared by dissolving 15 gm. of potassium-free sodium nitrite (Merck) in 30 ce. of water. The contents of the tube are thoroughly mixed and allowed to stand for 5 minutes.2. Water is added to 4 cc. and the contents are again mixed. 2 cc. of the sodium cobalti-nitrite reagent are added drop by drop. The contents of the tube are mixed and allowed to stand for a half hour, then centrifuged for 7 minutes at about 1,300 revolutions per minute. The precipitate will then be found at the bottom of the tube. All but 0.2 to 0.3 ce. of the supernatant fluid is removed. This is accomplished by means of the following apparatus. Through one opening of a two-holed cork is inserted a glass tube by means of which a positive pressure can be made in the centrifuge tube. Through the other hole is a tube which reaches to about 3 or 4 mm. above the precipitate. The lower end of this tube is drawn out to a 21f the sodium nitrite is not added, it will be found that the precipi- tate obtained on the addition of the cobalti-nitrite reagent will float on the surface of the fluid and adhere to the sides of the tubes. The precip- itate will also adhere to the sides unless the tubes have been previously cleaned with the use of a brush, washed out with a strong cleaning fluid (commercial H.SO, and dichromate) and then thoroughly rinsed with distilled water. Low results will be obtained unless these procedures are carried out. 226 Inorganic Elements of Blood bore of about 1 mm. and curved so that the opening is directed upward. By fitting the cork to the centrifuge tube and blowing through thé first opening the supernatant fluid can be readily re- moved without disturbing the precipitate. 5 cc. of water are allowed to run down the side of the tube which is then gently agitated so that the added water is mixed thoroughly with the residual reagent. Care should be taken that the precipitate itself is disturbed as little as possible. This may be accomplished by holding the tube vertically and gently hitting the lower end with a circular motion. The brown fluid may be seen to rise and mix with the supernatant fluid. The tube is then centri- fuged for 5 minutes. The procedure is repeated three times so that the precipitate is washed four times in all. The superna- tant fluid from the last washing should be perfectly clear. After the removal of the fluid from the final washing the precipitate is ready to be titrated. Titration.—An excess of 0.02 N potassium permanganate is added (1.6 to 2 ec. are sufficient for normal blood), followed by 1 ec. of approximately 4 n sulfuric acid. The precipitate is then thoroughly mixed with the fluid by means of a glass rod. The sample is heated in the boiling water bath for 45 to 60 seconds at the end of which time the solution should be clear and still pink. If all the precipitate is not oxidized, the contents will be cloudy and the intensity of the color will be seen to diminish. Heating should then be continued until the solution is clear but still pink. When the heating is continued too long, the contents again become cloudy and have a brownish color. If this is allowed to happen, the sample must be discarded as high results will be obtained. An amount of 0.01 nN sodium oxalate sufficient to decolorize the solution completely (generally 2 ec.) is promptly added. The excess of oxalate is then determined by titrating to a definite pink color with 0.02 N potassium per- manganate delivered from a micro-burette graduated in 0.02 ce. The details for the calculation of the amount of potassium present in the sample and also the methods for the preparation of the reagents are given in a former paper (9). For the determination of potassium in solutions of blood ash we placed a quantity of fluid equal to 0.1 or 0.2 ce. of blood in a graduated centrifuge tube, added water to 2 cc., and then ht Aigo _ a B. Kramer and F. F. Tisdall 22. slowly added 1 ce. of the cobalti-nitrite reagent. The subsequent steps were the same as described for the determination of this element in serum (5). Calcium Method. 4 ec. of the material prepared as outlined above are placed in a graduated centrifuge tube previously cleaned with commer- cial H,SO, and potassium dichromate. 1 ce. of saturated ammo- nium oxalate is added, followed by 2 ce. of a filtered saturated solution of sodium acetate. The contents are mixed and allowed to stand for 1 hour. The volume is made up to 8 ec. with dis- tilled water, mixed, and then centrifuged for 15 minutes at about 1,300 revolutions per minute. This throws all the calcium oxalate precipitate to the bottom of the tube. All but 0.3 ce. of the supernatant fluid is removed by means of the apparatus described under the potassium method. The remaining fluid and the precipitate are mixed by tapping the tube. Enough 2 per cent ammonia (2 ce. of concentrated ammonia diluted to 100 ec.) is then added to bring the volume to 4 cc., care being taken to wash the sides of the centrifuge tube free from adherent oxalic acid. The tube is then centrifuged for 5 minutes. This procedure is repeated twice, thus making three washings in all. After the third washing the supernatant fluid is removed, the tube is shaken to suspend the precipitate, 2 cc. of approximately n sulfuric acid are added and the tube is warmed in the boiling water bath for a few minutes and titrated with 0.01 n potassium permanganate until a definite pink color persists for at least 1 minute when viewed under a good light against a white back- ground. The strength of the permanganate solution is determined by titrating against a 0.01 N sodium oxalate (Sérensen). The details for the calculation of the amount of calcium present in the sample and also the methods for the preparation of the reagents are given in a former paper (9). For the determination of calcium on 0.1 n HCl solutions of blood ash the procedure is identical with that described above. The blood ash solution was made up so that 1 cc. corresponded to 1 ce. of blood. It was allowed to stand for 2 or 3 weeks to allow the sediment to settle. 2 ce. of this solution were used for each determination. 228 Inorganic Elements of Blood The Magnesium Method. 5 ec. of the supernatant fluid from the calcium determination are measured into a 30 ec. beaker, 1 ce. of (NH4)2HPO, solution is added and then 2 ec. of concentrated ammonia. The next day the sample is filtered through a well packed Gooch crucible, washed ten times with 5 ce. of 10 parts of concentrated ammonia to 90 parts of water, then twice with 95 per cent aleohol made alkaline with ammonia. The crucible is returned to the beaker and dried for a few minutes at 80° C. in the oven. 10 ce. of 0.01 n HCl are added to the crucible and after a few hours the entire material is transferred to a test-tube, centrifuged, and 5 ce. of the supernatant fluid are measured into a flat bot- tomed colorimeter tube graduated for 10 ec., which contains 2 cc. of the iron thiocyanate solution. The volume is then made up to 10 ee. with 0.01 x HCl, a rubber stopper inserted, and the fluid mixed. A series of standards is prepared by adding varying amounts of a known NHiMgPO,j solution in 0.01 x HCl, to 2 ce. samples of thiocyanate solution and bringing the volume up to 10 cc. as in the unknown samples. The color is compared by looking through the entire length of the quid column against a white background. Calculation —The calculation is the same as in the original method: Reading (cc. of standard solution) X0 .01 X2 X8/5 X 100 ce. blood used in Ca determination cc. of blood. | = mg. of magnesium in 100 Preparations of Reagents. 1. Ammonium Magnesium. Phosphate Standard—This_ solu- tion is made by dissolving 0.102 gm. of air-dried magnesium ammonium phosphate (MgNH,PO,.6H.2O) in 100 cz. of 0.1 N hydrochloric acid and diluting to 1 liter with water. Of this © solution 1 ce. is equivalent to 0.01 mg. of magnesium. Mag- nesium ammonium phosphate loses water of crystallization when heated and must therefore be dried at room temperature. Commercial preparations of the salt are generally unreliable; it should be prepared by precipitation of pure solutions (10). B. Kramer and F. F. Tisdall 229 2. Ammonium Phosphate Solution—Ammonium phosphate solution is made as follows: 25 gm. of (NH,4)2HPO, are dissolved in 250 ec. of H.O. 25 ce. of concentrated ammonia are added ‘and the mixture is allowed to stand over night. The following day it is filtered, the filtrate is boiled to remove the excess of ammonia, cooled, and made up to 250 cc. This solution is diluted five times with water. 3. The Ferric Thiocyanate Solution——This solution is made from two solutions which are mixed an hour before use. Solu- tion A is 0.3 per cent ammonium thiocyanate. Solution B is 0.3 per cent ferric chloride, made up from the salt with its con- tained water of crystallization, adding a few drops of acid, if necessary, to clear the solution. 5 cc. portions of Solutions A and B are mixed and the whole is diluted to 40 cc. with water. 4. 10 Per Cent Ammonia.—100 cc. of concentrated ammonia are diluted to 1 liter. Protocols. We have previously shown (5) that sodium may be precipi- tated quantitatively, as sodium pyroantimonate, from solutions of blood salts. Since the composition of the supernatant fluid after deproteinizing blood with trichloroacetic acid is comparable, except for the small amount of residual protein and the non- protein nitrogenous constituents, to some of the solutions of blood salts which we have analyzed, we have considered it unnecessary to repeat this demonstration here. Table I shows that the sodium determinations when performed on the deproteinized solutions yield results practically identical with those obtained on a solution of the whole blood ash. The absolute values vary from 170 to 225 mg. of sodium per 100 ce. of blood. Table II. The concentration of potassium seems to vary considerably in the blood of different animals. Bunge found 213 mg. of potassium in 100 ce. of pig’s blood, 227 mg. in that of the horse, but only 34 mg. in the same volume of cow’s blood and 20 mg. per 100 cc. of dog’s blood. The lowest figure which Abderhalden reports is 21 mg. of potassium per 100 cc. of dog’s blood while the highest figure is 227 mg. per 100 cc. of horse’s blood. We have found that the potassium content of human 230 Inorganic Elements of Blood blood varies from 153 to 202 mg. of potassium per 100 cc. It varies with the percentage of corpuscles. It might be mentioned that the cobalti-nitrite reagent gives no precipitate when added to ferric chloride or trichloroacetic acid. We have shown else- - where that none of the constituents of serum except potassium TABLE I. Sodium Determinations on Blood. Na per 100 ce. of blood) Na per 100 ec. of blood Sample. Plasma. ashed. deproteinized. per cent mg. . mg. 1 65 225 216 2 55 170 175 3 65 207 207 4 58 187 186 5 56 185 193 6 60 198 260 Average....... 60 195 196 TABLE II. Potassium Determinations on Blood. Determinations on blood treated with Determinations on ashed blood. enidhlarotiontiguinnll Sample. Plasma. a as Sample. Plasma. K Pee mg. mg. 1 61 172 of 61 180 2 60 187 8 62 175 3 57 188 9 57 202 4 68 153 10 59 193 5 58 186 11 65 164 6 57 200 12 65 169 13 56 201 Average... 60 181 =: 61 183 yields demonstrable amounts of insoluble nitrites with this reagent (6). Table III. The concentration of calcium in serum or plasma is singularly constant (7). On the other hand the concentration of this element in blood varies inversely as the corpuscular con- . B. Kramer and F. F. Tisdall Dai tent. The results which we obtained varied from 5.3 to 6.8 mg. of calcium per 100 cc. of blood. The individuals from whom the samples were obtained were all normal adults. We have found that the addition of ferric chloride to a solution of blood salts TABLE III. Calcium Determinations on Blood. Determinations on ashed blood. Deteunins fons ou Dios eeaoe with samote | Pama. | CR] smote | Phone. | CEP per cent mg. per cent mg. 1 58 5.3 8 6.3 2 57 5.3 9 5.3 3 72 6.7 10 6.1 4 59 6.2 11 5.3 5 58 5.3 12 5.7 6 65 5.9 13 6.4 7 57 5.9 Average... 5.7 5.8 TABLE IV. Magnesium Determinations on Blood. Determinations on blood treated with Determinations on ashed blood. roe roncetiondid. sample, | ME-PRIDof | Sample, | Meng IID co mg. mg. 1 2.8 5 ZAG 2 2.8 6 4.0 3 3.8 0 3.8 4 2.3 8 3.8 PAWET APC IN. ociei- 2.9 3.5 does not interfere with the quantitative determination of calcium. The results obtained on the deproteinized material and on the solutions of blood ash are practically identical. Table IV. The concentration of magnesium in the blood of various animals has been found by Bunge and Abderhalden 232 Inorganic Elements of Blood to be fairly constant, varying only from 2 to 4 mg. per 100 ce. of blood (2). We have found that the concentration of this element in the blood of the adult male varies from 2.3 to 4.0 mg. per 100 ce. CONCLUSIONS. 1. A method has been described by means of which sodium, potassium, calcium, and magnesium may be quantitatively deter- mined on only 7 ce. of blood. 2. The basis of this method is deproteinization by means of trichloroacetic acid. The quantitative determination of each of these elements is then made on aliquots of the supernatant fluid by modifications of procedures recently described for serum. 3. The results obtained by these methods on deproteinized blood agree well with those obtained on solutions of blood ash. 4. We have found the concentration of these elements in 100 ec. of human blood to be as follows: sodium, 170 to 225 mg.; potassium, 153 to 201 mg.; calcium, 5.3 to 6.8 mg.; and mag- nesium, 2.3 to 4 mg. 5. The concentration of these elements in normal blood varies more than in normal serum. This is due to the variations in the corpuscular content of the blood. BIBLIOGRAPHY. 1. Bunge, G., Z. Biol., 1876, xii, 191. 2. Abderhalden, E., Z. physiol. Chem., 1897, xxiii, 521; 1898, xxv, 65. 3. Greenwald, I., J. Pharmacol. and Exp. Therap., 1918, xi, 281. Green- wald, I., and Gross, J., Proc. Soc. Exp. Biol. and Med., 1919-20, xvii, 50. 4. Greenwald, I., J. Biol. Chem., 1915, xxi, 61. 5. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 1921, xlvi, 467. 6. Kramer, B., and Tisdall, F. F., J: Biol. Chem., 1921, xlvi, 339. How- land, J., and Marriott, W. McK., Quart. J. Med., 1917-18, xi, 289. Kramer, B., and Howland, J., J. Biol. Chem., 1920, xliii, 35. 7. Kramer, B., and Tisdall, F. F., Bull. Johns Hopkins Hosp., 1921, Xxxli, 44, 8. Greenwald, I., J. Biol. Chem., 1915, xxi, 29. Marriott, W. McK., and Howland, J., J. Biol. Chem., 1917, xxxii, 233. Bloor, W. R., J. Biol. Chem., 1918, xxxvi, 49. Feigl, J., Biochem. Z., 1917, 1xxxi, 380. Iversen, P., Biochem. Z., 1920, cix, 211. Kleiman, H., Biochem. Z., 1919. xcix, 19. 9. Tisdall. F. F., and Kramer; B., J. Biol. Chem., 1921, xlviii, 1. 10. Jones, W., J. Biol. Chem., 1916, xxv, 87. PHOSPHORIC ESTERS OF SOME SUBSTITUTED GLU- COSES AND THEIR RATE OF HYDROLYSIS. By P. A. LEVENE anp G. M. MEYER. WITH THE ASSISTANCE OF [. WEBER. (From the Laboratories of The Rockefeller Institute for Medical Research. ) (Received for publication, July 7, 1921.) The purpose of the preparation of the substance to be described in this communication was developed in a previous publication by Levene and Yamagawa. The interest, as was explained, is both of a theoretical and a practical nature. The question to be answered is the following. Does the stability of the inorganic radicle in a phosphoric ester of a sugar (free or substituted) depend on the position of the inorganic radicle? The answer to this question is given in the following table of the constants of hydrolysis of three 1-2-acetone phosphoric acid glucoses. K 1-2-Monoacetone phosphoric acid glucose............... 44 (107%) 1-2-Monoacetone-6-phosphoric acid glucose............. 58 (107%) 1-2-Monoacetone-3- or 5-phosphorie acid glucose!....... 24 (107%) Substance 1 was prepared from 1-2-acetone glucose, hence the position of the phosphoric acid in it may be in any position from 3 to 6 (inclusive). Substance 2 was prepared in the course of preparation of the derivative from 1-2-3-5-diacetone, hence it is in the main 1-2-diacetone-6-phosphoric acid glucose. The third substance was prepared from 1-2-3-5-diacetone-6-benzoyl glu- cose. This was converted into 1-2-acetone-6-benzoyl glucose, this further into 1-2-acetone-3- or 5-phosphoric acid-6-benzoy] glucose, and finally the latter into 1-2-acetone-3- or 5-phosphoric 1 The second sentence of the last paragraph on p. 324, J. Biol. Chem.., xlill, should read: Whereas the fourth was obtained by the action of phos- phorus oxychloride on monoacetone glucose, the fifth is formed as a by-product by the action of phosphorus oxychloride on diacetone glucose. 233 234 Phosphoric Esters of Glucoses acid glucose. Thus in Substance 2 the phosphoric acid radicle is attached to carbon atom 6, and in Substance 3 in position 3 or 5. The differences in the rate of hydrolysis are far beyond the limits of error of the method. It is interesting to note that the rate of hydrolysis of Substance 3 is practically the same as found in the earlier experiments of Levene and Yamagawa on 1-2-acetone-6-benzoyl phosphoric acid. This similarity is easily understood, since the benzoyl group being in position 6 is very labile, hence, in the course of hydrolysis the benzoyl derivative is soon transformed into the monoacetone phosphoric acid glu- cose. In the same manner is explained the fact that 1-2-acetone- 6-phosphoric acid glucose, and 1-2-3-5-diacetone-6-phosphoric acid hydrolyze with the same velocity. : The analytical data on the barium salts of phosphoric esters, here reported, call for a special note. They are not so satisfac- tory as is desired. However, it has been the experience of this laboratory that when barium salts of pure phosphoric esters are obtainable only in an amorphous form, the analytical results are often not perfect. Thus it was found practically impossible to obtain a perfect agreement between the found and required elementary composition of the amorphous barium salt prepared from the erystalline adenosinphosphoric acid. The observations on the phosphoric esters of the 1-2-3-5- methyl glucose and of the 1-3-5-6-methyl glucose are in harmony with those on the esters of the 1-2-acetone glucose. Thus the rate of hydrolysis of 1-2-3-5-methyl-6-phosphoric acid glucose proceeded normally, giving an average ratio, K = 44 (10-*), which is not far removed from that of the acetone glucose ester having the inorganic radicle in position 6, where. K = 56 (107%). On the other hand, the rate of hydrolysis of 1-3-5-6-methyl-2-phosphoric acid glucose proceeded abnormally. There was noted a rapid hydrolysis at the beginning which reached a maximum after about 6 hours, and the further progress was very slow. The explanation of this result may be the following. The 1-3-5-6-methyl glucose is apparently very difficult to prepare in pure form. The material employed for coupling with phos- phoric acid may have contained as an impurity some trimethyl glucose with either carbon atom 1 or carbon atom 6 free. Thus the material derived from this product may be a mixture of two Pp. A. Levene and G. M. Meyer 235 phosphoric esters, one with an inorganic radicle in position 1 or 6, which is labile, and the other in position 2 which is very stable. The first is decomposed rapidly, whereas the second proceeds at the low rate indicated in the table. The estimations of the rate of hydrolysis were carried out by Miss I. Weber. EXPERIMENTAL. a -Methyl Glucosidophosphoric Acid. Portions of 10 gm. of dried methyl glucoside are dissolved in 50 ec. of warm, water-free pyridine. This solution is cooled to — 20°C. and to it is added, in small portions, a solution of 7.8 gm. (1 mol) phosphorus oxychloride in 20 ce. of pyridine, also cooled to —20°C. _ It is important that the pyridine as well as the methyl gluco- side should be absolutely dry. For this purpose the substance is dried over phosphorus pentoxide at 100°C. under a pressure ot about 1 mm. The pyridine is boiled with reflux over barium oxide for 5 hours and then distilled. The pyridine and phosphorus oxychloride should be well cooled before mixing, and if the pyridine is dry no pyridine hydrochloride will settle out even after standing for some time. After the phosphorus oxychloride in pyridine has been added to the solution of the glucoside, the reaction mixture is allowed to remain at —20°C. for several hours, during which time a con- siderable amount of pyridine hydrochloride separates. ‘The reaction mixture is now diluted with 20 ce. of ice water and allowed to come to room temperature when it is further diluted by pouring it into 200 cc. of cold water. An excess of barium hydrate (100 gm.) is added and the pyridine removed by distillation under diminished pressure. The temperature of the bath should not be above +30°C. When all the pyridine has been removed the solution is made just acid to Congo red with sulfuric acid. The hydrochlo- ric acid is now removed by the addition of 30 gm. of silver sulfate. After shaking this mixture for half an hour it is filteredand the silver removed from the filtrate by hydrogen sulfide and the latter removed by a current of air. 236 Phosphoric Esters of Glucoses An excess of barium hydrate is added and carbon dioxide passed into the solution until the reaction is neutral to litmus. The solu- tion is now filtered and concentrated under diminished pressure and low temperature. When the volume has been reduced to one half of the original, the solution is filtered and the filtrate con- centrated to a small volume which is then poured into a large volume of absolute alcohol. For purification the precipitate is dissolved in a small quantity of water, filtered, and reprecipi- tated with alcohol. The yield was 9.0 gm. For analysis the substance is dried under diminished pressure at the temperature of water vapor. 0.1042 gm. of substance gave on combustion 0.0866 gm. of CO: and 0.0310 gm. of H.O. 0.2757 gm. of substance gave 0.0746 gm. of Mg2P20;. O10982 7 se nO U4550 eo seas Oys C7Hi;06H2PO3. Calculated. C 30.7, H 4.8, P 11.0. Found (calculated Ba-free). C 31.8, H 4.68, P 10.6. . The optical rotation in water was found: » +1.32°X 100 2 ee ee [al asenleis) oe 1-2-3-5-Diacetone Glucose. Fischer’s latest method for the preparation of diacetone from 6-glucose? in a somewhat modified form was employed. 75 gm. of dried $-glucose, which is readily prepared by the method of Behrend, are placed in an ordinary glass-stoppered acid bottle with 1,500 cc. of dried acetone containing 1 per cent hydro- chlorie acid. The bottle was shaken for 24 hours at 30°C. The contents are now dark-colored and nearly all the 6-glucose has dissolved. The solution is filtered and neutralized to litmus with sodium methylate, which is prepared by dissolving 4 gm. of sodium in 400 ce. of dry methyl alcohol. When neutral the acetone solu- tions assume a much lighter color. The solution is again filtered and concentrated to dryness under diminished pressure. A solid cake.is formed and repeatedly extracted with warm petroleum ether from which on cooling, diace- ? Fischer, E., and Rund, C., Ber. chem. Ges., 1916, xlix, 93. | P. A. Levene and G. M. Meyer 237 tone glucose crystallizes. The diacetone glucose is further purified by the method described by Fischer. 600 gm. of 8-glucose under proper conditions yield 350 gm. of diacetone glucose. Although some monoacetone is also obtained the yield of this substance is comparatively small. 1-2-3-5-Diacetone-6-Phosphoric Acid Glucoside. The action of phosphorus oxychloride on diacetone glucose depending upon certain conditions yields either a diacetone glu- cose phosphoric acid, the barium salt of which is soluble in absolute alcohol, or a monoacetone phosphoric acid, the barium salt of which is insoluble in alcohol. 10 gm. of thoroughly dried diacetone glucose are dissolved in 50 ce. of dry pyridine and cooled to —35°. This low temperature is readily obtained by using a mixture of CaCl+6H,O and ice. To the sugar solution are added in small amounts 5.4 gm. phos- phorus oxychloride in 20 ce. of dry pyridine, likewise cooled to —35°. The mixture is now transferred to a bath of —10° and kept therein for 2 hours. During this time crystals of pyridine hydrochloride have separated. The reaction mixture is again cooled to —35°C. and about 20 ec. of moist pyridine, also cooled to —35°C., added in such small quantities that the temperature will not rise above —10°. ‘This is then followed by the addition of a cold solution of 10 cc. each of pyridine and water and finally a small cake of ice. The reaction product is now allowed to come to room tempera- ture. An excess of barium hydrate is added, the pyridine removed under diminished pressure, and the hydrochloric acid removed by shaking with silver sulfate, and further treated as previously described. The barium salt of diacetone phosphoric acid is very soluble in alcohol and is not precipitated by ether or acetone. On allow- ing the alcoholic solution to evaporate to dryness the barium salt is obtained as a fine white powder which when dry is no longer hygroscopic. It does not reduce Fehling’s solution until after hydrolysis with acid. For analysis the substance was dried to constant weight under diminished pressure at the temperature of water vapor. 238 Phosphoric Esters of Glucoses 0.11383 gm. of substance gave on combustion 0.1331 gm. of CO: and 0.0482 gm. of H.O. .0923 gm’. of substance gave 0.0341 gm. of BaSO,. WeT20 +5 “ 0.0625 “ “ MgsP.0;. Cy2Hi9OsH2POs. Calculated. C 42.25, H 6.18, P 9.10. Found (calculated Ba-free). C 41.5, H 6.18, P 8.90. The rotation in water was found: 12 — 11° 100 [als = ———~—_-= ~2.48° 1 X 4.472 1-2-Monoacetone Phosphoric Acid Glucoside from Diacetone Glucose. This substance is obtained by the action of phosphorus oxy- chloride on diacetone glucose when the temperature of the reac- tion mixture is allowed to rise above +10° during the process of destroying the unutilized phosphorus oxychloride. 10 gm. of dried diacetone glucose are dissolved in 50 ce. of dry pyri- dine and cooled to —20°. To this is added 5.4 gm. of phosphorus oxychloride dissolved in 20 ec. of pyridine also cooled to —20°. The mixture is allowed to remain at —20° for several hours and then 20 ce. of ice cold water are added. The temperature of the mixture rises to about +30°. After allowing it to stand at room temperature for a while it is further diluted with water. An excess of barium hydrate is added and the pyridine removed by distillation under diminished pressure. The product is treated with silver sulfate and barium hydrate as previously described. The final residue is soluble in water which on the addition of alcohol forms a gelatinous mass. The barium salt is precipitated from its aqueous solution by pouring it into a large volume of dry acetone. The salt is obtained as a fine white powder. It does not reduce Fehling’s solution until after hydrolysis with acid. For analysis the substance was dried under diminished pres- sure at the temperature of water vapor. 0.1032 gm. of substance gave on combustion 0.1016 gm. of COs and 0.0394 gm. of H.O. 0.2679 gm. of substance gave 0.0653 gm. of Mg,P207. 0.0893 “ * “ rep aee. * i BaSoe C,H,,O;H2PO;. Calculated. C 35.75, H 6.00, P 10.28. Found (calculated Ba-free). C 35.9, H 5.61, P 9.30. P. A. Levene and G. M. Meyer _.’ 239 The optical rotation in water was: 2 0.35° X 100 = = + 6.8” labs Sgseq ate 1-2-Monoacetone Phosphoric Acid Glucoside from Monoacetone Glucose. 10 gm. of dry monoacetone glucose are dissolved in 50 ec. of dry pyridine and cooled to —30°. To this is added 4.4 gm. of phosphorus oxychloride dissolved in 20 ee. of dry pyridine. The reaction mixture is allowed to stand for some time at —20° and treated as described in the previous preparations. The residue is soluble in 95 per cent alcohol which on pouring into a large excess of dry ether precipitates the barium salt of monoace- tone phosphoric acid glucoside. The yield was 9 gm. The substance is dried for analysis under diminished pressure at the temperature of xylene vapor. 0.1103 gm. of substance gave on combustion 0.1056 gm. of CO, and 0.0400 gm. of H,0. 0.2940 gm. of substance gave 0.0744 gm. of Mg.P2O;. O:0980" “Sf a O03 8)" Sb aSOx. C.Hi,0;H2PO;. Calculated. C 35.75, H 6.00, P 10.28. Found (caleulated Ba-free). C 35.4, H 5.42, P 9.55. The optical rotation found was: yo +20° X 100 ie eee 5 0 leis scanene 3 1-2-Monoacetone-6-Benzoyl Phosphoric Acid Glucoside. The benzoyl monoacetone glucose was prepared from benzoyl diacetone glucose according to the method of Fischer. 10 gm. of dried benzoyl monoacetone glucose are dissolved in 50 ce. of dry pyridine and cooled to —30°. To this are added in small amounts 4.7 gm. of phosphorus oxychloride dissolved in 20 ce. of dry pyridine, also cooled to ~30°. The mixture is allowed to stand at —30° for several hours during which time crystals of pyridine hydrochloride have separated. Moist pyridine, cooled to —20°, is now added, followed by a cake of ice. The mixture is allowed to come to room temperature, diluted with water, 240 Phosphoric Esters of Glucoses and treated as previously described. The residue is soluble in absolute aleohol from which the barium salt is precipitated by pouring it into a large volume of dry ether. The yield was 10 gm. For analysis the substance was dried under diminished pres- sure at the temperature of chloroform vapor. 0.1097 gm. of substance gave on combustion 0.1316 gm. of CO, and 0.0426 gm. of H20. 0.2941 gm. of substance gave 0.0642 gm. of Mg»P.0;. O:0980 = 3 = O10394 = => BasOz C,sH2007H2PO;3. Calculated. C 47.4, H 4.95, P 7.64. Found (calculated Ba-free). C 42.9, H 5.3, P 8.06. The optical rotation was found: « _ +0.26° X 100 [aly = [Mb 1g ee From the analysis of the substance and from the fact that it is non-reducing it was supposed that in the process of preparation a small part of the benzoic acid radicle was removed. This assumption was corroborated by the estimation of the benzoic acid on hydrolysis of the substance. 1.000 gm. of barium salt was hydrolyzed with dilute sulfuric acid. The solution was extracted with ether, the ethereal solution dried with anhydrous sodium sulfate and concentrated. The residue consisted of benzoic acid and weighed 0.1376 gm. or 13.76 per cent. Theory required 22.6 per cent. 1-2-Monoacetone Phosphoric Acid Glucose from 1-2-Monoacetone-6-Benzoyl Phosphoric Acid Gtucose. 15 gm. of the barium salt of the phosphorated sugar were dis- solved in 150 ce. of water and made just acid to Congo red with sulfuric acid. An additional 10 cc. of 2 Nn sulfuric acid were added and all made up to 200 ec. This was heated in a water bath at 50°C. for 70 minutes. The solution was cooled, filtered, and extracted with ether. The aqueous solution was made alkaline with barium hydrate and filtered from the barium phos- phate. Carbon dioxide was passed until the reaction was neutral , tolitmus. The solution was again filtered and concentrated under diminished pressure at 30°C. The residue was soluble in 95 per P. A. Levene and G. M. Meyer 241 cent alcohol and the barium salt precipitated by pouring it into a large volume of dry ether. The yield was 6 gm. For analysis the substance was dried under diminished pressure at the temperature of water vapor. 0.10°1 gm. of substanee gave on combustion 0.0979 gm. of CO, and 0.0420 gm. of H.0. 0.2811 gm. of Se ee gave 0.0658 gm. of Mg2.P2O7. Moose f “ pean 0: 0446.05 0% BaS@, Co9Hi1g06H2POs. Calculated. C S15) 75, H 6.00, P 10.28. Found (calculated Ba-free). C 35.67, H 6.24, P 9.48. The substance does not reduce Fehling’s solution until after hydrolysis with dilute acid. The optical rotation found was: 0.25° X 100 | 1X 0.4000 ~O lel, = +6 25 2-3-5-Trimethyl-M ethyl Glucoside. The trimethyl glucoside was prepared by the method of Haworth.? Methyl glucoside was methylated in portions of 20 gm. each. After 300 gm. of glucoside had been methylated the combined product consisting of a mixture of the methylated glucosides was subjected to a fractional distillation. The progress of fractionation was controlled by means of the index of refrac- tion as well as by methoxy determinations. The product was separated into the following fractions: Fraction. n No CHO Boiling point P= 0.15 mm. per cent it OF I 46°56. 5’ 1.4449 60.95 85 II > AGC512 5" 1.4450 61.08 90 JADE 46°41 .5’ 1.4446 | 60.00 95 IV 46°01.5’ 1.4502 56.67 105 V Ale au 1.4550 52.30 110 The theory for tetramethyl-methyl glucoside requires 61.75 per cent CH;O0 and trimethyl-methy! glucoside calls for 52.54 3 Haworth, W.N., J. Chem. Soc., 1915, evii, 8. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 1 242 Phosphorie Esters of Glucoses per cent CH,O. Fractions IV and V were again distilled, the main fraction boiling at 108°C., P = 0.07 mm., n = 44° 43’, N2? = 1.45786, D?? = 1.1477. Found M, = 56.06. Theory requires 55.917. The yield of this fraction was 137 gm. Methoxy determination: 0.1803 gm. of substance gave 0.7145 Agl (factor = 13.2) = 52.30 per cent of CH,0. Theory requires 52.54 per cent. 2-3-5-Trimethyl-6-Phosphoric Acid Methyl Glucoside. Portions of 10 gm. of the glucoside are dissolved in 50 ce. of dry pyridine and cooled to —30°. To this mixture is added a solution of 6.5 gm. of phosphorus oxychloride in 20 ce. of pyri- dine, also cooled to —30°C. The reaction is rather feeble and only after some time pyridine hydrochloride begins to separate. The reaction mixture is allowed to stand for several hours at —20° and then 20 cc. of cold moist pyridine are added. The product is now allowed. to come to room temperature and further diluted with ice cold water. After making alkaline with 60 gm. of solid barium hydrate, the pyridine is removed by distillation under diminished pressure. The reaction product is then treated with silver sulfate and barium hydrate. The barium salt of this substance is very soluble in alcohol and acetone. It was purified by dissolving it in a small quantity of acetone and precipitating it in a large volume of dry ether. The salt is obtained as a white hygroscopic powder. For analysis it is dried under diminished pressure at the temperature of xylene vapor. 0.1122 gm. of substance gave on combustion 0.0462 gm. of H,O and 0.1164 gm. of COs. 0.0908 gm. of substance gave 0.0382 gm. of BaSO,. Oia. “0.0705 “ “ Mg2P:O;. C;9H190.-H2PO3. Calculated. Cc 37.97, H 6.68, P 9.80. Found (calculated Ba-free). C 37.50, H 6.12, P 9.45. The rotation in water was found: lal” _ —2.36° X 100 | 4.77.07° CUD eRe wenn hr _P. A. Levene and G. M. Meyer 243 3-5-6-Trimethyl-1-2-A cetone Glucose. 3-5-6-Trimethyl-methyl glucoside was prepared by methylating 1-2-acetone glucose and converting the product into the glucoside. The methylation of monoacetone glucose had been attempted by Irvine and Scott* using the silver oxide and methyl] iodide method. The results by this method are not satisfactory. Attempts to methylate the trimethyl glucose obtained after cleaving off the acetone were also unsuccessful. The following method was finally adopted. Portions of 25 gm. of monoacetone glucose were methylated with an excess of dimethyl sulfate by the method of Haworth. The temperature of the bath should not exceed 55°; the methyla- tion should proceed rather slowly, and the alkali should be always present in a slight excess. To 25 gm. of monoacetone there were used 150 ce. of 30 per cent sodium hydroxide and 90 ce. of freshly distilled dimethyl sulfate. After all the reagents had been added the stirring was continued for one hour at the same temperature. The solution was then cooled and ammonium hydroxide added to destroy any unutilized dimethyl sulfate. The solution was extracted with chloroform and the chloroform extract, after drying over anhydrous sodium sulfate, concentrated under dimin- ished pressure to a syrup. The product of five such methylations was combined, dissolved in ether, dried with anhydrous sodium sulfate, and subjected to a fractional distillation. The largest fraction amounting to 90 gm. boiled at 88-90° at a pressure of 0.03 mm. 0.1025 gm. of substance gave on combustion 0.2042 gm. of CO, and 0.0780 gm. of HO. Cy2H20¢. Calculated. ¢ 54.96, H 8.17. Found. C 54.33, H 8.5. The optical rotation was: wale, % 100 > 1X4.034 ° lal, == — 28.5 * Irvine, ‘J. C., and Scott, J. P., J. Chem. Soc., 1913, ciii, 573. 244 Phosphoric Esters of Glucoses 3-5-6-Trimethyl Glucose. Hydrolysis of the trimethyl acetone glucose was carried out in 75 per cent alcoholic solution containing 0.5 per cent hydrochloric acid and heating for 1 hour at 100°. The solution was shaken with silver carbonate, extracted with ether, and the ethereal solution dried and concentrated to a syrup. This was distilled and a fraction obtained boiling at 147° and P = 0.05 mm. 0.1150 gm. of substance gave on combustion 0.2080 gm. of CO: and 0.0872 gm. of H,0. CsH;.905. Calculated. C 48.75, H 8.11. Found. C 49.32, H 8.48. The optical rotation in alcohol was: Initial. Final. » —0.75° X 100 5 Sa scie ES peepee oa 2 RON TEN, = Spey Dee [ap 1 X 6.874 2 lal, 1 X 6.874 It was attempted to convert this product into its methyl glucoside by all the known methods and by several modifications as regards the concentration of catalyst and time of reaction. In every experiment the unchanged material was recovered. 3-5-6-Trimethyl-M ethyl Glucoside. The conversion of trimethyl monoacetone glucose to the gluco- side was obtained finally by the following process: 50 gm. of trimethyl acetone glucose were dissolved in 100 ce. of dry methyl alcohol containing 0.1 per cent hydrochloric acid and heated in sealed tubes for 24 hours at 100°C. The hydro- chlorie acid was removed by shaking with moist silver carbonate and the solution concentrated under diminished pressure to a syrup. This was taken up in ether and dried with anhydrous sodium sulfate. The ethereal solution was concentrated and the syrup fractionated. The larger part distils at 135°, P = 0.035 mm. The yield was 20 gm. 0.1510 gm. of substance gave on combustion 0.2758 gm. of CO, and 0.1174 gm. of HO. CioH210¢. Calculated. C 50.85, H 8.47. Found. 24933" 87. P. A. Levene and G. M. Meyer 245 Repeated attempts to obtain this glucoside uncontaminated with traces of free sugar were unsuccessful. The product always produced a slight reduetion of Fehling’s solution. 3-5-6-Trimethyl-2-Phosphoric Acid Methyl Glucoside. 10 gm. of 3-5-6-trimethyl-methyl glucoside were dissolved in dry pyridine, cooled to — 20°, and a solution of 5.1 gm. of phospho- rus oxychloride in 20 ce. of pyridine, also cooled to —20°, was slowly added. ‘There was a slight rise in temperature and after standing at —10° for some time, crystals of pyridine hydrochlo- ride separated. The barium salt, prepared by the method pre- . viously outlined, was found to be soluble in alcohol, ether, and acetone. The alcoholic solution was allowed to evaporate. The barium salt which was obtained as a white powder did not reduce Fehling’s solution until after hydrolysis with acid. For analysis the substance was dried under diminished pres- sure at the temperature of water vapor. 0.1070 gm. of substance gave on combustion 0.1258 gm. of CO: and 0.0570 gm. of H20. 0.2918 gm. of substance gave 0.0717 gm. of Mg,P.0:. 097s “ =O 0274 * * BaSO,. C1 0Hi1905H2PO3. Calculated. C 37.97, H 6.68, P 9.80. Found (calculated Ba-free). C 38.6, H 7.15, P 8.25. The barium salt was in the main an acid barium salt, since analysis showed 16.5 per cent of barium; theory requires for the acid salt 17.85 per cent, and for the neutral salt 30.5 per cent barium. The optical rotation in water was: = 0.71° X 100 len Seca wore Rates of Hydrolysis. 1-2-Monoacetone-3- or 5-Phosphoric Acid Glucose. 1.658 gm. of the barium salt of this substance were dissolved in a small volume of warm water and made up to 50 cc. Of this solution 3 cc., equivalent to 0.030 gm. of P, were put into glass tubes together with 2.832 ec. of 0.1-N H.SO, and 0.168 ce. of water and sealed. The tubes were heated in an oil bath at 100 ce. 246 Phosphoric Esters of Glucoses for the intervals indicated in the following tables. The method of analysis:was as described in the paper of Levene and Yamagawa.® 3-5-6-Trimethyl-2-Phosphoric Acid Methyl Glucoside. 3.651 gm. of the barium salt of this substance were dissolved in a little warm water and made up to 25 cc. Of this solution 3 ec., equivalent to 0.030 gm. of P, were put into glass tubes together with 1.227 cc. of 0.1 N HeSO, and 0.773 ce. of water and sealed. The tubes were heated at 100° for the intervals indicated in the following tables. 1-2-M onoacetone-3- or 5-Phosphoric Acid Glucose. Time. Mg,P.0, Average. P in Mg,P,O,. | P in free acid. | P of total P. hrs. gm. gm. gm. per cent per cent 0.0014 1 0.0013 0.0009 0.27 3.02 0.0012 0.0030 , 2 0.0032 0.0022 0.67 7.42 0.0034 0.0062 4 0.0063 0.0044 1.33 14.61 0.0064 | 0.0082 6 0.0083 0.0059 1.75 19.25 0.0083 . 0.0099 8 0.0100 0.0070 2.11 23.19 0.0100 0..0160 : 16 0.0161 0.0115 3.47 38.21 0.0162 0.0199 24 0.0199 0.0139 | 4,20 46.16 0.0198 * Levene, P. A., and Yamagawa, M., J. Biol. Chem., 1920, xliii, 323. 16 bo 0.0344 0.0346 0.0096 P. A. Levene and G. M. Meyer 247 3-5-6-Trimethyl-2-Phosphoric Acid Methyl Glucoside. Mg,P.0, Average. Pin Mg,P,O, | P in free acid.| P of total P. gm. gm. gm. per cent per cent 0.0178 0.0179 0.0125 3.37 41.53 0.0179 0.0210 0.0213 0.0151 4.09 50.31 0.0214 0.0210 0.0210 0.0146 3.96 48.71 0.0209 0.0232 0.0233 0.0162 4.39 54.05 0.0234 0.0207 0.0208 0.0145 3.92 48.25 0.0208 0.0212 0.0213 0.0148 4.01 49.41 0.0214 Sample 2. 0.0303 0.0302 0.0084 4.46 70.04 0.0301 0.0303 0.0305 0.0085 4.50 70.74 — 0.0307 0.0320 0.0324 0.0090 4.89 76.90 0.0328 0.0348 oert 80.26 248 Phosphoric Esters of Glucoses 1-2-Monoacetone-3- or 5-Phosphoric Acid Glucose. F } Mg2P207 (2) a-x 360 480 960 1,440 Average a = 0.1077 _ 3-5-6-Trimethyl-2-Phosphoric Acid Methyl Glucoside. T Meg2P207 (2) a-x + log oa min. gm. 60 0.0448 0.0629 0.0038 120 0.0530 0.0547 0.0024 240 0.0525 0.0552 0.0012 360 0.0583 0.0494 0.0009 480 0.0520 0.0557 0.0006 960 0.0533 0.0533 0.0003 1,440 0.0692 0.0385 0.0003 a = 0.1077 3-5-6-Trimethyl-2-Phosphoric Acid Methyl Glucoside. Sample 2. 1 a th Mege2P207 (x) a-z — log — t o-2z min. ; gm. 60 0.0755 0.0322 0.0087 120 0.0763 0.0314 0.0044 240 0.0829 0.0248 0.0026 360 0.0865 0.0212 0.0017 HOW DO YOU KNOW When You Purchase a Drying Oven DE KHOTINSKY TRIPLE WALLED OVEN SHOWING RECORDER The attempt to answer such questions as these led our testing department to devise a recording arrangement (shown attached to the oven in the illustration) which automatically records any variations in temperature to one-fou th degree centigrade over a period of twenty-four hours. For every oven which we sell, one or more such records is made and kept in our files under the serial number of the oven as a permanent record of its perform- ance. Should the record show a greater variation in tem- perature than plus or minus one-half degree centigrade from the test setting, the regulator is sent back to the con- stant temperature department and a new one installed. how many degrees the tempera- ture may vary from that which you desire to maintain? The manufacturer may tell you that the temperature will remain con- stant within one degree but just how does he arrive at this state- ment? By reading a thermom- eter? At whatintervals of time? During the night also? What assurance does he or do you have as to the amount of variation in the interval between readings? A curve showing how the temperature line smoothed itself out after proper adjustment was made. Remember that there is a vast difference between constancy of regulation and uni- formity of temperature. An oven may maintain its temperature constant to one degree centigrade and still have a difference of twenty degrees from top to bottom. In an early issue we shall take up the question of temperature difference between parts of the oven space or lack of uniformi-y and discuss the performance of the De Khotinsky Oven in this respect. For Complete Description of the de Khotinsky Oven, send for Bulletin 61K CENTRAL, SCIENTIFIC, ComPANT’ LADORATORY (ENC ~/ UPPUIEK7 Chicaco. aratusr wx 460 E.Obio St., ap LY Chemicals Ch le ae. UF. BOCK-BENEDICT COLORIMETER Devised by Dr. Joseph C. Bock and Dr. Stanley R. Benedict of the Depart- ment of Chemistry, Cornell University Medical College, New York, and fully described in the August number of The Journal of Biological Chemistry, 1918 Simple in construction and highly ac- curate, this new form of a Colorimeter is an ideal instrument for the iaboratory worker and scientific clinician as well as for the student. It is reasonably priced and can be favorably compared with other more expensive colorimeters All parts are easily replaced at small cost. Cells and cups are made to resist acids, alkalies, and heat. $40.00 a KLETT MFG. CO., Inc. Send for Descriptive Circular and Reprint 202 E. 46th St. New York . Hay Fever Memoranda Series III Late Summer Type. Patients whose hay fever develops in mid- August and continues until frost should be tested with the pollens of such weeds as ragweed, goldenrod, and the related sunflower. ~- Also with the pollen of the one important late flowering grass, viz., corn, if exposed to same. Together with any pollen of local importance—such as alfalfa in some sections—or cocklebur in others. Patients whose hay fever continues beyond the pollinating seasons— even into the winter—should be tested with bacteria! proteins to locate a possible secondary sensitization of this type. For those who react to bacterial proteins, specific bacterial vaccines are indicated as supplementary treatment. Arlco-Pollen Extracts For Cutaneous Tests and Treatment cover Early and Late Spring, also Summer and Autumn.. Literature and List of Pollen Extracts and Specific Bacterial Vaccines on request. The Arlington Chemical Company Yonkers, N. Y. 2 ‘*Eternal Vigilance is the Price of Accuracy’”’ POR TY YEARS The Traditions and Standards developed in our shops since 1881 are reflected in the accuracy and quality of each piece of apparatus made by us. The scientific worker cannot afford to overlook the painstaking care and pride of workmanship summed up in the trade mark riil 18 Sie fs We are specialists in the manufacture of apparatus made to order. 12 | We are the original manufacturers of the Van Slyke COz apparatus and amino nitrogen apparatus, the Van Slyke-Cullen Urea apparatus, Folin’s tubes and pipettes, The Fredericia apparatus and many other types now widely used. If you are not already one of our many customers WHY NOT? FULL LINE OF LABORATORY SUPPLIES THE EMIL GREINER COMPANY ESTABLISHED 1881 Foun ood Esa GREINER oO 55 Fulton Street (5 New York, N. Y. Pipette Pron HENRY HEIL CHEMICAL CO. 210-214 South 4th Street St. Louis, Mo. IMPORTERS AND MANUFACTURERS OF Chemical Apparatus, Chemicals, Laboratory and Assay Supplies Generally We have the stock and can make prompt delivery SPECIALTIES: Best Grade of American Glassware, ‘‘Non-Sol,’’ ‘‘Perfection,’’ ‘‘Pyrex’’ and “Fry”’ Munktell, Whatman and H. H. C. Co’s. crimped white Filtering Paper, low in ash and filtering rapidly, specially manufactured for us in America Coor’s Best American Porcelain Ware Troemner & Becker Balances and Weights, Platinum, Wire, Foil and Ware 3 Fused Silica Laboratory Utensils ‘‘Vitreosil’”’ Hanovia Pure Transparent Quartz Glass, Alundum Ware . Hoskins’ Electric Furnaces Chatelier’s Pyrometers. Bausch and Lomb Microscopes and Accessories Hydrometers and Thermometers of every description Chemically Pure Acids and Ammonia, Molybdie Acid and Molybdate of Ammonia Merck’s, Baker & Adamson’s and J. T. Baker Chemical Co.’s Chemicals and Reagents Caustic Potash and Soda, purified in sticks and pure by alcohol, Acetate of Lead, Cyanide Mixture, Soda Ash and Commercial Caustic Soda We carry a very large and completestock of everything used in the laboratory and solicit your orders or inquiries. There is nothing in the chemical line which we cannot furnish. You will find us prompt, courteous and reliable. In our Glassblowing Establishment we can produce any ' Apparatus, no matter how complicated SS 5) BOVIE POTENTIOMETER A semi-automatic recording potentiometer directly reading and recording H-ion concentrations (and the resulting Sérensen pH values) without computation of voltage values or the use of transformation tables ‘Originally described in Journal of Medical Research, $3: 295, 1915, by Dr. W. T. Bovie, assistant professor of biophysics, Harvard University, and designed and constructed by Leeds and Northrup Company. This is a semi-automatic recording potentiometer by means of which H-ion concentrations are directly plotted as a function of some measurable, variable quantity, with which the H-ion concentration is chang- ing. The variable quantity may, for example, be amount of titrating solution added or elapsed time. Records are made on a special cross-section paper by means of a marking device automatically moved along the H-ion scale as the potentiometer is balanced. This feature makes the instrument particularly adapted to research problems in which a record of the change in H-ion concentration is desirable as the charts can be filed away with the observer's experiments. In courses of physical chemistry, biochemistry, etc., this graphic method of emphasizing the true meaning of acidity and alkalinity and of the difference between actual teaction and total reaction seems particularly commendable. A reflecting galvanometer, especially designed for gas chain work is built into the case with the instru- ment, as 1s also the standard cell against which the working current is adjusted. The necessary tapping keys are mounted for convenient manipulation, and binding posts are provided for connection to circuit for operating the galvanometer lamp, to the gas chain consisting of a saturated KCl calomel electrode and hydrogen electrode; and to the working current for the potentiometer, usually two dry cells. A rheostat for adjusting the working current is mounted in the instrument and a dial corrects the instrument reading for temperature changes in the gas chain. The galvanometer is sensitive to changes of 0.01 ofa pH unit; its accuracy, depending somewhat on the gas chain resistance, is less, but on the same order of magnitude. 4890. Bovie Potentiometer, L. &. N., as above described, with 25 sheets of semi-multiple logarithmic codrdinate paper and one spare lamp, diagram of connections and instructions for use; with resistance for galvanometer lamp for operation on 110-volt circuit...................0.- ror arch aif .. . 250.00 4891. ditto, but with resistance for 220-volt circuit ................2ee++-++ + 250-00 Descriptive literature on request. Orders accepted now for October delivery. ARTHUR H. THOMAS COMPANY WHOLESALE, RETAIL AND EXPORT MERCHANTS LABORATORY APPARATUS AND REAGENTS WEST WASHINGTON SQUARE PHILADELPHIA, U. S. A. THE QUANTITATIVE DETERMINATION OF AMINO- ACIDS OF FEEDS. By T. S. HAMILTON, W. B. NEVENS, ann H. S. GRINDLEY. (From the Department of Animal Husbandry, University of Illinois, Urbana.) (Received for publication, July 12, 1921.) INTRODUCTION. The need and the importance of knowledge concerning the amino-acid content of foods and feeds require no emphasis. Grindley, Joseph, and Slater (1) in 1915 were the first to publish data on the quantitative determination of amino-acids in feeds. About 1 month later Nollau (2) published results on the amino- acid contents of certain commercial feeds. Later in the same year Grindley and Slater (3), in a second paper, published additional results.on the same problem. Both Grindley and associates and Nollau used in their work the Van Slyke method, but the ‘results from the two laboratories do not agree well. As is stated in more detail later, the lack of concordant results, in general, may be explained by differences in the procedures used. Criticism has been made, however, of the application of amethod, designed entirely for the purpose of analyzing pure isolated pro- teins, to the analysis of heterogeneous mixtures such as feeds. Among several difficulties mentioned, the effects of the non- protein nitrogenous constituents and of the carbohydrates on the results of the Van Slyke analysis were unknown. In order to determine the effect of the non-protein nitrogenous material, Grindley and Eckstein (4) made a study of the non-protein con- stituents extracted from various feeds with cold water. Hart and Bentley (5) made a similar study but used hot water instead of cold water as their extracting fluid. From the fact that most of the non-protein nitrogen of the feeds examined was in the form of ammonia or a-amino-acid nitrogen, free or combined, Grindley and Eckstein in part conclude: 249 , THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 250 Determination of Amino-Acids of Feeds “ , , it seems quite evident that only a small part, if any, of the ngeecete in nitrogenous constituents of foods and feeding stuffs can in anyway interfere with the application of the Van Slyke method for the determination of the chemical groups characteristic of the different amino acids of protein to the estimation of the free and combined amino acids and amides of feeding stuffs.’’ And that no claim to perfection is made for the results published by Grindley and his associates is shown by the statement of Grindley (6): ‘Further it is also quite evident that the results so far obtained in this work are only approximately accurate and at present are to be considered of comparative value only.” The chief source of error in the method of analysis used by Grindley and associates was thought to be caused by the presence of the carbohydrates in the feeds during the hydrolysis and subse- quent analysis. Gortner and his associates (7, 8) have made an extensive study of the formation of humin in the presence of carbohydrates during acid hydrolysis. These authors have shown that the formation of humin depends to a large extent on the presence of carbohydrates and that the quantity of humin formed on hydrolysis of pure proteins is greatly increased by the addition of carbohydrate material. Attempting to reduce the quantity of humin formed during hydrolysis, Eckstein and Grindley (9) made two decided improvements on the older method. The first was the removal of some of the non-protein nitrogenous constituents by extractions with ether and cold absolute alcohol. The second was “the conversion, as far as possible, of the insoluble carbohydrates into soluble carbohydrates by boiling the feeding- stuffs with 0.1 per cent hydrochloric acid.’’ In this manner it was possible to separate a large part of the carbohydrates from the main portion of the proteins before the latter are hydrolyzed. The quantity of humin nitrogen obtained by this method compared very favorably with that formed in the analysis of some of the pure proteins. While this method of Eckstein and Grindley was a decided im- provement over the first method used by Grindley and associates, it was far from perfect. The perfection of a method for the quanti- tative determination of the amino-acid content of feeds has been the aim ofan extensive investigation conducted in this laboratory. > Hamilton, Nevens, and Grindley 251 Method. The method outlined below is the culmination of 146 experi- ments designed to separate the proteins of feeds quantitatively, either as such or as hydrolyzed proteins, from the other constit- uents of the sample which would interfere with the determina- tion of amino-acids by the Van Slyke method (10,11). The completeness with which this has been accomplished is shown by an examination of Tables I and II. The analysis is divided into two distinct parts: First, the treatment of a sample of feed so that all the proteins are obtained in solutions sufficiently free from interfering substances so that the Van Slyke method for the estimation of certain amino-acids may be applied; and second, the quantitative estimation of these amino-acids. f The first part of the procedure consists of a series of extractions with various solvents, and, in those extracts in which it is neces- sary, the separation of the proteins, by various methods, from the interfering substances. The residue remaining after the last extraction consists chiefly of crude fiber and contains very little nitrogen. The essential features of this part of the procedure are as follows: 1. The non-protein nitrogenous constituents are extracted from a weighed quantity of the finely ground feed, equivalent to ap- proximately 6 gm. of protein, by extracting with anhydrous ether, cold absolute ethyl alcohol, and cold 1.0 per cent trichloroacetic acid, in the order named. These extractions as well as all other extractions in the cold are carried out in the following manner: The sample is placed in a 500 ce. centrifuge bottle, 100 to 200 ce. of the extracting liquid are added, and the bottle is placed on a shaker arrangement which rolls the bottle back and forth continu- ously. Usually but two extractions are made each 24 hours; one extraction for a 7 to 8 hour period is made during the day and a second extraction for a 14 to 15 hour period is made during the night. As a rule six or seven extractions with each solvent are necessary to insure complete extractions in the cold. After each extraction period the solution is centrifuged and the super- natant liquid decanted. The small amount of protein extracted by the cold trichloroace- tic acid is recovered by precipitation with colloidal ferric hydrate. 252 Determination of Amino-Acids of Feeds 2. The main portion of the proteins is next extracted with cold dilute sodium hydroxide solution on the shaker. 2.32 cc. N, 20°, 762 mm. (Van Slyke). (C4H,O;N)2CU3°8H2O. Calculated. H2O 22.9, Cu 30.4, N 4.47 Found. H.O 22.0, Cu 30.4, N 4.31 Zine Salt (Anti)—This salt closely resembles the para salt. It is sparingly soluble and contains three equivalents of zine to one of the acid. Analysis. 0.1984 gm. substance:0.0397 gm. H.O, 0.0782 gm. ZnO. (C4H4,O;N)2Zn3:7H.O. Calculated. H.O 20.5, Zn 31.9. Found. H.O 20.0, Zn 31.7. Silver and Lead Salts (Anti).—-The silver salt was obtained as a white insoluble curdy precipitate containing 59.7 per cent of silver (calculated 59.8). The normal lead salt is obtained as a dense white heavy precipitate on adding lead acetate to a neutral solution of the sodium or ammonium salt of the anti acid. It contained no water of crystallization and gave on analysis 58.1 per cent of lead (calculated 58.3). On adding lead acetate to a solution of the free acid a microcrystalline insoluble salt of com- plex composition (48.3 per cent lead) separates out, and on washing with water is slowly converted into the normal salt. Action of Nitrous Acid on. Anti- and Para-Hydroxyaspartic Acids.—In each case the anti or para acid (1.49 gm.) was dis- solved in 150 cc. of water and 3 ce. of concentrated hydrochloric acid. The solutions were kept at room temperature and sodium nitrite (1 gm.) was added by degrees in the course of a day. Nitrogen was freely evolved on agitating the solutions. The following day the solutions were almost neutralized with ammonia 290 Para- and Anti-Hydroxyaspartie Acids using litmus as indicator, and an excess of calcium acetate was added. The precipitated calcium salts which separated rather slowly were in each case filtered off, dissolved. in a few drops of hydrochloric acid, and _ reprecipitated with ammonia. The anti-hydroxyaspartic acid gave a granular calcium salt, composed of small prisms and contained close to 3 molecules of water of crystallization, which it lost at 170° and appeared identical in every respect with calcium mesotartrate prepared for comparison. Analysis. 0.1463 gm. substance:0.0331 gm. H.O, 0.0789 gm. CaSOx. C,H,O,Ca-3H.0. Calculated. H,O 22.3, Ca 16.5. > Found. H.O 22.5, Ca 16.0. The para-hydroxyaspartic acid gave a calcium salt which was slightly more soluble than that from the antz acid and was made up of needles. Few if any of the mesotartrate prisms were found on microscopic examination. The salt contained close to 4 molecules of water which were removed at 170° and appeared identical with calcium racemate prepared for comparison. Analysis. 0.1838 gm. substance:0.0498 gm. HO, 0.0989 gm. CaSO,. C,H,0,Ca-4H,.O: Calculated. H:O 27.7, Ca 15.4. Found. HO 27.1, Ca 15.8. The yield of mesotartarie and racemic acids was only equiva- lent to about 25 per cent of the theoretical amount. Action of Aniline on Chloromalic Acid—In connection with the study of other bases than ammonia upon chloromalic acid, the following two derivatives were obtained by the action of aniline. Chloromalic acid (1 mol) with aniline (4 mols) were heated for 3 hours in a flask placed in a paraffin bath at 130°. The sticky mass was well washed with dilute hydrochloric acid to remove excess of aniline and then heated with alcohol. A small amount of sparingly soluble substance which proved to be an ‘fanil”’ derivative of phenylaminomalic acid was filtered off and subsequently recrystallized from glacial acetic acid in which itis readily soluble when hot but sparingly soluble at room tem- perature. It erystallizes in bright yellow plates and melts at 238-239° (uncorrected). From the alcoholic filtrate a dianilide of phenylaminomalic acid was obtained which was recrystallized from 90 per cent methyl alcohol. It crystallizes in nodular clumps H. D. Dakin 201 of bright yellow needles and on heating softens above 200° and melts at 210—211° (uncorrected). The yield of the latter substance is considerably greater than that of the ‘‘anil’’. Its reaction may be represented as follows: COOH CO-NH C,H; co | CHCl CH-NH C,H; VA SAGAS O20 ST. — | — O;H;N | ai CHOH Ny Chor COOH CO-NH C,H; CO Chloromalic acid. Dianilide of Phenylaminomalic acid anil. phenylaminomalic acid. Analyses. 0.1102 gm. anilide:0.2850 gm. COs, 0.0543 gm. H.O 0.1000 ‘“‘ = 50.0112. .° N (jeldahi): C22H2103N3. Calculated. G 70.7, H 5.58, N M22 Found. C 70.6, H 5.50, N 11.2. 0.1169 gm. anil: 0.2901 gm. COs, 0.0537 gm. H.0. CigH1403No. Calculated. Cc 68.0, Et 5:0: Found. Croan ElaoslO: The hydrolysis of the preceding derivatives with formation of phenylaminomalic acid has not yet been satisfactorily accomp- lished. BIBLIOGRAPHY. . Skraup, Z. H., Z. physiol. Chem., 1904, xlii, 274. . Erlenmeyer, E., Ann. Chem., 1905, eecxxxvil, 218. . Neuberg, C., and Silbermann, M., Z. physiol. Chem., 1905, xliv, 147. . Lossen, W., Ann. Chem., 1906, ecexlviii, 306. . Fischer, E., and Leuchs, H., Ber. chem. Ges., 1902, xxxv, 3787. oR Whe 2a STUDIES IN INORGANIC BLOOD PHOSPHATE. By EDWIN P. LEHMAN. (From the Department of Surgery, Washington University School of Medicine, Saint Louis.) (Received for publication, July 18, 1921.) As preliminary to a series of experiments to be noted in the latter part of this paper, a number of inorganic phosphate deter- minations in the whole blood of rabbits under normal and experi- mental conditions has been made. These covered first the inor- ganic phosphate of the blood of normal rabbits fed on the usual laboratory diet of hay, bread, and oats; and second, the curve of inorganic blood phosphate following the intravenous injection of phosphate solutions. Normal Bloods. No series of observations on the inorganic phosphate of the blood of normal rabbits has been found in the literature. Iver- sen’s (1) observations on the ‘‘acid-soluble” phosphate in normal bloods are prefaced by remarks on his technique in which he states that the investigated bloods (rabbit, guinea pig, rat, cat, etc.) showed 5 to 7 mg. of phosphorus as inorganic phosphate per 100 ce. of blood, whereas in his experimental reports the total ‘‘acid-soluble”’ phosphate is given us about 30 mg. of P per 100 ec. of blood. The method of Bell and Doisy (2) for the estimation of inorganic blood phosphate was used under the personal direction of Doctor Doisy to whom acknowledgment is gratefully given. The method, a recent one, is simple and as judged by a number of duplicate determinations, accurate. Occasionally a sample of blood was met with in which the color reaction of phosphomolyb- die acid failed to appear. Possibly the quantity of ammonium 293 294 Inorganic Blood Phosphate molybdate added was insufficient to combine with the phosphate in the usual manner. We have often noted a turbidity which is not phosphomolybdate when the ammonium molybdate is added. This undoubtedly indicates another insoluble compound of molyb- dic acid with the probable failure of the formation of ammonium phosphomolybdate—an essential upon which the determination hinges. It has been found that these anomalous results can be corrected by doubling the quantity of ammonium molybdate added; 7.e., 1 ee. of 10 per cent ammonium molybdate in 2 N H.SOs. Oxalated blood was obtained by nicking the marginal ear vein and collecting in amounts of a little over 2 cc. by allowing it to run into a calibrated test-tube containing a few crystals of potassium oxalate. From this tube 2 cc. were pipetted off for estimation. The potassium oxalate was tested for phosphate and was found free from this impurity. The results of twenty-six readings of normal rabbit. blood were as follows: Mg. per 100 ee. of blood. 4.1 5.8 4.5 4.4 4.2 6.0 5.3 4.5 4.5 5.1 thal 3.9 5.4 5.4 6.8 4.5 4.3 5.0 5.7 2.6 5.3 4.6 4.5 4.2 4.7 4.3 Average...... 4.87 mg. of P as inorganic phosphate per 100 ce. of biood. The striking fact in these figures is the uniform level of this substance in the normal blood. All but three of these readings lie between 4 and 6 mg. of P per.100 cc. of blood. The average is seen to be 4.87 mg. The high reading is 7.1 mg. and the low, 2.6 mg. Eleven of these determinations were made in duplicate. Several of the determinations were carried out on each of three different rabbits over periods up to 502 hours. EK. P. Lehman 295 Rabbit No. Date. Time. er eee ty Oe: 1 Oct. 11 1.30 p. m. 4.1 80), 4.2 Boal): & 4.5 Oct. 12 10s Tb yaceng: 5.4 3.00 p.m 4.3 Oct. 13 10.15 a. m aoe 2 Oct. 12 ELS ae mn 5.8 2.00 p. m 6.0 BRD -& Hil Oct. 13 105.2. mM 5.4 ble! 2.00 p. m 5.0 3 Oct. 14 2.00 p. m. 4.6 ay 4.7 Oct. 15 Boy 4.5 Here again a striking constancy of inorganic phosphate level is shown. Experimental Injection of Phosphate Solutions. The literature of this field of the present study offers a greater number of previous reports, largely because of interest in the phosphorus-calcium balance in the blood and its relation to tetany. The most important work in this connection is that of Binger (3), who plotted a curve of toxicity of injected ortho- phosphates varying with their pH. He found that in dogs as much as 250 mg. of P per kilo of body weight in the form of ortho- phosphoric acid could be injected intravenously without develop- ment of tetany, whereas 200 mg. in the form of NasHPO, and 150 mg. in the form of Na3PO, each caused tetany. He further plotted a curve of serum phosphorus, using Marriott and Haess- ler’s method of estimation, following injection. This curve shows the same prompt fall to normal as the present experiments show. He presents also the inverse curve of calcium that blood analyses in various forms of tetany indicate.. Iversen (1) found in the rabbit a similar curve to those about to be reported. His injections were made over a prolonged period. In a later paper (4) he found that both in vitro and in vivo the red corpuscles take 296 Inorganic Blood Phosphate up gradually a proportion of the acid-soluble phosphate present in the plasma. Jn vivo this means that following the injection of phosphates, the curve of acid-soluble phosphate in the cells falls more slowly than that in the plasma. All of the present series of experiments were done in the same manner with the exception of the solutions and amounts used. The rabbit, on the usual diet, was weighed and a sample of blood was taken for estimation of inorganic phosphorus as described in the first portion of this paper. The solution at room tempera- ture was then injected as rapidly as possible by gravity from a burette into the marginal vein of the ear not previously employed for the procuring of a blood sample. ‘This injection took about 5 to 8 minutes on the average. Observations for toxic effects were made and blood samples were withdrawn at varying inter- vals. The solutions used were: (1) M/15 NaH»PO,; and (2) a mixture of NaH,PO,-4H,O and Na;HPO,.-2H20 so calculated as to present a solution of approximately pH 7.3 and approximately isotonic with the blood. The curves (Figs. 1 to 8) present the results of the estimations following the injection in 8 of the below 10 experiments. Experiment No. Solution 1. Solution 2. ce. cc. 4 100 5 75 6 15) 7 75 8 75 9 75 10 50 11 : 50 19 75 20 75 Experiments 4 and 9 present no curves on account of technical errors in collecting the blood samples. They are included in this report because the former succumbed with positive symptoms of tetany and the latter was the only animal to die without symptoms of tetany. 119. f per 100 cc. Blood Eee Pieces a cee: Mme eis beet le ORL MIO CH 52 G2 ae ST OUP S Fic. 1. Experiment 5. 75 mg. of P per kilo—NaH.POs, solution. No symptoms. The reading marked * is probably erroneous. The color reaction resembled that mentioned in the text as occasionally disturbing the {estimations. ro Nie DEE Ee we LN Sa |ITIN eS aS <7 Lt N10 eae 89 ‘Skea aye |p| aa Size ae ie eit eee Fae ce aa 2s | OQOl246 F535 6 F-#E249 LL OUPS Fig. 2. Experiment 6. 75 mg. of P per kilo—NaH2PO, solution. Sal- ivation; cyanosis. Survived. oe 7 298 Inorganic Blood Phosphate The protocols of Experiments 15 and 18 have been omitted on account of the occurrence of the phenomenon mentioned in the discussion of the Bell and Doisy method. This obviously inval- 19 19 18 16 m7 Mig 16 ib N 19 Ve] rn /4 ha iN NB NEG N % NS ~ & fee N Sl S M/ = \ 10 g < \X 9 S 9 “ +S S Ng Na a S 7 We NS 6 V6 S e; SF, 4 4 3 of 2 ke i a Ore. TlOuTS TOUTS Fra. 3. Fria. 4. Fie. 3. Experiment 7. 75 mg. of P per kilo—NaH PO, solution. Tet- any; salivation. Dead in about 3 hours. Fic. 4. Experiment 8. 75 mg. of P per kilo—NaH.PO, solution. Tet- any; salivation. Dead in 18 hours. Fic. 5. Experiment 10. 50 mg. of P per kilo—NaH,PO, solution. No Symptoms. idated the results which showed in the former instance a late rise of blood phosphate and in the latter, an experiment on a dog, no rise at all. E. P.- Lehman 299 There is in these curves a demonstration of the strong tendency of the rabbit’s body to maintain the constant level of inorganic blood phosphate found in the estimations on hormal rabbits given above. The rapidity, with which concentrations of blood LT OUTS Fia. 8. Fig. 6. Experiment 11. 50 mg. of P per kilo—NaH PO, solution. No symptoms. Fig. 7. Experiment 19. 75 mg. of P per kilo—mixed phosphate solution. No symptoms. Fig. 8. Experiment 20. 75 mg. of P per kilo—mixed phosphate solution. Slight tetanic symptoms. Survived. phosphate four to five times the normal disappear, is extreme. In this connection it must be remembered that the first post injection estimation is made at about 1 hour. Previous to the end of this hour the phosphate concentration must be enormously 300 Inorganic Blood Phosphate higher. Within 4 hours after injection the normal level has been reached. No attempt was made to follow the blood calcium. Somewhat similar experiments on the calcium content of the whole blood of rabbits are reported by Clark (5), with similar results. It is clear that the phosphate-calcium balance is main- tained by the organism at a constant level, and that any deviation from this level will be promptly corrected. \ The symptoms observed following the injection of massive doses of phosphates deserve mention on account of the interest in the toxicity of these substances. When 100 mg. of P per kilo of NaH»PO, were injected (Experiment 4) there was the development of tremors followed by distinct tetanic convulsions and death in 3 hours. These convulsions could be brought on in the rigid extensors of the leg by handling or attempting to flex. Similar observations were made in Experiments 7 and 8 with death in 3 and 18 hours respectively. These two rabbits received 75 mg. of P per kilo of the same substance. Rabbit 9 which also re- ceived the last named dosage, died about 12 hours after injection without symptoms of tetany. The only symptoms noted were cyanosis and some form of circulatory collapse which prevented the free flow of blood from the vein. One rabbit (Experiment 20) which received 75 mg. of P per kilo of the mixed phosphate solution showed dyspnea and a questionable spasm of the back and neck muscles. There were no convulsions. An interesting symptom which has for lack of a better name been noted in the curves as ‘‘salivation’’ occurred in several instances. This consisted in a free flow of thin mucoid material from the mouth in the first 2 to 3 hours following injection. Whether or not this represents simply an attempt on the part of the organism to dispose of the excess fluid injected is not clear. No estimation of phosphate in this secretion was made. Nor were controls with the injection of salt solution carried out. Cod Liver Ol Feeding. In connection with recent clinical and experimental reports (6, 7, 8) on the calcification of bone in rickets following the feeding of cod liver oil, it was thought that possibly some light might be thrown on the phenomenon by a similar series of experiments, substituting cod liver oil per os for the injection of phosphates. These experiments were entirely negative. E. P. Lehman 301 The same general method was employed. Cod liver oil was given by stomach tube in 20 cc. amounts and the inorganic blood phosphate was followed at somewhat longer intervals to allow of absorption. In three such experiments in the rabbit and one in the dog no alteration of inorganic blood phosphate was observed. Blood Phosphate and the Calcification of Callus. The purpose for which the above experiments were carried out was to form a groundwork on which a surgical problem with biochemical aspects could be carried out. As this work presented negative results it will be but briefly mentioned here. Provided that the periosteal cell reaction about a fractured bone is normally active, the length of time during which the patient is incapacitated depends primarily on the rate of deposi- tion of calcium salts in the soft callus. With this idea as a basis many experimental and clinical attempts have been made to increase the speed of this calcification. The present attempt has been based on the following facts. If one adds zn vitro a solution of phosphates to a solution contain- ing calcium and corresponding in composition to the inorganic composition of the blood, there is precipitated a calctum compound corresponding to the composition of bone (9). Furthermore, the injection of phosphates intravenously results in an immediate fall in the calcium content of the blood, which stays low until the excess phosphate disappears (3). This latter fact suggested the possibility that the reaction definitely known to occur in vitro might also occur in vivo. The fate of the calcium which dis- appears from the blood stream is unknown, but it seemed reason- able to assume that it might be directed to a site where there is under ordinary circumstances a tendency for calcium to be deposited. Such a site is, of course, furnished by a soft callus. The experiments were therefore conducted on the following lines: A bone defect was made with a saw and after varying in- tervals allowing for the periosteal reaction, the phosphate con- tent of the blood was increased by intravenous injection. The calcification of the fracture was then followed by the x-ray in both the experimental and control animals. 302 Inorganic Blood Phosphate The rabbits were taken in pairs from the same litter and the fore legs x-rayed. A transverse segment of bone about 3 mm. in thickness was then removed from the ulna of each rabbit with the Albee saw, with care to disturb the remaining periosteum as little as possible. Ether anesthesia was used and the wound closed with silk. At the end of an interval varying from 7 to 20 days, the rabbits were again x-rayed. The experimental rabbit then received an injection in the marginal ear vein of a m/15 solution of acid sodium phosphate in the proportion of 50 mg. of phosphorus per kilo of body weight. None of the rabbits showed signs of distress nor was there evidence of tetany. X-rays were taken within the next 6 to 8 hours and again in most instances at later intervals. Eleven pairs of rabbits were sub- jected to this type of experiment without infection of the wounds. One experiment was conducted by feeding the rabbit cod liver oil instead of injecting phosphate. The results of the experiments showed in no case an undoubted increase of the speed of calci- fication in the injected rabbits. Two of the above eleven experiments were conducted with the addition of the intraperi- toneal injection of a vital stain, sodium alizarin sulfonate (10), as a measure of calcium deposit but in neither of these ‘did this method add anything to the x-ray evidence. Clark (5), already quoted above, found that an increase of calcium in the blood stream by injection quickly reached the normal level; and that the ingestion of calcium did not affect the normal level in the blood. From these observations one is led to believe that the repetition of the present experiments with an attempt to increase the available calcium would be equally unsuccessful. SUMMARY. 1. The average of twenty-six estimations of the normal inor- ganic phosphate in the whole blood of rabbits is 4.87 mg. of P per 100 ce. 2. The normal inorganic phosphate of rabbit’s blood is practi- cally—that is within biological limits—a constant. 3. An increase by four or five times in the concentration of inorganic phosphate in rabbit’s blood returns to normal within 4 hours. | E. P. Lehman 303 _ 4. The intravenous injection of rabbits with 75 mg. of P per kilo of body weight in the form of NaH»PO, will cause tetany in a certain proportion of individuals. 5. On the basis of few experiments, the ingestion of cod liver oil causes no change in the level of inorganic blood phosphate in the rabbit and dog. 6. The intravenous injection in the rabbit of a single massive dose of phosphate has no demonstrable effect on the calcification of callus. NOOR wWNe BIBLIOGRAPHY. . Iversen, P., Biochem. Z., 1920, cix, 211. . Bell, R. D., and Doisy, E. A., J. Biol. Chem., 1920, xliv, 55. Binger, C., J. Pharmacol. and Exp. Therap., 1917-18, x, 105. . Iversen, P., Biochem. Z., 1921, exiv, 297. Clark, G. W., J. Biol. Chem., 1920, xliii, 89. . Phemister, D. B., J. Am. Med. Assn., 1918, Ixx, 1737. . Phemister, D. B., Miller, E. M., and Bonar, B. E., J. Am. Med. Assn., 1921, Ixxvi, 850. . Shipley, P. G., Park, E. A., McCollum, E. V., Simmonds, N., and Parsons, H. T., J. Biol. Chem., 1920-21, xlv, 343. . Marriott, W. McK., personal communication. . Brooks, B., Ann. Surg., 1917, lxv, 704. CAN “HOME GROWN RATIONS” SUPPLY PROTEINS OF ADEQUATE QUALITY AND QUANTITY FOR HIGH MILK PRODUCTION? III.* By E. B. HART anp G. C. HUMPHREY. With THE CooPERATION oF J. H. JONEs. (From the Departments of Agricultural Chemistry and Animal Husbandry, c University of Wisconsin, Madison.) (Received for publication, July 25, 1921.) Supplementary to previous work! we have continued our studies on the possibility of furnishing an adequate protein supply to high milk-producing cows from home grown sources. In the work done in 1919-20! with alfalfa hay, we maintained by the use of corn starch a like energy supply in the several rations when a change in the grain mixture was made. For example, when ground oats were substituted for ground corn meal the lowered net energy value of the ration was made good by the use of a definite amount of corn starch adjusted according to Arms- by’s? data in which he gives the net energy value of 100 pounds of corn meal as 89.16 therms and of 100 pounds of whole oats as 67.56 therms. When this was done, as our records showed, it was entirely possible to maintainsnitrogen equilibrium and high milk production with these liberal milking animals over a period of 16 weeks. The possible effect of a lowered energy intake through the substitution of ground whole oats for ground corn meal in the ration with maintenance of a constant protein level but with no starch additions was now studied. * Published with the permission of the Director of the Wisconsin Agricultural Experiment Station. 1 Hart, E. B., and Humphrey, G. C., J. Biol. Chem., 1919, xxxvii, 515; 1920, xliv, 189. 2 Putney, F. S., and Armsby, H. P., Pennsylvania State College Bull. 143, 1916. 305 306 Home Grown Rations. III Such an experiment would touch practice more closely than our earlier experiments did, as the common farm procedure would be to use either a single grain such as whole oats or a mixture of grains, but without the addition of starch. EXPERIMENTAL. In these experiments we worked with the corn, barley, and oat grains, used singly and supplemented with corn silage and alfalfa hay. The alfalfa was grown in southern Wisconsin and was taken from the second cutting. Since the protein content of barley was Watered to that of the corn and oat grains used, we chose the barley ration as the standard to which the other rations must conform with re- spect to protein. For example, 50 pounds of the barley grain ration consisted of 10 pounds of grain, 10 pounds of alfalfa, and 30 pounds of corn silage. The corn grain mixture consisted of 10.6 pounds of corn meal, 10 pounds of alfalfa, and 30 pounds of silage and contained the same amount of total protein as the barley ration. The oat grain ration consisted of 9.1 pounds of whole oats, 10 pounds of alfalfa, and 30 pounds of silage and fur- nished the same amount of protein (N X 6.25) as the other rations. The 50 pounds of barley ration furnished 16.37 therms; the 50.6 pounds of corn ration furnished 17.24 therms; while the 49.1 pounds of oat grain ration furnished but 14.43 therms. Much to our surprise these differences in the net energy values of the rations, particularly the oat ration as compared with the others, were sufficient to determine whether these high milk- producing cows would be in negative or positive nitrogen balance. The corn and barley rations were ample both in protein and net energy content for high milk production over the periods of observation; but the oat ration was not generous enough in its net energy content for these lactating animals—the effect being manifested by distinct negative nitrogen balances during the period. of 4 weeks observation. Evidently protein was being de- stroyed as a source of energy during the feeding of this lower therm- containing ration. Cow 3 weighed approximately 1,000 pounds and was producing daily 40 pounds of milk containing 3 per cent of fat. According to Armsby’s standard such a cow would require for maintenance el E. B. Hart and G. C. Humphrey 307 and the production of this amount of milk a daily intake of 14.4 therms. Actually she was receiving 14.43 therms per day, but with that amount of energy, nitrogen equilibrium was not main- tained; for while Armsby’s standard requires 2.22 pounds daily of digestible true protein for a cow with the producing capacity of No. 3, she was receiving but 1.68 pounds of digestible true protein. The two other animals involved in this inquiry showed similar negative nitrogen balances on the whole oat ration but positive balances or equilibrium on the barley and corn rations, in which the protein content was the same as in the oat ration but the net energy supply 2 to 3 therms higher per day. Animal 1 was a pure bred Holstein, No. 2 a grade Jersey, and No. 3 a grade Guernsey. They weighed respectively as follows: No. 1, initial weight 1,502 pounds, final weight 1,465 pounds; No. 2, initial weight 997 pounds, final weight 1,034 pounds; No. 3, initial weight 1,038 pounds, final weight 995 pounds. The methods of analysis and quantitative collection of excreta have been described in earlier publications. Each ration was fed for a period of 4 weeks with a preliminary feeding period of 5 days before quantitative collection of excreta was begun. There was no preliminary feeding period when the rations were changed. Each cow was allowed what she would completely consume of the mixed ration. The ration used contained the following percentages of nitrogen: Nitrogen. per cent CO TEER ST oa Oe eae a or a a oy RS iLGY/ LD TE EAD HITDS Sae ee ae ac) ee eae ee > 1.82 EET COG NI Pam oe 0 See MN ee | a 1.66 OT LR ln Tn Be ea, re a 0.36 AL IEDILID Ty ek nS eee I oe aaa a 2.65 The data on nitrogen balances are presented in Tables I to III inclusive. . From the data presented in the tables it is clearly evident that there was not only a distinct negative nitrogen balance during the oat grain period of feeding, when the therm intake was reduced, but that there was a marked increased destruction of protein in this period as shown in the greatly increased urinary nitrogen output exhibited by each animal. No. 1 increased her urinary 308 Home Grown Rations. III © nitrogen output 50 per cent above the urinary nitrogen output on the corn grain ration and increases of nearly similar magnitude were shown by Nos. 2 and 3. These data are not to be interpreted as indicating an inferiority of the oat proteins as compared with those of the corn or barley grain since all available evidence! points towards an approximately equal supplementary efficiency for the cereal grain proteins. TABLE I. Record of Nitrogen Balance, Milk Production, Etc., in Animal I. Nitrogen. Date. F Intake. Feces. | Urine. | Milk. | Balance. poe, Barley grain ration. | gm. gm. gm. gm. gm. tee: Deew 4-20. a. eee 1,712.9] 647.7 | 570.9 | 585.8} —91.5| 248 BO hixtick a acs A229) doe? 540.2 547.2 —90.2 236 ce 28=) ANS see 1,712.9) 672.8 458 .2 550.3 +31.6 241 Yant.4A—1 Oe ea, oe eee 1,850.1) 698.3 423.1 | 559.0 | +169.7 244 Corn grain ration. Jams 11=17 .22ci04.0-<) 1, 850.1) 728.3-| 47229: 6970 | 7-79 2Cie eae SEIS 4 eh Ace 1,850.1| 788.9 | 486.8 | 569.6 | +54.8) 257 et Oat tees Sree 1,850.1) 702.5 484.5 586.1 +77.0 258 Reb slave aces ee 1, 850.1) 702.5 | 525.6] 571.7 | +650.3) 252 Oat grain ration. Feb. 3 4. 1,850.1) 583.4| 668.9| 555.9| +41.9| 244 BE OL ee eee 1,850.1 616.5 | 707.0} 556.9 | -—30.3) 244 ES BO et nee 1,850.1} 622.9 | 793.0 | 539.7 | —105.5) 236 Mar-vl=7. 5 Ween cee 1,850.1) 651.8 817.3 540.1 | —159.1 232 Another important point coming from the collection of such data as here presented are differences in urinary nitrogen elimina- tion which are often observed with approximately similar absorp- tion from the intestine. Apparently in individuals different rates of deamination and destruction of important carbon nuclei are taking place which may be closely related to the often observed differences in the efficiency of a protein mixture with different animals. For example, in these trials No. 2 during the corn EK. B. Hart and G. C. Humphrey 309 ration period was absorbing approximately 1,000 gm. of nitrogen per week and eliminating 480 gm. in the urine, while Animal 3 during the same period was absorbing practically the same amount of nitrogen but eliminating only 410 gm. per week; pre- sumably the energy requirement was amply covered in both. in- dividuals. It is in this direction; namely, the rates of inter- mediary nitrogen metabolism and the special tissues involved in producing these different rates, that we must look for an TABLE II. Record of Nitrogen Balance, Milk Production, Etc., in Animal II. Nitrogen. Date. ; Intake. Feces. | Urine. | Milk. Balance. Leer Barley grain ration. gm. gm. gm. gm. gm. lbs. WMecwtA-J0i te. oes k a 1,575.7| 549.6 | 409.2 |) 491.3 | +125.6) 185 Rae ers. stu. Sa 1,575.7) 588.1 | 524.3 | 444.5 | +18.8) 167 SE 2S ALIN ay). ator 1,575.7; 602.0} 449.0 | 440.2 +84.5| 188 mes —1 ees os | 1, 5/on4\) 200350) | 45027 | 445-0 | =-77.0) 7104 Corn grain ration. los rr 1,575.7| 560.1 | 444.8 | 439.1 | +131.7| 180 U2 a? re 1,575.7) 620.9 492.8 434.9 +27.1 160 OF GT Ie eee 1,575.7| 567.4 | 497.8 | 4389.8 | +-70.7| 185 ING) 0-1 / Gi gont epee eee 1,575.7, 563.6 | 476.6 | 4387.3 | +98.2 180 Oat grain ration. 122) oy ca eee IL SYSS TAL GOB aE | 628.9 | 433.3 +9.9} 177 22° G4 ES aie Sen 1,575.7| 488.9 | 614.1 | 431.1} +41.6| 173 2 ODE eae 1,575.7| 494.0 | 663:6 | 428.5 | -—10.4) 174 IWS (cscs stctens ceie yokes 1,575.7| 446.6 | 713.5 | 439.3 | —23.7| 175 explanation of differences in the efficiency of individuals in respect to protein utilization. While protein constitution is of primary importance in this respect, the additional factor of the individual rate of intermediary metabolism will come into play especially in the application to practice of a mathematical standard. This factor would assume special importance where the protein allowance was not liberal and was reduced to a standard formulated from data obtained with the ‘‘best”’ individuals. © TABLE III. Record of Nitrogen Balance, Milk Production, Etc., in Animal III. Nitrogen. Date. 7 ; Intake. | Feces. | Urine. | Milk. | Balance. Bela. ; Barley grain ration. gm. gm. gm. gm. qm. lbs. Deo. 14-20)... 225 ok 1,712.9} 664.2 | 415.5] 638.7) —5.3 316 are LOC Bethke rns 1,712.9| 661.2 | 387.8 | 612.8 | -+49.1 308 cS 28—-Jamnss se 1,712.9) 689.1] 318.6 | 639.7 | +65.5 341 Jan AO exces tee ee 1,712.9} 688.1 | 3892.5 | 640.9 | +41.4 334 Corn grain ration. dois THES Sonn niin Ace 1, 712:9|' 731.8 | 366.7 | 628.1 | —13°7 317 70 Gogh Goce Gap am Agee a, SOU 1,712.9} 746.3 | 437.8] 588.3 | —59.5 321 Sl As a eae 1,712.9] 682.0 | 447.4] 570.3 | +13.2 319 Heb tsl= erence 1,719.9} 698.4 | 397.0] 565.5 | +52.0 308 Oat grain ration. : | RebiSal4iiwn 8 eee 1,712.9| 580.8 | 558.6} 572.1 | +1.4 302 ae 2 Aa epee 1,712.9} 590.0 | 564.3} 562.1 —3.5 291 Set DDR ee i eta 1,712.9) 570.2 | 647.0 | 553.9 | —58.2 278 10 EA Ey fe Rs eed 1,712.9} 549.7 | 642.6] 555.4 | —34.8 279 TABLE IV. Composition of Milk and Average Daily Flow in a Selected Week of Each Period. Dec. 28 Jan, 25 | Feb. 22 Animal I. Total somdsieper Cent... i 6 cies: 12.98 12.03 11.85 Rat ser Cente Lec ariscc cee soe 3.60 3.40 3.55 Nitrogen.) pemcentenemcr cei acter 0.51 0.50 0.49 MIKO aily 0 seie catertets eke cree 35.00 35.00 34.00 Animal IT. Total ‘solids; ger cent. 1c oa ere 14.38 13.92 13.39 Bat; ver cent. 2 ee eee eee - 4.80 4.70 4.70 Nitrogen, pericent?.2 3) anaes eee 0.52 0.53 0.51 Milk daily, [bs; #4? wd. ecee 27.00 26.00 25.00 Animal ITT. Total solids, per cent........2...>- 11.75 10.79 10.84 Bat... per ‘cents. a:. 4... eee ee 3.00 2.80 - 3.008 Nitrogen, per cents. .o...%s eee 0.42 0.38 0.39 Milk daily, ths. 220223 Sc See 48.00 45.00 40.00 310 -E. B. Hart and G. C. Humphrey 311 In Table IV are recorded the composition of the milk and the average daily flow in a selected week of each period. The milk composition as well as flow was well maintained for the period of observation, but there can be no doubt but that a long continued feeding period on the oat ration, involving inadequate energy intake, would ultimately have affected milk secretion and milk composition. SUMMARY. 1. Data are presented which show that it is entirely possible when feeding equal but limited amounts of protein to maintain nitrogen equilibrium and high milk production in dairy cows with a ration composed of either barley or corn supplemented with corn silage and alfalfa hay, but not with the whole oat grain so supplemented. 2. Previous records had indicated this possibility with all cereal grains, but only when the deficient net energy content of the oat grain ration was made good by the use of corn starch. 3. The oat grain ration contained 14.43 therms per 49.1 pounds; the corn ration contained 17.24 therms per 50.6 pounds; both rations contained exactly the same amount of protein; yet this difference in the energy supply of the two rations was sufficient to produce a positive nitrogen balance on the 17.24 therms but a negative nitrogen balance on the 14.43 therms. 4. In practice, a mixture of the corn grain or the barley grain with the oat grain, 50 per cent of each, would very probably make up this deficiency in net energy. er rr wre STUDIES ON BLOOD SUGAR. THE TOTAL AMOUNT OF CIRCULATING SUGAR IN THE BLOOD IN DIABETES MELLITUS AND OTHER CONDITIONS.* By REGINALD FITZ anp ARLIE V. BOCK. (From the Medical Laboratory of the Massachusetts General Hospital, Boston.) (Received for publication, July 18, 1921.) In this paper observations are reported on the total amount of sugar in the circulating blood, and on its relative distribution between plasma and corpuscles in a series of normal persons, of diabetic patients, and of patients with other diseases. Numerous experimenters have studied the distribution of sugar per unit volume of plasma and corpuscles. The subject was carefully reviewed by Gradwohl and Blaivas, in 1917, and more recently by Wishart. According to the statements of certain previous workers, the corpuscles sometimes contain little or no sugar; according to others, the sugar content of the corpuscles does not differ greatly from that of the plasma; another belief has been that the corpuscles take up sugar more slowly and retain it longer than does the plasma so that the corpuscular sugar is low in the early stages of hyperglycemia, but above that of the plasma in the declining stages. The preponderance of experience is that the sugar content per unit volume of corpuscles is usually a little below that. of the plasma. According to Wishart the discrepancy in favor of the plasma generally becomes greater as the blood sugar rises. The desirability of knowing the total amount of circulating sugar and its distribution between the total volume of plasma * This paper is Number 19 of a series of studies, on the physiology and pathology of the blood, from the Harvard Medical School and allied hos- pitals, a part of the expense of which has been defrayed from a grant from the Proctor Fund of the Harvard Medical School for the study of chronic diseases. 313 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLYIII, NO. 2 314 Studies on Blood Sugar and corpuscles was suggested by Epstein and Baehr in this country in 1914. .These observers produced rapid changes in the blood volume of experimental animals. By diluting the blood the percentage concentration of sugar diminished, although the total amount of circulating sugar remained constant or was in- creased. They concluded, therefore, that in diabetes the total amount of circulating sugar must bear a more definite relation to glycosuria than the mere concentration of sugar for each unit volume of blood. Sansum and Woodyatt, in 1917, also determined the importance of the total blood sugar in establishing and re- gulating the rate of glycosuria and that the concentration of sugar in the plasma was of but little importance in this respect. No data have heretofore been published, however, which record the normal amount of circulating sugar in man, nor the variations from the normal that may occur in diabetes and other conditions. We obtained data on the subject by the following method. The total volume of the circulating blood and the total cor- puscular and plasma volume were determined by the vital red method of Keith, Rowntree, and Geraghty. One of us (Bock) has recently published a discussion of the advantages and dis- advantages of this method. The sugar concentration of oxalated whole blood and oxalated unhemolyzed plasma for each 100 ce. was obtained at the same time by the method of Folin and Wu. Knowing the amount of sugar to 100 ec. of blood and plasma, and the total volume of circulating blood and plasma, the total amount of sugar in the whole blood and plasma was readily ob- tained. The differences between the total amount of sugar and that found in the plasma represented the total corpuscular sugar. The results of our observations are incorporated in Tables I to IV. In Table I is recorded the total amount of sugar in the blood of seven normal persons; the amount varied between 7.54 and 2.50 gm. The plasma sugar varied between 4.85 and 1.64 gm. The corpuscular sugar varied between 3.85 and 0.69 gm. The average total amount of blood sugar for the group was 5.18 gm. The average plasma sugar was 3.29 gm. The average corpuscular sugar was 1.89 gm. ‘The marked variations encoun- tered in the individual cases probably depended, among other reasons, on wide differences in the area of body surface, and in weight, and on the fact that the estimations were made with Rea bitz-and Ax Ve Bock ay Ua) disregard for the time or nature of the previous meal. However, it seems logical to conclude from these data that by the methods employed the normal total amount of blood sugar does not ex- ceed 7.50 gm., and that the normal plasma sugar is usually con- siderably greater than the corpuscular sugar, but does not exceed 4.85 gm. In Table II are recorded similar observations in a group of nine diabetic patients. As was to be expected, in diabetics, as well as in normal persons, there were fluctuations in the amount of circulating sugar. The highest amount of sugar in the blood was 15 gm., ‘ +ha lowest 6.81 gm. The plasma, relatively TABLE I. Normal Persons. 3 g ic} Blood sugar 2 g E mg. per 100 ce. = z Z ¥ eran i EOE oe ee 'o) D = aa) a Oo ea) oy a oc a kg cc ce ce gm gm gm. 1 M. 82 6, 218) 3,731] 2,487) 120 130 | 7.54 | 4.85 2.69 2 M. 63 5, 758] 3, 628] 2,130} 130 100 | 7.48 | 3.63 3.85 3 12, 55 5, 750| 3,970) 1,780} 100 Cl) |) Gy. 706 |) S555 2.20 4 1 60 | 4,500} 3, 200} 1,300; 100 100 | 4.50 | 3.20 1.30 5 1a 60 4,460) 3,120) 1, 330 90 100) 32815 || 3-12) > (O869 6 | F. | 60 | 4,230] 2,750] 1,480] 110] 110 | 4.65| 3.02] 1.63 7 2, 60 3, 570} 2, 340} 1, 230 70 a 2.50 | 1.64 0.86 PAGVET ACO rc oetete eines US || Bi 248, 1.89 contained much more sugar than did the corpuscles. Thus the highest plasma sugar content was 10.78 gm., and the lowest 4.75 gm., while the highest corpuscular sugar content was 4.22 gm., and the lowest was 1.09 gm. The average total amount of blood sugar for the group was 8.95 gm. The average total plasma sugar was 6.72 gm. The average corpuscular sugar content was 2.23 gm. These findings are evidence that the blood, especially the plasma in diabetes, is a vehicle for the transportation of sugar from the body cells which are unable to burn or store it, to the kidney which excretes it. The blood corpuscles, as a whole, are but 316 Studies on Blood Sugar little concerned with such transportation of sugar and do not contain an increase in sugar proportional to that found ‘n the plasma. Whether this depends largely, as Wishart has suggested, on the fact that glucose is more freely so'uble in the plasma than in the corpuscular substance, or on other considerations, is un- certain. In any event, it seems logical to conclude with Wish- art that analyses of plasma of the total amount of sugar and of its concentration for each 100 ee. are preferable to those of whole blood. TABLE II. Patients with Diabetes. 3, 340) 2,140) 1,200; 240} 260] 8.02) 5.56) 2.46 2, oe 2,200} 730} 330) 390 | 9.67 : 3 rc Blood sugar 2 £ 2 E mg. per 100 ce. co 4 3 fo) & = 5 ore S E 2 S.| 8 & + S e | 33 2 Sit | ee g "so 8 5 aso \ tie f | ah | e881 se a bs o 2 & 65 2 a oa oa om 6) n = aa) x Oo za) cv a a o kg. cc ce. ce: gm gm gm 1 | F. | 70.0 | 4,690; 2,990| 1,700) 320] 360 | 15.00] 10.78] 4.22 2 M. | 47.0 | 4,030) 2,500} 1,530; 170] 190 | 6.86) 4.75) 2.11 3 | M. | 43.0 | 3,880] 2,440] 1,440] 190] 220| 7.37] 5.36] 2.01 4 M. | 41.5 | 3,784! 2,365} 1,419} 180 220 6.81) 5.20) 1.61 5 M. | 62.0 | 3,760). 2,480) 1,280} 220 240 8.24} 5.95) 2.29 6 M. | 54.0 | 3,590; 2,370) 1,220) 200 250 TAS| "5 9215 eb (6 Wats |) abe) |) ae Stasi eee 200, 1,163} 340 | 380 | 11.43} 8.36] 3.07 50.0 3.9 MGR BBE. vice) Bleu. 8.95 The findings shown in Tab!e II demonstrate, moreover, that blood sugar concentration expressed as mg. per 100 cc. of blood or plasma may give misleading information with regard to the total amount of circulating sugar, as Epstein and Baehr and San- sum and Woodyatt have suggested. For example, in Cases 1, 7, and 9, respectively, the sugar concentrations were 360, 380, and 390 mg. of sugar per 100 cc. of plasma. In Case 1 the plasma sugar concentration was the lowest of the three, with 10.8 gm. of sugar in the total plasma. In Case 7 the sugar in the total plasma was 8.36 gm., and in Case 9, in which the sugar concen- tration was the highest of the series, the sugar in the total plasma was only 8.6 gm. er R. Fitz and A. V. Bock a07 t The relation between the total amount of plasma sugar and the total amount of urine sugar was studied in a few cases. For this purpose the urine was collected for the 24 hour period in the midd'e of which the blood volume and sugar determinations were made. The sugar excretion was titrated by the Benedict me- thod on the urine in cases which yielded a positive reaction to Benedict’s qual tative test. The findings are recorded in Table WEL. The thresho!d at which glucose appeared in the urine of patients with diabetes seemed to le between 5.2 and 5.36 gm. of total plasma sugar. One diabetic patient whose plasma sugar concentration was 190 gm. per 100 cc. had a low plasma volume TABLE III. The Relation between Total Plasma Sugar and Sugar Excretion. oa Eee eee Case. Diagnosis. me per Dae Ww rene per liter. 4 hours. wve gm. kg. gm. gm. 1 Normal. 130 4.85 | 82.0 0 a0) 2 Diabetes. 190 4.75 | 47.0 0 0 3 aS 220 5.20 | 41.5 0 0) 4 ? 220 Recta || 4870) | abiesverey, || Bras very. 5 a 250 D292) wade O 5.00 27.50 1.14 6 o _ 390 8.58 39.95 13.90 21.54 2.26 Uf eS 360 10.78 70.0 29.40 52.92 4.10 and thus a low total amount of sugar. This probably explains why the patient did not excrete sugar in the urine with so high a glycemia. Three patients excreted titratable amounts of sugar. The total sugar excretion did not have any obvious connection with either the concentration of sugar in the plasma or with the total amount. The sugar excretion was therefore estimated according to the formula which Ambard has used in studying urea and chloride excretions and which Fitz and Van Slyke used as the basis of their acid excretion formula. Sugar excretion recorded in this manner was not proportional to the plasma sugar concentration. The total plasma sugar, however, appeared to be related to sugar excretion expressed in this form (Chart 1). . 318 Studies on Blood Sugar These observations are too few to be very conclusive. They suggest, however, that the total amount of plasma sugar offers a more rational basis of comparison with sugar excretion than does the plasma sugar concentration alone, and that it may be possible to work out a formula which will express mathematically the relationship between the sugar circulating in the blood and that excreted in the urine. Urine suger Plasma we 12.0 : TET eas 4.0 SRE REE eee ase BRR RRRER A. 10.8 BERRA Hi bet 3.0 BRE oe 9.0 2.0 7.5 |—— f, 10 640 BU G@ERARE |. ae ie Pleas aE eral 4.5 = "gS FP a a Pr 0 Cuartl. The relationship between total plasma sugar and the excre- tion of sugar in the urine. The most striking feature of the miscellaneous cases tabulated in Table IV is the great difference in the estimation of total blood sugar. Thus a patient with polycythemia had 16.20 gm. of sugar in the blood, while a patient with nephritis had only 2.06 gm. of sugar in the blood. The average for this group of patients with miscellaneous diseases as a whole is lower than the average for normal persons (Table I), probably on account of the a of anemic patients who were studied. R. Fitz and A. V. Bock 319 It is of especial interest that the patient with polycythemia had more sugar circulating in the blood and more in the corpuscles than any of the diabetic patients, although the urine was normal. The total plasma sugar, on the other hand, was within normal limits. This suggests that sugar contained in the corpuscles is tightly bound to them in some manner and has little effect on the production of glycosuria. In all three groups of cases the corpuscular sugar content was parallel with the corpuscular volume. Apparently, therefore, TABLE IV. Patients with Miscellaneous Diseases. Blood | 5 g be ¢ 3 Diagnosis. E E = ere 8 Di 5 “S is = B PaaS = Be Pe g e) 5 EF |eag/S/8| 3 |s2| a» a| 8 | 28 £ s 52 /2/8/.5 |oa| oa Sm | a= ca) ay }/ 16) Sia} ae |e a kg. cc cc Ce. gm gm.| gm. 1 | M. |60.5| Polycythemia. 8, 540|3, 240/5, 300/190|150)16. 20/4. 85/11. 35 2 45.3| Leukemia. 5, 060/3, 460}1, 590) 90} 80} 4.55)2.77| 1.78 3 | F. |60.5) Secondary anemia. |4, 470/3, 400/1, 070/110/110) 4.92)3.76} 1.16 4 | M. |65.0| Nephritis. 4, 390}3, 170)1, 270|110} 80} 4.83)2.54| 2.29 5 50.0} Leukemia. 4, 030/3, 140} 890)100/100) 4.03/3.14| 0.89 6 | F. |43.5}) Pernicious anemia. |3,070|2,730} 340/120/120} 3.68/3.28| 0.40 Gan Bs, 43.5 ct i 3, 050/2, 600} 460)120)120) 3.66|/2.60) 1.06 SeiPEeio4. 5 ne e 2, 740|2, 330} 410)/120/120} 3.29/2.80) 0.49 9 | M. |54.5| Nephritis. 2, 680/2, 140} 530) 80} 90) 2.14/1.92) 0.22 10 | M. |45.0 2, 580)2, 200} 380) 80} 80} 2.06/1.76| 0.30 hae Pernicious anemia. |2, 460}2, 180) 280/100} 90} 2.46|1.96| 0.50 PAW ELE Chine ne sn rae eae See 4.71|2.85] 1.86 all blood corpuscles contain a certain amount of sugar which is fixed within rough hmits. If the number of corpuscles is greatly increased, the total corpuscular sugar is also increased; the cor- puscular content of sugar is low when the number of corpuscles is diminished; when sugar is added to the circulating blood it is found largely in the plasma and to a much less extent in the corpuscles. Glycosuria does not occur unless the total plasma sugar is above a certain limit, regardless of what the sugar content of the corpuscles or whole blood may be. 320 Studies on Blood Sugar SUMMARY. The total amount of sugar in the blood of seven normal persons varied but did not exceed 7.5 gm. The plasma sugar was almost always considerably greater than the corpuscular sugar, but it did not exceed 4.85 gm. The total amount of sugar in the blood of nine diabetic patients also varied considerably. The highest blood sugar content estimated was 15 gm., and the highest plasma sugar was 10.78 gm. The plasma of the diabetic bloods, relatively, contained much more sugar than did the corpuscles. This suggests that the plasma in diabetes is a vehicle for the transportation of sugar from the body cells, which are unable to burn or store it, to the kidney which excretes it, and that the blood corpuscles are but little concerned with such transportation of sugar, a statement which is supported by the fact that the sugar content of the individual corpuscle tends to be fixed within rough limits. If the number of corpuscles is increased, as in polycythemia, the total corpuscular sugar is increased. Jf the number of corpuscles is much diminished, as in anemia, the amount of corpuscular sugar is diminished. Gly- cosuria does not occur unless the plasma sugar exceeds a certain threshold. Blood sugar concentration expressed as mg.’ per 100 cc. of blood or plasma may give misleading information with regard to the total amount of circulating sugar. The threshold at which glucose appeared in the urine of the diabetic patients of this series seemed to lie between 5.20 and 5.36 gm. of total plasma sugar. The total plasma sugar offered a more rational basis of comparison with sugar excretion than did the plasma sugar concentration alone. BIBLIOGRAPHY. Ambard, L., Physiologie normale et pathologique des reins, Paris, 1920. Bock, A. V., The constancy of the volume of the blood plasma, Arch. Int. Med., 1921, xxvii, 83. Epstein, A. A., and Baehr, G., Certain new principles concerning the mechanism of hyperglycaemia and glycosuria, J. Biol. Chem., 1914, xviii, 21. : Fitz, R., and Van Slyke, D. D., Studies of acidosis. IV. The relationship between alkaline reserve and acid excretion, J. Biol. Chem., 1917, xxx, 389. R. Fitz and A. V. Bock aval Folin, O., and Wu, H., A system of blood analysis, J. Biol. Chem., 1919, Xxxvill, 81. Gradwohl, R. B. H., and Blaivas, A. J., The distribution of the blood sugar as regards corpuscles, plasma, and whole blood in health and disease in man, J. Lab. and Clin. Med., 1916-17, ii, 416. Keith, N. M., Rowntree, L. G., and Geraghty, J. T., A method for the determination of plasma and blood volume, Arch. Int. Med., 1915, xvi, 547. Sansum, W. D., and Woodyatt, R. T., Studies on the theory of diabetes. VIII. Timed intravenous injections of glucose at lower rates, J. Biol. Chem., 1917, xxx, 155. Wishart, M. B., Experiments on carbohydrate metabolism and diabetes. III. The permeability of blood corpuscles to sugar, J. Biol. Chem., 1920, xliv, 563. ee re ek, 2 Ly a a 5 pe \ , OP a ee ee Fas D> + Bay Mere c lt i a ‘a ere Z oA" ~~ oe ae a : i = j x obi at ‘ j Z Leet ot lt : pile Th Pier -t) Adel Law's . vows | | . ae a al ot oo utee Gear ’ "4 oe i moth) 7 : rep SMe ivy>« > 4: Mw : hh . Vital Cow epee “ om EFFECT OF HEATING THE ANTISCORBUTIC VITAMINE IN THE PRESENCE OF INVERTASE. By ERMA SMITH anp GRACE MEDES. (From the Physiological Laboratory of Vassar College.) (Received for publication, July 13, 1921.) A number of papers which have appeared recently have suggested that the presence of enzymes in food may influence the stability of the antiscorbutic enzyme. Givens and McClugage (1) state: “The influence of heat upon the antiscorbutic vitamine appears to be related not only to the degree of temperature but to the duration of the treatment, the reaction, the enzymes present, and the manner of heating.”’ They base their conclusion as to this enzyme action partly upon experiments showing that potatoes baked for a short time at high temperature and then dried at 35-40°C. retain a greater amount of the antiscorbutic vitamine than those dried at 35-40°C. without previous heating. , In this same article, Givens and McClugage refer especially to oxidases present in the potato. Ellis, Steenbock, and Hart (2) have shown that drying cabbage in an atmosphere of CO, for 35 hours at 65°C. does not prevent the destruction of the antiscorbutic vitamine. Oxidizing agents such as hydrogen peroxide and potassium permanganate cause its destruction. Anderson, Dutcher, Eckles, and Wilbur (3) state that oxida- tion is a more important factor than heat in the destruction of the antiscorbutic vitamine. Bubbling air through cow’s milk at 145°F. for 30 minutes causes some destruction, but the destruc- tion is more marked when oxygen or hydrogen peroxide is used. These experiments, together with the fact, as shown by Harden and Robinson (4) and Givens and Macy (5), that dehydrated orange juice retains its antiscorbutic value after 2 years, whereas untreated orange juice deteriorates appreciably within 3 months (6) indicate strongly that enzyme action may be an important factor in the gradual destruction of the antiscorbutic vitamine. 323 324 Heating the Antiscorbutie Vitamine EXPERIMENTAL. In the present study the authors have heated orange juice, both in the presence and absence of an enzyme. The tempera- tures chosen were 38°, 55°, and 76°C. If enzyme activity causes destruction of the. vitamine, presumably disappearance of the latter would be most rapid at 55°C., the temperature at which the activity of the enzyme is greatest. The enzyme selected was invertase, since it 1s present in the natural orange juice. The experiments conducted show conclu- sively that this enzyme does not decrease the value of the anti- scorbutic vitamine. This does not preclude the possibility that other enzymes may have such an effect, and it is hoped that experi- ments along this line may be continued with an oxidizing enzyme. The antiscorbutic vitamine was separated from orange juice! by the method of Hess and Unger (6) with 96 per cent alcohol which precipitated the enzyme and thus separated it from the vitamine. The extract was tested on a solution of sucrose and gave a negative test for invertase. The invertase was prepared by a method described by Hudson and Paine (7). It was dried on filter paper and 25 mg. were added to each 100 cc. of orange extract. 4 ee. of orange extract, prepared fresh about every 10 days, were fed to each guinea pig daily for the first 46 days of the ex- periment. As none of the guinea pigs receiving the extract showed any symptoms of scurvy at that time, the amount fed was re- duced to 3 ce. for the following 18 days. As there were still no indications of scurvy in any of the animals, and as the experi- ment had to be terminated within another month, the amount fed daily was reduced to 1.5 ce. per animal. Table I gives the amount fed for the various periods, and the time each portion was heated. The basal diet used, consisted of equal parts by weight of alfalfa meal and wheat flour, with powdered milk which had previously been heated to 95°C. for 1 hour. These three were mixed thoroughly together and made into a soft paste with water. 1 The oranges in these experiments were generously supplied by the tesearch Laboratory of the California Fruit Growers Exchange, Corona, California. E. Smith and G. Medes . 325 The allowance of milk was about 3 cc. per day per animal. Cracked oats were kept constantly before the animals. Table II gives the diets fed the various guinea pigs. The animals were weighed every morning before feeding. In Table III are recorded the weights for every fifth day. TABLE I. Treatment of Orange Extract. Time heated. Amount fed. Period fed. hrs. ae ie S72 a ee days 4 4 11 2 4 21 4 4 15 4 3 18 4 IL 2p TABLE II. Diets. No. of animal. Basal diet. Invertase. aanee Temes oC; 29 + — _ 25 = se = 27 ae = == 33 and 34 -- — 38 31 and 37 =F == = 38 28 and 35 _- -- + 55 32 and 36 + ae =F 55 30 and 38 — — — 76 26 and 39 a5 == =F 76 SUMMARY. Invertase does not contribute to the destruction of the anti- scorbutic vitamine when heated with the vitamine for 4 hours at 76>, 55°, or 38°C. Heating for 4 hours at a temperature of 76°C. either in the presence of invertase or in its absence, causes a more rapid de- struction of the vitamine than heating at 55°C. Heating for 4 hours at 38°C., does not cause an appreciably greater loss of antiscorbutic value than keeping at room temperature. Heating the Antiscorbutic Vitamine 326 T9& SEP Té¢ 89¢ TZS 99¢ OLS 09¢ Tvs 08S sI¢ 96F LLY 6SP LPP PoP 60F 98& L8E 68 °ON CLG ee O&F GSP PoP 9cF c9F Shr LEP LEP LIP SCF LOF 60F €68 62 LLE 8oé LSE 9&8V 6IP OF 968 | 6LE Goce | SPE a nnn EE ‘Burkq |'surAq SEs TLE GOP 8FS GOS vgs PES 9&¢ ‘pred ‘prod GSE P9E OLE CoP PIP COE €8€ PLE 9¢ “ON “peed. | Ped oes 6G& LLE 9LE cor lev GOS Gés¢ Tg¢ 8é¢ LE¢ 86g GES LOS L¥S GLY LEg GLY €0¢ LLY GES GLY LIS 9ST L8¥ LYY COP CGP SEP F6E OCP OLE 98E €gé 9LE lane 8 ‘ON 0€ ON | 9€ ON ZEON | GE ON TE “ON | #8 ON GZ ON 62 ON ‘shigq pauiny fo syybra 44 TH ATAVL E. Smith and G. Medes 327 In all cases except Guinea pig 32 which was fed on orange extract plus invertase heated at 55°C., the animals receiving orange extract heated in the presence of the enzyme were in a less advanced stage of scurvy at the close of the experiment than those receiving orange extract similarly heated without the enzyme. The authors have at present no suggestion as to the significance of this fact. BIBLIOGRAPHY. 1. Givens, M. H., and McClugage, H. B., J. Biol. Chem., 1920, xlii, 491. 2. Ellis, N. R., Steenbock, H., and Hart, E. B., J. Biol. Chem., 1921, xlvi, 367. 3. Anderson, E. V., Dutcher, R. A., Eckles, C. H., and Wilbur, J. W., Science, 1921, liii, 446. . Harden, A., and Robinson, R., Biochem. J., 1920, xiv, 171. . Givens, M. H., and Macy, I. G., J. Biol. Chem., 1921, xlvi, p. xi. . Hess, A. F., and Unger, L. J., J. Biol. Chem., 1918, xxxv, 489. . Hudson, C. 8., and Paine, H.S., U. S. Chem. Bureau, Circular 55, 1910. “IQ Or THE EFFECT OF CERTAIN STIMULATING SUBSTANCES ON THE INVERTASE ACTIVITY OF YEAST. By ELIZABETH W. MILLER. (From the Hull Laboratories of Physiological Chemistry and Pharmacology, University of Chicago, Chicago.) (Received for publication, August 2, 1921.) The remarkable effect on yeast growth produced by the addi- tion of an alcohol or water extract of yeast to a synthetic medium has been noted by several investigators. Wildiers' in 1901 first observed this fact. More recently others have confirmed Wildier’s observations. Euler? in studying invertase formation in yeast, found that the ‘‘Generationsdauer’’—that is, the time required for yeast cells to double in number—was only one-half as long when a yeast extract was added to the nutrient medium as when other sources of nitrogen were used. Equivalent amounts of asparagine, glycocoll, alanine, cystine, and tyrosine were added singly or in combination, and all proved less favorable for yeast growth than the yeast extract. Williams? presented evidence that the constituent of yeast which stimulates reproduction of yeast cells is identical with the antineuritic vitamine, and that the rate of reproduction of yeast cells might be used as a quantitative method for determining this substance. Almost simultaneously, Abderhalden and Koeh- ler,* using the same method—microscopic observation of the growth of single yeast cells in hanging drops—found that the addition of dilute extracts of yeast markedly accelerated the rate of repro- duction. Similar results were obtained with two other organisms, Colpoda cucullus and the alga, Ulothrix. These authors also 1 Wildiers, E., La Cellule, 1901, xviii, 313. 2 Kuler, H., Biochem. Z., 1918, lxxxv, 406. 3 Williams, R. J., J. Biol. Chem., 1919, xxxvili, 465. 4 Abderhalden, E., and Koehler, A., Arch. ges. Physiol., 1919, clxxvi, 209. 029 330 Invertase Activity of Yeast suggest that the active substance is the antineuritic vitamine and that yeast or some other simple organism might be used to deter- mine its presence. It is reasonable to suppose that a substance so potent in its effect on higher organisms as the antineuritic vitamine is known to be, must also play a significant réle in the life processes of the yeast cell itself. The effect of the presence or absence of such a necessary substance on a simple cell like yeast can be more easily studied than in the more complex forms of life, and a knowl- edge of its function in the yeast cell undoubtedly is of fundamental importance for an understanding of its mode of action in higher organisms. Abderhalden and Schaumann?’ have suggested the significance of this phase of yeast nutrition. They prepared an active extract by treating yeast with 10 per cent ,H2SO,; for 24 hours. After filtering and removing the H.SO, with barium the solution was evaporated to dryness and the residue extracted with absolute alcohol five times. The combined alcohol extracts were evaporated under reduced pressure and the residue again extracted with absolute alcohol. This process was repeated until the residue was entirely soluble in alcohol. The alcohol solution was finally evaporated and the residue taken up in water. Such a yeast extract contained 8.95 per cent dry substance, 0.87 per cent ash, 0.19 per cent nitrogen, and 0.093 per cent phosphorus. Additions of 5 to 10 cc. of this extract to 250 ec. of a sugar solution in which living yeast was suspended accelerated greatly the fermentation of glucose, fructose, galactose, sucrose, and maltose. This action seemed to be specific for some unknown substance in the yeast extract in that amino-acids and phosphorus compounds, added to the fermentation mixtures, were entirely inactive. The Ht concentration was kept constant so as to eliminate that as a factor in the increased rate of fermentation. A more highly purified preparation which they call ‘ Eutonin” was precipitated from the alcohol extract by acetone. This was entirely phosphorus-free, but was still effective in augmenting fermentation by living yeast, although it was not so active as the original extract. These results indicate that in the ‘‘crude”’ extract more than one substance was active. ® Abderhalden, E., and Schaumann, H., Fermentforschung, 1919, ii, 120. a oles. “teeta é E. W. Miller Be These questions arise: To-what is the action of the yeast extract due? Does its effect lie solely in accelerating cell reproduction? Does it stimulate the formation of one or more of the enzymes? Or, finally, does it act directly on the enzymes as an activator or as a coenzyme? Abderhalden and Schaumann state that the addition of yeast extract had some effect in accelerating fermentation when dried yeast or a maceration juice was substituted for the living yeast. On the basis of their results they conclude that there is a substance in the yeast extract which acts as a coenzyme or activator of an enzyme. However, their protocols show that in the experi- ments in which dried yeast or maceration juice was used, only slight increases in the rate of fermentation followed the addition of the yeast extract. As the determination of loss in weight was the method used for measuring the rate of fermentation the small increases in fermentation which were found in these experiments might reasonably come within the limits of experi- mental error. Also, there was opportunity for bacterial growth during the long period in which these mixtures were allowed to ferment without the addition of toluene. When living yeast was used there was undoubtedly a remark- able increase in the rate of fermentation with the addition of yeast extract to the fermentation mixture. This increase oc- curred, too, after the tenth hour, during the period of most rapid growth of yeast.6 It appears then that the chief effect of the active substance in yeast extract was on the living yeast, either in promoting growth or in stimulating the production of one or more of the enzymes. Euler’? and other workers have reported the variations in inver- tase activity of yeast grown in different media. Not only the substrate, sucrose, and the reaction products, glucose and fruc- tose, but other hexoses as well, especially mannose, increase the formation of invertase. Euler? also investigated the effect of nitrogen nutrition on in- vertase formation in yeast. He used (NHa,).SO,, asparagine, glycocoll, alanine, tyrosine, and cystine as sources of nitrogen, 6 Huler, H., and Lindner, P., Chemie der Hefe und der alkoholischen Giarung, Leipsic, 1915, 254. 7 Kuler, H., and Cramér, H., Biochem. Z., 1913-14, lviii, 467. 332 Invertase Activity of Yeast , the amino-acids having been added to the medium both singly and in combination. All these nitrogen compounds were about equal in their effect on growth and on the invertase activity of the yeast. However, when an equivalent amount of a water extract from yeast was used as a_ source of nitrogen, there was a decided increase both in the rate of growth and in the formation of invertase. Euler ascribes this effect to a nitrogenous substance in the yeast extract which is in the most available form for yeast nutrition. But here again it is obvious that some other stimulating substance present in the yeast extract may be responsible for these results. The experiments reported in this paper were undertaken prior to the writer’s knowledge of the Abderhalden work, and were devised to obtain further information concerning the effect of yeast extract on the enzymes of yeast. To begin with, invertase was chosen as the enzyme, the rate of formation of which was to be followed, because its activity is so easily measured and because it is so important in yeast action. As the work progressed the question soon came up as to whether the substance which promotes growth of yeast also accel- erates invertase formation. Yeast was grown with and without addition of solutions known to contain the growth stimulant. Various methods were used to separate this substance from other constituents and these partially purified preparations were also added to the standard medium. The yeast grown in these dif- ferent media was filtered off and the invertase activity thereof determined. EXPERIMENTAL. Method for Determining Invertase in Small Amounts of Yeast. For this work it was first necessary to devise a method for extracting invertase quantitatively from small amounts of yeast. Euler’s method was as follows.2. The filtered yeast was drained afew minutes on a porous plate, 0.25 gm. portions were weighed out and suspended in 10 ce. of 1 per cent NaH»PO, solution, and after standing 10 minutes this suspension was added to 20 ce. of 20 per cent cane-sugar solution. At stated times samples were removed to determine the rotation. The inversion was checked: SSK ee ie A ATO EK. W. Miller 330 and multirotation accelerated by adding 10 ce. of 5 per cent NaOH solution. Lévgren’ used practically the same method. A serious objection to this method is that the yeast cells are not killed and when inversion was allowed to continue several hours some growth and fermentation might occur. To obviate this, various methods of killing the yeast cell and extracting the enzyme were tried. Small amounts of yeast, 0.1 to 0.3 gm., were dried at 37° for 2 to 4 hours, then shaken with water to which a few drops of toluene were added. Other samples were ground in a mortar with fine sand or other abrasive material before treat- _ ing with water. None of these methods gave uniform results. Finally the method of Willstitter, Oppenheimer, and Steibelt® for the quantitative determination of maltase was adopted for our purpose. Fresh yeast was shaken with water and toluene. Willstatter and coworkers obtained almost complete extraction of maltase in 24 hours. There was a slight increase by 48 hours treatment. Because maltase is rapidly destroyed by acid, it was necessary to keep the extract neutral by frequent additions of NH,OH. With invertase this was unnecessary since this enzyme is more resistant to acid and is also most active in faint acidity. Invertase is more readily extracted by this method than is maltase. Fresh samples of Fleischmann’s yeast were weighed out, suspended in 25 ec. of water, 0.5 ec. of acid-free toluene was added, and the mixtures were shaken moderately at 30° for 4, 8, 12, and 24 hours respectively. At the end of the stated time each mixture was transferred to a 100 cc. volumetric flask. 50 ec. of 20 per cent sucrose solution, 0.4 ec. of 0.1 N HCl, and water to fill to the mark, were added. This was put into a 500 ce. Erlenmeyer flask and mixed well. 50 cc. were then removed, and 2 drops of NH,OH (sp. gr. 0.90) added to check invertase action and to hasten multirotation. Before filtering it was necessary to add a small amount of taleum in order to obtain a perfectly clear filtrate. The rotation of the clear filtrate was then read in a 2 dm. tube at 20-25°C. 8 Lovgren, 8., Fermentforschung, 1919-20, iii, 221. 9 Willstaitter, R., Oppenheimer, T., and Steibelt, W., Z. physiol. Chem., 1920, cx, 232. 334 Invertase Activity of Yeast The remaining 50 ec. were moderately shaken at 30°C. for 7 hours, after which the solution was made alkaline, filtered, and the rotation noted in the same way. The results are given in Table I. It is evident that most of the activity was obtained by a preliminary treatment of 4 hours. There was a slight increase up to 12 hours, but a marked diminu- tion in 24 hours. é; TABLE I. ge a ae Time of shaking. Change in @ in7 hrs. gm. hrs 0.2 4 11.88 OgZ 8 11.84 0.2 12 ; 12.38 0.2 24 8.36 0.3 4 14.90 0.3 8 14.98 0.3 12 15.24 0.3 24 17 As a final check on the method the invertase activity was determined on samples of yeast which were weighed out by another person. These results are found in Table II. TABLE II. Mie gibed essere, at Time of shaking. Change in @ in 7 hrs. gm. hrs. 0.15 2 8.7 0.2 12 12.36 0.3 12 15.26 For the subsequent determinations the yeast was shaken with water and toluene at 30°C. for 12 hours—from 8.00 p.m. to 8.00 a.m.—then added to the sugar solution, and the change in rota- tion taken at the end of 7 hours. The toluene prevented any bacterial action. It was found that the invertase extract was optically inactive. For convenience in taking the initial reading a blank was made up of 50 ec. of a 20 per cent sucrose solution plus 0.2 ec. 0.1 N HCl and water to make 100 cc. The rotation of this solution EK. W. Miller 339 was immediately taken for the initial reading. In the earliest studies the solutions were brought to a temperature of 20° before the rotations were read. The differences obtained in the readings with different samples of yeast were so great, however, that the slight errors due to a difference of 2 or 3° temperature were in- significant. Subsequent determinations were made at room temperature, which varied from 22—25°. Changes in Invertase Activity of Yeast Grown with the Addition of an Alcohol Extract of Yeast.—An alcohol extract of yeast was prepared as follows: 3 pounds of starch-free Fleischmann’s yeast were broken into fine pieces and dried in a current of warm air for 48 hours. The dry yeast was then covered with ether and heated on a water bath under a reflex condenser. This was repeated three times.. Five extractions with 70 per cent alcohol were made in the same way ‘The alcohol extraction was continued over 2 days. The combined alcohol extracts were equal to 1,850 ce. 200 ce. of this alcohol extract were evaporated on the steam bath and the residue extracted with warm water four times. There was considerable lipin material present, so that in order to obtain a clear filtrate the water extract was run through a - Berkefeld filter. The filtrate was made up to 200 cc. in water and labeled Alcohol Extract I. Portions of this preparation were added to the medium in which yeast was grown to determine whether such an addition actually does increase the invertase activity of the yeast. The medium used for the growth of the yeast was that adopted by Williams. It contained per liter 20.0 gm. of sucrose. 1.5 “ “ asparagine. ain) ONDE SIO. Pee ee KPO: O22bea, = CaCl: Os25e a) MgSO): 500 ec. portions of this medium were introduced into each: of four 2 liter conical flasks. This exposed a large surface and insured sufficient oxygen for vigorous growth. To two of the flasks - were added 2.5 ec. of the alcohol extract plus 2.5 ec. of water, and to the other flasks 5 ec. of the same alcohol extract. These mixtures were sterilized at 10 pounds pressure for 15 minutes. 336 Invertase Activity of Yeast A yeast suspension was prepared by shaking 0.2 gm. of Fleisch- mann’s yeast in a liter of distilled water. 5 cc. of this suspension were then introduced into each of the flasks with a sterile pipette. The flasks were then incubated at 30° for 24 hours. It was evident that growth was much greater in the flasks to which 5 ec. of alcohol extract had been added. The yeast was then filtered by suction, onto alundum crucibles, washed twice with distilled water, and sucked dry. To facilitate the filtering, the contents of the flasks were first centrifuged and TABLE III. Changes in Invertase Activity of Yeast Grown with the Addition of Alcohol Extract I. 0.2 Gm. (Moist Weight) Used in Each Invertase Test. Yeast grown with addition of extract. Change in @in7 hrs. ce. 2.5 4.31 4.54 5 8.91 5 9.25 9.73 229 5.38 5.50 2.9 4.51 4.36 5 7.08 6.93 5 7.38 6.79 most of the supernatant liquid was poured off. The yeast was then washed into the crucibles. 0.2 gm. portions of each sample of yeast were weighed out in duplicate, washed into Erlenmeyer flasks with 25 ec. of water, 0.5 ec. of toluene was added, and the mixtures were shaken 12 hours. The invertase preparation was then combined with the 20 per cent sucrose solution, 0.4 ec. of 0.1 N HCl, and water to make 100 ce. The initial rotation and the rotation at the end of 7 hours were taken as described. These results are given in Table III. There is no doubt that the alcohol extract not only increases the rate of growth, but HK. W. Miller 337 also the invertase activity per unit weight of yeast. In this case when twice the amount of alcohol extract was added to the medium the invertase activity was doubled. This experi- ment was repeated a second time with similar results. Dry weights of these yeast samples were not obtained. However, the differences in moisture content could not account for the great differences in invertase activity of the different prepara- tions. In subsequent experiments dry weights were determined and were found to be quite uniform in a single experiment. The next step was to determine whether the substance which stimulates cell reproduction is identical with the substance which stimulates invertase formation. McCollum and Simmonds" have shown that the water-soluble vitamine is soluble in benzene after it has once been dissolved in alcohol. This suggested itself as a method of obtaining evidence as to whether the growth-pro- moting substance was also responsible for the increase in inver- tase formation. Invertase Activity of Yeast Grown with the Addition of Benzene- Soluble Material. 200 ec. of the alcohol extract of yeast, equivalent to 2.74 gm. of total solid, were evaporated at 37° on 15 gm. of starch. This material was extracted with anhydrous ether for 6 hours to remove hpins. After this it was extracted with three portions of redis- tilled benzene (Kahlbaum’s) for periods of 6, 7, and 8 hours. These combined benzene extracts were evaporated on a water bath and the residue was extracted with 200 ec. of water. This was called Benzene Extract I. It contained only 0.019 per cent of total solids and 0.0014 per cent of nitrogen. Yet when compared with Alcohol Extract I it was shown to be fully one-half as active in promoting growth (see Table IV). 5 and 10 ec. portions of the aqueous solution of this benzene extract were added to 590 ce. of the standard medium and 5 ce. of a yeast suspension introduced. The 24 hour growth of yeast was tested for its invertase activity as described above. The results are found in Table V. 10 McCollum, E. V., and Simmonds, N., J. Biol. Chem., 1918, xxxiii, 55. 338 Invertase Activity of Yeast TABLE IV. Comparison of Growth-Promoting Activity of Various Preparations. Growth of yeast cells per Extract used. lee. aAdad to 30 ce. of medium. Aléobolibxtractalc< co: Geri re eed ee eee es eee 469 «“ iO erh Sote tee. Pera bla meee oe anaes mH a Benzene = Cuart 2. The preliminary diet was the same as in Experiment 1 but at the point indicated (x) B. coli were added to the ration. The loss of weight was not stopped. 382 Days oy LOR Si .20< (25, 30: Soe Cuart 3. At the point marked (x) 20 gm. of B. subtilis were added to each 100 gm. of ration but the animals continued to lose weight. DISCUSSION. As already pointed out certain observers have apparently demonstrated the presence of a growth-promoting principle in bacteria. The standards by which they determined the presence or absence of this substance are not comparable however. So far as the author is aware the only way of measuring vitamine B that is not open to objection is by feeding the substance that is to be tested to rats that are being maintained on a diet that is devoid of this factor. The experimental data presented above indicate that so far as Bacillus paratyphosus B, Bacilius coli, and Bacillus subtilis, are concerned there is no production of vitamine by these organisms. The objection may be raised that the quantity of bacteria added to the diet was not sufficient to affect the growth of the rats but it should be borne in mind that excessively small amounts of substances containing vitamine have been shown to produce a marked effect. 384 Bacteria as a Source of Vitamine Regarding Thjétta’s seeming demonstration of the production of a growth-promoting principle by mucoid bacilli and Bacillus proteus a further report will be made in a subsequent paper. CONCLUSION. Bacillus paratyphosus B, Bacillus coli, and Bacillus subtilis do not produce the growth-promoting principle known as water- soluble B vitamine. The author desires to acknowledge the receipt of helpful suggestions from Professor F. P. Gorham. BIBLIOGRAPHY. 1. Pacini, A. J. P., and Russell, D. W., J. Biol. Chem., 1918, xxxiv, 48. 2. Thjétta, T., J. Exp. Med., 1921, xxxiii, 763. : 3. Cooper, E. A., J. Hyg., 1914, xiv, 20. 4. McCollum, ‘E. V., Simmonds, N., and Pitz, W., J. Biol. Chem., 1917, XKIX,, O21. 5. Osborne, T. B., and Mendel, L. B., Carnegie Inst. Washington, Pub. 156, 1911, 32. THE CHARACTERISTICS OF CERTAIN PENTOSE-DE- STORYING BACTERIA, ESPECIALLY AS CONCERNS THEIR ACTION ON ARABINOSE AND XYLOSE.* By E. B. FRED, W. H. PETERSON, ann J. A. ANDERSON. (From the Departments of Agricultural Bacteriology and Agricultural Chemistry, University of Wisconsin, Madison.) PLATES 1 AND 2. (Received for publication, August 2, 1921.) Pentoses and pentose-yielding substances are found wherever the conditions are suitable for the growth of higher plants. A study of the changes these substances undergo indicates that there are present everywhere microorganisms which are able to bring about decomposition of the pentoses and related compounds. The almost universal distribution of the five-carbon compounds and the large quantities of plant products stored up in this form indi- cate the importance of studying the factors concerned in their decomposition. The great family of organisms termed the lactic acid bacteria includes many forms which possess the power of splitting vigor- ously the pentose sugars. Perhaps the first recognition of the role of the lactic acid bacteria in the fermentation of pentoses was in 1894 when Kayser (1) isolated from sauerkraut an organism which fermented arabinose and xylose with the production of lac- tic acid. Since this time many reports of the fermentation of pentoses have appeared, Grimbert (2), Bertrand (3), and Bendix (4). Gayon and Dubourg (5) studied the carbohydrate metabolism of the “‘mannit-forming bacteria” in considerable detail. Du- bourg (6) reported additional studies of these organisms. Miiller- Thurgau and Osterwalder (7, 8) greatly expanded the studies of Gayon and Dubourg, isolating and describing many new strains of pentose-fermenting bacteria of the general group called “man- nit-forming bacteria.’”? They also investigated the nature of the * This work was in part supported by a grant from the special research fund of the University of Wisconsin. 380 386 Pentose-Destroying Bacteria products obtained from this fermentation. Henneberg (9) de- scribed many types of pentose-fermenting bacteria of the lactic acid group. In general, the properties of the organisms are not described in sufficient detail to follow in the identification of un- known forms of lactic acid bacteria. Orla-Jensen (10) in an exhaustive treatise on the lactic acid bacteria gives the results of a careful study of the protein metab- olism, and the fermentation characters of 330 strains of lactic acid bacteria isolated from sour cabbage, beets, sliced potatoes, mash, and dough, and also from the excrement of cows, calves, and human beings. We have shown in preceding papers (11, 12, 18) that a certain group of the lactic acid bacteria break down the pentose sugars with a high acid production, and the hexose sugars with a low acid and a high alcohol production. Since these first reports concerning the acid fermentation of xylose, our studies have been extended until at present we have results to show that there are other types of pentose-fermenting lactic acid bacteria commonly present in silage, sauerkraut, and related substances. The present paper deals with the fermentative ability and general characteristics of a few members of the lactic acid family which destroy pentoses. It was the hope that such an investiga- tion would give an insight into the physiology, the distribution, and the importance of these organisms in nature. The cultures were obtained from various samples of corn silage and sauerkraut taken at different stages of their fermentation, usually between the 10th and 21st day. The structure of the colonies on the plates was studied and different types picked off into litmus milk and into measured amounts of 1 per cent xylose-yeast water and 1 per cent glucose-yeast water. From those showing acid production in xylose-yeast water, or a curd in litmus milk after 10 days, 12 were selected and replated. The selection of 12 cultures from the large number was based chiefly on the amount of acid formed from xylose, on the change noted in litmus milk, and on the source of the culture. It was planned to have representative types of the pentose fermenters which showed decided differences in degree of acid formation. These 12 cultures fall into two groups; the organisms of Group I are readily distinguishable from those of Group II by their action Fred, Peterson, and Anderson 387 on milk and fructose. All of the strains of Group I coagulate milk slowly and do not form mannitol from fructose. Those of Group II form mannitol from fructose and do not coagulate milk. On the basis of fermentation reactions the bacteria of Group I may be divided into at least three strains, which differ with respect to the fermentation of certain carbohydrates. It is believed that the groups herein described are fairly representative of the rod forms of lactic acid bacteria that take part in the fermentation of pen- tose sugars. Morphology of the Cultures. Although morphology is of little value in subdividing any group of organisms, it is possible by such a study to divide the lactic acid bacteria into coccus forms and short and long rods. All microscopical examinations were made from 24 hour glucose or xylose-yeast water agar slants or liquid-yeast water cultures in- cubated at 28°C. Chinese ink preparations are especially useful in the study of morphology. It was noted that the organisms of the different groups and strains varied but slightly in size and shape. They are non-motile, usually blunt ended rods which occur. singly or in twos, although long filaments or chains are sometimes noted, especially in liquid media. The different strains vary some- what in size, especially in width of cells, from 0.5 to 0.8 » wide and 1.25 to 3.00 uw long; Cultures 102, 31, and 32 are smaller, about 0.5 to 0.6 » wide by 1.2 to 2.00 » long. Even a single culture exhibits a wide variation in the size of the cells, hence morphology is of little varietal significance. Spores are not formed. Thee organisms stain easily with the ordinary aniline dyes and are Gram-positive. Photomicrographs of representative fields from microscopic preparations of the various groups are shown in Plates 1 and 2, Fig. 1, 2, 3, 4, and 5. In accordance with the results of Beijerinck (14), these various groups of lactic acid bacteria were found catalase-negative; 7.e., without the ability to break down hydrogen peroxide. The cultural characteristics of these organisms did not bring out any well defined differences. Briefly: They all grow best in a medium in which the source of nitrogen is yeast-water ex- tract. Colonies are small and reach their maximum growth in 388 Pentose-Destroying Bacteria 3 to 4 days. In stab cultures growth is moderate and uniform along the line of inoculation; on slants, scanty and beaded. Gela- tin is not liquefied nor is casein digested. Growth in the acid range for both groups is stopped at a hydrogen ion concentration of about pH 3.5, while in the alkaline range for Group I it is stopped at about pH 9.0 to 9.4, and for Group II at pH 8.6 to 8.8. The optimum temperature for most of the organisms is about 30°C. and their thermal death-point is between 60 and 65°C. The group, strain, and laboratory number, the source and the behavior in milk of these cultures are shown in TableI. For the sake of comparison, cultures of Bacillus lactis acidi and Bacillus bulgaricus are included. The value of milk in dividing these groups of bacteria is well illustrated in the figures of this table. Perhaps no other single physiological test so clearly defines the groups of lactic acid bacteria that ferment pentose sugars. As compared with Bacillus lactis acidi or Bacillus bulgaricus, none of these organisms forms large amounts of acid in milk; about 0.5 per cent of lactic acid is the maximum production after 2 weeks - at 28°C. The time of curdling varies with the different cultures and with the temperature. At 38°C. curdling was more rapid than at 28°C. Group I, Cultures 29, 102-1, 124-1, 102, 31, and 32 require from 8 to 18 days, while Group II, Cultures 52, 52-7, 14, 57, and 118-8 fail to curdle milk. Unlike Bacillus lactis acidt the organisms of Group I did not reduce litmus until after curdling. None of the lactobacilli of the pentose-fermenting group pro- duced a firm curd so characteristic of the lactics commonly found in milk. On the contrary, the curd is soft and flocculent and it ‘sinks leaving a ? inch layer of whey on top. Sources of Nitrogen All attempts to grow these various strains of lactic acid bacteria in peptone-phosphate medium (0.5 per cent each of dipotassium phosphate, peptone, and xylose), and in meat infusions resulted in a slow and scanty growth. Yeast water extract is far more suitable for growth and acid production than any other medium tested and for this reason it was made use of in all of these studies. To insure a medium low in fermentable carbohydrates and also low in organic acids, only fresh yeast was used and each batch of medium was carefully analyzed. A representative analysis was as follows: 0.15 cc. of Nn volatile acid, 0.75 cc. of N non-volatile acid, and 0.0508 gm. of nitrogen in 100 ce. of yeast water. 389 Fred, Peterson, and Anderson ‘doy = oy + uo AoYM JNOYPIM ‘ULI (6 8°SZT *snavwpbing “J ‘doy “BUI[PINd 9LOJoq paonpoy | Uo AoYM JnoyBM SULIT] z 8°28 “Upraw $1490) “| ”? ) ”? ”? ” L-GG ” ” ” ” “qNBIFLION’S GG ? ” ? ? ‘odRlIS uo‘) Lg RMR Vide. ‘ornuvut dooyg | 8-SIT | * II “YIIUL UL Y} MOIS ON ‘OSB[IS ULOD ial ‘doy 48 soynueis o71yM pus Surl[pinod TO4 JB poeonpey ”? ”? ” ”? 6 GLE ” ? GS ‘do} 48 soynuevasd ogy M 0) I pus ”? ? ? ” ”) ” ”? 6 0 OF ‘OSUTIS uloy) like ‘SUI[PINd 104 Je poonpoyy ‘doy uo AvyM “Qjog SI Q'ST “‘qINVIyIONVG ZOL ” ”? ” ” ”? ” ” SI 0' FG ” G-¥GT “SUI[PInd 194Je poonpoy ‘doz uo AoyM “4jog 6 0°9F ‘qneiyloneg | [-$Z1 a Ti ” ” ” ” ” ” ” 6 9°¢¢ ” T-20r “SUI[PpANd 104Je Poonpoyy ‘doy uo AoyM “4jJog FI 8°OF “‘qnVIyIONVG 62 V I shop 29 ‘sNUHZI] UO OG ‘pana Jo puryy Jo ary, [PBN TO Z ‘sorngno Jo vo1n0g Sains ‘ureyg | ‘dnoi5y “Oo86 #8 ATTA "YL UL SLOIUIULIAY ASOJUAT 94} [O S914SLLAJIDLOY) IY], ‘I ATAVL 390 Pentose-Destroying Bacteria PARTI. The Fermentation of Carbohydrates and Related Substances. The question of the constancy of the acid fermentation of sugars by bacteria has received much study. The value of this test, as well as its limitations, has been the object of so many careful investigations that no attempt is made in this report to review the literature. The results of various investigations (15-19) indicate that the power of an organism to form acid from carbo- hydrates or related substances is a characteristic not easily lost or acquired. Although in this work no extensive study has been made of variations in fermentation reactions, it has been noted that cul- tures of Lactobacillus pentoaceticus which have been carried on glucose yeast-water agar for more than 2 years have not shown any well defined change of their fermentation reactions. The acid fermentation of sugars is without doubt the best possible means of differentiating members of the lactic acid group, provided a uniform and reliable method of determining fermenting power is used. In a study of the acid production of these bacteria, 2 per cent solutions in yeast water of the carbohydrate or related substance to be fermented were employed. The 10 day cultures were titrated for total acidity, at which time a portion of the culture was removed for hydrogen ion determination and another por- tion for sugar analysis. The colorimetric method for the meas- urement of hydrogen ion concentration and the Shaffer and Hartmann (20) method of sugar analysis were used. To over- come as much as possible the decomposition of the sugar by sterilization, the more unstable compounds, arabinose and xylose, were sterilized in water solutions and added to the yeast water by means of sterilized pipettes. The xylose was prepared from corn cobs according to the method of Monroe (21) and recrys- tallized from alcohol until the correct specific rotation and free- dom from heavy metals had been obtained. The other sugars, alcohols, ete., were Difco or Pfanstiehl preparations and were assumed to be true to label. In these yeast water cultures, determinations of total acidity proved far more valuable as a measure of the degree of fermenta- Fred, Peterson, and Anderson 391 tion than determinations of the hydrogen ion concentration. Within a certain range, a change in the concentration of hydrogen ions is proportional to the acid formed, but does not give a true picture of the degree of utilization of the carbon compound. This criticism may also be directed towards the titration of total acidity but to a much less degree. These two acid determina- tions plus the sugar analysis give a fairly accurate evaluation of the nature of the fermentation. Support for these statements will be found in the tables that follow. The Fermentation of Arabinose, X ylose, and Rhamnose. In Table IIT are assembled the analyses for acid production and sugar consumption from the breaking down of arabinose, xylose, and in the case of rhamnose, acid production alone. Where the acidity is below 0.5 to 0.6 per cent of normal acid it was assumed that there was no fermentation. A small amount of acid may be due to the action of the bacteria on the substance present in yeast water and to a slight decomposition of the carbohydrate during sterilization. In every case the figures given in this and in other tables are the result of subtracting this acid from that in the fer- mented culture. , All of the bacteria described in this paper ferment arabinose with the production of considerable amounts of acid. The various strains show a well defined difference in their ability to form acid; 10 days after inoculation cultures of the organisms of Strain A contain about 5.0 per cent, Strain C about 8.9 per cent, and Strain B about 11.3 per cent of normal acid, Table II. The high acid production which is characteristic of Strain B is also noted in the case of all of the organisms of Group II. A point of special interest shown in the results presented in this table is the close agreement between sugar fermented and acid formed. Perhaps in no other table is this correlation so evident. These results indicate that the chief products of the fermentation are acids rather than neutral or highly volatile bodies. Bacillus lac- tis acidi did not ferment arabinose, xylose, or rhamnose. On the basis of total acid formed from xylose, the bacteria of Group I may be arranged in three divisions: Strain A and B, low acid, and Strain C, no acid, or only a trace. All organisms of Group II are high acid formers. vel SL 8°F 6S vv Lg “UPLoD 81190) *_ v9 eg! P2480 ae 3 T'F01 096 °0 Ge OTT L-GS v'S GL ¥L8°0 Lan G POL 0¢6 0 ans 8° SOL og 3 99 8°9 P28 0 ve 0° 609°0 | rE | Z°1OT | 16:0 | He | F'Zer ie “ub EV | “m6 “20 a *pojJuoUr “ple *poyuoUL “prose fale -lej iesng ze N]Q |-aay sesng Hd NTO “9sOpONA ‘asouUBy]y 9°€ €E8"0 9°¢ ots 8'é ars 862°0 8°é as vs 499°0 9°¢ 98Z°0 v's 9°¢ 9°¢ £980 vs *‘poyuour -1d} IBvsNg Hd *980}0B [BH 060°T 060°T OF6 0 *poyuoUL -lo} 1Bsng asoon[y Gol “ple NTO “aprov son} “J L-@G Gg ZS 8-STT PI (as Tg col o-FGT L-¥el T-| S622) 932G0|25045 || aan 32 112.6] 3.2 | 83.8 | 3.4 Coal 78.0 |-3.6 | 33.5 4.4 14 6.8) 5.6 | 22) | 620) 4358) i238) 55-0 7.2 118-8 7.6) (O260 |) V5.0ssbeSuleAaeO 5950) |) (0).74 7.2 o7 6.4, 5.8 | 5.0] 5.8] 41.8 4.0 | 6.2 7.2 52 8.0) 5.4 | 15.2 | 5.6 | 4552 3.2 | 5.6 Uc? 52-7 5.6) 6.2} 6.4/5.6] 46.2 2.4 | 6.4 7.2 B. lactis acidi. 9.6) 5.1 6.2 | 5.6 16.7 21.6 | 4.2 7.2 not attack a-methyl glucoside. Because of the decided difference in the fermentation of a-methyl glucoside by the lactic acid bac- teria isolated from fermenting plant tissue, it is important in sub- dividing the various strains of these organisms. It is not attacked by the bacteria of Strain C. All the organisms of Group II form acid from a-methyl glucoside. The two glucosides, salicin and esculin, are decomposed by the organisms of Strains A, B, and C, but are resistant to the organ- Fred, Peterson, and Anderson 399 isms of Group IJ. An exception to this statement is seen in the ease of Culture 14 where a slight fermentation is noted. In every case the total acid formed from esculin is far less than that from salicin. Fermentation of Mannitol, Glycerol, and Dulcitol. The polyhydric alcohols, mannitol, glycerol, and dulcitol fur- nish sources of carbon for the separation of the lactic acid bac- TABLE VI. Fermentation of Polyhydric Alcohols. ; Calculated for 100 cc. of culture. Culture No. Mannitol. Glycerol. Dulcitol. 0.12 0. S10, aa pH neh pH eat pH ce Rea Cos 29 42.0 S85 36.0 3.8 6.2 5.4 102-1 56.0 3.5 Sone 3.9 6.0 5.4 124-1 49.0 336 25.6 4.0 39.8 3.8 124-2 58.0 Boo Zia 4.0 36.4 3.8 102 46.0 Balh 17.6 4.4 6.0 5.4 31 57.0 3.6 14.6 4.0 4.6 5.8 32 55.0 3.6 13.8 4.2 5.6 5.6 . 14 20.0 4.4 8.6 5.8 4.0 6.6 118-8 15.2 4.3 5.4 6.0 3.6 6.2 57 15.6 4.4 (0) 6.2 3.4 6.6 52 GHZ 4.2 4.8 6.0 3.8 6.6 52-7 19.0 4.2 3.8 6.0 33(0 6.4 B. lactis acidt. 46.0 3.8 7.4 5.6 teria into groups, as shown in Table VI. Of the three alcohols dulcitol is by far the most important for a study of fermentation reactions. It is not attacked by any of the lactic acid organisms used in this study except Cultures 124-1 and 124-2. In this respect our results agree with those of Orla-Jensen (10) who says that lactic acid bacteria which ferment dulcitol are extremely rare. According to Winslow and his associates (23) dulcitol occupies a unique position in the fermentation test in that it does not corre- 400 Pentose-Destroying Bacteria late with the other carbohydrates. From our results it appears that the fermentation of dulcitol is highly specific and may be used to separate closely related strains of lactic acid bacteria. Mannitol is fermented by all of the organisms of both groups, although much more slowly by those of Group II. Glycerol is fermented even more slowly than mannitol and very slightly by the organisms of Group II. In addition to the substances already described fermentation tests were carried out with starch, dextrin, and inulin, but no appreciable acid production was noted. Distinctive Fermentation Characteristics. The fermentation of certain carbohydrates and related com- pounds furnishes a means of separating into well defined groups the lactic acid bacteria that ferment the pentose sugars. Arabin- ose, xylose, a-methyl glucoside, melezitose, and dulcitol have been found especially useful in the separation of the different strains of Group I. 1 per cent xylose-yeast water is easily the most valuable medium in the separation of these lactic organisms into different groups. According to the amounts of acid formed these organisms naturally fall into three, and possibly four, divi- sions. The separation of Strains A and B, is based solely on the variation in the amount of acid produced, approximately 4.4 per cent of normal acid for Strain A, and 5.8 per cent for Strain B. It is only fair to say that the fermentation of xylose does not fur- nish a clear-cut separation of the organisms of these two groups. The production of acid from dulcitol is an easily distinguishable characteristic of the organisms of Strain B, while those of Strain A will not attack this polyhydric alcohol. The bacteria of Strain C are readily separated from the other organisms of both groups by the fermentation of melezitose. The principal results of the fermentation tests are grouped in Table VII. The figures of this table bring out clearly the most interesting facts obtained in this preliminary study of the pen- tose-fermenting lactic acid bacteria. Here the fermentation of the test substance is indicated by numbers. The data are ar- ranged to bring out the degree of fermentation. For example, in the case of Culture 29 the figure 3 for arabinose and xylose indi- cates a fair acid production while 6 for hexoses and disaccharides indicates a strong fermentation. the approximate amount of acid formed by the several organisms Fred, Peterson, and Anderson 401 The figures of this table show from the breaking down of the various carbohydrates and related compounds. TABLE VII. General Fermentation Characters of the Pentose Bacteria. Group I. Carbon compound. a Es e I PATADINOSE® ..c1Aci PPevatinNOses. 2.5 se eee Geleoneon lou on ono 13. a-Methyl] glucoside........ 1*) 1*| 3*| 4*|/07) 0*| 0* AMES AUITCIM eta ee, Nore ee tats 34 Doon eon lon Onipon|ro iG}, LBRO bin laeceeeee See an ie eee BN DE) BAN SE a Wy 14 Gee Vianmitolems.tee wcnte ceas | 33 i) Sh) BP | Ss |) Bs |S MPU GEL ONS. -o 2,2 sees sere clase ctw Dole | ed On OO Loe CibOlesseeee see ee ee | Os mOne oe 22 Os OF) OF COM SCONO OFA wR NwWHMOae [14 Group II. SOHSOWSOOOR NH HH WH OD @ | 118-8 57 SCOFPOCONOCOC OWN KRKF WROD 52 SCOrFMOCONOCOCOORRF RHE WhO OD * These figures represent the fermentation tests especially useful separating the different strains of Group I. Pp The arbitrary standards adopted were as follows: OQ equals 0—15 ec. of 0.1 N acid. 1 “c 15—30 “ “QO. “ « D) “ 30—45 “< “oO. “ « S) “ 45—60 << “O41 “ « 4 “ce 60—75 “ “c O. 1 “ “ 5 “ 75—90 “ “oy « 6 “ 90 or above ce. of 0.1 N acid. 62-7 SCOFPOOWOC COR OR HE WHO DO in 402 Pentose-Destroying Bacteria Among the compounds most valuable for differentiation are lactose, fructose, melezitose, the pentoses, and the higher alcohol, duleitol. This separation is not dependent upon acid formation alone but also on the production of neutral bodies, for example, mannitol from fructose. It is realized that the grouping adopted in this paper may bring together bacteria related in only one character but not in other characters; however, it is the best means at hand to separate the great complex of lactic acid bacteria. PARI un. Quantitative Determination and Identification of the Products Formed from Arabinose and Xylose. Arabinose and xylose are easily destroyed when sterilized in a slightly alkaline solution, and undergo a small amount of decom- position even when sterilized in yeast water. To avoid this change an 8 per cent water solution of each pentose was sterilized and then by means of a sterilized pipette added to the yeast water until the concentration was about 2 per cent. The exact strength was determined by analysis, which usually gave from 1.91 to 2.04 per cent of the pentose. The fermentation flask was equipped with a carbon dioxide absorption bottle similar to that described in a previous paper (12). Sterilized brom-cresol purple was added to the fermenting solution at the time of inocu- lation to indicate the formation of acids. Whenever a strong acid reaction was apparent, sterilized N sodium hydroxide was added until the solutions were approximately neutral. In this way, it was possible to measure the rate of fermentation and also to determine when fermentation had ceased. Occasionally fresh additions of brom-cresol purple were necessary as the indicator is partly destroyed in the fermentation process. When acid forma- tion ceased, usually after 10 to 14 days, the cultures were analyzed for carbon dioxide, unfermented sugar, volatile and non-volatile acids, and alcohol. Methods of Analysis —Carbon dioxide, volatile and non-vola- tile acids, and alcohol were determined as described in previous publications (11, 12). The distillate was analyzed by the method of von Firth and Charnass (24) for lactic acid carried over with Fred, Peterson, and Anderson 403 the volatile acid, but in no case were more than a few milligrams found. A correction corresponding to the quantity found has been applied to the values for acetic and lactic acids. Sugars were determined by the titration method of Shaffer and Hartmann (20). This is a rapid volumetric method that gives practically the same accuracy as the longer gravimetric methods. The method was tested with purified xylose and arabinose and found to give quantitative recovery of the sugars both from water and from culture solutions. Analyses were made of the uninoculated medium for volatile and non-volatile acids, blanks were run on the reagents, and the values thus obtained were subtracted from the corresponding determinations of the fermented cultures. In the regular procedure 400 cc. of an approximately 2 per cent sugar solution were fermented. Due to the addition of n NaOH to neutralize the acids formed, the volume at the end of the fer- mentation was in the neighborhood of 475 cc. Of this, 200 cc. were used for the determination of volatile and non-volatile acids, 100 cc. for alcohol, 50 ec. for sugar, 5 to 10 cc. for carbon dioxide, and the remainder kept in the ice box as a reserve in case a deter- mination should be lost. The fermentation of the pentoses was rapid and usually com- plete in about 14 days. Of the two, arabinose was more rapidly fermented than xylose. Very little sugar remained unfermented, rarely more than 0.1 gm. and sometimes as little as 0.05 gm. to 100 cc. of culture. Of the fermented sugar about 95 per cent is accounted for by the products. The volatile and non-volatile acids, which later will be shown to be acetic and lactic acid re- spectively, comprise 98 per cent or more of the totalproducts. No measurable quantity of aleohol was found and the carbon dioxide was never more than 0.046 gm. for 100 cc. of culture. The acetic acid and lactic acid are produced at approximately the ratio of 1 molecule of acetic for 1 molecule of lactic. The ratio of their molecular weights is 1:1.50 and the ratios found vary from 1:1.34 to 1:1.52. Somewhat lower ratios were obtained from the mannitol-forming group, the bacteria of which have the power of slowly fermenting lactic acid to acetic acid and carbon dioxide. When all, or nearly all, of the sugar has been fermented, it is probable that the bacteria attack the lactic acid formed and 404 Pentose-Destroying Bacteria so increase the proportion of acetic acid at the expense of the lactic acid. . The absence of any appreciable amount of arabinose or xylose (less than 0.05 gm. to 100 cc. of culture) and a rather high production of carbon dioxide lends support to this view. The data are given in Table VIII. TABLE VIII. Total Fermentation Products from Arabinose and Xylose. Calculated for 100 ce. of culture. oe Sueur. Weight | Sugar ac- ARE of sugar] Acetic Lactic Ratio of Carbon | counted fer- acid. acid. |acetic to lactic. | dioxide.| for by mented. products. ; gm. gm. gm. gm. per cent 29 Arabinose. 1.73 | 0.653 | 0.992 eae 52 0.023 96 29 Xylose. 1.79 | 0.695 | 1.022 1:1.48 0.023 97 124-2 Arabinose. 1.81 | 0.688 | 1.004 1:1.46 0.017 90 124-2 Xylose. 1.73 | 0.644 | 0.936 1:1.45 0.024 93 102 Arabinose. 1.97 | 0.714 | 1.035 1:31.45 0.023 90 31 5s 1.95 | 0.660 | 0.968 | 1:1.47 | 0.031 | 85 14 Hi 1.97 | 0.705 | 1.015 | 1:1.44 | 0.047] 90 14 | Xylose. 1.94] 0.771 | 1.043 | 1:1.36 | 0.030] 95 118-8* 4 1.41 | 0.545 | 0.732 | 1:1.34 91 52-7 | “ 1.94 || 0.780 |.1.082.|. . 11:39 1/0. 020sleeag * From J. Biol. Chem., 1919, xxxix, 368. Aside from this somewhat lower ratio between acetic and lactic acids for the mannitol-forming group, no essential difference manifests itself in the splitting of the two pentoses by the differ- ent bacteria. The sugars are almost completely fermented in all cases; carbon dioxide is produced only in minute quantities, and acetic and lactic acids constitute almost the entire amount of measurable products. On the basis of these data the fermentation equation may be represented as: C;H,,.0; = C.H,0; + C;H,O; ot (22.0 calories) Arabinose or xylose Acetic acid Lactic acid 561.3 calories 209.6 calories 329.7 calories Fred, Peterson, and Anderson 405 A certain quantity of the sugar is incorporated in the cells of the bacteria and a small quantity is consumed for their develop- ment, but the foregoing equation is as nearly quantitative as can be expected of a biological process. Identification of Products. Volatile Acid—The volatile acid from the various fermentations was subjected to a Duclaux distillation and the distilling constant calculated from the titration data. The constants obtained are given in Table IX. For comparison the distilling constant ob- tained with our apparatus for acetic acid made from recrystallized barium acetate and Duclaux’s original constant for acetic acid are also given in this table. The results indicate that the vola- tile acid is practically pure acetic acid in all cases, although the constants are slightly higher than for acetic acid. The difference is within the range of experimental error. Slight variation in the constants is to be expected with different pieces of apparatus, and different methods of heating. Additional evidence for the ab- sence of a higher fatty acid such as propionic acid was obtained by fractionating a volatile acid distillate, Arabinose Culture 14, con- taining 200 cc. of 0.1 N acid. The barium salt was dissolved in 100 cc. of water, 50 ec. of 0.1 N sulfuric acid were added, and the partially freed volatile acid was distilled with steam. The higher fatty acids such as propionic and butyric would, if present, be concentrated in the distillate leaving the lower acids in the distilling flask. The fractional distillate was submitted to a Duclaux analysis, but proved to have the same distilling constant as the unfractioned distillate. Since there was no change in the distilling constant, it is evident that the first distillate contained a single volatile acid and not a mixture. As a check on the Duclaux analysis the barium content of the volatile acid was determined. The barium salt was dried to constant weight in a platinum dish at 130°C. and then ignited with an excess of sulfuric acid. The weights of barium sulfate found and the calculated quantity that should have been present if the salt was barium acetate are given in Table X. The close agreement between the found and theoretical values proves that the volatile acid was acetic and corroborates the conclusions of the Duclaux analysis. . 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In most cases a slight precipitate of some amorphous material was formed. A similar precipitate obtained by ex- tracting the uninoculated control showed it to be an impurity from the yeast water and not the barium salt of some non-volatile acid produced by fermentation. A portion of the alcohol solution was evaporated to dryness and dried at 130°C., and the barium content of the residue determined as in the case of the volatile acid. The data are given in Table XI and agree satisfactorily TABLE X. Composition of Barium Salts of the Volatile Acids. a7 ae Barium sulfate equivalent. Sugar fermented. Culture No. SRSA Ue. the volatile acid. set 0 Calculated for ‘ gm. gm. gm. Arabinose. 29 0.5680 0.5132 0.5189 Xylose. 29 0.3128 0.2812 0.2858 Arabinose. 124-2 0.5983 0.5448 0.5466 Xylose. 124-2 0.7002 0.6370 0.6397 Arabinose. 102 0.3880 0.3530 0.3545 ss i 31 0.6910 0.6288 0.6314 os 14 0.7560 0.6890 0.6908 Xylose. 14 0.3354 0.3026 0.3065 with those required for lactic acid. The optical form of the lactic acid was ascertained by preparing the zinc lactate and deter- mining its water of crystallization. Inactive zinc lactate crys- - tallizes with 3 molecules of water, or 18.17 per cent, while the active form contains only 2 molecules, or 12.9 per cent. The percentages of water found are given in Table XII and prove that all of these different bacteria produce inactive lactic acid. In previous publications (11, 12, 13) it has been shown that the mannitol-forming bacteria of Group II as far as has been deter- mined always form a racemic mixture of lactic acid. This is true for the fermentation of both the pentoses and hexoses and also holds for mannitol. The non-mannitol-forming bacteria, Group 408 Pentose-Destroying Bacteria I, exhibit this same characteristic toward pentoses. The kind of lactic acid produced from hexoses and hexahydric alcohols re- mains to be determined. TABLE XI. Composition of the Barium Salts of the Non-Volatile Acids. . Bariurhanlt Barium sulfate equivalent. Sugar fermented. Culture No. of the non- eo volatile acid. Rowand! peer gm. gm. gm. Arabinose. 29 0.3376 0. 2456 0.2498 Xylose. 29 0.2756 0.2024 0.2039 Arabinose. 124-2 0.7104 0.5268 0.5257 Xylose. 124-2 0.5156 0.3750 0.3815 Arabinose. 102 0.8040 0.5852 0.5949 Es 31 0.8996 0.6578 0.6657 ee 14 0.3426 0. 2464 0.2535 Xylose. 14 0.3232 0.2372 0. 2392 TABLE XII. Water of Crystallization Contained in the Zinc Lactates. Weight of Water in Culture No. Source of salt. zinc lactate Water lost. Zn (C3HsOs)2 used. + 3H20. gm. gm. per cent 29 Arabinose. 0.7466 0.13842 18.0 18.17 29 ok 0.3854 0.0694 18.0 18.17 29 Xylose. 0.6024 0.1072 17.6 18.17 124-2 Arabinose. 1.2256 0.2220 18.1 1S 124-2 Xy lose. 1.1942 0.2162 18.1 18.17 102 Arabinose. 2.2206 0.4042 18.2 18.17 31 aS 1.7082 0.3094 18.1 18.17 14 “g 1.5862 0.2870 18.1 18.17 14 Xylose. 0.6864 0.1232 18.0 18.17 SUMMARY. The pentose sugars, arabinose and xylose, are readily fermented by various strains of the lactic acid bacteria. These pentose- destroying bacteria are widely distributed in nature, occurring in large numbers in silage, sauerkraut, and related products. At different stages of the fermentation of corn silage and sauer- Fred, Peterson, and Anderson 409 kraut, pure cultures of these lactic acid bacteria were isolated and their general characteristics studied. It was found that these organisms are usually short, blunt ended rod forms occurring as single cells or long filaments. From a large number of cul- tures isolated 12 were selected for special study. This choice of 12 cultures was based chiefly on the amount of acid formed from arabinose and xylose, on the change noted in litmus milk, and on the source of the culture. According to their behavior in milk the lactic acid bacteria which ferment pentoses may be arranged in two groups, the one group which slowly causes the milk to coagulate, the other which fails to bring about any noticeable change. Because of the very slight variation in morphology, the separa- tion of these organisms into groups depends upon characters other than cell structure. Measurements of the fermentative ability are undoubtedly the best means of separating the various groups and strains of the lactic acid bacteria. Among the com- pounds most valuable for differentiation are xylose, arabinose, fructose, lactose, melezitose, dulcitol, and a-methyl glucoside. The organisms studied naturally fall into two great groups; those of Group I ferment fructose without forming mannitol, and those of Group II ferment fructose with the production of mannitol. Aside from the two main divisions these organisms may be ar- ranged into several subdivisions or strains, depending upon differences in kind of sugars fermented and amount of acid formed. The following group of reactions indicates the nature of these strains: Group I—Strain A ferments arabinose, xylose, and lactose, but does not ferment melezitose or dulcitol. Strain B ferments arabinose, xylose, lactose, and dulcitol, but does not ferment melezitose. Strain C ferments arabinose, lactose, and melezi- tose, but does not ferment xylose or dulcitol. Group II —All strains ferment arabinose and xylose, but do not ferment lactose, melezitose, or dulcitol. No doubt some of these forms have been described previously; however, the characters reported are not in sufficient detail to insure identification. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 410 Pentose-Destroying Bacteria The authors suggest the following names for these various types of lactic bacteria: Group I-—Strain A—Cultures 29, 102-1, Lactobacillus pentosus, n. sp. Strain B—Cultures 124-1, 124-2, Lactobacillus pentosus, n. Sp. The authors feel that the difference between Strains A and B is not sufficient to warrant a separate name for Strain B. Strain C—Cultures 102, 31, and 32. Lactobacillus arabinosus, n. Sp. Group II.—This group contains closely related organisms be- longing to the Lactobacillus pentoaceticus type. The fermentation of arabinose and xylose by certain of the lac- tic acid bacteria results in the production, mainly, of acetic acid and lactic acid. These two compounds are equivalent to about 90 per cent of the sugar destroyed and 98 per cent of the isolated products. The ratio of the two acids to one another is approxi- mately 1 molecule of acetic to 1 molecule of lactic. The theoreti- eal ratio of their molecular weights is 1:1.50, while the ratios found varied from 1:1.34 to 1:1.52. The mannitol-forming bacteria slowly ferment lactic acid to acetic acid and carbon diox- ide. The secondary fermentation results in a deviation from the theoretical ratio in the direction of the lower values. The only other product that could be identified was carbon dioxide. This is produced in minute quantities; from 10 to 20 mg. are formed per gm. of sugar fermented. On the basis of the almost complete fermentation of the pentoses and the high percentage of sugar accounted for by the products, it appears that the main line of the fermentation is simple cleavage into acetic and lactic acids. The pentose-fermenting organisms studied represent a closely related family which may be divided into groups and strains according to their fermentative ability. Although these organ- isms possess differential fermentation characters, the products in the breaking down of arabinose and xylose by them are identi- cal, and in the same proportions. NOoPwhe Fred, Peterson, and Anderson 411 BIBLIOGRAPHY. . Kayser, E., Ann. Inst. Pasteur, 1894, viii, 737. . Grimbert, L., Compt. rend. Soc. biol., 1896, xlviii, 191. . Bertrand, G., Compt. rend. Acad., 1898, exxvii, 124. Bendix, E., Z. diétet. u. physik. Therap., 1900, iti, 587. Gayon, U., and Dubourg, E., Ann. Inst. Pasteur, 1901, xv, 527. . Dubourg, E., Ann. Inst. Pasteur, 1912, xxvi, 923. . Miller-Thurgau, H., and Osterwalder, A., Centr. Bakt., 2te Abt., 1912- 113}, Fosayst, WE) . Miller-Thurgau, H., and Osterwalder, A., Centr. Bakt., 2te Abt., 1917- 18, xlviii, 1. . Henneberg, W., Garungsbakteriologisches Praktikum, Betriebsun- tersuchungen und Pilzkunde, Berlin, 1909, 443. . Orla-Jensen, S., Mem. Acad. Roy. Sc. et Lettres Danemark, 1919, v, 81. . Fred, E. B., Peterson, W. H., and Davenport, A., J. Biol. Chem., 1919, XXXIX, 347. . Peterson, W. H., and Fred, E. B., J. Biol. Chem., 1920, xli, 438. . Peterson, W. H., and Fred, E. B., J. Biol. Chem., 1920, xlii, 273. . Beijerinck, M. W., Arch. Neerland Sc. exactes et naturelles, 1901, series 2, vi, 212. . Andrewes, F. W., and Horder, T. J., Lancet, 1906, ii, 708, 775, 852. . Gordon, M. H., Gt. Britain Local Gov. Bd., Suppl. Rep. Med. Off., 1903- 04, xxxiil, 388. . MacConkey, A., J. Hyg., 1906, vi, 385. . Rogers, L. A., Clark, W. M., and Evans, A. C., J. Infect. Dis., 1914, xv, 100. . Rogers, L. A., and Davis, B. J., U. S. Dept. Agric., Bureau Animal Industry, Bull. 154, 1912. . Shaffer, P. A., and Hartmann, A. F., J. Biol. Chem., 1920-21, xlv, 365. . Monroe, K. P., J. Am. Chem. Soc., 1919, xli, 1002. . Gayon, U., and Dubourg, E., Ann. Inst. Pasteur, 1894, viii, 108. . Winslow, C.-E. A., Kligler, I. J., and Rothberg, W., J. Bact., 1919, iv, 429. . von Furth, O., and Charnass, D., Biochem. Z., 1910, xxvi, 199. . Méslinger, Z., Z. Untersuch. Nahrungs- u. Genussmittel., 1901, iv, 1120. 412 Pentose-Destroying Bacteria EXPLANATION OF PLATES. PLATE 1. Fig. 1. Culture 29. A 24 hour culture on arabinose yeast agar. Stained with fuchsin. xX _ 1,000. Fig. 2. Culture 124-2. A 24 hour culture on arabinose yeast agar. Stained with fuchsin. X 1,200. PLATE 2. Fia. 3. Culture 102. A 24 hour culture on arabinose yeast agar. Stained with fuchsin. X 1,200. Fia. 4. Culture 31. A 24 hour culture on arabinose yeast agar. Stained with fuchsin. X_ 1,000. Fig. 5. Culture 14. A 24 hour culture on arabinose yeast agar. Stained with fuchsin. X 1,000. — = THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII. PLATE 14 \ 33 “\ ‘eae ‘ / \) df q. ‘ 4 i - ‘“ oN =~ ~oe z a — 1 7 ‘ Ser A? w\ v Nos \ a “oo A sn Fie@s 1 7 ~ oe 2 a - «A > wo | \ .¥ Fie. 2. (Fred, Peterson, and Anderson: Pentose-destroying bacteria.) ee THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL, XLVIII. é A Me 4 os 64 Fia. 3 no x X ~ ? F ee | B 2 f & sy & ¢ ae 2 a ‘% a si ve i 4 ues by PEATE 2: ‘| (Fred, Peterson, and Anderson: Pentose-destroying bacteria.) THE EXCRETION OF ACETONE FROM THE LUNGS. By A. P. BRIGGS anp PHILIP A. SHAFFER. (From the Laboratory of Biological Chemistry, Washington University Medical School, St. Louis.) (Received for publication, July 18, 1921.) It is well known that under conditions which lead to the formation and accumulation of the ‘‘acetone bodies,” acetone is present in the breath. It occurred to one of us that this ex- cretion of acetone from the lungs might be the result merely of evaporation from the blood plasma into the alveolar air, condi- tioned by the distribution coefficient of acetone between plasma and air at the body temperature. To learn whether this surmise was correct we have determined the distribution coefficients of acetone between water and air, and blood and air outside the body at different temperatures, and with these data have com- pared results, and coefficients obtained therefrom, from human subjects of natural ketonemia and from dogs after the injection of acetone solutions intravenously. Our experiments lead to the conclusion that acetone is excreted from the lungs by the simple process of diffusion and volatilization, since the ratio between the concentrations in blood and alveolar air is the same as the distribution coefficient determined in vitro at body temperature. A smaller number of experinrents indicate that the concentration of acetone in urine is equal to and parallels its concentration in blood, and the excretion of acetone by the kidneys also thus appears to be the result of simple diffusion. At the time of beginning our experiments about 2 years ago, we were under the impression that the question had not been taken up from the point of view mentioned, but this impression proved to be incorrect when the literature was more carefully searched. The characteristic odor of the breath of patients showing symp- toms of diabetic coma, and the fact that this odor is due at 413 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 414 Excretion of Acetone from the Lungs least in part to acetone, noted by Petters (1) in 1857, is the be- ginning of practically all of our knowledge concerning the acetone bodies. Although most of the host of workers who have con- tributed to the subject have devoted their attention to the ex- cretion of these substances by the kidneys there have been many observations on the excretion of acetone by the lungs. The first quantitative determinations of the amount thus excreted seem to have been made in 1897. In that year Nebelthau (2) recorded the excretion of 150 mg. per hour by the lungs by a subject of chronic inanition who excreted about one-tenth that amount in the urine. The same year Geelmuyden (3) found that on giving acetone by mouth or subcutaneously to rabbits and dogs it was in great part excreted by the lungs; and Schwarz (4) showed that dogs and rabbits excrete only a few per cent of in- jected or fed acetone in the urine while from 50 to 76 per cent appears in the breath. In the same volume in which Schwarz’ paper was published, Miiller (5) describes and pictures a simple apparatus with which he determined the amount of acetone excreted in the breath. The exhaled air was bubbled through ice water and the absorbed acetone titrated by the Messinger method. With normal well fed subjects he found 1.3 to 3.3 mg. of acetone per hour, with diabetics up to 20 mg. per hour, and after giving by mouth 3.8 gm. of acetone to a normal subject, 130 mg. per hour in the exhaled air. Although he records no blood analyses and no individual determinations, he concluded that the amount of acetone in the breath depends upon (1) the content of acetone (or acetoacetic acid) in the blood or tissues, and (2) the volume of respired air, the evaporation in the lung capillaries being faster the lower the acetone tension in the al- veolar air and vice versa. It is evident that Miiller had in mind that the pulmonary excretion of acetone is determined by its partial pressure in blood and alveolar air, though he offered no data to prove that such is the case. In 1910 Cushny (6), without it seems being aware of Miiller’s work, reported data from three experiments on cats which had received acetone intravenously, showing that the excretion in the breath was fairly constant over a 3 hour period, from 8.5 to 14 per cent being excreted within that time, depending upon the amount injected. He states that the “percentage of that injected which is excreted in a given time A. P. Briggs and P. A. Shaffer 415 thus rises with the dose.’’ He also performed one experiment in which measured volumes of air were drawn through an acetone solution (containing chloroform as well) of known concentration and found that the rate of evaporation of acetone was similar to the rate of its excretion in exhaled air by a cat which had been injected with acetone solution. Although he had from these observations data from which the distribution coefficient of acetone might have been calculated approximately, the results were not considered from this point of view. Indeed near the close of his paper, in discussing the reason for the different rates of exhalation of chloroform, methyl and ethyl alcohol, and acetone, he suggests that the different behavior is determined by their solubility in and miscibility with plasma, and states that ‘‘acetone while completely miscible with water is not so nearly related in constitution and its evaporation and exhalation are thus less dependent on its concentration in the blood.”’ We find, on the contrary, that the rate of evaporation of acetone, at any rate, and its exhalation from the lungs is directly dependent upon its concentration in the blood, which in turn determines the partial pressure in the (alveolar) air with which the solution (blood) is in equilibrium. Acetone in dilute aqueous solutions, in the body as well as in vitro, appears to behave in accord with Henry’s law and it is perhaps not unlikely that such other substances as chloroform and alcohol would be found to behave in the same way. Cushny, nevertheless, concluded that the ‘‘exhalation of volatile substances from the lungs is exactly analogous to their evaporation from solutions in water,’’ which as regards acetone is confirmed by our results. Finally, in a recent paper Widmark (7) has reported experi- ments covering almost the identical ground as our own and leading to the same conclusions. He determined the distribution co- efficient of acetone between water and air at 38°C. and found concentration in solution to be 406 and 389 by two the ratio, ——a concentration in air different methods. In one case only the concentration in air, 1 —__.__» the reciprocal of the relation, which solution we have preferred to use. We have recalculated his data for comparison with our own. 1 Widmark gives the ratio 416 Excretion of Acetone from the Lungs and in the other only the concentration in solution was determined, the other concentration being calculated or assumed in both eases. For blood serum he gives 392 and for whole beef blood 323 and 304 as values of the same ratio. On making simultan- eous analyses of blood and alveolar air of a normal subject after taking various amounts of acetone by mouth, he found as values concentration in blood —- — from 394 to 334, thus demonstrat- concentration in alveolar air ing approximately the same relationship as found in vitro with blood. In another paper Widmark (8) showed that the concen- tration of acetone in urine very closely parallels its concentration in blood and that the amount excreted by the kidneys in a given time thus depends only upon the blood content and the urine volume. Distribution of Acetone between Water and Air, in Vitro. For the determination of the distribution coefficient of acetone we have used several different procedures of which only one need be described in detail. In all cases we have directly determined by the iodoform titration the amount of acetone both in the air and in the solution with which the air was in equilibrium. In one method about 2 liters of air were shaken with about 500 ce. of acetone solution in bottles at room temperature (laboratory, warm room, and cold room), and in another a measured volume of air was bubbled through successive tubes of acetone solution placed in a water thermostat. Observations were made over a considerable range of temperature and with solutions of different concentration. The arrangement of apparatus usually used is shown in Fig. 1. Tubes, A, B, and C and the bottle, D, were nearly filled with the same acetone solution and were covered by the water of the thermostat which was stirred and maintained within 0.1° of the desired temperature. A thermometer in the bottle indicated the temperature at which the air and solution were in final equili- brium, and an additional exit tube was connected with a water manometer (/), to insure atmospheric pressure. Air was bubbled through, using just enough pressure at the inlet and suction at the outlet to maintain a steady flow at atmospheric pressure A. P. Briggs and P. A. Shaffer 417 in the bottle, D. Two tubes each containing about 60 cc. of cold water, and surrounded by water and ice, outside the thermo- stat, absorbed the acetone from the air,? the volume of which was measured at room temperature by the water dis- placed from the siphon bottle, 7. The volume was calculated to the temperature of the bottle, D, at which equilibrium was es- tablished, making correction for temperature and water vapor. Usually about 2 liters of air were taken for a determination. Sam- ples of the solution were withdrawn from the bottle, D, from time to time and analyzed. The concentration in D changes very Fig. 1. Apparatus used for determining distribution coefficient of acetone between solution and air. slowly since the air receives its acetone from the earlier tubes. The following example illustrates the data determined and the method of calculation. 2 That the acetone was for practical purposes completely absorbed in the two tubes of ice water was proved by the following experiment. 4 liters of air passed through the tubes of the thermostat which contained an acetone solution of approximately 0.1 per cent. The acetone was collected in a series of three tubes of ice water (4.0°C.). The acetone in the first two tubes was determined by titration to be 42.7 mg. The amount of acetone in the third tube was determined nephelometrically to be 0.0118 sees at 5° is about r mg. or about 0.003 mg. per liter of air. The ratio 1,900. 418 Excretion of Acetone from the Lungs Temperature (of solution during equilibration) = 37.8°C. Barometric pressure =750. 2,000 ce. of air measured at 27.4°C. contained 6.85 mg. of acetone. 10 cc. of acetone solution from the last tube in the thermostat contained 10.64 mg. of acetone, or 1,064 mg. per liter. (Vapor tension, H,O at 27.4° C. = 27 mm. at 37.8° = 49 mm. of Hg) 273 + 37.8 _. 750 — 27 2,000 = Se ite 273 + 27.4 750 — 49 a xX 6.85 = 3.21 mg. of acetone per liter of air. = 2,134 ce. at 37.8° Ratio of concentrations, aaa ae 39] 7 oe": The interest being in the values of the distribution coefficient at body temperature, only the results between 37° and 38°C. are given (Table I). These results and many others at higher and lower temperatures show that the ratio between solution and air is quite independent of concentration over a wide range. The distribution coefficient was determined also for blood serum (beef) with added acetone using an additional tube with glass-wool as a trap to catch foam. It will be seen that the values are practically the same as with pure solutions of acetone. The average of results give the following values: solution 37-38" = for pure solutions, 334; for serum, 337. alr A few determinations were made also with defibrinated blood (beef), but more difficulty was experienced from foaming, and the results were not quite reliable. They indicated roughly the same values as obtained with serum. The Ratio of Acetone in Blood and Alveolar Air.—In order to determine acetone in alveolar air it was desired to have the animals or subjects rebreathe into a bag as in the Plesch method for COs. A rubber bag was found unsuitable due to a rapid loss of acetone vapor, probably by solution in the rubber. The concentration of acetone in the air decreased about 30 per cent in a half hour. A satisfactory bag was made from sheets of adhesive plaster, to the gummed side of which glazed tracing paper was attached. Two sheets of this fabric 8 by 10 inches were glued and clamped together along three sides; into the open end of the bag thus formed, a covered rubber stopper carrying a glass outlet tube was fitted and glued in place. In order to absorb moisture from expired A. P. Briggs and P. A. Shaffer 419 air, and prevent its condensation inside the bag, a short calcium chloride tube was attached to the outlet tube of the bag, fresh lumps of CaCl, being. used for each experiment. When air was bubbled through an acetone solution of known concen- tration in the thermostat apparatus into this bag and subsequently drawn from the bag through ice water, the amount of acetone TABLE I. Distribution of Acetone between Solutions and Air. Acetone per liter. | Ratio. Temperature. Soluti Salationt JES. | Boluver. Air Acetone in distilled water. mg. | mq. 37.0 2, 143 6.65 323 Bi(e| 2) 143 6.74 318 sie 7 | 1, 220 3.67 332 3728) 1,120 308 328 BT 1, 115 3.29 33! erie 209 0.748 341 Shae 1, 064 ay-A0)7/ 347 37.5 1, 064 3.08 346 37.9 1, 064 3.20 339 3/.8 1, 064 es 329 37.8 5, 326 16.06 352 Avyerage........ 37.50 334 Acetone in blood serum. 37.6 3, 020 | 8.69 347 37-0 3, 020 9.22 328 37.6 3, 020 9.12 332 3/.4 3.020 8.99 336 Average........37.4 337 obtained from a measured volume was in agreement with that computed from the concentration of the solution and the dis- tribution coefficient at the observed temperature. With this bag, thus shown to be relatively impermeable to acetone vapor, we determined the concentration of acetone in the alveolar air of a number of human subjects and of experimental animals. 420 ‘Exeretion of Acetone from the Lungs After rebreathing from the bag for a period of 30 to 60 seconds, the air was passed through two tubes of ice water, surrounded by water and ice, the volume of air being measured as in the thermostat experiments at room temperature and calculated to its volume, moist, at body temperature. The acetone absorbed by the tubes of ice water was determined by titration with 0.01 or 0.002 'N iodine and thiosulfate. The determination of acetone in blood was accomplished by aerating the Folin-Wu blood filtrates. Oxalated blood was precipitated by tungstic acid according to the Folin-Wu directions (9), using small flasks to minimize the volume of air in contact with the solution. The mixtures were filtered on funnels with long stems, and covered by watch-glasses to minimize loss during filtration. 10 cc. of the filtrate, equivalent to 1 ce. of blood, were measured into large test-tubes containing about 5.0 gm. of NaCl, and the solution was aerated into another large test- tube containing 50 ce. of water, 2 ec. of 5N NaOH, and 10 ce. of 0.002 N iodine. After 10 minutes of slow aeration, an additional 10 cc. of iodine were added, and the rate of aeration increased and continued 15 minutes. Normal blood to which were added known amounts of acetone, when analyzed under these conditions, gave results within 2 or 3 per cent of the calculated value.* Experiments on Dogs after Injection of Acetone Solutions. Experiment 1.4—A female dog of 7.5 kilos was given 4 gm. of urethane in 200 ec. of water by mouth. A tracheal cannula was inserted under local anesthesia (benzyl aleohol). The bladder was emptied by catheter and control samples of blood and alveolar air were taken, after which 75 ec. of 14.5 per cent acetone solution (10.87 gm. or 1.45 gm. per kilo of body weight) were injected intravenously. Samples of blood, alveolar air, and urine were collected as stated in the protocol. 10.21 a.m. Control air, 760 ce. at 28° = 805 ce. at 37.6° = 0.046 mg. of acetone. Control urine, 13 ce. 10.24 a.m. Bladder emptied. 10.27 a.m. Blood control. 10.50 a.m. 75 ec..of 15 per cent acetone solution intra- venously. 10.52 a.m. Urine 1 = 3.3 cc. 10.59 a.m. Air 1 = 680 ce. at ’ Air under pressure was used in aeration. After 30 minutes aeration, 20 ee. of 0.01 n iodine showed a loss of about 0.15 ec. and 20 cc. of 0.002 N iodine a loss of about 0.25 ce. 4 The thanks of the authors are due Dr. E. K. Marshall, Jr., and Dr. W. H. Olmsted for assistance in conducting several of the experiments. A. P. Briggs and P. A. Shaffer 421 28° = 720 cc. at 37.6° = 5.02 mg. of acetone. 11.00 a.m. Blood 1 = 15 ce. 11.06 a.m. Urine 2 = 5.2 cc. + 20 ec. of wash water. 11.11 a.m. Rectal temperature = 37.6°. 11.15 a.m. Air 2 = 650 cc. at 28° = 689 ec. at 37.6° = 4.41 mg. of acetone. 11.24 a.m. Blood 2. 11.28 am. Air 3 = 660 ce. at 28° = 700 ce. at 37.6° = 4.07 mg. of acetone. 11.33 a.m. Blood 3. 11.35 a.m. Urine 3 = 21 ce. + 20 ce. of wash water. 11.45 a.m. Air 4 = 730 ce. at 28° = 774 cc. at 37.6° = 4.38 mg. of acetone. 11.48 a.m. Blood 4. 11.58 a.m. Urine 4 = 10 cc. + 20 ce. of wash water. 12.02 p.m. Air5 = 650 ce. at 28° = €89 cc. at 37.6° = 3.63 mg. of acetone. 12.06 p.m. Blood 5. 12.14 p.m. Urine 5=6.5 ec. + 20 cc. of wash water. 12.28 p.m. Air 6 = 750 cc. at 28° = 795 ce. at 37.6° = 3.84 mg. of acetone. 12.30 p.m. Blood 6. 12.37 p.m. Urine 6 = 3.5 ec. + 20 ee. of wash water. 12.56 p.m. Blood 7. 1.00 p.m. Air 7 = 695 cc. at 28° = 737 cc. at 37.6° = 3.62 mg. of acetone. 1.06 p.m. Urine 7 = 6 cc. 2.01 p.m. Blood 8. Air 8 = 688 cc. at 28° = 729 cc. at 37.6° = 3.39 mg. of acetone. 2.02 p.m. Urine 8 = 5.8 ce. The analytical results of this experiment are given in Table II and have been plotted in time curves shown in Fig. 2. For the calculation of the ratios between concentration of acetone in blood and in alveolar air, given in the last column of the table, a curve of the concentration in blood plasma was plotted and values (in parentheses in table) read off corresponding to the time of taking the air samples. The ratio of concentrations pisns varies from 320 to 350, the average being 336, or nearly air the same as observed in the in vitro experiments (337). The variation is irregular and is doubtless due to accumulated errors in the procedure. The curves show that the concentration of acetone in both alveolar air and urine follows very closely that in blood, the amount in urine and blood being almost identical. The initial rise in the urine is probably in response to a much higher concentration in the blood immediately after the injection and before the first blood sample was drawn. With the fall of the amount in blood due in large part to its passage into the tissues, the concentration in urine fell to or slightly below that in blood. It is of interest to learn the relative amounts of acetone ex- creted by this animal from the lungs and kidneys. The total urine secreted during 3 hours and 10 minutes was 61 cc., contain- ing 114 mg. of acetone. The total excretion by the lungs was 422 Exeretion of Acetone from the Lungs not determined but may be very roughly calculated as follows. The respirations varied from 13 to 25 per minute of from 100 to 130 ec., giving a total volume per minute of 18 & 115 = 2,070 ec. The alveolar air may be taken as containing about 5 mg. per liter and the tidal air perhaps 4.0 mg. per liter. This gives a total excretion by the lungs during the experiment of 1.56 Pease esa [| ll ea ae Ae eed Os Be hd We o45| ¢ -——— alveolar air IGEMne Seema cee Ha | ac Sake alo ge | eB Bes FO Pe FP ithe alate Dla) Cali tall LSet a SAUNE RPS eee of Sst Veal cl] 0) sal ok Sia aa Pies SSS 71S i ee RES kale PEE Ree RRSS Sek hal fas asf BEER ORES | Let | tim] | tee | | te] tT Fig. 2. Curves showing parallel changes in concentration of acetone in blood, alveolar air, and urine of dog after intravenous injection (Experi- ment 1). ele milli grams per lo gm. or roughly fifteen times the excretion by the kidneys during the same time. Since the concentration in urine is independent of the urine volume, the amount thus excreted will, of course, depend upon the amount of urine secreted as well as upon the blood concentration. And similarly the amount excreted in a given time by the lungs is determined by the volume of respired air which aerates the acetone from the blood. A. P. Briggs and P. A. Shaffer 423 TABLE II. Experiment 1. Acetone per 100 ce. Ratio. Time. r i eee eda ee eee a.m. mg. | mg. mg. mq. 10.21 1.8 0.0057 10.27 10.50 Acetone injected. : 10.52 106 10.59 (242) 0.698 347 11.00 236 | 242 11.06 264 11.15 (205) 0.640 320 11.24 198 196 fai -28 (194) 0.571 340 11.33 188 191 11.35 217 11.45 (185) 0.566 327 11.48 186 184 11.58 168 p.m. . 12302. (179) 0.527 340 12.06 177 176 12.14 175 12.28 (170) 0.483 350 12.30 168 169 12.37 159 12.56 165 161 1.00 (160) 0.491 326 1.06 *147 2.01 163 169 0.465 | 342 2.02 | (agen | AVWORT RD. Ao eee aA coor eo no neeaee mee eO ero. SE 336 Experiment 2.—Under ether anesthesia a tracheal cannula was inserted in a dog weighing 10 kilos. Urethane was injected intraperitoneally and the ether discontinued. Blood and air samples were taken before and after the injection of acetone solution as stated in the protocol. The analytical results are given in Table III. 2.45 p.m. 10 gm. of urethane intraperitoneally. 3.00 p.m. Control samples of blood and alveolar air. 3.05 p.m. 25 cc. of acetone solution 424 Excretion of Acetone from the Lungs a. (50 per cent) injected into the peritoneal cavity. 3.23 p.m. Blood sample 1. 3.27 p.m. Air sample 1 = 395 cc. at 27°. 3.55 p.m. Blood sample 2. 4.00 p.m. Air sample 2 = 410 ce. at 27°. 4.10 p.m. Blood’ sample 3. 4.14 p.m. Air sample 3 = 330 cc. Rectal Temperature 97°. 4.25 p.m. Blood sample 4. 4.30 p.m. Air sample 4 = 400 cc. at 27°. 4.40 p.m. Blood sample 5. 4.43 p.m. Air sample 5 = 295 cc. at 27°. 5.00 p.m. Blood sample 6. 5.05 p.m. Air sample 6 = 380 cc. at 27°. 5.15 p.m. Blood sample 7. 5.25 p.m. Air sample 7 = 520 cc. at 27°. 5.35 p.m. Blood sample 8. 5.40 p.m. Air sample 8 = 250 ce. Rectal Temperature = 97°. Dog killed with chloroform. TABLE III. Experiment 2 Acetone per 100 ce. Ratio. Time. Blood serum. Alveolar air. eee eR tet eet p.m 3.05 Acetone solution injected (25 ec. of 50 per cent). B58} 157 One, (156) 0.453 344 3.55 149 4.00 (145) 0.471 307 4.10 132 . 4.14 (131) 0.3590 336 4.25 128 4.30 (127) 0.392 324 4.40 125 4.43 (125) OFS7 329 5.00 123 5.05 0.328 369 515 114 O25 en Os308 310 DoD 113 5.40 0.357 317 ANVCRE GC 0s 5 Felis Poe 330 serum The ratios vary considerably, from 307 to 369, alveolar air the average being 330. It will be noted that the acetone (12.5 or 0.8 gm. per kilo) was injected into the peritoneal cavity from which it rapidly passed into the blood and from the blood | was more slowly distributed and excreted. A. P. Briggs and P. A. Shaffer 425 Four other experiments of similar character yielded results which gave ratios, pu a lal from about 280 to 380, most of alveolar air which were of doubtful accuracy and for that reason will not be recorded. The acceptable results leave no doubt that distri- bution of acetone between blood and air in the lungs of dogs is substantially the same as the distribution found in vitro. Distribution of Acetone between Blood and Alveolar Air in Diabetic Acidosis. The determinations in blood and alveolar air were carried out as above described, at once after collecting samples. The results obtained are given in Table IV in which is also included for com- parison a summary of the results on animals and a few of the results of zn vitro experiments. Results are recorded also of two determinations on normal subjects on the morning of the third day of fast, when acetone bodies were being formed in small amounts. For the determinations of acetone in alveolar air of these normal subjects 5 liters of air, separately equilibrated by rebreathing 1 liter portions, were taken for analysis. The ratio between blood and air was evidently the same as in the diabetic subjects with marked acidosis. These results from diabetic and normal subjects show conclus- ively that the concentration of acetone in alveolar air bears a constant relationship to the concentration of free, preformed acetone in the blood, and that this relationship is expressed by the distribution coefficient of acetone between the air and its solution in blood plasma. From this fact it follows that one may learn the amount of’ acetone in blood by determining the amount in alveolar air and multiplying the result by a factor, the value of which according to our data is about 340. And if there were also a constant relation between the amount of acetone, and of the related acetoacetic and hydroxybutyric acids it would be possible to calculate also the latter values. In Table V are given the results on a few blood analyses showing separately the amounts of acetone, and acetoacetic and hydroxybutyric acids. Although these results are too few to justify generalization, they indicate that the relative amounts of free acetone in blood 426 Excretion of Acetone from the Lungs vary at least from 13 to 26 per cent of the sum of acetone, and acetoacetic and hydroxybutyric acids. One may, therefore, perhaps get a rough approximation of the amount of total acetone bodies (expressed as acetone) present in blood by multiplying the concentration in alveolar air by, say 1,700, (340 « 4),”) Such a calculation is not to be recommended as a substitute for TABLE IV. Summary of Results, Ratio of Acetone in Blood and Alveolar Air, and Com- parison with Results in Vitro. Acetone per liter. Ratio Subject. Condition. Blood. Alveolar air. ipod Air Whole blood. mg. PLR: Diabetic acidosis. 112 0.524 345 On: sf a 96 0.278 345 F. M. of “ 148 0.436 340 Peters. i. ca 199 0.560 355 K. Normal, 3 days of fast. 64 0.163 392 C. a Seah eiedice Fas 23 0.066 350 | Acetone injection. Blood plasma.) - Dog. | Experiment 1. 2,420 to 1,590 | 6.98 to 4.65 336 << 2. 1,570 to 1,130 | 4.53 to 3.57 | 330 In vitro experiments. Water solutions. Solution. Air. Solana Air Maximum concentration. .......... 5, 326 16.06 - 332 Minimum €e Fae MANO: 255 0.748 341 PVCTA RECON cite ons oe 334 Biood! serum=.3 5s eee eee ee | 3, 020 8.96 337 the direct determination in blood. The excretion of acetone by the lungs is nevertheless of real value as affording a very simple means of detecting and roughly determining the extent of ketosis. The subject is asked merely to exhale for 1 or 2 minutes through a glass tube into a large test-tube of ice cold distilled water. At the end of the period of exhalation 10 or 20 ee. of Scott Wilson reagent are added and if ketosis exists a faint to deep opalescence Ea, A. P. Briggs and P. A. Shaffer 427 or precipitate appears after a few moments, the amount being roughly proportional to the amount of acetone exhaled. This test 1s quite sensitive and appears even before the urine shows a positive reaction with ferric chloride.for acetoacetic acid. TABLE V. Acetone Bodies in Blood. od 2 Per cent of ag as eS total. ea serliaes ga = | 2 ee paps, Subject. § 8 Es = 3 2 os Remarks. ad We ESS ae = la |s8 a}< | Ere esl Seles ann a | 8 |8s| 2% : As acetone in 100 ce. $| 2 |vs| Se S of blood. |< ig*|o> ii es ee 1*|“‘K,”’ Severe diabetic.|17.6)10.4| 45.2) 73.2] 24) 14 62'56.7, Not in coma. BF e “ 3.3] 7.4| 15.5) 24,5) 14) Siirb5i15- 9) = ee Bal ¢ 1V.0) 7.0) 49-5) G74) 16) 10) 74i22-1) & » GI oun 9061 ‘oyeq ‘poi0[oo-yaep ‘oui “IOATY POTD ‘AB snulpog “Avg Aa10}Uoyy “U01yRyg 432 Carbohydrate of King Salmon Tissues were analyzed by the method of Pfliiger and the glycogen was hydrolyzed and determined as glucose by the copper sulfate colori- metric method. The hydrolysis was used to guard against any loss by possible autolysis occurring during the preservation. The determinations are comparatively few but we have had no oppor- tunity to add to the series. There are data enough in Table I, which gives the entire set of determinations for the series of samples, to indicate the average glycogen content of salmon tissues and the contrasts at the extremes of the fast. Muscle. Samples were taken of the great lateral muscle in a vertical . band in the region of the anterior dorsal fin. In Fish 732, Mon- terey Bay, July 18, the duplicate samples show 0.015 and 0.016 per cent of glucose. These are voraciously feeding fish and are to be contrasted with Fishes 754 and 765 from Bolinus Bay near the mouth of the Golden Gate. The Bolinus fish are in the very prime of condition with the high store of 18 per cent of muscle fats.’ They represent the highest nutritive value of any fish in this series, greater than the average for feeding fish at Monterey and of higher value than the migrating fish at tide- water on the Sacramento River. Fish at Bolinus Bay at this time had practically ceased feeding (judged by the absence of food content in the stomach). Muscle samples were not obtained for glucose analysis from the Black Diamond fish. The muscles of spawning fish are without glucose as shown by the analyses from four females and three males. Not a single sample yielded glucose. The tissues were perfectly fresh, often alive when minced. Liver. The salmon livers in fish from the Monterey feeding grounds show a variable glucose content, from 0.19 to 0.66 per cent. Livers from these fish examined histologically were crowded with fat droplets. Chemical analyses showed fat as high as 16.6 per cent. The glucose content is from ten to forty times greater than that 7 Greene, C. W., Tr. Am. Fisheries Soc., December, 1915. C. W. Greene 433 of the muscle in the Monterey fish. The two Bolinus Bay fish vary as much as the extremes of the six examined at Monterey; 2.e., 0.18 and 0.70 per cent of glucose. Samples of livers from six fish were analyzed from Baird. These livers from spawning fish contain with two exceptions less than 0.1 per cent of glycogen. The average is 0.057 per cent. This average is strikingly lower than the glycogen content of the Monterey or Bolinus Bay fishes. It indicates a depletion of the glycogen and a decrease of the part played by carbohydrate in liver metabolism during the fast. Ovaries and Eggs. The ovaries of the series of six fish are remarkably uniform in glucose content, whatever the stages of the journey. The percentages are from 0.08 to 0.18. This is a glucose content not above the average found in vertebrate blood.’ One fish, No. 946, was a mature female from which spawning eggs were obtained. The analysis of these eggs, free from ovarian tissue and fluids, gave 0.09 per cent of glucose. This is near the average for the ovary of the series of immature fish. It demonstrates that the carbohydrate content, in this growing tissue is independent of the stage of development and of the duration of the fast. Tichomiroff® has given analyses of invertebrate eggs showing that in the eggs of Bombyx the glycogen amounts to 1.98 per cent. The total dry substance in these eggs is 35.51 per cent, somewhat less than salmon eggs, which average 45 per cent or more in total solids. Octopus eggs have been examined by Henze,!° who found as high as 1 per cent of glucose in the fresh eggs (5.4 per cent in the dry residue). Kojo!! found 0.272 per cent of glucose in the yolk and 0.55 per cent in the whites of the hen’s egg. Testes. Three samples of testes were examined for glucose from the Baird salmon. The salmon were nearly mature but not one contained glucose. 8 Macleod, J. J. R., Physiol. Rev., 1921, i, 208. 9 Tichomiroff, A., Z. physiol. Chem., 1885, 1x, 566. 10 Henze, M., Z. physiol. Chem., 1908, lv, 485. U Kojo, K., Z. physiol. Chem., 1911, Ixxv, 1. 434 Carbohydrate of King Salmon Tissues Other Organs. Samples'‘of the skin and of the stomach-intestinal mass were analyzed. The skin sample of ish 765 from Bolinus Bay contained 0.038 per cent of glucose. ‘Three samples of skin from spawning fish contained no glucose. Of the three stomach-intestinal samples, two were from Mon- terey. One gave a trace of glucose, the other none. The specimen from Bolinus Bay contained 0.041 per cent of glucose. However, the pyloric ceca and the intestine of both Monterey and Bolinus Bay fish contained a variable quantity of mucus and unabsorbed food products. These are retained in part in the ground up total mass used for the.sample, hence the 0.041 per cent of glucose from the Bolinus Bay specimen might have come from the food remnants, though it is improbable. The stomach and intestines of McCloud River fish are greatly atrophied and small. However, no glucose analyses were obtained. DISCUSSION. Kalborn and Macleod? have emphasized the comparatively low content of glycogen in invertebrates and fishes. ‘The four fish species examined by them were dogfish, chimera, carp, and lake trout. In their Table IV they present analyses of muscle, heart, and liver of dogfish, of carp, and of trout and give deter- minations for the liver of the chimera. In the dogfish their . average for body muscle is 0.018 per cent, for carp it is 0.29 per cent, and in lake trout muscle there is only a trace of glucose. Our analyses of the sea run salmon check against these determina- tions, 0.015 per cent in the salmon muscle in comparison with 0.018 in the dogfish and 0.29 in the carp. On the other hand, the liver determinations of Kilborn and Macleod show as much as 5.6 per cent of glucose in the carp liver but only a maximum of 0.16 per cent for dogfish liver and 0.055 per cent.for the liver of lake trout. The lake trout figure checks with that of our fasting salmon. They find a trace of glucose in lake trout muscle (probably fasting) while we find none in the fasting salmon. They have presented no analyses of the glucose content of either eggs or ovaries of fishes. In fact we have not been able to find such in the literature. C. W. Greene 435 No previous comparisons have been made of the glucose content of tissues during long starvation, comparisons for which the salmon migratory habit without food lends a rare opportunity. A study of our data will show that the muscle glucose, presumably elycogen, is present in low amount during the feeding period, but drops to a trace at the beginning, and disappears entirely during the migration. Since it has been shown in several of my earlier articles! that the large store of fats progressively decreases with the migration, one can scarcely escape the view that the carbohydrates play little part in supplying kinetic energy for the migration; in fact the small amount of glucose in the muscles of feeding salmon may well be present by virtue of the digestive and anabolic processes going on at that time. That glycogen is not entirely absent in salmon metabolism during the migratory journey is indicated by its constant presence, though in small amount, in the liver and ovaries. In view of the well known liver glycogenic function in vertebrates, and especially in mammals, it is surprising to find so small a percentage present in the salmon liver even under the most favorable conditions of feeding. In our determinations it has never exceeded 0.70 per cent of the wet weight of the organ. Livers from the Monterey feeding fish, in which the glycogen reaches its highest amount, invariably contain a considerable percentage of fat. For example, in the liver of No. 765 from Bolinus Bay unpublished analyses reveal as much as 25.8 per cent of fat, which is the highest liver fat observed. In general the normally small percentage of glyco- gen is greatly diminished in the spawning salmon. If we contrast the Monterey fish with the Baird spawning fish the averages for the liver are 0.405 and 0.057, respectively. We are most pleased by the discovery of the constant composition of the ovaries in glucose (glycogen). The average for the entire series is 0.096 per cent. It seems to us to point directly to a uniform synthetic and storage process in this tissue, removed as it is from any part in energy production during the migration, but constantly growing and actively storing foods. This uniformity of com- position as regards glucose adds one more point in evidence to the unpublished data of the author showing uniformity of composition of the food-loaded protoplasm of the egg cell at whatever stage of its growth it be considered. 436 Carbohydrate of King Salmon Tissues The protein and fat percentages of fish from this series were published in 1915.7 We reproduce the averages of the table in that paper: but with columns for glucose introduced. Unfor- tunately we have no glucose determinations for tide-water fish. Both proteins and fats are stored in the mature salmon muscle in quite large excess. This storage of proteins and fats is also true for salmon ovaries.” The muscle protein excess is 6 per cent of the tissue figured on the protoplasmic basis. ,The fat is stored TABLE II. Protein, fat, and glucose in wet muscle samples giving average per- centages for Monterey Bay, tide-water on the Sacramento River, and the spawning beds on the McCloud River. Station. Protein.* Fat.* Glucose. Gineoee soe per cent per cent re per cent per cent per cent Monterey Bay........... 15.6 18.0 0.015 0.606 0.13 ide-waten. a. - ose scese 16.9 14.6 McCloud River.......... 14.4 1G 0.000 0.130 0.09 * Protein and fat taken from Greene’s’ table. to 18 and more per cent of the moist tissue (25 to 30 per cent of the dark muscle). No such large storage of carbohydrate occurs in any salmon tissue. Carbohydrate is never present in more than 0.70 per cent even in the livers of feeding salmon. Carbohydrate is always present in the growing ovary, is in small amount in all the tissues of the feeding salmon, but disappears from the muscles and drops to a lower level in the liver during the migratory fast. 12 Greene, C. W., J. Biol. Chem., 1921, xlviii, 59. VITAMINE REQUIREMENTS OF CERTAIN YEASTS AND BACTERIA. By CASIMIR FUNK anp HARRY E. DUBIN. (From the Research Laboratory of H. A. Metz, New York.) (Received for publication, July 30, 1921.) In a previous publication (1) we described a practical method for testing the vitamine requirements of yeast, based upon the work of Ide and his coworkers, and also of Williams, of Bach- mann, and of Eddy. At that time we thought our test was specific for antiberi-beri vitamine; however, because of certain differences, the question was left open. In this connection, Emmett and Stockholm (2) suggested that the vitamine necessary for the growth of yeast had nothing to do with antiberi-beri vitamine. Later Fulmer, Nelson, and Sher- wood (3), Souza and McCollum (4), and MacDonald and Mc- Collum (5), claimed that by improving the medium, results could be obtained similar to those noted after vitamine addition. These claims have been disproved by the work of Eddy, Heft, Stevenson, and Johnson (6), and our own findings are in accord with their conclusions in this particular. Our present results show rather conclusively that yeast requires for growth a different substance than that needed by animals, since we were able to separate from autolyzed yeast one sub- stance active for yeast and another for rats and pigeons. This separation will enable us to study each substance individually. At the same time, in agreement with our findings, Ide (7) shows that by improving the medium one can actually obtain a slightly better growth, not to be compared in magnitude, however, with the action of the specific vitamine-like substance. Although the work thus far does not shed immediate light on the test for the antiberi-beri vitamine, it is of importance in the study of the vitamine requirements of yeasts and bacteria. We believe that we are dealing here with a specific substance—either 437 438 Certain Yeasts and Bacteria a new vitamine or a cleavage product of antiberi-beri, or vitamine B. We wish to point out that although our yeast test previously described does not specifically indicate the vitamine B activity, still it does show, to a certain extent, the relative richness in water- soluble vitamines when we are concerned with naturally occurring foodstuffs. Simultaneous experiments with yeast and a strain of strepto- coecus obtained from Mueller (8), and following his method of testing, tend to show a close, if not fully established relationship between their nutritive requirements. It would be very attrac- tive to consider these nutritive elements as one substance, but although there are many points in common, there are still many differences to be reconciled, so that the question must be left open for the present.! Another thing worthy of note is that different strains of yeast behave differently as regards vitamine requirement. Some of them, as shown by Nelson, Fulmer, and Cessna (9), seem to be able to synthesize their own vitamine, the initial inoculation pro- viding the first impulse, while others require the addition of extra vitamine.? This difference in the vitamine requirements of various strains of yeast may shed some light on the ultimate physiology of yeast cells. These lower organisms having greater synthetic power appear to be able to utilize the simplest type of vitamine, so that chemically it might be advantageous to study the structure of vitamines in this way. ; EXPERIMENTAL. Differentiation between the Substance Active for Yeast and That Active for Pigeons and Rats—In our previous paper, we showed that much larger quantities of fullers’ earth were necessary to remove from autolyzed yeast the substance necessary for the ‘ Detailed experiments along this line are being conducted in our labo- ratory by L. Freedman, and the results will be presented in a later publi- cation. * Peters (10) believed at first that protozoa can live and divide on purely inorganic material, but he found subsequently that they became smaller and smaller and appeared to live at the expense of their own protoplasm; he thinks, therefore, that addition of vitamines is necegsary for proper growth. C. Funk and H. E. Dubin 439 growth of yeast, than those which are known to be sufficient for the removal of vitamine B; this is in agreement with the findings of Emmett and Stockholm (2). Our present results show that the substance active for yeast may be removed almost quantitatively from autolyzed yeast by two successive shakings each with TABLE I. No. Substance tested.* nee Paraves mm. Pr eA ULbOly Zed, Yeasts. vce. 2:22. She ae eee 14.5 Positive. 2 § “shaken with fullers’ earth (Osermuperwlten) kiss? ose oes a ee 12.0 Negative. 2a| Fullers’ earth from No. 2 decomposed with DAY Cars oe FS 90) ORAS ot ai hea lee. amet 4.0 | Positive. 3 | Autolyzed yeast (filtrate from No. 2) shaken with fullers’ earth (100 gm. per liter)....... 6.0 Negative. 3a| Fullers’ earth from No. 3 decomposed with Damby Gates o/c tyareat on bats oe can ees Pee 3.9 “6 4 | Autolyzed yeast (filtrate from No. 3) shaken with fullers’ earth (100 gm. per liter)....... 0.5 He 4a| Fullers’ earth from No. 4 decomposed with [ECT Ui MOD Mee Mae Lely UE Co Een os curd ere, 0 a 5 | Autolyzed yeast shaken with norit (50 gm. Pete Lavery fei, ih 505 ae Re ose te ae 13.5 = 5a| Norit from No. 5 decomposed with glacial ACETIC. ACIG. "v/a yreten bale a meee ee meas 3.0 Positive. 6 | Autolyzed yeast (filtrate from No. 5) shaken ; Withenorit. (O0foams persliten) seen eee 3.0 Negative. 6a| Norit from No. 6 decomposed with glacial CCLICT ACIE At eee nese Cane eR ce 3.0 os 7 | Autolyzed yeast (filtrate from No. 6) shaken Wibhnoriie dOOlems ner liter aera eee eeee OfSan ss 7a| Norit from No. 7 decomposed with glacial A COLIC ACI iy wa yanet ee ake neers or een ee 0 is *In each case the amount tested was 0.05 cc., so that the results are quite comparable. - 100 gm. of fullers’ earth or of norit, per liter. It is essential that with every lot of autolyzed yeast, controls must be run to deter- mine the degree of separation. The fullers’ earth and norit were decomposed with baryta and glacial acetic acid respectively, according to the method of Seidell, and of Eddy and coworkers. Norit, extracted with baryta, did not yield any active substance. 440 Certain Yeasts and Bacteria The various fractions were also tested on rats and pigeons with concordant results, both preventive and curative experiments being performed. Specificity —The following experiment shows that by improving the medium either in its inorganic or organic moiety (glucose, proteins) the growth of yeast cells can sometimes be improved; however, the magnitude of the resulting response is of an entirely different order than that obtained by vitamine addition. Using the inorganic medium of Fulmer, Nelson, and Sherwood (Medium F) we did not obtain any more growth than on our Nageli solu- tion. Using Medium F and autolyzed yeast we had even lower results than with Niigeli solution. This corroborates the finding of Eddy, Heft, Stevenson, and Johnson (6). No. Medium. actinty. > aout mm. mm, 1 | Blank determination (Medium F).............. 3.0 2 s of (Nigel) #5. 22574 ee ae 3.0 3 | Medium F plus 0.05 ec. of autolyzed yeast plus Yeast Suspension . <6... a): eee ee eee 12.5 9.5 4 | Nigeli plus 0.05 ce. of autolyzed yeast plus VeRSL BUNPENSIONs:<..ccin cee eee 14.0° |. SEO 5 |Medium F plus 0.05 cc. of autolyzed yeast. 0 6 | Nigeli plus 0.05 cc. of autolyzed yeast......... 0 The results with glucose have shown that an addition of the sugar has little or no effect, contrary to the findings of MacDon- ald and McCollum (5). It seems that the slight effect obtained with glucose can be eliminated by shaking the sugar solution with an adsorbent. Ground up meat was extracted and autoclaved till a watery extract no longer showed any vitamine activity. The meat was then subjected to hydrochloric acid hydrolysis, neutralized, and tested again. There was a small but definite activity mani- fested. The same was true of casein and gelatin but not of zein, egg albumin, or serum albumin. These experiments are being continued. The addition of glucose to the blank determination does not affect the result. C. Funk and H. E. Dubin 441 No. Medium. Yeast Net activity. activity. mm. mm. Hi0>.cc. of autolyzed yeast...2. 2. s02on0 5 peaee as 12.5 10.0 2 | 0.05 cc. of autolyzed yeast plus 1 ce. of 10 per COMUPONICOSC!. ccc ckcver ie Wee Oe CP aes 13.5 11.0 3 | 0.05 cc. of autolyzed yeast plus 1 ce. of 10 per cent glucose shaken with 10 per cent charcoal (COROT) AS el 5 ee Se OR A tC 133.6 i) 4 | 0.05 ce. of autolyzed yeast plus 1 ce. of 10 per cent glucose shaken with 10 per cent fullers’ 5 | 0.05 cc. of autolyzed yeast plus 1 ce. of 10 per cent glucose shaken with 10 per cent Lloyd’s MOA OMG ee ey Cee teat Nese? os. te lin sh tos vo ee 12.5 10.0 6 | Blank (without autolyzed yeast)............... 2.5 (aaplacceonl Onpermcent, elicoseim. see eeee seeders 2.5 0 Regarding the activity of bakers’ and brewers’ yeast we have found, in agreement with Williams (11) that brewers’ yeast extract is more potent than bakers’ yeast extract in affecting the growth of brewers’ yeast. On the other hand, contrary to Williams (11) we have noted that bakers’ yeast extract does not stimulate the growth of bakers’ yeast as much as does brewers’ yeast extract. Goy (12) claims to have isolated a nitrogen-free acid which stimulates the growth of bacteria and yeast, but a closer examina- tion of his results does not lend support to his claims, the sub- stance actually isolated being entirely inactive. Preliminary work shows that there are many points of similarity and dissimilarity between the substance stimulating the growth of yeast and that stimulating the growth of streptococcus. We have found for example that undecolorized heart infusion exhibits a very marked yeast growth-stimulating activity, while the de- colorized infusion (decolorized with norit) shows only a very negligible activity. Autolyzed brewers’ yeast also showed marked stimulating action on the growth of streptococcus. Peptone added to the medium gives growth both with yeast and with streptococcus. Casein hydrolysate acts slightly on yeast growth and more so on streptococcus. Hydrolysates of some other proteins did not act on yeast but were active for 442 Certain Yeasts and Bacteria streptococcus. It is just such discrepancies as these that make it imperative to obtain more data before reaching definite con- clusions. The method of separation from vitamine B of the sub- stance stimulating yeast growth, and which we will provisionally eall “vitamine D,’’ will facilitate further work and may help to clear up the question of the identity of vitamine D with the sub- stance stimulating the growth of streptococcus. CONCLUSIONS. We have separated from vitamine B a substance which we shall call provisionally vitamine D and which acts on microorganisms. Vitamine D appears to be a definite and specific substance stimulating the growth of yeast. Streptococcus is more difficult to study because apparently it needs at least two substances for growth. Although vitamine D has been obtained free from vitamine B, as far as our animal experiments have shown, the reverse is not true. It is evident, therefore, that most animal tests conducted up to the present were carried out with a mixture of vitamines B and D and will consequently have to be repeated as soon as a clear separation of the two substances can be effected. It may develop that the vitamine D, obtained from yeast, and the vita- mine-like substance obtained from proteins, such as casein, may have some special function in the body, and such experiments are now being planned. Regarding the possible identity of the substance promoting the growth of yeast with that influencing the growth of streptococcus, our present data are insufficient to venture a definite statement. BIBLIOGRAPHY. 1. Funk, C., and Dubin, H. E., J. Biol. Chem., 1920, xliv, 487. 2. Emmett, A. D., and Stockholm, M., J. Biol. Chem., 1920, xliii, 287. 3. Fulmer, E. I., Nelson, V. E., and Sherwood, F. F., J. Am. Chem. Soc., 1921, xliii, 186, 191. . Souza, G. deP., and McCollum, E. V., J. Biol. Chem., 1920, xliv, 113. 5. MacDonald, M. B., and McCollum, E. V., J. Biol. Chem., 1920-21, xlv, 307. 6. Eddy, W. H., Heft, H. L., Stevenson, H. C., and Johnson, R., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 138; J. Biol. Chem., 1921, xlvii, 249. us —————— eS oy Funk and H. KE. Dubin | 443 . Ide, M., J. Biol. Chem., 1921, xlvi, 521. . Mueller, J. H., Proc. Soc. Exp. Biol. and Med., 1920-21, xviii, 14. . Nelson, V. E., Fulmer, E. I., and Cessna, R., J. Biol. Chem., 1921, xd hvaly (fle . Peters, R. A., J. Physiol., 1919-20, liii, p. eviii; 1920, liv, p. 1; 1921, lv, 1. . Williams, R. J., J.. Biol. Chem., 1921, xliii, 43. . Goy, P., Compt. rend. Acad., 1921, elxxii, 242. THE EFFECT OF SUBCUTANEOUS INJECTIONS OF SOLUTIONS OF POTASSIUM CYANIDE ON THE CATALASE CONTENT OF THE BLOOD. By WILLIAM H. WELKER anp J. L. BOLLMAN. (From the Laboratory of Physiological Chemistry, College of Medicine, University of Illinois, Chicago.) (Received for publication, June 20, 1921.) In 1889 Geppert! concluded on the basis of an extensive series of experiments that potassium cyanide acted on the living organ- ism through the mechanism of making the cells lose their power of oxygen utilization. He states that the picture is one of internal suffocation in the presence of excess oxygen. His experimental results showed that the oxygen consumption was very markedly diminished, and also that the carbon dioxide formed was very markedly diminished. Scientific investigators seem to have accepted Geppert’s results and interpretation as final, since no experiments have been carried out since that time along precisely the same lines. In recent years a theory has been built up to the effect that catalase of the blood (by which we understand the catalytic activity which greatly accelerates the breaking down of hydrogen peroxide) follows the oxidative capacity. Further, that anything which affects the catalase content of the blood must necessarily have a similar effect on the oxidative process in the organism. It appeared to us that potassium cyanide would be a satisfactory substance to apply, in testing out this theory. The methods that had been used. for the estimation of catalase at the time this work was planned, appeared to be rather crude, and so an apparatus was built in which the liberated gas could be measured under conditions corrected for pressure and where the shaking could be done mechanically under constant conditions. 1Geppert, J., Z. klin. Med., 1889, xv, 307. 445 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 446 KCN on Catalase Content of Blood 90 acy 80 | 10 60 | (exe, of oxyden liberated Ds © Time in minutes Fia. 1. W. H. Welker and J. L. Bollman 447 A description? of this apparatus is being published in another journal. Our determinations were carried out in triplicate and practically all the results given are the averages of closely agreed triplicates. In all but one of our series of experiments, samples of 0.5 cc. of oxalated blood were used. The exception was in the case of Dog 8 in Fig. 1, in which samples of 0.25 cc. were used. The sample was placed into a small aluminium vessel and floated on top of 75 cc. of equal volumes of hydrogen peroxide and dis- tilled water. The stoppers were then carefully placed into the bottles, the gas levels adjusted, the stop-cocks on the bottles closed, and the machine was started. The volumes of gas, liberated, were read at 5 minute intervals, run off by a timer, and the shaking was continued until the curves became practically flat. From an examination of the curves obtained from normal bloods (Fig. 2) it is apparent that final readings taken at the end of a 10 minute period do not give results that can be satisfactorily compared. This 10 minute period has been used by a number of investigators in the estimation of catalase. These curves also show that there is quite a marked variation between different normal bloods. In these experiments potassium cyanide was injected subcutaneously in such amounts as to produce death. The blood samples were removed from the femoral artery by means of a cannula. A small quantity of powdered potassium oxalate was used to prevent clotting. The starred curve for Dog 4 in Fig. 3 was obtained from blood mixed with potassium cyanide in vitro in the same proportion as was injected. From an examina- tion of the curves from our eight experiments (Figs. 1, 3, 4) it becomes apparent that there is a diminution in the catalase content of the blood in only one case. In these figures the curves marked N were obtained from normal blood and those marked C from blood after injection of potassium cyanide. This is true even when the blood was taken during coma, and in two cases where the blood was tested after death. From the experimental results we conclude that lethal doses of potassium cyanide injected subcutaneously have practically no effect on the catalase content of the blood; secondly, that if the generally accepted theory of Geppert as to the mode of action of 2 Welker, W. H., J. Lab. and Clin. Med., 1921, vii, in press. 448 KCN on Catalase Content of Blood potassium cyanide is correct, there can be no connection between oxidase and catalase. 90 80 T0 S10) am Cc. of oxygen liberated Normal) bloods {5 20 30 Time in minutes rq; 2; Cc. of oxygen liberated W. H. Welker and J. L. Bollman 449 90 Brae ose 80 [ en : A ee 60 a z ——} ues N [ 7] 20 | 7 = = Dog ff A NO oe 10 yj 10 15 20 Zo 30 Time in minutes Fia. 3. 450 KCN on Catalase Content of Blood Protocols. Dog 1.—Two samples of normal blood were taken at an interval of 15 minutes. 3 mg. per kilo of 5 per cent KCN solution injected at 11.50 a.m. Second injection of same dose at 12.35 p.m. Blood sample taken at 12.40 p.m. 6 mg. per kilo of 5 per cent KCN solution injected at 12.55 p.m, 1 ea — ee Pelee 4@) 10) 3 © 50 © 2 « 40] © Od a ~% 30 ae O 5 20 % 10 6 10 1 20 20 30 Time in minutes Ere Blood sample taken at 1.05 p.m. Blood sample taken at 1.55 p.m. The animal was in convulsions at this point. Death occurred at 2.00 p.m. Another sample of blood was taken at this time. Dog 2.—Blood taken for normal sample. 8 mg. per kilo of 5 per cent KCN solution injected at 1.20p.m. Injection repeated at 1.30 p.m. Death occurred at 1.45 p.m. Blood removed from the heart at 1.48 p.m. W. H. Welker and J. L. Bollman 451 Dog 3.—Blood taken for normal sample. 10 mg. per kilo of 5 per cent KCN solution injected at 12.35 p.m. Death occurred at 1.00 p.m. Sample of blood removed from the heart within 30 seconds after the occurrence of death. Dog 4.—Blood taken for normal sample. 8 mg. per kilo of 5 per cent KCN solution injected at 2.15 p.m. Injection repeated at 2.30 p.m. Blood sample taken at 2.40 p.m. Death occurred at 2.50 p.m. Dog 5.—Blood taken for normal sample. 10 mg. per kilo of 5 per cent KCN solution injected at 11.40 a.m. Convulsions at 11.43 a.m. Blood taken at 12 noon. Death occurred at 12.20 p.m. Dog 6.—Blood taken for normal sample. 8 mg. per kilo of 5 per cent KCN solution injected at 11.11 a.m. Convulsions and labored breathing at 11.25 a.m. Blood sample removed at 11.38 a.m. and 1.55 p.m. Death occurred at 2.20 p.m. Dog 7.—Blood taken for normal sample. 8 mg. per kilo of 5 per cent KCN solution injected at 10.15 a.m. Blood removed at 11.05 a.m. The excitement stage had reached its maximum at this point. Death occurred at 2.00 p.m. Dog 8.—Blood taken for normal sample. 10 mg. per kilo of 5 per cent KCN solution injected at 4.05 p.m. 6 mg. per kilo of 5 per cent KCN solution injected at 4.30 p.m. Marked convulsions at 4.37 p.m. Blood sample taken at the point of death at 4.45 p.m. . . Ate fark “oy fe mT is sila i. | Walgers Wal esp eee raat hee “7 LR . wie ogy PLELE vy omy. {hyp i") Bex), ule ys Away : % a iy raabpdie ri, hah Bie) OFA GRY Se ph ree we | #83 we) gS ee ir , Jil ae a Revolt es ge yh > nae ny Leas bee) Pe Se or ahe NED OR “m ? A “a x¢ . PLS iP ae ites, Se lies ; pat os, A are . , . 1 vi ‘aihebetet iis rt ‘ PT. tbe vid Wal = Ateg petit? 4k ie eile a ua ul VAC ; AK parse: sae : » Ag med ~ tye eae as pny" ‘ ith Lal 00 ie y 7" 5 f é? p if CITRIC ACID CONTENT OF MILK AND MILK PRODUCTS. By G. C. SUPPLEE ann B. BELLIS. (From the Research Laboratory of The Dry Milk Company, New York.) (Received for publication, July 23, 1921.) Citric acid has been recognized as one of the normal constitu- ents of milk for many years, but there is still some disagreement as to the forms in which it exists in this product. Soldner (1) claims the presence of potassium, magnesium, and calcium citrates, whereas Van Slyke and Bosworth (2) state that only sodium and potassium salts of this acid are present. Regardless of the exact form in which this constituent is found in milk, quantitative determinations have shown that there is normally between 0.1 and 0.2 per cent citric acid combined in one form or another. Interest has been directed to the parallelism between the citric acid content of fruit juices and their antiscorbutic properties, and to the similar association of these factors in milk. If only natural products, unheated and without subjection to processing for preservation, were considered, the existence of a definite relationship between the two factors would be more acceptable. Reliable experimental evidence, however, shows that the anti- scorbutic properties of natural foods are destroyed by heat where- as the citric acid content of the same products is not quantitatively affected by the application of heat even in excess of that required for the destruction of the antiscorbutic property. Sommer and Hart (3) have shown that milk may be autoclaved at 15 pounds pressure for 1 hour without causing a diminution in its citric acid content. They have also shown that the citrates of milk are not rendered insoluble by autoclaving for 20 minutes at 15 pounds pressure. Since comparatively recent investigations by Hess (4, 5), Hart (6, 7), and Dutcher (8) and their coworkers have made available additional data on the antiscorbutic potency of milk as affected 453 454 Citric Acid Content of Milk by the feed of the lactating animal, and by what appears to be the method of processing in the preparation of various concentrated milk products, it has been considered desirable to record the variations in citric acid content of milk from individual cows while on a normal winter ration and when on a norma! summer or pasture ration; also to determine the citric acid content of some well known concentrated milk products. EXPERIMENTAL. Estimation of Citric Acid in. Milk. Due to the small amount of citrie acid present in milk and be- cause of the possible variations which it was desirable to detect, it has been deemed advisable to incorporate details of the well known methods used in this series of analyses. Determination of Citric Acid in Milk.—50 ee. of milk are treated with 10 ec. of dilute sulfuric acid (1:1) and thoroughly agitated. 2 ec. of 40 per cent potassium bromide solution and 20 ce. of a solution of phosphotungstie acid are then added. After a thorough mixing, the precipitate is separated by filtration. To the perfectly clear filtrate in an Erlenmeyer flask is added an excess of freshly prepared saturated bromine water (usually between 5 and 10 ce.). The mixture is then placed on the water bath at a temper- ature of from 48-50°C. for about 5 minutes. After removing from the bath, add rapidly from a burette 25 ec. of potassium permanganate solution (5 per cent) drop by drop with frequent interruptions, and with con- stant and vigorous shaking, avoiding a temperature during the oxidation exceeding 55°C. Set the flask aside until the hydrated peroxide of manga- nese begins to settle. The supernatant liquid should be dark brown showing an excess of permanganate. Add more permanganate if an excess is not indicated. When the precipitation assumes a yellow color and most of it is dissolved, add drop by drop a clear solution of ferrous sulfate until the hydrated peroxide of manganese and excess of bromine are removed. Allow the solution to cool, shaking occasionally. Allow the mixture to stand over night. Collect by means of gentle suction on a tared Gooch crucible pro- vided with a thin pad of asbestos previously dried over sulfuric in a vacuum desiccator; wash with water slightly acidified with sulfuric acid and finally wash twice with water. Dry the precipitate to constant weight over sul- furie acid in a vacuum desiccator protecting the precipitate from strong light. The weight of the precipitate multiplied by the factor 0.424 gives the equivalent weight of anhydrous citric acid in the sample. Determination of Citric Acid in Milk Powder.—Weigh 5 gm. of powder into a beaker and reconstitute with 45 ec. of warm water. Mix thoroughly and proceed as with liquid milk. G. C. Supplee and B. Bellis 455 Determination of Citric Acid in Sweetened Condensed Milk.—Weigh out 25 gm. of the sample and add 200 ce. of 95 per cent alcohol. Mix thoroughly and filter. To the filtrate add enough 0.25 n barium hydroxide to almost neutralize the solution and then 5 ce. of 50 per cent barium acetate in order to insure an excess of barium. Add about 150 ec. of 95 per cent alcohol and reflux until the precipitate settles readily after being shaken. Filter and thoroughly wash the precipitate in the flask and on the paper with 95 per cent alcohol. Transfer the precipitate from the filter to the flask with a jet of hot water. Boil until alcohol can no longer be detected by odor and add enough sulfuric acid (1:5) to precipitate all of the barium originally present and to allow 2 cc. in excess. Evaporate to a volume of 60 or 70 cc.; cool and add an excess of bromine water. Filter and add 10 ee. of potas- sium bromide, then place on the water bath at a temperature of 48-50° C. and proceed as with liquid milk. TABLE I. Percentage of Citric Acid Recovered from Milk Products. Liquid milk | Evaporated | Condensed Liquid milk. sand sugar. milk. milk. No. 1 |.No. 2 | No.1] No. 2 | No.1] No. 2| No.1] No. 2 Girigmmalen 2. o.)...- 2.2 «= (0.13210: 12910; 13110 13010-2202 0.204 0.096,0.090 After adding 0.02 per cent .. 0.1100. 104 os se OZ05" <¢ 0.179,0. 180.0. 182)0.179/0. 252 0.253. 0.1430.148 e By (OST eeey we 0.190/0.197 a 0.15“ “ ~ |0.279/0.27910. 27910. 275 | The relative accuracy of these methods is shown in Table I in which is given the results of duplicate determinations on liquid milk with and without sugar, on evaporated milk, and on sweetenea condensed milk; also duplicate results from each of these prod- ucts after known amounts of citric acid in the form of sodium citrate had been added. It will be noted that the maximum variation in duplicate results does not exceed 0.006 per cent; it is believed therefore, that any significant variations occurring in the products examined were easily detected by the methods used. Citric Acid in Milk as Affected by Feed. In view of the work reported by Hess, Unger, and Sup- plee (4) in which it was shown that the milk produced from a highly concentrated ration contained less citric acid than that produced during pasture feeding, it has seemed desirable to obtain further 456 Citric Acid Content of Milk data on the amount of this constituent found in the milk of the same herd while receiving a normal winter ration and again during pasture feeding. Accordingly, samples were analyzed late in February and again late in June; the results are shown in Table II. From the results in Table II it is evident that there is a wide variation shown in the milk from individual animals receiving the same feed as represented by the difference between 0.121 and 0.182 per cent(Herd I). While the evidence pointing toward a variation TABLE II. Citric Acid Content of Milk from Winter Ration and from: Pasture Feeding. Winter ration. ~ Summer pasture. Herd. | Cow ae = ee Feed. eee Feed. 2 per cent per ey 1 | 0.173) Hay, distiller’s grains, en- | 0.174, Fresh grass only. I 2 | 0.121) silage, corn stover, mo- | 0.114 “ “ “ 3 | 0.182) lasses. 0.156 MC ee II | 1 | 0.145) Hay straw, cottonseed, co* 5 cele meme 2 | 0.155} meal. 0.148 es ac 3 | 0.106 : 0.130. «“ “ “ III 1 | 0.139 Hay, oil meal, corn-meal, | 0.164 ‘“ “« « 2 0. 119 bran. 0. 138 “ (74 “ 3 0. 139 (0). 160 “ “ “ ——— in citric acid content as affected by the different feeds is not con- clusive, there is, nevertheless, a tendency toward a higher per- centage of this constituent in the winter milk of cows receiving ensilage and corn stover than in the milk of those herds receiving only hay as roughage. When summer and winter milks from each herd are compared there is a significant difference only in the case of Herd III in which the milk from pastured cows contains a uniformly higher citric acid content. The average citric acid content of the milk from all cows on a winter ration was 0.142 per cent and from all cows when on pasture was 0.148 per cent. G. C. Supplee and B. Bellis 457 Citrie Acid Content of Concentrated Milk Products. Since one of the purposes of this paper’is to furnish analytical data showing the citric acid content of concentrated milk products, it is desirable to briefly mention that in the manufacture of con- densed, evaporated, and desiccated milks heat is applied in amounts varying from 110-112°C. for a few seconds in the manu- facture of powdered milk by the Just process, to sterilization under steam pressure in the case of evaporated milk. Therefore, the results from the products analyzed will adequately cover the temperature range to which concentrated milk products are subjected during process of manufacture. TABLE III. Citric Acid Content of Condensed and Evaporated Milks. Citric acid Sample. Milk. oe naa milk basis. per cent per cent 1 Evaporated: ..;,«.% cccctaescth eee eek pen eceee 0.168 0.084 2 He neo Rcem aie Ata chal a NNO IRR cal dy tae 0.302 0.151 3 MPTP Rein dos 0 5 ee a eam, Aen 0.295 0.147 4 tN RES RCW x caste ota 2 1 0.211 0.105 5 SO | | GASPS ER ERR Pe cee te Aa 0.255 0.127 6 MEER SREPIAE eB i irreceserins 3 os ao SRO Aa Bote 0.203 0.101 Haalnoweetened Condensed). .-.o40s ene enemies dene 0.094 0.078 8 fe Siete: LAER ST a cat Eh hd CaP Mea ar 0.124 0.103 The citric acid content of six different brands of evaporated milk and two brands of sweetened condensed milk are shown in Table III; also included in this table is the citric acid content calculated to the original liquid milk basis assuming a concentra- tion ration of 2 to 1. The citric acid content of milk powder made by the spray process is shown in Table IV. The concentration ratio used for calculating to the liquid milk basis is 1 to 8.5 and 1 to 12 respectively for whole milk and skimmed milk. It has been possible to check up very closely on the citric acid content of powder made by the Just process by determining the citric acid and total solids before drying, for comparison with the dried product and with the reconstituted milk correctly diluted 458 Citrie Acid Content of Milk on the basis of the data obtained from the total solids determina- tions. The results of these determinations are shown in Table V. The results from the different concentrated and desiccated milks do not show any variation in citric acid content which could TABLE IV. Citric Acid Content of Milk Powder (Spray Process). 2 ea es acid 7 : ‘itric acid | caleulated Sample. Milk. in product.| to liquid milk basis. per cent per cent 1 Wholeimilk powdery: tes. eaeeeee oe 1-26 0.148 Zi ae of SO) i nee Ss x 5 aia ae ee eae 1.22 0.143 3 os oe aera SE CNIAT FEE init ARS oT EN 1.23 0.144 4 >| Skimmed :milkipowder.<--s-2e-- 22> eee ee 1.70 0.141 5 sf es simi Str eee RAEN hl Le eis 1.50 0.125 6 sf rf A ea ey ee CH RRS 1.45 0.121 TABLE V. Citric Acid Content of Milk Powder (Just Process). : heal P Citric acid Citric acid 2 ~ x = - Uk Cit . id in accu- Sample Milk. ‘before | in powder. | MCN eg Tying. powder. per cent per cent per cent If” |fiskabortea soli son adeaeuccaccccocase 0.139 LB ¥/ 0.135 2 oS Fae aed tera ae OEE 0.105 1205 0.104 Ss a Aen) rhsadteer Ce eee 0.165 1.90 0.166 4 | -Partiskimmedimuill Dai br (OFS ee 4 itl Gia if ¢ Retires hee Pe Re Ry iv ear Ta eT eAL bb Fire 4 272 “eo (rheG , vite oho es O20 sol) Ee: hae eee ee | . A Heselyus £0 DOLeiey Dae ie TT one 4 dite Ss | 0206; 0:07 0.14 | 0.02] 0.02 0.06 0.09 | 0.02] 0.00 0.03} 0.01 | 0.00 0.16 |} 0.04) 0.00 0.10 0.11 | 0:03) 0-01 0.19 0.18 | 0.04] 0.01 0.07 | 0.01 |} 0.00 0.18 |} 0.03 | 0.00 0.04 0.01 0.00 0.07 ORZI 02035 0201 0.08 |} 0.01} 0.00 0.08 OF10= 02027" OF 00 0.28 |* 0.03 | 0.01 0.06 | 0.03} 0.01 Blood after stand- ing on ice previous to addition of carbonate-oxalate. 0.08 * Results are given for 100 cc. of blood. { This blood stood at room temperature throughout. again after 30 minutes, and again after a further 30 minute interval. The results are given in Table IV. The figures reported in Table IV show that egg albumin does not decompose to yield ammonia under the conditions which ~~ 476 Ammonia Content of Blood we used for blood. These results lend some support to the view | that the quantities of ammonia found in blood are preformed, . and are not decomposition products formed during the analysis. This latter view remains, however, a possibility. ; a TABLE III. Ammonia in Whole Blood and Plasma: Effect of Standing. NH:-N in 100 ec. Blood. Plasma, Dog. Source of blood. SSS Ss Second | Third Second | Third aeration | aeration ree aeration | aeration First : . e : 30 min, | 30 min. ; 30 min. | 30 min. aeration.| “ after after |#eration.| “a tter after first. second. first. | second, mg mg mg mg. mg mg 1 Vena cava. 0.18 |} 0.05} 0.00} 0.2@ | 0:00} 0.00 2 Carotid. 0.11 0.03 | 0.01 0.13 |} 0.00} 0.00 3 Femoral artery. 0.07 |} 0.03} 0.00} 0.08; 0.01} 0.00 4 Mixed systemic. 0.12! 0.03; 0:01 | 0.10} 0.02) 0:00 Syl Carotid: 0.10} 0.02) 0.01/ 0.12] 0.00| 0.00 TABLE IV. Experiment 2. Ammonia in Egg Albumin Mixtures. NH:-N in 100 ce. No. Second | Third aera- Remarks. First aeration |tion 30 min. aeration. 30° min. after: after first. second. 1 0.05 0.00 26 cc. of fresh egg albumin diluted to 44 ec. 2 0.00 0.01 | Fresh egg albumin diluted four times. 3 0.07 0.00 0.00 as ef oa os to double volume. 4 0.03 0.00 0.00 es < a The next point studied was concerned with the question as to whether minute amounts of ammonia added to shed blood can be recovered quantitatively. This question is directly related to the broader question as to whether ammonia may be trans- ported in the blood in a complex combination from which the ammonia cannot be liberated by simple treatment with carbonate- T. P. Nash, Jr. and S. R. Benedict A477 oxalate mixture. While this latter question is obviously difficult to study directly, it seems probable that if the blood possesses the power of combining ammonia, then minute amounts of am- monia added to blood should not be completely recovered. In connection with any theory of a complex ammonia combina- tion in blood it should be borne in mind that any combination of ammonia from which the base could not be liberated by treat- ment with sodium carbonate would presumably defeat the object of ammonia formation in the organism, if we assume that the ammonia is produced for the purpose of acid neutralization. In testing the recovery of added ammonia we made use of ox blood. Simultaneous analyses were carried out with and with- out the addition of small amounts of ammonia nitrogen. The figures given in Table V show that ammonia added to blood can be completely recovered within the limits of accuracy of the method. The next experiment was planned to find out whether the blood can yield ammonia to neutralize added acid. Ammonia in such a combination would be readily available for the needs of the or- ganism. The following typical experiment shows that blood does not yield ammonia to neutralize added acid. Fresh ox blood gave on analysis 0.08 mg. of ammonia nitrogen per 100 ce. To 10 cc. of this blood was added 1 ee. of ammonia-free isotonic saline solution, and to a second 10 ee. portion of blood was added 1 cc. of a 10 per cent lactic acid solution. The two mixtures were then incubated in a water bath at 88-40°C. Atthe end of 30 minutes the blood-saline mixture gave 0.08 mg. of NH3-N per 100 ce., and the blood-acid mixture gave 0.09 mg. of NH3-N per 100 cc. 30 minutes later the values were 0.09 and 0.08, respectively. A study of the blood ammonia concentration in animals where the urinary ammonia output is higher than normal was made upon phlorhizinized dogs which were available from other experiments. These dogs had received daily injections of 1 gm. of phlorhizin in oil over long periods, and blood was taken for analysis when the animals were in the late stages of the poisoning. The results given in Table VI show that even where the ammonia content of the urine is markedly increased, there is no increase in the ammonia content of the blood. (Compare figures on normal dogs, Table II.) THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 A478 Ammonia Content of Blood TABLE V. Recovery of Ammonia Added to Blood. Results Given in Terms of NH3-N per 100 cc. of Blood. Differ- No. | Original.*| Added. Found. as Remarks. mg. mg. mg. mg. 1 0.08 0.15 0.23 0.00 | Added NH,OH. Aerated im- mediately. 1 0.075 | 0.075 | 0.18 0.03 ss G3 ee 1 OL075n Onto 0.23 0.005 | Added NH,Cl. Aerated im- mediately. 2 0.20 0.20 0.37 0.03 | Added NH,OH in isotonic saline solution. Aerated at once. 2 0.20 0.20 0.39 0.01 | Added NH,Cl in isotonic saline solution. Aerated at once. 2 0.20 0.20 0.33 0.07 | Added NH,OH in isotonic saline solution. Stood 1 hour at room temperature before aeration. 2 0.20 0.20 OF37 0.03 | Added NH,Cl in isotonic saline solution. Stood 1 hour at room temperature before aeration. 2 0.26 |, 0.20 0.37 0.09 | Added NH,OH in isotonie saline solution. Stood 2 hours at room temperature before aera- tion. 0.20 0.45 0.08 | Added NH,OH in isotonic saline solution. Stood 24 hours in ice box before aeration. 3 0.20 0.20 0.38 0.02 | Added NH,OH in isotonic saline solution. Aerated at once. os 0220 0.20 0.42 0.02 | Added NH,OH in isotonic saline solution. Stood 1 hour at room temperature before aeration. 4 0.36 0.25 0.66 0.05 | Added NH,OH. Stood 30 min- utes at room temperature be- fore aeration. 4 0.36 1.00 1.41 0.05 | Added NH;Cl. Stood 30 min- utes at room temperature be- fore aeration. 5 0.09 | 0.094; 0.20 0.016 | Added NH,-lactate in isotonic saline solution. Aerated at once. 5 0.09 | 0.188 | 0.26 | 0.018 “3 “ ee bo = 0 ot) T. P. Nash, Jr. and 8. R. Benedict 479 TABLE V--Continued. ioe aan Remarks. No. | Original.*} Added. | Found. mg. mg. mg. mg. 5 0.10 0.094 | 0.22 0.026 | Added NHgz-lactate in isotonic saline solution. Stood 30 min- utes at 38-40° C. before aera- tion. 5 0.11 0.094 | 0.22 | 0.016 | Added NHz-lactate in isotonic saline solution. Stood 1 hour at 38-40° C. before aeration. * The values in this column do not represent the average preformed am- monia nitrogen in fresh ox blood. The blood used was brought to the lab- oratory from a slaughter house, and the higher values found undoubtedly are to be attributed to bacterial action or to the presence of foreign ma- terial. We have found as the average value for 15 slaughter house bloods, when the analyses were made as soon as received, 0.13 mg. of ammonia nitrogen per 100 cc., but the conditions of handling and drawing this blood were not rigidly controlled. TABLE VI. Experiment 6. Ammonia in the Blood of Phlorhizinized Dogs. NH3-N NH3-N F Dog. Source of Blood. in 100 iy of ca erate aS te blood. 24 hour urine. mg. per cent vol. per cent 6 Femoral artery. 0.075 11.0 9 Carotid. 0.07 6.3 38.6 23 Femoral artery. 0.07 6.5 47.6 26 Jugular. 0.09 4.0 38.1 27 i 0.05 4.6 54.1 \ It seemed desirable next to test the question as to whether there is accumulation of ammonia in the blood following double nephrec- tomy, or after ligation of both ureters. Similar experiments have been reported for dogs by Winterberg (41) and for goats by Henri- ques and Christiansen (31). These investigators failed to find increased ammonia in the blood under the conditions cited. Since with the kidneys extirpated (or after ligation of both ureters) acid formation must be going forward, while ammonia cannot leave the organism, we deemed this question of sufficient import- 480 Ammonia Content of Blood ance to warrant full repetition of the experiments on dogs. Our experiments in this connection were carried out as follows. The dogs of this series were operated under ether anesthesia. Through a midline incision in the abdominal wall the renal vessels were ligated and the kidneys removed; in other cases only the ureters were tied off. We observed that those dogs, in which only the ureters were ligated and the kidneys left, survived the operation somewhat longer than those dogs whose kidneys were extirpated. No convulsions or other typical ‘‘uremic’’ symptoms were observed in the operated animals other than slight tremors in the extremities, rapid and shallow breathing, and rapid and labored heart action. Blood was taken for analysis in most animals when it appeared that the animal was near death. In the oxalated blood the corpuscles settled with extraordinary rapidity, leaving a plasma of light yellow color. An inspection of the results given in Table VII shows (in agreement with the previous investigators above cited) that in spite of total absence of kidney function in dogs there is no ac- cumulation of ammonia in the blood. In fact some of these experimental animals gave us the lowest figures for ammonia in the blood which we have found. This failure to find any accumu- lation of ammonia in the blood after extirpation of the kidneys is, we believe, a unique finding for this substance as compared with any other constituent of both blood and urine, and we believe that this finding necessitates the conclusion drawn later in this paper concerning the origin of ammonia in the organism. In view of the facts shown above, especially the findings that there is no accumulation of ammonia in the blood in phlorhizinized dogs, or in dogs without functioning kidneys, we were led to the conclusion that the kidneys themselves must produce the urinary ammonia. That the kidneys may perform an active synthetic function is not a new idea (Bunge and Schmiedeberg, 43), but so far as we know the production of ammonia has not hitherto been ascribed to the kidney. It seemed probable that if ammonia production takes place in the kidney, this organ would not excrete every trace of the ammonia formed, and we might then expect to find the blood of § In this we do not agree with Jackson (42). T. P. Nash, Jr. and 8. R. Benedict 481 the renal vein richer in ammonia than the systemic blood. We have therefore carried out experiments in which we compared the ammonia content of the carotid blood with that of the renal vein. In most cases we also included determinations of the am- monia content of blood from the vena cava taken posterior to where the renal veins enter this vessel. In collecting the several bloods for analysis in this experiment we have proceeded ‘as follows: The animal was anesthetized with ether, and a cannula placed in the carotid artery. In some cases the arterial blood was drawn first, in other cases last; we have not TABLE VII. Experiment 7. The Blood Ammonia in Nephrectomized Dogs, and in Dogs with Ligated Ureters. Time | Per 100 ce. of Dog. Source of blood. a Bicod: 6or" Remarks. tion. (NHeN|N-P-N| hrs mg, my: ais nt 7 | Femoral artery. 44 | 0.06} 200 Kidneys extirpated. 8 ae cs 46 | 0.08 | 182 Ureters tied. 8 cd ie (ies ee) 197 se se 14 < a 42 | 0.03 | 172 | 38.2 | Kidneys extirpated. 18 | Carotid. 47 | 0.03 | 162 | 57.1 | Ureters tied. Autop- sy disclosed severe hemorrhage due to bursting of capsule of one kidney. 21 ss Aa Oke, 186 | 36.2 | Ureters tied. found that the order of taking the blood is material. The abdo- men was opened by a midline incision, and the vena cava and the renal vein on one side exposed. Blood was then taken from the renal vein (we found the most convenient method of taking this blood was by the use of a curved needle attached by a piece of rubber tubing to a 25 ce. pipette containing potassium oxalate, the blood being drawn into the pipette by carefully regulated suc- tion). The point of the needle was introduced into the vein toward the kidney. The renal vein was tied off (to prevent hemorrhage), and a ligature placed around the vena cava just behind the renal veins. Blood was now quickly drawn from the vena cava, caudad 482 Ammonia Content of Blood to the ligature, using a similar technique to that described for the renal vein. ; It will be noted from the experiments reported in Table VIII that the blood of the renal vein is invariably much higher in ammonia content than the systemic arterial or venous blood. TABLE VIII. Experiment 8. Comparison of Ammonia Content of Systemic and Renal Venous Blood. j NHs-N in 100 ce. of blood. Subject. Sex. rr ————SSS Remarks. Pag Md Renal vein. mg. mg. mg. Cat 11] Male. 0.08 0.20 Ether anesthesia. «2! Female. | 0.08 0.26 Chloretone anesthesia. Cat pregnant. « ~~ 3.| Male. Omi2 a0 a2 O22 Ether anesthesia. «< 4} Female. | 0.11 | 0.10 0.27 ye - Cat preg- nant. “65 | Male. 0.12 | 0.12 0.18 Ether anesthesia. Average..........| 0.102) 0.113} 0.226 Dog 10| Female. | 0.08 0.22 (L)| Ether anesthesia. 0.18 (R) “ 11] Male. 0.06 | 0.03 | 0.09 oy ‘f ee [2 Sf 0.075] 0.16 | 0.12 ee <6 aig} =< O05 OO 74) OOK GR) 0.18 (L) “ “ 0S ‘ 0.07 | 0.08 0.21 sé % « 24! Female. | 0.14 0.25 id ee Dog in last stages of phlorhizin poisoning. “ 95 cc 0.13 0.18 “ce “ “ Cy - 0.05* 10:44 iG ae % Average..........| 0:088} 0.085] 0.176 * Blood of jugular vein. The blood from the renal vein averages twice as much ammonia as does the blood from other sources. These differences are so marked as to admit of only one inter- pretation; viz., that the kidney, instead of excreting ammonia from the blood, forms the ammonia which it excretes, while at T. P. Nash, Jr. and 8. R. Benedict 483 the same time it contributes a small amount of ammonia to the blood. No theory of concentration (by abstracting water) can possibly explain the differences in ammonia content which we have found, since to explain the ammonia increase in the renal vein on such a basis we should have to assume a 50 per cent concentration of the blood in a single passage through the kidney. As a corollary of the view that the kidney is the center of ammonia production in the body we should expect that acid or alkali introduction into the organism should have no detectable effect on the ammonia content of the systemic blood, while the ammonia content of the blood of the renal vein might be expected to show a slight increase in ammonia after acid introduction and a slight decrease after alkali ingestion. These results would be expected because as the kidney makes more or less ammonia we might expect some corresponding change in the ammonia escaping into the renal blood. There is noreason to believe~that the slight changes which might be produced in the ammonia con- centration of the blood of the renal vein should be reflected in the blood of the general circulation, for we must assume that the am— monia of the systemic blood represents an equilibrium state between the ammonia which comes into the circulation by way of the renal veins (and possibly traces of ammonia from the intestinal _ circulation which pass the liver) and the transformation of this ammonia into urea. If ammonia were formed in the organism in appreciable amounts elsewhere than in the kidney, we should expect injection of acid into the circulation to be followed by a definite increase in the ammonia of the general systemic blood. The opposite change might be expected as a result of alkali treatment. The experiments conducted in this connection were as follows. Effect of Acid Injection. The procedure was as follows: The animal was etherized and cannule were placed in the carotid artery and jugular vein. A sample of carotid blood was taken and the ammonia determined. 1.0 n hydrochloric acid was then run from a burette into the jugular vein at the rate of about 1 ec. per minute, until the animal showed severe symptoms of dyspnea. As rapidly as possible then blood was again taken from the carotid artery, and from 484. Ammonia Content of Blood the renal vein and vena cava. The ammonia in each of these bloods was determined, and in the carotid blood, the plasma CO,-combining power was also determined. Effect of Alkali Injection. In one series of dogs we attempted to eliminate ammonia from the urine by means of sodium bicarbonate in olive oil injected subcutaneously. The urinary ammonia was determined in daily catheter specimens, and while it did not completely disappear it fell on several days to very low values. The ammonia and CO,- combining power of the jugular blood was followed during the course of the injections, the specimens of blood being taken through a needle inserted through the skin into the vein. TABLE IX. Experiment 9. Effect of Acid Injection on Blood Ammonia. NH:-N in 100 ce. of blood. Before. After. Dog. Remarks. 4 Carotid Carotid. | Carotid. ene Hepa plasma 16 0.16 0.075 | 0.06 0.28 13.4 | 30 ce. of 1.0 n HCl injected. 17 0.075 0.085 0.10 O25 1855, | 45ce or 10 Nee injected. In one dog, blood taken from the jugular vein gave 0.07 mg. of ammonia nitrogen per 100 cc. The dog was then etherized and 2 per cent NaHCO; solution run into the vein through a cannula. After 2 hours, when 196 ec. of the bicarbonate solution had. been injected, the urine (by catheter) was ammonia-free. At this time, femoral arterial blood gave 0.07 mg., and renal venous blood 0.18 mg. of ammonia nitrogen per 100 cc. From Tables IX and X it is plain that acid or alkali injection have no influence on the ammonia content of the systemic blood. There is some evidence of an effect on the ammonia content of the blood of the renal vein, but further experiments would be required to warrant any definite conclusion ‘here. i. PB. Nash, Jr-and 3S: RK: Benedict 485 TABLE X. Experiment 9. Effect of Alkali Injection on Blood Ammonia. NaHCO; in Olive Oil. Urine. : Blood. Sean le a ee Date. ; NE in 100 ce. in 100 ce. azn neuen NH3-N of renal | of vena to litmus. LON NH3-N venous cava ~*"* 1 in 100 ce. | blood. *| blood. Dog 19. per cent |vol. percent mg. mg. mg. gm. May 5 Basie. 0.038 | 47.7 0.10 2 oot 1:6 < 0.010 60.0 0.05 3 eer sf 0.115 O2098 | nOLOd 2 SS ct 0.030 a3 0.06 0.13 0.08 Dog 22 May 20 Acid. 0.152 Dy, 23 Basic. 0.068 66.8 0.07 2.5 eno. Acid. 0.050 FD Basic. 0.040 2509 SSG “ 0.036 4 SPP f 0.004 + ce 28 3 0.014 80.2 0.07 0.16 0.06 * CO.-combining power of carotid blood plasma, under ether anesthesia, was 39.3 volumes per cent. DISCUSSION. If we accept the view that ammonia production takes place in the kidney as part of its excretory function, it would seem that certain facts in regard to acidosis may be more readily understood. Under the commonly accepted view that neutralization of acids - by ammonia is a function of the organism in general, or of the liver, it would seem very difficult to understand such acidosis (depletion of the alkali reserve) as frequently occurs in nephritis, where there is no marked increase in acid production. Even the increase in acid phosphate in the blood reported in some of these cases affords no explanation of depletion of the alkali reserve, since ammonia should be available for neutralization of any circu- lating acid. 486 Ammonia Content of Blood If, however, we look upon the kidney as the seat of ammonia production, depletion of the alkali reserve becomes readily under- standable under certain definite conditions. If ammonia is not available within the organism the acids must be transported wholly in combination with the fixed bases, or with protein. A depletion of the alkali reserve of the blood could therefore arise under any one of three definite conditions. 1. Introduction of acid radicles into the blood stream more rapidly than the normal kidney can eliminate them, or can make ammonia to combine with them while eliminating them. 2. If the kidney becomes defective in its power to eliminate acid radicles, and thus to maintain them at a minimal level in the blood, a depletion of the alkali reserve would result, since the acid radicles would remain in the circulation in abnormal amounts, and would have to be neutralized by the fixed bases or protein. This condition might well result with a kidney still normal in its power of ammonia production. Such ammonia is available for the needs of the organism only as acid radicles are excreted. 3. A depletion of the alkali reserve of the blood would result should the kidney become defective in its power of ammonia formation. Even should such a kidney remain normal in its power of excreting acid radicles, the organism would lose base excessively during the excretion of the acid. It would appear that the first of these three forms of acidosis occurs, if at all, in diabetes. Very probably either or both of the two latter forms occur in nephritis. It seems from our results that acidosis in the sense of depletion of the alkali reserve is primarily a kidney disease. Although Wakeman and Dakin (44) came to the conclusion that the formation of urea in the animal body is an irreversible process, we believe that urea is the most probable precursor of ammonia in the kidney. It has been frequently demonstrated that the urinary ammonia is increased at the expense of urea, and unless we assume a conversion of urea into ammonia by the kidney we should have to assume the transportation in the blood of some intermediate product between urea and ammonia,.or of some ‘‘complex an- monia compound.” Our work has rendered either of these views very improbable. It is, of course, also possible that the kidney is active in deamination of amino-acids, and that excreted ammonia is supplied from this source. Ye —_- De EP. Nash; rand S. ik: Benedict 487 BIBLIOGRAPHY. . Folin, O., and Denis, W., J. Biol. Chem., 1912, xi, 161, 527. . MacCallum, W. G., and Voegtlin, C., J. Exp. Med., 1909, xi, 143. . Myers, V. C., Practical chemical analysis of blood, New York, 1921, 20. . Nencki, M., and Zaleski, J., Arch. exp. Path. u. Pharmacol., 1895, XXXVI, 385. . Boussingault, quoted from Folin, O., Z. physiol. Chem., 1902-03, xxxvii, 161. . Wurster, C., Centralbl. Physiol., 1887, i, 485. . Minkowski, O., Arch. exp. Path. u. Pharmacol., 1886, xxi, 41. . von Schréder, W., Arch. exp. Path. u. Pharmacol., 1881-82, xv, 364; 1885, xix, 373. . Nencki, M., Pawlow, J. P., and Zaleski, J., Arch. exp. Path. u. Pharm- acol., 1895-6, xxxvli, 26. . Nencki, M., and Pawlow, J. P., Arch. exp. Path. u. Pharmacol., 1896- 7, XXXvill, 215. . Salaskin, S., Z. physiol. Chem., 1898, xxv, 449. . Salaskin, S., and Zaleski, J., Z. physiol. Chem., 1900, xxix, 517. . Biedl, A., and Winterberg, H., Arch. ges. Physiol., 1901-02, lxxxviii, 140. . Nencki, M., and Zaleski, J., Z. physiol. Chem., 1901, xxxiii, 193. . Horodyfiski, W., Salaskin, S., and Zaleski, J., Z. physiol. Chem., 1902, xxxv, 246. . Folin, O., Z. physiol. Chem., 1902-03, xxxvii, 161. . Beceari, L., Biochem. Centralbl., 1906-07, v, 460. . Voegtlin, C., and King, I., J. Biol. Chem., 1909, vi, p. xxviii. . Wolf, C. G. L., and Marriott, W. McK., Biochem. Z., 1910, xxvi, 165. . Carlson, A. J., and Jacobson, C., Am. J. Physiol., 1909-10, xxv, 403. . Greenwald, I., Am. J. Physiol., 1911, xxviii, 103. . Carlson, A. J., and Jacobson, C., Am. J. Physiol., 1911, xxviii, 133. . Hopkins, R., and Denis, W., J. Biol. Chem., 1911-12, x, 407. . Medwedew, A., Z. physiol. Chem., 1911, Ixxii, 410. . Matthews, S. A., and Miller, E. M., J. Biol. Chem., 1913, xv, 87. . Denis, W., J. Biol. Chem., 1913-14, xvi, 389. . Jacobson, C., J. Biol. Chem., 1914, xviii, 133. Pehohde: AL, Ji, Brole Chemsa | Olonexxreo25: . Gettler, A: O., and Baker, W., J. Biol. Chem., 1916, xxv, 211. . Bang, I., Biochem. Z., 1916, Ixxii, 144. . Henriques, V., and Christiansen, E., Biochem. Z., 1917, Ixxviii, 165; Ixxx, 297. . Gad-Andersen, K. L., J. Biol. Chem., 1919, xxxix, 267. . Morgulis, S., and Jahr, H. M., J. Biol. Chem., 1919, xxxviii, 435. . Myers, R. G., J. Biol. Chem., 1920, xli, 119, 137. . Barnett, G. D., J. Biol. Chem., 1917, xxix, 459. . Barnett, G. D., and Addis, T., J. Biol. Chem., 1917, xxx, 41. 488 Ammonia Content of Blood . Burton-Opitz, R., and Edwards, D. J., Am. J. Physiol., 1917, xliii, 408. . Salkowski, Centralbl. med. Wissensch., 1880, xviii, 689. : Folin, O., J. Biol. Chem., 1919, xxxix, 259. . Bock, J. C., and Benedict, S. R., J. Biol. Chem., 1915, xx, 47. . Winterberg, H., Z. klin. Med., 1898, xxxv, 389. . Jackson, H. C., J. Biol. Chem., 1911, ix, p. xxvii. . Bunge, G., and Schmiedeberg, O., Arch. exp. Path. u. Pharmacol., 1876-7, vi, 233. tng . Wakeman, A. J., and Dakin, H. D., J. Biol. Chem., 1911, ix, 327. THE MECHANISM OF REDUCTION OF NITRATES AND NITRITES IN PROCESSES OF ASSIMILATION. By OSKAR BAUDISCH. (From the Department of Chemistry, Yale University, New Haven.) (Received for publication, July 30, 1921.) The mechanism of reduction of nitrates of the alkali metals to ammonia and the formation of amino nitrogen in biochemical syntheses from inorganic nitrogen compounds have not been explained satisfactorily. Some investigators explain the trans- formation by assuming a direct reduction of the nitrates to am- monia by action of nascent hydrogen, while others assume an intermediate reduction of the nitrate to nitrite, from which, as the reduction .proceeds, ammonia is produced. The exact chemical procedure by which bacteria or molds are able to produce ammonia or nitrites from alkali nitrates also has not been satisfactorily explained. Schimper,! the botanist, has been able to demonstrate experi- mentally that the reduction of nitrates in green leaves is connected intimately in some manner, not only with the influence of light, but also with the action of iron compounds in the leaves. This observation was so interesting to the writer that it led him to an investigation of- the question whether iron actually takes part in the reduction of nitrates by means of bacteria. This work has proved very productive and, as a matter of fact, experiments have shown conclusively that the cholera bacillus, which possesses extraordinary reducing power for nitrates, has the ability to accumulate iron? in its organism, and its reducing power may possibly be a function, not only of its ability to absorb oxygen by respiration, but also of its iron content. The data revealed through this biological research, which was interrupted by the War and political disturbances in Europe, 1 Schimper,A. F. W., Bot. Z., 1890, xlvi, 73. 2 Unpublished data. 489 490 Nitrates and Nitrites in Assimilation warranted a purely chemical investigation of the reduction of inorganic nitrates by means of iron salts. The opportunity to continue this work has now been offered to me, and, as a result, the study of this interesting problem has been taken up.* An interesting paper by Menaul which recently appeared in this Journal,‘ brings up for discussion a very important biochemical change. This investigator describes the action of formaldehyde on saltpeter in aqueous solution when exposed to sunlight, and the observation is made by him that in such solutions small quantities of hydrocyanic acid can be detected easily. This quite remarkable action of sunlight on nitrates was observed by the writer several years ago® and this preliminary paper is now contributed to bring the results of this work before the American reader, and at the same time to present a summary of the principal results of his earlier investigations on the photochemical reduction of nitrates and the reduction of nitrates and nitrites with iron salts, which have appeared in various scientific publications during the last 10 years.6 This summary will be presented in three parts as follows: (1) Reduction of nitrates of alkali metals by means of light and also iron salts; (2) Reduction of nitrites of alkalh metals by means of light and also iron salts; and (3) Synthesis of organic compounds containing nitrogen from inorganic compounds of nitrogen. The Reduction of Nitrates. In the course of researches on the photochemical decomposi- tion and synthesis of nitrates and nitrites, the observation was made, for the first time, that one oxygen atom in nitrates must be bound in the molecule in a manner quite different from that of the oxygen atoms in nitrites. The writer has used the specific term ‘‘nitrate oxygen atom’? to designate that oxygen atom which Through cooperation with Prof. Treat B. Johnson, who has supplied rare research material, it has been possible to extend the field of investi- gation into the pyrimidine and purine series. The results of these re- searches, which are of immediate biochemical interest, will be published at a later date. 4Menaul, P., J. Biol. Chem., 1921, xlvi, 297. 5 Baudisch, O., Ber. chem. Ges., 1916, xlix, 1151. 6 Baudisch, O., Ber chem. Ges., 1911, xliv, 1009. 7 Baudisch, O., Ber. chem. Ges., 1912, xlv, 2879; 1916, xlix, 1176. O. Baudisch 491 is easily split off from such salts either under the influence of light or of metallic iron. That we are dealing here with such a labile oxygen linking in nitrates is demonstrated to us by nature, in that the various nitrifying and denitrifying bacteria have the power to differentiate between nitrate and nitrite oxygen. The question naturally arises, under what influence or by means of what power an atom of oxygen can be split out of potassium nitrate, for ex- ample, with the formation of a nitrite. This problem was first attacked from a purely chemical standpoint, but it is now proposed to continue the study also from a biological point of view. In explanation of the photochemical reduction of nitrates in aqueous solution, we may assume, according to Werner’s theory® of reaction, an activation of the residual valence of an oxygen atom of the nitrate and of the oxygen atom of the water, resulting in the attraction of molecules of water into the inner sphere of the nitrate molecule. There follows a dissociation of the nitrate molecule with the formation of oxygen as is expressed by the following equation: SL ] K; N~..0--- | ——— K| N~~0.---.0H, | > KNO. +0 + H.0. | | Porno | | | O ro In other words, this reduction process takes place under the influ- ence of light even in an atmosphere of oxygen, and stops at the nitrite stage. The reverse process can also take place and nitrite is readily formed from ammonia by photochemical oxidation without, however, any production of nitrate. It is apparent from these facts that light can readily split off an atom of oxygen from nitrates of the alkali metals, without the presence or influence of either nascent or molecular hydrogen. This dissociation of nitrate into oxygen and nitrite can also be brought about by means of metallic iron as well as under the influence of the energy of light. If a neutral oxygen-free solution of potassium nitrate be shaken in a vacuum with active iron pre- pared by reduction with hydrogen, the supernatant liquor obtained 8 Werner, A., Neuere Auschauungen auf dem Gebiete der anorganische Chemie, Brunswick, 4th edition, 1920. 492 Nitrates and Nitrites in Assimilation after the iron powder has been allowed to settle will give every reaction applicable for the detection of nitrous acid.® In other words, metallic iron will easily reduce potassium nitrate to potas- sium nitrite in the cold in the absence of every trace of oxygen, and under conditions such that neither the action on the iron by water nor the effect of nascent hydrogen can possibly come into consideration. These results lead to the assumption, therefore, that iron readily splits off an oxygen atom of the nitrate after having first entered into a loose combination with it. This change may be expressed by the following equation: Regs POX KI N~n~A0-- | + Fe —> K] N ----0-----Fe | ——> KNO.+Fe0. z { From these examples it is seen that the photochemical reduction of nitrates to nitrites and their reduction by means of metallic iron are similar in nature, and in both cases there occurs either an activation or mobilization of the valence energy leading to the formation of an unstable addition product, which finally breaks down into the final products of reaction. Although it has been possible to show a relation between the photochemical reduction of nitrates to nitrites and the corresponding biological reduction in green leaves, it has not yet been possible to connect known chemical reduction processes with the biological reductions occurring naturally in bacteria or in molds. It formerly appeared scarcely possible to attach any biological importance to ferrous hydroxide in these reduction processes because, as was commonly believed, the reaction proceeded stoichiometrically and ferric hydroxide was formed at the expense of the oxygen split off from the nitrate. In biological processes, however, one cannot speak of stoichiometrical reactions in connec- tion with metals, because, as is well known, they are present only in traces. Their action must be explained, on the contrary, by a particular energy inherent in their molecules, and consequently the writer has been accustomed to consider the metals as function- ing in biological processes either as “mobilizers’” or “catalysts,” 9 Baudisch, O., Ber. chem. Ges., 1921, liv, 406. ‘Sian O. Baudisch 493 having the power to bring into play the latent energy of cer- tain organic molecules. i From the point of view of Werner’s coordination theory, it is possible to conceive of a relation between the mobilizing power of such a catalytic agent, and the peculiar power expressed by residual valency, which, as is well known, has the ability to draw new atoms, molecules, and radicals into the sphere of action of the internal nucleus of the metal, resulting in the most varied types of reactions. In fact, it is well known that extremely finely divided metals, such as platinum, palladium, or iron, possess these valence powers to a large degree, and their specific action has often been placed in parallel with purely biological processes. The reduction of nitrates with ferrous hydroxide assumed a new interest when it was discovered that this reagent alone does not split off nitrate oxygen as had been assumed, but reduces nitrates only under the influence of oxygen.!° As itis well known that white ferrous hydroxide is converted instantaneously into green ferrous hydroxide peroxide by the oxygen of the air, it is reasonable to say, therefore, that this polymolecular combina- tion or peroxide is the active reagent which brings about this transformation of nitrates into nitrites. Oz Fe (OH) 2 (OH2)s (Coordination formula for ferrous hydroxide peroxide.) The mechanism of this reduction has not yet been explained, but the attempt will be made here to show that the free energy of ferrous hydroxide is increased enormously by a loose combina- tion with an oxygen molecule, and that this increase in energy makes itself apparent both physically and chemically. The strik- ing effect on the color of white ferrous hydroxide, which is caused by the smallest trace of oxygen, shows that the oxygen enters into the inner sphere of the iron nucleus. Schafer! has proved spectroscopically that such extraordinary alterations in color, either in the visible or in the invisible part of the spectrum, can only take place simultaneously with changes in the inner sphere 1° Baudisch, O., Ber. chem. Ges., 1921, liv, 410. 11 Schafer, K., Z. anorg. Chem., 1918, lxxxvi, 221. 494 Nitrates and Nitrites in Assimilation of the molecule. It has, however, not yet been shown how many oxygen molecules are present in such a molecular combination. It may be possible that the observation of Meyer,” who dis- covered that strongly magnetic substances were rich in absorp- tion bands, whereas diamagnetic substances were poor in absorp- tion bands, has some connection with these facts. This coincides completely with the action of the above mentioned iron compound, because, while the white ferrous hydroxide has practically no mag- netic properties, the green to black ferrous hydroxide peroxide pos- sesses magnetic properties which are almost equal to that of me- tallic iron. According to Hilpert,” when a stream of air or oxy- gen is led through a precipitate of ferrous hydroxide, the magnetic properties of the precipitate increase rapidly. Quartaroli“ has also shown that oxidation with air converts the ordinary non-magnetic ferrous hydroxide into mixed ferro-ferri oxides, which possess a sus- ceptibility almost a hundred times greater than ferric salts. In fact the magnetic susceptibility of Fe;0, approaches that of the metal iron itself. It may be concluded, therefore, from our present knowledge, that there is a very close relationship between the phys- ical properties of metallic iron and those of ferrous hydroxide peroxide, and it will be of the greatest interest and importance to determine whether there is also any direct relationship between the peculiar chemical properties of this peroxide and those of finely divided metals. The simplest explanation, therefore, for the reduc- tion of nitrates is the assumption that freshly precipitated, colloi- dal ferrous hydroxide peroxide aets catalytically as a finely divided metal. It seems very probable that the peculiar properties resulting from the colloidal nature of ferrous hydroxide peroxide and the properties of the metal resulting from its position as the central atom of a complex system actually coalesce. Furthermore, these characteristic properties apply only to the peroxide and not to ferrous hydroxide, because the latter compound is not only unable to bring about a reduction of nitrates, but also will not react to form polynuclear compounds. Not until brought under the - influence of oxygen does the central iron nucleus of ferrous hy- 12 Meyer, S., Wied. Ann., 1899, Ixviii, 325. 13 Hilpert, S., Ber. chem. Ges., 1909, xlii, 2248. 44 Quartaroli, A., Chem. Zentr., 1917, i, 729. O. Baudisch 495 droxide or other ferrous salts acquire the property of attracting new molecules of ferrous hydroxide into its inner sphere. This coordination combination exists, according to Werner, not between iron and iron, but between the active hydroxyl oxygen atoms of the ferrous hydroxide, which are attracted to the central iron nucleus of the peroxide by its residual valency. It is also possible for the iron atoms of ferrous hydroxide to be held.in combination through the peroxide oxygen atoms, as has been demonstrated in the case of cobalt compounds by the classical researches of Werner. The mechanism of the autooxidation of ferrous hydroxide and the formation of strongly magnetic Fe,04.cH,O may be expressed by the following formulas: Oz [recor | (OH). + 0, —— Ec | (OH), + 2Fe (OH), (OH2)s Af) HO r( Yr) (OH)> HO” Bente The structure of this polynuclear combination may be expressed graphically as follows: i iid Or O OH Fe ; de oe = Fe,0, ++ cH.0 + O. HO ‘6 : On | | H H From these graphic representations it is seen that ferrous hydroxide is converted by the absorption of oxygen into a per- oxide of greater potential energy, whose iron nucleus, as experience has shown, possesses the property of intensifying the activity of and of entering into loose combination with the residual valence of oxygen atoms in other molecules of ferrous hydroxide present. 496 Nitrates and Nitrites in Assimilation With the coordination theory of Werner as a basis, it becomes ap- parent from the foregoing that ferrous hydroxide may be trans- formed by the absorption of oxygen into a complex salt whose iron nucleus, just as finely divided metals, may enter into a wide range of reactions. A nitrate oxygen atom may be split off as well by means of light as by means of metallic iron or by ferrous hydroxide peroxide. All three of these processes of reduction may be considered to depend upon the same principle; namely, the mobilization or activation of the energy in the residual valence of the reacting materials. Ferrous hydroxide peroxide reacts most probably in a very similar manner to finely divided iron or platinum. The Reduction of Nitrites. Aqueous solutions of potassium nitrite containing easily oxidi- zable substances, such as alcohols, aldehydes, sugars, starches, etc., suffer a comparatively rapid reduction and decomposition under the influence of diffused daylight, and the change may be expressed very simply as follows: KNO, = KNO + O. The presence of potassium nitrosyl in solution may be detected by means of its condensation reaction with aldehydes (Angeli’s aldehyde reaction).!° Hydroxamie acids are formed as products of reaction, and as is well known, these acids give characteristic complex salts with iron which are colored a deep reddish violet. This reduction of potassium nitrite to potassium nitrosyl can also be accomplished by means of complex iron salts. The smooth reduction of potassium nitrite via potassium nitrosyl to ammonia by means of glucose, in the presence of very small quantities of iron, possesses particular biological interest. The system, glucose + iron + alkali, which is a fundamentally new reducing combination, does not attack in the least the alkali salts of nitric acid.'® It is therefore possible to make a quantitative separation 15 Angeli, A., Samml. Chem. u. chem. Techn. Vortr., 1908, xiii. 16 A method, based on this observation, has been developed for the quantitative determination of nitrites and nitrates in the presence of other nitrogen compounds in soil extracts. Pfeiffer, T., and Simmerbacher, W., Land. Versuchsstat., 1916, xciii, 65; J. Soc. Chem. Ind., 1919, xxxviii, 507. O. Baudisch 497 between nitrate and nitrite by means of a grape sugar-iron-alkali solution. Grape sugar, which is absolutely free from iron, does not cause the slightest reduction, even on heating, of nitrites; and also chrysarobin (1,8-dioxy-3-methyl-anthranol), which occurs quite widely in the vegetable kingdom, has not the ability of reducing the salts of nitrous acid. On the other hand, the addition of traces of any iron salt to an alkaline solution of either of thesesubstances enables them to reduce immediately the nitrites by way of nitrosyl to free ammonia. The particular part that iron plays in these reactions remained a mystery for a long time, but seems now to have been explained quite satisfactorily. To explain this reduction process andits application for the quan- titative separation of nitrates and nitrites, we may assume that the unsaturated, trivalent nitrogen atom of the nitrite molecule enters, through its activated residual valency, into a loose com- bination with the central iron nucleus of whatever complex salt is present, and then dissociates through the intermediate forma- tion of nitrosyl into NO and K. The residual valency of the nitrate oxygen atom is not sufficiently active or powerful to dis- place the molecules or radicals already present in the inner sphere of the iron nucleus, and therefore these compounds are not at- tacked. That these reductions may all be considered as complex salt reactions, or in other words ‘‘nuclear exchange or displace- ment reactions’? may be shown by the following example: A solution of 1 gm. of K,Fe (CN)., 1 gm. of NaNQOs, and 5 gm. of sodium carbonate in 200 cc. of water, is distilled in a stream of oxygen. After interrupting the stream of oxygen, it is possible to detect nitrous acid in the distillate. The oxygen, under the influence of heat, has displaced the cyanogen group from the inner sphere, and in its place a molecule of nitrite has entered. The nitrite, however, decomposes and its scission product, NO, which at first takes its place in the inner sphere of the iron nucleus, is in turn displaced by the oxygen, and finally passes over into the distillate where it is easily detected as nitrous acid. The fact that this remarkable reaction may not only be in- fluenced by daylight, but in some cases will not take place except under the influence of daylight, is of particular chemical and biological interest. For example, if a freshly prepared solution of potassium ferrocyanide be treated with an aqueous alcohol 498 Nitrates and Nitrites in Assimilation solution of nitrosobenzene and placed under the influence of dif- fused daylight, the solution which in the beginning possesses a weak, greenish yellow color, changes in a few minutes to a deep reddish violet. ‘The mechanism of the reaction may be expressed as follows: (CN)s | Fecow | Ky, +t C.;H;NO —?; E C;-H;NO | K; + KCN. On treating the aqueous solution of this reddish violet compound with an excess of potassium nitrite and again placing the solution under the influence of diffused daylight the following decomposi- tions take place: KNO; ——_ > “KNO-- oO KNO -———> _ KeENO HO EEK OM (CN)s (CN)s Fe K; a KNO, 7 Fe K; . C,H;NO. C.sH;NO NO ' The deep violet color of the solution disappears very rapidly and the reddish yellow or potassium nitroprusside takes its place. By means of this reaction, the reduction of sodium nitrite by means of a complex iron salt and light is demonstrated. These processes of reduction of nitrites by way of nitrosyl to ammonia, may be drawn into intimate relation with biological reductions of nitrite, particularly as sugar, or its products of decomposition, and iron constantly accompany the nitrates in plants or in bacteria. Kostyschew and Tswekowa"™ state that the reduction of nitrate to nitrous acid takes place without the presence of any sugar, but that the further conversion of the nitrous acid, at leastin the case of Mucor racemosus, is accomplished only in the presence of sugar. It seems likely, from investiga- tions with cholera bacteria, that nitrates are reduced to nitrites by way of nitrosyl, because it was possible to detect the alkaline decomposition products of nitrosyl, for example, NO and NHs; (the latter as a reduction product of NO), in the volatile portions of alkaline cholera peptone cultures.'8 17 Kostyschew, S., and Tswekowa, E., Z. physiol. Chem., 1920, exv, 171. 18 Baudisch, O., Ber. chem. Ges., 1916, xlix, 1148. O. Baudisech 499 The assumption that nitrite is converted into nitrosyl finds a further support in the qualitative and quantitative composition of the gases which are produced during the photochemical reduc- tion of nitrites in the presence of formaldehyde and during the biological reduction. For example, those bacteria which have the property of decomposing nitrates, produce a fermentation gas which consists of about 65 to 72 per cent N2O.° In addition to the nitrous oxide, there is also always formed a little nitrogen, traces of NO, andalso of prussic acid. A formaldehyde-potassium nitrite solution produces a gas under the influence of diffused daylight, which contains 64 per cent N2O, as. well as very small quantities of NO and HCN. The latter two gases were detected qualitatively by sensitive reactions. Clawson and Young!® have detected prussic acid in cultures of Bacillus pyocyaneus and of other bacteria. To summarize, the photochemical reduction of the alkaline nitrites proceeds by way of the reactive intermediate product, potassium nitrosyl, which may be detected by means of aldehydes, as well in the case of the photochemical reduction of nitrites as in the case of a reduction with the system, grape sugar + iron + alkali. Certain complex iron salts possess the property of reducing nitrites, whereas under these conditions nitrates remain unchanged. The reduction of the alkali nitrites by means of complex iron salts depends most likely upon the residual valency of the central iron nucleus and in all these changes light as well as heat exercises a very fundamental influence. Synthesis of Organic Compounds Containing Nitrogen from Inorganic Compounds of Nitrogen. Nitrosyl, which is formed in the reduction of alkali nitrates, interacts readily with formaldehyde with formation of formhy- droxamic acid (Angeli’s aldehyde reaction). This reaction proceeds, as was shown by the writer and Coert,?° through the intermediate formation of nitroso methyl alcohol. Formhydrox- amic acid is then formed from this by molecular rearrangement, and may be detected easily by means of its characteristic iron and copper salts, which are highly colored. 19 Clawson, B. J., and Young, C. C., J. Biol. Chem., 1913, xv, 419. 20 Baudisch, O., and Coert, J. H., Ber. chem. Ges., 1912, xlv, 1775. 500 Nitrates and Nitrites in Assimilation H NO OH hee Le H:€HO+HNO ——> CG —> HC ites \ H OH NOH Formhydroxamie acid, under the influence of light, loses an atom of oxygen and is converted into formaldoxime, which is also characterized by its great reactivity. The stable form of this compound is altered under the influence of light and also alkali, and is transformed into an extremely labile modification. This transformation may be expressed as follows :*! H H On al ate See OH + H.0 Hat steal | H H H = Qe a acer. C=NOH+KOH |. mel} lie H lig H As is well known, formaldoxime exhibits a strong tendency to polymerize with the formation of three-carbon chain compounds. Its labile form is also capable of reacting further with aldehydes with the formation of three-carbon compounds, and furthermore under the influence of light combines with formaldehyde to form cyclic combinations containing both nitrogen and carbon. Under the influence of light formaldoxime undergoes, in part, a Beck- mann rearrangement with formation of formamide, and, in part, a complete dissociation into prussic acid, water, and ammonia. These changes are expressed below: H H ~ H.C=NOH —— |!CH,=N OH — C=NH+H,0 H OH H- CONE a 7 ; “a ~ HCN + H.0 *t Baudisch, O., Ber. chem. Ges., 1916, xlix, 1159. O. Baudisch 501 The small quantities of prussic acid that are always found accompanying the treatment of formaldehyde in nitrate solutions with light, or by the reduction of nitrates with bacteria, may have been formed in accordance with the above reaction from the aldoximes. The photochemical formation of nitroso methyl aleohol (or hydroxamie acid) from formaldehyde, methyl alco- hol, and nitrosyl may be the chemical counterpart of a possible biochemical formation of carbon-nitrogen-containing organic substances from inorganic nitrogen. The next step to amino nitrogen is simpler and may either take place by reduction or, as in the case of aldoxime, by simple rearrangement. It seems probable that the proof of the biological importance of nitroso methyl alcohol or formhydroxamic acid is found in its marked reactivity and in the pronounced tendency which it has to rear- rangement, to polymerization, and to the formation of complex salts, particularly of iron. It seems extremely possible to intro- duce amino nitrogen into the higher alcohols, sugars, starches, etc., by means of nitrosyl which is formed photochemically from the nitrites and which is capable of entering into such widely different reactions. KNO, + H.O + CH;O0H SSSR Fe(OH), and air x N reduction oxidation KNO. HCHO. x BS Vis \ 4 synthesis and H.C = NOH Recent work’? has revealed the fact that light may be replaced in certain cases by means of ferrous hydroxide and oxygen. Fer- 502 Nitrates and Nitrites in Assimilation rous hydroxide peroxide oxidizes alcohols to aldehydes simul- taneously with the reduction of nitrites to nitrosyl, and therefore synthesis of formhydroxamic acid or formaldoxime takes place. In fact, in these solutions of ferrous sulfate, containing bicarbon- ate and nitrite, three dissimilar reactions proceed simultaneously— oxidation, reduction, and synthesis. To summarize, nitrosyl is formed from alkali nitrites photo- chemically and by reduction with glucose in the presence of iron and by reduction with ferrous hydroxide in the presence of oxygen. The formation of carbon and nitrogen organic compounds in green plants and bacterial cultures from inorganic nitrogen, and the production of NsO, Ne, NO, and HCN during fermentation and photochemical reduction may be explained by the inter- mediate formation of nitrosyl H {NO and its subsequent reaction with aldehydic combinations. 22 Bracket indicates labile character. | STUDIES ON THE PHYSIOLOGICAL ACTION OF SOME PROTEIN DERIVATIVES. VII. THE INFLUENCE OF VARIOUS PROTEIN SPLIT PRODUCTS ON THE METABOLISM OF FASTING DOGS. By MICHAEL RINGER anp FRANK P. UNDERHILL. (From the Department of Pharmacology and Toxicology, Yale University, New Haven.) (Received for publication, August 1, 1921.) It is the design of this study to extend certain recently developed aspects of the problem of proteose intoxication. The toxic properties of protein split products, at the proteose stage of hydrolysis, have called forth a wide literature (1). This has been primarily concerned with the clinical effects of a rapid intravenous injection of a concentrated solution of proteose. The chief signs emphasized were, the depression of blood pressure and respiration, the delayed coagulability of the blood, the lympha- goguic effect, the constitutional depression or ‘‘peptone shock,”’ the gastrointestinal irritation, the diminished response to subse- quent injections or ‘‘immunity,’”’ the leucopenia, the marked concentration of the blood,! the severe acidosis,! and the rapidly fatal issue. These constitute a syndrome rivalling in violence any that we know. Naturally proteoses were assigned the toxic agency in diseases where injury of tissue was a factor. In the absence of a more tangible cause it was widely assumed that ‘the absorption of toxic proteoses’”’ explained an acute intoxi- cation. Recently, these assumptions have been given experi- mental ground. Vaughan (2) and his coworkers have prepared toxic split pro- ducts from a variety of proteins possessing many of the properties of proteoses. They concluded from their experiments that all 1 Author’s unpublished data. 503 504 Action of Protein Derivatives. VII proteins contain a toxic nucleus which when released gives rise to an intoxication identical with an infectious process. They have offeréd an attractive theory of infection and immunity on the basis of this work. Bied] and Kraus (3) showed the physiological resemblance between peptone shock? and anaphylactic shock, and urged the view that the formation of digestion products identical with those present in the market peptone were responsible for anaphylactic shock. Indeed the mechanism of anaphylactic shock as presented in the work of Jobling, Peterson, and Eggstein (4) is essentially a proteose intoxication, by proteose derived from the host’s serum as a result of the disturbance of the ferment antiferment balance. And more to our point Whipple and Cooke (5) and their coworkers in a series of extensive experiments have demonstrated that the intoxication of intestinal obstruction in dogs is due to the absorp- tion of a toxic proteose produced in the obstructed gut. They have isolated and purified a proteose and clinically reproduced the disease by injection of the substance in normal dogs. More- over they have shown that such injections cause a very large in- crease in the output of urinary nitrogen and a significant rise in non-protein nitrogen of the blood. But they have gone further and it is with this phase that the present work is concerned. In a series of papers on proteose intoxications and injury of body protein (6) these authors showed that inflammatory processes initiated either through bacterial agency or by aseptic means also call forth an increase in urinary nitrogen excretion and blood non-protein nitrogen. Hence they offer the suggestion that perhaps every inflammatory process is fundamentally a proteose intoxication. According to this view an inflammatory process may so injure body cells that toxic proteoses are formed which injure other cells, etc., so that a vicious circle is established. This hypothesis is certainly highly suggestive and if correct would render all inflammatory reactions simple of explanation and reduce the whole process to a single simple reaction. We have been working for some time on proteose intoxication and have been particularly interested in these view-points. We have asked ourselves first of all whether the catabolic reactions called forth by Whipple’s toxic proteose are specific or whether * Referring to commercial ‘Witte pepton”’ ae of a mixture of proteoses and peptone. M. Ringer and F. P. Underhill 505 such reactions are common to all proteoses. And, secondly, are there any other protein split products that are likely to occur in tissue injury capable of the same catabolic efféct? Technique. The plan of the experiments was to follow the urinary nitrogen, creatine, and phosphorus in fasting dogs until a nitrogen level had been reached, to inject intravenously a solution of the substance under consideration in normal saline solution, then to continue the experiment until these excretory products had returned to normal. Usually 4 days of fasting sufficed to bring the dogs to a basal nitrogen level. On the 4th day the injection was made into the jugular vein, under light ether anesthesia. The rate of injection was usually slow enough to avoid acute shock (in 5 to 15 minutes). The preparations will be described in their places. The animals were kept in standard metabolism cages and the urine collected by catheterization every 24 hours. They were allowed to drink water according to their desire. The dogs were usually house-broken so that we had little trouble from contamina- tions of the urine with feces or vomitus. When such occurred, they were included in the 24 hour samples. Nitrogen was done by the Kjeldahl method, creatine and creatinine by Folin’s methods, and total phosphorus by the uranium acetate method. We con- fined ourselves to these urinary constituents because we found early that they were the only ones to show any significant change. The clinical effects were typical and are not recorded because they have been sufficiently described elsewhere. Control Experiments. In Table I and Chart 1 are given the results of several control experiments. Dog 1 was fasted for 7 days. It will be observed the constituents maintain a comparable level. Dog 2 received an intravenous injection of normal saline solution under iden- tical conditions with the other experiments. The technique em- ployed apparently has no influence on the course of metabol- ism. In Dogs 14, 15, and 16 we tried to show that the injection of a colloidal solution was in itself unable to affect A 506 Action of Protein Derivatives. VII | metabolism. This was of importance, because most of the sub- -j stances subsequently injected were colloidal in character. The TABLE I. Influence of Fasting, Injection of Normal Saline Solution, and Non-Protein Colloids. Dog. Day. |Weight. sees heb oie P:05 Remarks. kg ce qm. mg mg 1 2 90 | 1.890) 20] 350 3 80 | 1.830) 35] 362 4 70 | 1.880) 49] 400 | Fasting only. 5 73 | 1.850) 62) 387 6 88 | 1.710) 54 ]|- 312 td 72 | 1.740] 401) 375 2 5 66 | 3.020) 29] 340 6 57 | 2.880) 35) 300 i, 8.5 60 | 3.030) 26] 359 8 56 | 2.920| 14] 347 | Injected 50 cc. of normal saline solution. 9 54 | 2.780| 14] 385 10 53: | 22710) £305 34t 14 3 |) 1520 74 | 3.380} 45] 490 4 90 | 3.250) 51 580 | Injected 50 cc. of solution of soluble starch. 5 78 | 3.040) 29]! 440 6 74 | 3.250} 50] 4388 a 72 | 2.960; 22] 420 GS i at! 110 | 3.620) 102] 663 5 | 10.4). 9920)|°32330) Bee) 675 6 90 | 2.840; 81] 525 | Injected 30 ce. of strong solution of inulin. 7 100 | 2.640}. 83] 565 16 4 7.8 90 | 2.950) 20} 338 5 92 | 2.205 4 337 6 96 | 1.890} .11 | 312 | Injected 50 cc. of 6 per cent gum acacia solution. a 92 | 1.890} 18} 262 8 72 | 1.830 influence of the physical state of the solution injected is neg- ative, from our view-point. 'M. Ringer and F. P. Underhill 507 Cuart 1. Control experiments. Influence of fasting, the injection of normal saline solution, and of non-protein colloids, on the daily excretion of nitrogen. Arrows show time of injection. Effect of Amino-Acids, Amines, and in Vitro Autolysates. At various times it has been claimed that specific amino-acids are responsible for the toxic effects of protein split products (7). This contention has been disposed of as far as the clinical effects are concerned (1). What is the influence of free amino-acids on tissue catabolism? In Table II and Chart 2 are given the results of the injection of various free amino-acids and mixtures. Dogs 17, 18, and 19 received pure amino-acids, leucine, glycocoll, and alanine. The results are entirely negative. The amount of nitrogen injected is excreted, but nomore. In Dog 18 the creatine showed a marked rise, but this effect could not be repeated in Dog 19. We may therefore disregard it. Dogs 20 and 21 received a mixture of amino-acids prepared from the hydrolysis of casein until biuret- free. These preparations contained most of the important amino- acids yet were entirely innocuous. It might be argued that the responsible amino-acid was not present in sufficient concentration. But it will be seen that one-half the amount of nitrogen here in- jected, also derived from casein but given in the form of a pro- 508 Action of Protein Derivatives. TABLE II. VII Influence of Amino-Acids, Histamine, and in Vitro Autolysate. ma or © 0 7. |Weight. volume. 9.0 Urine | Total sj 2.475 Crea- tine. mg. 72 26 24 135 193 115 P205 460 490 540 340 370 775 550 600 Remarks. Injected 50 ce. solution of glycocoll (0.288 gm. N). Injected 50 ce. solution of leucine (0.270 gm. N). Injected 100 cc. solution of alanine (0.75 gm. N). Injected 110 ce. solution of alanine (1.87 gm. N). Injected 65 cc. rapidly, biuret-free casein diges- tion product (0.665 gm. N). Injected 80 cc. of mixture of amino-acids (biuret- free casein digest, 0.489 gm. N). M. Ringer and F. P. Underhill 509 TABLE I1—Continued. Dog. | Day. |Weight. Blac ool ol P205 Remarks. kg ce. gm mg mg. 22 2 148 | 3.704; 42] 730 3 | 16.8 | 100 | 3.424 0} 720 4 130 | 3.160; 87 | 720 | Injected 0.5 mg. of hista- mine per kilo. 5 | 16.22) . 84] 3.000} 12) 600 6 114 | 2.880; 25 | 740 | Injected 1 mg. of hista- mine per kilo. a 92 | 2.736 0 | 420 39 3 65 | 1.680} 69} 340 + 6.7 55 | 1.560} 40) 280 5 345 | 1.320} 78 | 425 | Injected 1 mg. of hista- mine per kilo. 6 94 | 1.440} 82) 180 27 3 90 | 1.995} 76} 400 4 6.7 80 | 1.860} 73] 308 5 440 | 2.250) 107 | 400 | Injected 15 ce. of dog mus- cle autolysate (0.432 gm. N). Coagulable pro- teins absent. Proteose trace. 6 124 | 1.800} 90] 175 28 4 110.5 | 205 | 2.580| 25 | 440 5 615 | 4.850) 258 | 975 | Injected 70 ce. of dog mus- cle autolysate (1.95 gm. N). Coagulable pro- teins absent. Proteose 3 trace. 6 253 | 2.780} 178 | 320 teose, was extremely destructive of tissue (Dog 24, Chart 3 and Table III). Hence, if any particular amino-acid is the toxic agent it had opportunity to assert itself. fore that the amino-acids themselves are incapable of producing destruction of tissue and are not contributing factors in this res- pect to the toxicity of proteoses. We may conclude there- Recently Dale and coworkers (8) demonstrated the similarity of histamine shock and peptone shock, and Abel and Kubota (9) THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 510 Action of Protein Derivatives. VII claimed that histamine is the responsible agent in the toxicity of Witte peptone. Does histamine, one of the most toxic of the amines, have any influence on tissue catabolism? In Dogs 22 \ ‘ . mst aoe ae eee Lee a Pe SS we Shae Ae He HE 12s See ey cae fae hae ote CuaArt 2. Influence of amino-acids and amines on the daily excretion of nitrogen. Arrows show time of injection. Dotted lines show amount of nitrogen injected. and 39 we injected histamine in doses sufficiently large to produce shock and yet the effect on the elimination of nitrogen, creatine, and phosphates was negative. These doses (1 mg. of base per kilo) constitute about 300 times the amount of histamine contained M. Ringer and F. P. Underhill 511 in a toxic dose of Witte peptone (Dog 5, Chart 3), according to the analysis of Hanke and Koessler (10). Hence the amine content of proteoses as represented by histamine cannot be the catabolic agent of proteoses. This agrees with the findings of Hanke and Koessler (10) who showed that histamine-free proteose gives the typical clinical toxic picture. ; Dogs 27 and 28 show that autolysate free from coagulable protein, rich in amino-acids but poor in proteose and peptone as shown by a faint biuret reaction, produced no increase in urinary nitrogen., This substance was prepared by autolyzing 1 kilo of dog’s muscle under toluene, for a month, precipitating the pro- teins, and concentrating the residue. Unfortunately the creatine and phosphates were not determined in the injected material. That the undoubtedly rich content of creatine and phosphates of the injected material would account for the large output of those constituents in Dog 28 which received the undiluted residue, seems to us more rational than to accept the indication that tissue is destroyed. This view agrees with the negative influence on the total nitrogen. These last two experiments are of interest in indicating that autolysis in vitro and autolysis in vivo although probably the result of the same mechanism are capable of contrary effects. The autolysis that obtains during septic or aseptic suppuration is toxic, for these are usually accompanied by fever, depression, and as Whipple has shown by tissue catabolism. The explanation it seems to us is to be sought in the general quantitative character of the end-products which the conditions of the autolysis deter- mine, rather than in the production of special products endowed with specific properties. Had the autolysate given to Dogs 27 and 28 contained a sufficient amount of the more complex protein derivatives like proteose, in other words had the digestion been less complete, it would undoubtedly have been toxic also as will be clear from the next series of experiments. The Influence of Ordinary Proteoses on Catabolism. As has been already stated, the question of prime importance is: Are the reactions called forth by Whipple’s toxic proteose specific, or are they common to other proteoses? Clinically 512 Action of Protein Derivatives. VII the question has been answered. All, with the possible exception of gelatoses, are active. proteoses induce tissue catabolism. Dog. Day. 5 2 3 4 5 6 3 4 5 6 i 8 9 4a, 2, 3 4 5 6 7 4b 3 4 5 6 Weight. 10.3 TABLE III. It remains to be seen whether other Influence of Ordinary Proteoses. Urine volume. cc, 110 102 455 182 90 76 143 Total N. gm. 3.480 3.370 5.040 5.900 2.325 2.393 3.060 10.2 9.3 . Remarks. Injected 0.4 gm. per kilo of Witte peptone (0.6 Injected slowly 50 ce. of Witte peptone—0.2 gm. per kilo (0.325 gm. N). Injected 100 ce. of Witte peptone solution — 0.3 gm. per kilo (0.59 gm. Injected rapidly 90 ce. of Witte peptone for im- munity—0.3 gm. per kilo (0.592 gm. N). Crea- rehas P20s mg. mg. 112 805 138 Ale 292 |1,383 gm. N). 205 | 997 91 550 108 450 182 650 280 450 69 475 71 | 440 62 425 18 330 20 | 720 N). 133 460 19 495 0 470 23 680 37 610 180 | 1,295 350 700 130 800 M. Ringer and F. P. Underhill 513 TABLE IlI—Continued. Dog. | Day. |Weight.| Urine | Total | Crea- | p.o, Remarks. kg. ce. gm. mg. mg. 23 4 120 | 3.020) 117) 587 5 | 11:6) 113') 2.640) 84 | 650 6 150 | 3.690} 149 |1,050 | Injected pure deutero- proteose egg albumin— 0.15 gm. per kilo (0.174 gm. N). a 135 | 2.650) 54) 525 24 4 140F 3750) a5 7 25 oF 6.3) |) 120) 132160) 600) 675 6 365 | 4.710) 320 |/1,150 | Injected pure deuteroca- seose—0.3 gm. per kilo (0.370 gm. N). 7 390 | 5.730) 529 | 800 8 188 | 3.130 31 675 25° 4 52 | 1.345 9 180 5 6.8 40) | 1-470). 435)) 307 6 1.4380) 57 | 450 | Injected deuteroproteose egg albumin—0.15 gm. per kilo (0.100 gm. N). 7 102 | 2.420 4 330 8 90 | 1.300 22 230 30 -t 130 | 4.620 31 625 5 17.3 170 | 5.025 50 THUS: 6 260 | 6.660) 173 | 2,400 | Injected 0.1 gm. per kilo Vaughan’s crude soluble poison in 50 ce. of saline solution (0.167 gm. N). a 900 | 9.850) 655 | 1,670 8 100 |14.700; 490 | 1,700 In Table III and Chart 3 are recorded experiments along this line. Dog 5 received a large dose of Witte peptone and it will be observed in the next 2 days he excreted about 2.5 gm. of extra nitrogen. The creatine was more than doubled and the P.O; almost doubled. The significance of an increased nitrogen out- put has already been discussed by Whipple (5,6) and is a clear argument for tissue destruction. The marked rise in creatine is 514 Action of Protein Derivatives. VII an added indication of the same, as has long been known. During fasting, the involution of the muscular uterus, muscular atrophy, aah Es ode ave la Nae Ne iz ipsitdl lis alia - | ° 0 oleae / apm nes wee ‘- ESE) to ie J ° ct vogs EP eas = ea ed 3| feet @ lee @ C1 Sears eer eae . ie aes ee ae RRERYR ees pes) Pa) \| 1 Pe a .s oer Bae C7) Se Baa te Mae Oludle Ppisox Crarr3. Influence of proteoses on daily excretion of nitrogen. Ar- rows show time of injection. Dotted lines show amount of nitrogen in- jected. = or wherever else muscle tissue is disintegrated extra creatine appears in the urine. But more than a confirmation of the evi- dence that the extra nitrogen gives, the large creatine output M. Ringer and F. P. Underhill 515 points to the seat of the catabolism, a question that Whipple raised but could not answer from his experiments; namely, the muscle tissue. The large output of extra phosphorus is an added indication of tissue catabolism and probably of muscle in particular. Forbes and Keith (11) in a review of the literature on this subject bring out the facts that the destruction of phosphorus-containing pro- teins as in general catabolism, violent exercise, the destruction of leucocytes, cancer, tuberculosis, septic conditions, fever, and acidosis, results in an excess output of phosphates. In peptone shock we have an acidosis but in these dogs shock was avoided by slow injection, hence it must be the tissue destruction factor which accounts for the extra phosphate. Since the other condi- tions mentioned are largely muscle wasters, the extra phosphate output here may be considered an index of muscular catabolism. Dog 6 received half the dose of Witte peptone of Dog 5. The response in the three directions indicated is not as marked but it. is definite. Dog 4a received a dose intermediate between Dogs 5 and 6 and the response was intermediate by all three criteria. We may say that the size of the dose is an important factor in the severity of the catabolism induced. This has long been known with regard to the clinical effects, small doses being innocuous and larger doses fatal. There is no way of accurately comparing the degree of response of our dogs with Whipple’s because this author does not state his dosage in terms of nitrogen so that judgment can be made of the amount of proteose given. On the whole, our effects are neither as great nor as prolonged, but certainly of the same kind. Dog 4b received the same dose a second time several weeks later to test for immunity but the response was much greater the second time. This may have been due to the rapidity of the injection which we know from the clinical reactions is an important factor. All three criteria of tissue catabolism are strongly present. In order to controvert the idea that Witte peptone is an impure proteose and therefore not a fair criterion of what other proteoses will do, to Dogs 23, 24, and 25 we gave pure proteoses prepared from egg white and casein.? The response of Dogs 23 and 24 3 The material formed from egg white and casein was prepared by pep- sin digestion then saturated with ammonium sulfate after neutralization 516 Action of Protein Derivatives. VII although characteristic was not marked. This can be accounted for by the small dose, 0.15 gm. per kilo, which was necessitated by the fact that larger doses killed several dogs from the acute effects (blood pressure depression, etc). Dog 24 received 0.3 gm. per kilo at the usual rate and the tissue catabolism was as great as with Witte peptone. Hence the purity or impurity is of small moment so long as there is sufficient proteose, present. It will be observed in our dogs that the excess nitrogen excretion is partially delayed to the 2nd day as with Whipple’s dogs. That is to say, the crest of the excretion comes on the 2nd day in most of the cases. Another point of identity is the diuresis, although that is frequently not marked. As evidence of how much more toxic than ordinary proteoses, other protein split products can be, there is the enormous catabolic influence of Vaughan’s crude soluble posion. Dog 30 received only 0.1 gm. per kilo. Witte peptone would have been harmless in that dosage. On the 2nd day the nitrogen output was doubled and on the 3rd day trebled. The creatine and phosphate kept pace. What are we to say of the relative toxicity of proteoses? It is true Vaughan’s crude soluble poison is not strictly a proteose but it is not far removed from one. It gives the clinical reactions of proteoses, but more intensely. It is partially precipitated by saturation with ammonium sulfate, both filtrate and precipitate being active. Itis nearer the protein end of the hydrolytic chain. But proteins as we shall see later although catabolic agents are not nearly as destructive as this substance or even Whipple’s toxic proteoses. So that it cannot be the position in the hydro- lytic scale that is the whole story. It seems to us reasonable to conclude that probably all true proteoses are catabolic agents, although there is considerable variability in the degree of injury induced. of the digestion mixture and removal of undigested residue and neutraliza- tion precipitate. The proteoses were dissolved in H.O and dialyzed free from (NH;).SO,. The mixture was filtered from the small precipitate of heteroproteose and the filtrate evaporated and saturated with NaCl which precipitated the so called protoproteose. Treatment of the filtrate from the NaCl saturation with acetic acid precipitated a mixture of proto- and deuteroproteose. The filtrate from the acetic treatment was dialyzed free from NaCl and constituted the yield of deuteroproteose. The solution freed from NaCl was concentrated to small yolume and treated with alcohol. The precipitate of deuteroproteoses was washed with boiling alcohol and treated with ether while hot, and ground toafine white powder. M. Ringer and F. P. Underhill 517 Influence of Proteins. TABLE IV. Remarks, Injected 150 cc. of solution of crystallized edestin (0.234 gm. N). Injected 40 cc. of excelsin solution (0.060 gm. N). Injected 35 cc. of solution of egg albumin (0.480 gm. Injected 47.7 cc. of solu- tion of egg albumin (0.411 gm. N). Injected 50 ce. of egg al- bumin solution (0.480. Dog | Day. |Weight.| Urine | Total | Crea- | p.o, kg. ce. gm. mg. mg. Uf 2 75 | 2.533| 42 | 360 3 8.95} 60] 2.493) 32] 370 4 1100) Sa753|) 35) |) 714. 5 95 | 3.466; 68] 290 6 53 | 3.106) 51 | 440 7 62) |) 32080) © 55" | 320 8 50 | 2.640} 36) 290 10 6 100 | 2.760 588 7 10.9 | 102 | 2.640} 78) 488 8 200 | 2.925] 121 662 9 130 | 2.850) 152) 413 10 92 | 2.520 425 8 2 154 | 4.180) 81 671 3 112 32972) econ Gs a 16.1 110 | 4.060} 70) 680 5 187 | 4.480} 118 | 998 N). 6 130 | 4.836) 81 | 560 7 108 | 4.040; 48} 482 8 14.9 94 | 3.920) 40; 540 9 o Uy son ie 0) a (On ley 0) 10 110 | 5.000) 421} 400 11 130 | 4.146) 24] 680 9 4 100 | 1.835) 73 | 325 5 6.7 86 | 1.845) 108 | 362 6 148 | 2.344) 125] 700 gm. N). 7 130 | 4.590) 323 | 500 130 | 3.375, 177 | 518 Action of Protein Derivatives. VII TABLE IV—Continued. Dog. | Day! |Weight.| Urine | Total | Crea- | po, Remarks. kg cc gm. mg mg 11 4 104 | 3.980 67 700 3 125 | 3.440 58 780 4 16.8 130 | 3.008 0 680. 5 400 | 3.320 1 | 925 | Injected 110 ce. of gelatin solution (0.498 gm. N). 120 | 3.048 3 580 vi 90 | 2.912 9 | 480 12 3 68 | 2.448 Q | 480 4 11.10 84 | 2.916) 11 580 5 110 | 3.030 665 | Injected rapidly 94 ce. of gelatin solution (0.580 gm. N). 6 90 | 3.156 20 | 454 rf 88 | 2.920). 520 13 4 Use 90 | 3.030} 80] 575 5 120 | 3.360, 57 700 | Injected 80 ce. of gelatin solution (0.407 gm. N). 6 110 | 3.015} 46] 388 7 95 | 2.580 9 75 Influence of Foreign Proteins. Although it has long been known that foreign proteins intraven- ously introduced produce an increased nitrogen output (12), fever (2), and other toxic symptoms, it seemed wise to repeat this type of experimentation in order to compare the degree of cata- bolic effect with the similar effect of protein split products. Ac- cordingly Table IV and Chart 4 contain the records of a few experiments. Dog 7 received a dilute solution of edestin. The influence on the nitrogen and phosphates was quite marked, but on creatine only slight. Dog 10 responded with slight extra output of nitrogen and phosphates and a marked extra output of creatine. In view of the very small dose the effect was decidedly positive. Dogs 8 and 9 received egg albumin solution. The effect is in all es- sentials indicative of a tissue destruction. M. Ringer and F. P. Underhill 519 Dogs 11, 12, and 13, aside from the slight effect on phosphates which cannot be stressed in itself, responded indifferently to large doses of gelatin solution. This failure to behave like other pro- teins is not contradictory to the general influence of proteins. mares aa Ja a Pike eeeEE E Se Beane aaea ® . Pace fa be i a i led ae Bs 5 uitatk al ogee ae rela rhea aera “RES RReS CoS S| Cuart 4. Influence of proteins on daily excretion of nitrogen. Arrows show time of injection. Dotted lines show amount of nitrogen injected. Gelatin is not anaphylactogenic (13), its gelatoses are innocuous, it is deficient in important amino-acids. It confirms, however, our view that the colloidal nature of the solution is of small moment in the effects under consideration. 520 Action of Protein Derivatives. VII On the whole although the proteins are decided catabolic agents they are so to no greater degree than the proteoses. Fried- mann and Isaac have shown that even homologous sera are toxic in this respect. Vaughan suggests that this is due to the fact that the proteins have become ‘‘foreign”’ in the process of coagulation. The same may be held for the injury to body protein during an inflammatory process. Injured protein is foreign protein and hence is a catabolic agent. In explaining then, the cause of tissue catabolism during inflammation, the attention must not be directed to the products of the injured proteins alone, but to the parent proteins as well. DISCUSSION. We believe we have answered the first question we set out to answer, to wit, that the catabolic reactions called forth by the toxic proteose of Whipple and his coworkers, are not specific but are common to this genus of compound, although to a variable degree. Whether there are other products capable of the same effect, we have already pointed to proteins themselves, and to Vaughan’s crude soluble poison. We may add in anticipation of the following paper, in which the subject is given in detail, that certain nucleic acids are also capable of this catabolic effect to a very marked degree. SUMMARY. 1. Ordinary proteoses induce catabolism of tissue in fasting dogs, as evidenced by a large output of urinary nitrogen, creatine, and phosphates. 2. Both pure and impure proteoses are effective. 3. Proteoses differ in the degree of their effect. The dosage and rate of injection are factors. 4, Proteins except gelatin are also capable of this effect. 5. The amino-acids, histamine, and in vitro autolysates, are without influence. mem Whe 12. 13. M. Ringer and F. P. Underhill 521 BIBLIOGRAPHY. . Underhill, F. P., and Hendrix, B. M., J. Biol. Chem., 1915, xxii, 443. . Vaughan, V. C., Protein split products, Philadelphia, 1913. . Biedl, A., and Kraus, R., Wien. klin. Woch., 1909, xxii, 363. . Jobling, J. W., Petersen, W., and Eggstein, A. A., J. Exp. Med., 1915, xxii, 401. . Cooke, J. V., Rodenbaugh, F. H., and Whipple, G. H., J. Exp. Med., 1916, xxiii, 717. Whipple, G. H., Rodenbaugh, F. H., and Kilgore, A. R., J. Exp. Med., 1916, xxii, 123. . Whipple, G. H., and Cooke, J. V., J. Hxp. Med., 1917, xxv, 461. Whip- ple, G. H., Cooke, J. V., and Stearns, T., J. Exp. Med., 1917, xxv, 479. Cooke, J. V., and Whipple, G. H., J. Exp. Med., 1918, xxviii, 223, 243. . von Knaffl-Lenz, E., Arch. exp. Path. u. Pharmacol., 1913, lxxii, 224. . Barger, C., and Dale, H. H., J. Physiol., 1910-11, xli, 499. Dale, H. H., and Laidlaw, P. P., J. Physiol., 1910-11, xli, 318. . Abel, J. J., and Kubota, S., J. Pharmacol. and Exp. Therap., 1919, xiii, 243. . Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1920, xliii, 567. . Forbes, E. B., and Keith, M.H., Ohio Agric. Exp. Station, Techn. Series, Bull. 5, 1914. Friedmann, U., and Isaac, S8., Z. exp. Path. u. Therap., 1907, iv, 830. Wells, H. G., J. Infect. Dis., 1908, v, 459. STUDIES ON THE PHYSIOLOGICAL ACTION OF SOME PROTEIN DERIVATIVES. VIII. THE INFLUENCE OF NUCLEIC ACIDS ON THE METABOLISM OF FASTING DOGS. By MICHAEL RINGER anp FRANK P. UNDERHILL. (From the Department of Pharmacology and Toxicology, Yale University, . New Haven.) (Received for publication, August 1, 1921.) Whipple and Cooke! have demonstrated that the toxic proteose recovered from obstructed intestines or isolated loops, induces a marked destruction of tissue upon injection into normal dogs, as evidenced by the large output of urinary nitrogen. In the pre- ceding paper we have shown that other proteoses possess the same property. In view of the marked similarity in physiological behavior between proteose and nucleic acid, with regard to the blood pressure depression, delayed coagulability, increased lymph flow, etc., as described by Mendel, Underhill, and White,” it seemed opportune to study whether this resemblance can be extended to the catabolic effect under discussion. Accordingly pursuing the same method of investigation as recorded in the preceding paper we gave fasting dogs, with a constant nitrogen output, intravenous injections of representative animal and plant nucleic acids and noted the effect on the urinary nitrogen, creatine, and phosphates. The animal nucleic acids were prepared from thymus of the calf, spleen of the steer, and spleen and pancreas of the dog, by the methods described by Jones? and Baumann.? The vegetable nucleic acid was prepared 1 Whipple, G. H., and Cooke, J. V., J. Exp. Med., 1917, xxv, 461. 2 Mendel, L. B., Underhill, F. P., and White, B., Am. J. Physiol., 1902-03, vill, 377. 3 Jones, W., Nucleic acids. Their Chemical properties and physiolog- ical conduct, New York and London, 2nd edition, 1920, 103. 4Baumann, E. J., Proc. Soc. Exp. Biol. and Med., 1919-20, xvii, 118. 523 524 Action of Protein Derivatives. VIII from fresh brewers’ yeast by the method of Baumann.®:= The substances were dissolved in normal saline solution and neutral- ized before injection. : Effect of Plant Nucleic Acid. In Chart 1 and Table I are recorded the results of experiments with yeast nucleic acid. Dog 32 received a standard dose slowly. The output of nitrogen, creatine, and phosphates was considerably increased over the basal level. Dog 33 received the same dose rapidly and responded with a much greater output of each of these constitu- ents. As is the case with the injection of proteose the rate of injection is a factor of importance. Dog 31 received a smaller dose than any of the others but the output of nitrogen and creatine was very much greater. This animal was very badly shocked. As is apparent from the experi- ments of Mendel, Underhill, and White? there is a considerable individual variation in response. The effect on Dogs 40 and 41 was characteristic. The nitrogen output in all of these animals increased. It varied from 1 to 4 gm. in excess of the basal level, on the first day of the injection. The excretion continued above normal for several days thereafter (Chart 1). This is a clear argument for a significant tissue destruction. The effect on the creatine output is a striking confirmation of the tissue destruction which the large nitrogen excretion indicates. It, moreover, points to the muscular tissue as the seat of this effect. The evidence of the phosphates is not at once apparent, for the output above the amount injected does not seem large—no more than a few hundred mg. But the true condition is really obscured. Consideration of the protocols of the succeeding experiments on animal nucleic acids will show that the results are entirely negative as far as the effect on tissue destruction is concerned. We may consider those experiments from our view- 6 Baumann, E. J., J. Biol. Chem., 1918, xxxiii, p. xiv. 6 Dr. Emil J. Baumann was kind enough to send us samples of spleen and yeast nucleic acids. M. Ringer and F. P. Underhill 525 point, as controls. There, it will be observed, the phosphorus injected is not entirely eliminated in the urine. Not more than half the amount given is excreted. Hence, since in the experi- pee Cc a : et ele ke Jo ie Cuart il. Influence of plant nucleic acid on the daily excretion of nitrogen. Dotted lines show amount of nitrogen injected. Action of Protein Derivatives. VIII 526 TABLE I. Influence of Plant Nucleic Acid. Dog. | Day. |Weight. Poe ae ee P2O5 Remarks. ume. kg. (ee gm. mg. mg. 32 3 122° | 3.225) ~ 172.) .1,,000 ‘A 13.4 130) | 3.315 70 990 5 350 | 4.410} 153 | 1,600) Injected slowly 125 ce. of yeast nucleic acid, 2 per cent solution (0.300 gm. N, 750 mg. of P05). 6 134 | 3.765} 126 680 33 3 104 | 3.180} 52 887 4 13.04) 92 | 3.645 42 962 5 340 | 5.040) 240 | 2,000) Injected rapidly 116 ce. of yeast nucleic acid, 2 per cent solution (0.302 gm. N, 760 mg. of P2O;). 6 130 | 4.950} 240 725 ii 110 | 4.635} 135 800 31 4 92 | 2.235) 132 413 5 11.00} 96] 1.825) 194 507 6 200 | 6.050) 237 | 1,250) Injected 50 cc. of 2 per cent yeast nucleic acid (0.120 mg. N, 320 mg. of P205). a 188 | 4.550) 462 500 40 3 80 | 1.970} 118 530 4 8:00) 100) || 22210) 15 505 5 700 | 3.950) 239 | 1,560) Injected 0.5 gm. per kilo of yeast nucleic acid (0.390 gm. N, 970 mg. of P2305). 6 150 | 4.170) 307 530 2 90 | 3.160 41 3 125 | 3.590) 1388 613 4 1257 100 | 3.590 65 760 5 685 | 5.150} 200 | 1,790) Injected 0.5 gm. per kilo of yeast nucleic acid (0.540 gm. N, 1,350 mg. of P.O;). 6 210 | 5.100} - 380 810 of 160 | 4.040 M. Ringer and F. P. Underhill 527 ments under discussion the output is not only as great as the amount injected but greater, we may conclude that there was a considerable excretion of phosphates above the basal level. This confirms the evidence derived from the consideration of the effect on the total nitrogen and the creatine. It may be concluded, therefore, on grounds similar to those taken in the preceding paper that plant nucleic acid induces a marked tissue destruction when introduced directly into the circulation. Effect of Animal Nucleic Acids. Contrary to our expectations we were unable to demonstrate a similar effect as a result of the injection of animal nucleic acids. In Table II and Chart 2 are recorded the results of these experi- ments. In the five experiments where pure preparations from various sources were used, the results were uniformly negative with regard to the output of nitrogen. The amount injected was excreted, no more. Dog 37 showed a slight rise, this result, however, could not be repeated in Dog 38. The phosphate out- put to which we have already alluded, is in conformity with this finding. In Dogs 34 and 35 there were significant increases in the output of creatine. It is difficult to interpret these findings standing alone, unsupported by evidence from the nitrogen and phosphate output. We are inclined to disregard them, especially in view of the fact that this increase did not occur uniformly. It may therefore be concluded that animal nucleic acid injected directly into the circulation unlike plant nucleic acid gives no evidence of inducing tissue destruction. In short, it is not a toxic substance. DISCUSSION. This investigation, as was stated in the preceding paper, was undertaken primarily in order to find substances other than pro- teoses that are capable of stimulating catabolism and that might be formed as a result of tissue injury. We have demonstrated that plant nucleic acid is such a substance. It is reasonable to believe in view of the fact that the bacterial cell is largely com- posed of nucleoprotein, that the nucleic acids resulting from the 528 Action of Protein Derivatives. TABLE II. Influence of Animal Nucleic Acids. Crea- tine. mg. 175 120 235 42 100 43 10 79 109 30 88 102 96 76 96 67 69 P205 mg. 463 ~ 463 960 600 600 835 675 900 Dog. | Day. |Weight. Urine | Total kg. Cc. gm. 34 4 110 | 1.800 5 | 10.5 | 110 | 1.945 6 2.190 ‘ 120 | 1.920 30 4 105 | 3.240 5 111.4] 110 | 3.150 6 90 | 2.955 7 109 | 3.000 36 4 97 | 3.340 5 | 11.5] 116 | 3.250 6 190 | 3.602 7 170 | 3.450 8 115 | 2.860 37 4 110 | 2.800 5 117.0] 135 | 2.940 6 224 | 3,550 | 137 | 3.420 8 115 | 2.820 mak 38 4 132 | 3.190 5 | 15.0] 114 | 2.920 6 400 | 3.450 7 135 | 2.880 150 650 VIII Remarks. Injected 0.3 gm. per kilo of thymus nucleic acid in 50 ec. (0.369 gm. N, 920 mg. of P.05). Injected 50 cc. of thymus nucleic acid, 0.25 gm. per kilo (0.360 gm. N, 900 mg. of P.O;). Injected 0.1 gm. per kilo of dog nucleic acid (pan- creas and spleen, 0.158 gm. N, 400 mg. of P05). Injected 3 per cent solu- tion of spleen nucleic acid (0.320 gm. N, 800 mg. of P05). Injected 0.4 gm. per kilo of spleen nucleic acid (0.562 gm. N, 1,400 mg. of P,0;). M. Ringer and F. P. Underhill 529 decomposition of this substance might, in septic processes, be a contributing agent in the general intoxication. However, this source of nucleic acid is conceivably not great. The host’s own nucleoproteins would be a much richer source, and as Whipple and Cooke and their coworkers have shown for proteoses, the destruction of the host’s tissues is the real source of the intoxicat- im Is Zan amacesesae (ORM as ed oe IS _ aS ae ae ig AE \ TT et teh | Dog B8. | Sple cle net ote Cuart2. Influence of animal nucleic acids on the daily excretion of nitrogen. Dotted lines show the amount of nitrogen injected. ing substance. Since we have demonstrated that animal nucleic acid is not toxic it is probable that this type of compound is not an important factor in the intoxication of inflammatory processes. Moreover, bacterial nucleic acid has been little studied and it is not clear whether it would behave like the animal or plant variety. Hence, little can be definitely argued for the réle of nucleic acids, in inflammatory intoxications. 530 Action of Protein Derivatives. VIII But our finding with regard to the failure of animal nucleic acid to injure tissue, opens the question of whether this compound resembles‘ proteoses in physiological behavior at all. Mendel, Underhill, and White? showed that the plant nucleic acid they used, like proteose, caused a fall in pressure, delayed coagulability of the blood, increased flow of lymph, etc. We have recently TABLE III. Acute Effects of Plant and Animal Nucleic Acids. Dog. | Time. | pres- | Clotting | Heme-| reserve Remarks. sure: ; ‘ |volume. min. \mm. lig per cent|per cent 30 0} 130| 3min.| 100] 65 1 Injected spleen nucleic acid, 2 60 0.3 gm. per kilo in normal 31-1302) .8cazs. 92 | 65 saline solution. ; 63 130 93 300 130 | 45 min.| 100] 59 32 0 97.| 12min.| 100] 58 1 Injected spleen nucleic acid, 2, 30 | 3 hrs. 92 | 50 0.5 gm. per kilo. 7 60 12 80 27 112 97 | 55 180 115 | 50 min.| 101 53 21 0 108 | 4min.} 100) 41 1 Injected yeast nucleic acid, 2 10 |-11 min.| 86 0.3 gm. per kilo. 12 15 92 | 28 32 5D) Sam. 96 37 92 22 115 20 152 10 128 1é ee been able to add a concentrated blood’ as measured by the increase in hemoglobin, and an acidosis’ as measured by the alkali reserve. On repeating this work with pure animal nucleic acid we found that the effect on the blood pressure was exceedingly fleeting and not very profound, that in some cases the coagulability was mark- edly delayed but that there was no effect on the blood concentra- 7 Authors’ unpublished data. M. Ringer and F. P. Underhill 5ol tion (hence probably none on the lymph flow) and no effect on the alkali reserve. Table III gives three typical protocols, two of animal nucleic acid and the other of plant nucleic acid for comparison. We used large doses and concentrated solutions of spleen nucleic acid neutralized before injection. Hence it must be concluded that animal nucleic acid differs also from plant nucleic acid in not showing the typical peptone type of shock. This finding is at variance with the work of Bang® who worked with guanylic acid of the pancreas. SUMMARY. 1. Yeast nucleic acid induces a destruction of tissue, when introduced directly into the circulation, as evidenced by an increased output of nitrogen, creatine, and phosphates. 2. Animal nucleic acid has no such effect. 3. Animal nucleic acid differs also from plant nucleic acid in failing to give typical peptone type of shock. 4. Nucleic acids probably play a small réle in the intoxication of inflammatory processes. 8 Bang, I., quoted by Kossel, A., Z. physiol. Chem., 1900-01, xxxi, 410; 1901, xxxu, 201. hgh: ois geal con cog eae is 1 istheadcehh eae bara Mb ns tice Full Siete iio wet tid dba at Y “AL { air 7 9 olga etehy wiv ene: aerate Kuli opi ft. Picea ey, ‘s vicen mas lie ‘kylie f 5 ‘ a Wot % iw, q £«€ , . ’ , ry : cohiel: here | 25, ame bs oem ai he os piri we - * a 148 é 49 “¥ an , 7 % be Toetth eas Ta wes 5 oni be Tod i: . ; Wie te hy ys her ay Bere Nah . > Wo i iH Paes a a _ 4 hho f seree® igus Loch \ i ~ “ io + dem 1 } A+ ins aol ih : . ‘a ut iy ha : ar anes wee el “ton i hob eri l wht / " ' . F % b oJ =< \ \ STUDIES ON THE PHYSIOLOGICAL ACTION OF SOME PROTEIN DERIVATIVES. IX. ALKALI RESERVE AND EXPERIMENTAL SHOCK. By FRANK P. UNDERHILL anp MICHAEL RINGER. (From the Department of Pharmacology and Toxicology, Yale University, New Haven.) (Received for publication, August 1, 1921.) The relation of acidosis to shock has been the subject of numer- ous investigations leading to a diversity of views. That a dimin- ished alkali reserve may occur in shock no one will deny. On the other hand, evidence of a depleted alkali reserve in shock may be lacking. From a survey of all the evidence one is forced to the conclusion that the condition of acidosis in shock should be viewed from the aspect of an accompanying factor rather than as a contributing cause to the condition. It should be emphasized, however, that with shock once established an accompanying acidosis may directly contribute to a fatal outcome. An investigation of shock induced by various protein deriva- tives has afforded us the opportunity of studying the relation of diminished alkali reserve to the shock-like condition evoked. Methods. The experiments were carried out upon full grown, well nour- ished dogs that were allowed to fast for a period of 24 hours previous to the experiment. Anesthesia was produced by a mixture of morphine sulfate (0.01 gm. per kilo), atropine sulfate (0.001 gm. per kilo), and ether. Blood pressure was recorded with a mercury manometer attached to the right carotid artery. Injections were made into a jugular vein. Unless other- wise specified all injections were made rapidly—within a minute— and the volume of fluid introduced did not exceed 50 ec. Blood 500 534 Action of Protein Derivatives. IX for analysis was drawn from a femoral artery and alkali reserve was determined by the method of Van Slyke. Witte peptone, purified proteoses, Vaughan’s crude soluble poison, and nucleic acid were injected in doses of 0.3 to 0.5 gm. per kilo. Histamine dosage varied from 1 to 2 mg. per kilo calculated as the base. The Relation of Alkali Reserve to Shock Produced by Various Protein Derivatives. As may be observed from the table a shock-like condition as measured by low blood pressure was induced by the intravenous injection either of Witte’s peptone, Vaughan’s crude soluble poison, purified proteoses from casein, nucleic acid, or histamine hydrochloride. The data are arranged to emphasize the relation of the height of blood pressure and duration of low pressure to the alkali reserve. Thus, the word “maintained” in the table indicates a continued very low pressure, varying from 10to30mm. of mercury. The word “temporary” designates a fall of pressure perhaps even to a very low point but with either a pro- gressive or rapid rise to near normal limits. In general it may be noted that in these experiments, especially with Witte peptone, where low pressure has been maintained for a significant interval the alkali reserve shows the greatest decline. This, however, is by no means invariable. Generally, also, even though the fall in pressure is only temporary there is some indication of a decrease in alkali reserve. A striking difference is shown between the influence of yeast nucleic! acid and that of animal origin. In other respects the same fundamental difference in the behavior of these two types of nucleic acid when introduced into the body has been observed.? Less influence upon alkali reserve is to be seen from the injection of histamine than from that of any other substances introduced. ‘ Kindly furnished by Dr. E. J. Baumann. ? Ringer, M., and Underhill, F. P., J. Biol. Chem., 1921, xlviii, 523. IF’. P. Underhill and M. Ringer 5a0 The Relation of Low Blood Pressure to Alkali Reserve. Alkali reserve Experiment. Low blood pressure. bvolumeeiper pen), Fate. Normal. Later. Witte peptone. 2 Maintained. 67 25 Died 4 se 59 43 +6 5 < 57 15 “ 6 “ec 58 94 “ 9 ss 68 30 ‘¢ 45 § 44 22 & 3 Temporary. 63 58 17 ss 46 37 43 ef 53 42 47 sé 51 33 Deuteroproteoses (casein). 33 Temporary. | 54 34 Vaughan’s crude soluble poison (casein). 19 Maintained. Ou |) Maser 24 ts 43 32 36 f¢ 50 46 18 Temporary. 48 33 35 ss 34 20 ‘ 52 36 38 59 43 Nucleic acid (yeast). 21 Maintained. 41 15 Nucleic acid (thymus). 32 Temporary. | 58 53 30 ss 65 59 29 cS 55 54 15 as 53 49 28 ss 50 35 Histamine (hydrochloride). 8 Maintained. 59 32 11 se 58 42 13 : 51 60 14 ss 50 50 536 Action of Protein Derivatives. IX Why is it that there is such a diversity of response in the experi- ments presented? Undoubtedly in all the observations cited, with the possible exception of thymus nucleic acid, a shock-like condition intervened. This shock-like condition is accompanied by disturbances in respiration and circulation which may give rise to the production of acid products. Sufficient production of acid substances in the time interval possible under the experi- mental conditions hardly seems an adequate explanation. It seems to the authors that a much more plausible explanation lies in a decrease in the capacity of the body to excrete acid whether formed at a normal or at an accelerated rate. Thus a characteristic of low pressure is the appearance of a condition of anuria with a consequent greatly diminished ability to eliminate acid. We would suggest therefore that one large factor for the decrease in alkali reserve in the observations submitted is the failure of the renal mechanism incident to the low pressure. Inspection of the data from this view-point reveals a fairly close correlation between maintained low pressure and decreased alkali reserve. An apparent lack of correlation is encountered with the low pressure induced by histamine. An analysis of the details of the blood pressure fluctuations demonstrates that the pressure in the histamine experiments could not be maintained below 30 mm. of Hg even though histamine were continually introduced. In many of the experiments with other substances employed especially Witte peptone, a lower pressure obtained. Cushny® states that urine may continue to be secreted at 30 to 40 mm. of Hg. We would therefore suggest that the fall of alkali reserve noted in these experimental conditions of shock is directly related to failure of the renal mechanism to excrete acid—this inability of the kidney being induced by a maintained low blood pressure. Further work in this direction is in progress. Addendum.—Since the above was written a paper has appeared by Eggstein (Eggstein, J. Lab. and Clin. Med., 1921, vi, 481) on alkali reserve and protein shock. Our own experiments in as far as they are comparable yield results in many respects in confirmation of those of Eggstein. ®Cushny, A. R., The secretion of the urine, New York and London, 1917. STUDIES ON THE PHYSIOLOGICAL ACTION OF SOME PROTEIN DERIVATIVES. X. THE INFLUENCE OF NUCLEIC ACID ON THE METABOLISM OF THE FASTING RABBIT.* By FRANK P. UNDERHILL ann MARY LOUISA LONG. (From the Department of Pharmacology and Toxicology, Yale University, New Haven.) (Received for publication, August 1, 1921.) In a previous paper! in this series it has been pointed out that the introduction of yeast nucleic acid into the circulation of the fasting dog induces a destruction of tissue as evidenced by an in- creased output of nitrogen, creatine, and phosphates. The acute effects of this nucleic acid resemble strongly those characteristic of peptone shock. Since rabbits are more or less refractory to peptone injection in this respect experiments similar to those for dogs previously reported have been carried through on rabbits with nucleic acid to determine whether the nucleic acid influence as seen in dogs is capable of being extended to the rabbit. Methods. The animals were fasted for 2 days previous to the injection. Urinary excretion was divided into 24 hour periods by pressure upon the bladder through the abdominal wall. Total nitrogen determinations were made in duplicate by the Kjeldahl method, preformed creatinine by Folin’s method, and total creatinine by the procedure of Benedict. Phosphates were estimated by titra- tion with uranium acetate. Blood drawn from an ear vein was precipitated and analyzed for non-protein nitrogen by the method * The data of this paper are taken from the essay by Mary Louisa Long in partial fulfillment of the requirements for the degree of Master of Science, Yale University, 1920. 1 Ringer, M., and Underhill, F. P., J. Biol. Chem., 1921, xlviii, 523. 537 538 Action of Protein Derivatives. X of Folin and Wu. Hemoglobin was determined by the procedure of Cohen and Smith. Nucleic acid, containing 12.2 per cent nitrogen and 8.2 per cent phosphorus was prepared from brewers’ yeast by the method of Baumann.’ A fresh 1 per cent solution was used for each injection made by dissolving the nucleic acid in hot 0.9 per cent NaCl solution with the aid of a few drops of concentrated KOH. The injections were made on the basis of nitrogen content of the solution. Amounts varying from 20 to 60 ec. according to the size of the animal were slowly injected into the circulation through the marginal ear vein. The immedi- ate effects of the injection were an accelerated respiration and a transitory shock-like condition. Control Experiments. The Influence of Fasting, Etc., wpon Nitrogen Excretion in Rabbits. To determine whether the increased nitrogen output caused by the injection of nucleic acid really means an increased tissue catabolism, or is due to other factors, such as fasting, rabbits in a fasting condition were injected with a slightly alkaline solution. The following experiments show that under these conditions the nitrogen rises slightly on the 2nd day of fasting, but afterwards keeps on a level or falls. The creatinine remains practically unchanged, but there is a slight rise in creatine. The phosphates show practically no change. The nitrogen gradually increases, showing a marked rise on the last 2 days of the experiment. This probably accounts for a secondary rise observed in some of the injected rabbits. The creatinine keeps on a level as in the saline injected animals, but the creatine rises very markedly, increasing to eight times its original amount on the last day of fasting. This no doubt indi- cates a destruction of muscular tissue, and was usually observed in very thin animals. The phosphates remained on a level as before. ? Baumann, E. J., Proc. Soc. Exp. Biol. and Med., 1919-20, xvii, 118. F. P: Underhill and M. L. Long 539 The Influence of Intravenous Administration of Nucleic Acid upon Nitrogen Excretion in the Rabbit. In testing the influence of the intravenous injection of nucleic acid upon nitrogen excretion, varying amounts were given in order to determine the most effective dose. Rabbits 8 and 9 TABLE I. The Influence of Fasting, Etc., wpon Nitrogen Output. Control Experiments. Urine. Water Date. . i Remarks. intake. Vol- pore Creati-| Crea- P20 ume. | “gen. | Dine. tine. Bok Rabbit 6. Body weight 1.8 kilos. 1920 Ge. (Ae gm. gm. gm. gm. Mar. 8} 200 55 | 0.584 0.050) 0.004) 0.150) Mar. 9, injection of 67 enol. - 90 90 | 0.714| 0.063) 0.011) 0.204) ce. of faintly alkaline “ 10} 37 105 | 0.602) 0.044) 0.010} 0.150} 0.9 per cent NaCl solu- Seely 80 72 | 0.882| 0.065) 0.030} 0.336] tion. > 12), 80 52 | 0.884 0.058) 0.043) 0.226 Rabbit 22. Body weight 1.85 kilos. Apr. 29} 40; 70 | 0.562] 0.069) 0.038) 0.202 Apr. 30, injection of 52 230) 55 60 | 0.942) 0.072 0.018 0.192) cc. of faintly alkaline May 1 0 50 | 0.850) 0.099 0.015) 0.258) 0.9 per cent NaCl solu- cD 0 34 | 0.872 0.084 0.006, 0.202) tion. ee Sh 40 23 | 0.854) 0.079, 0.011 0.182 | Rabbit 23. Body weight 1.62 kilos. May 2| 170| 75 | 1.110! 0.108| 0.013] 0.162| May 3, injection of 40 «“ 100 | 98 | 0.874! 0.069| 0.008| 0.180! cc. of faintly alkaline 80 | 100 | 1.035) 0.075, 2? | 0.293) 0.9 per cent NaCl solu- 32 | 0.756, 0.065, 0.036, 0.222) tion. 0! 30 0.765) 0.073, 0.026, 0.190 & ore & i=) were given very small doses of nucleic acid, 0.05 gm. per kilo . of body weight with practically no effect, as shown in Table III. The picture is the same as shown in the control experiments. The fact that the nitrogen rises to a maximum the day after the injection and then returns to normal might indicate a slight destructive action of a small dose. 540 Action of Protein Derivatives. X Table IV shows the effect of increasing the amount of nucleic acid injected. Rabbit 7 received 0.1 gm. per kilo of body weight. The nitrogen rose 0.4 gm., an amount not to be accounted for by TABLE II. Effect of Plain Fasting on Nitrogen Output. Urine. Wat Date. Are re Total elle Remarks. ume. | ™!*T0- | nine. | tine. P20s ge Rabbit 19. Body weight 1.68 kilos. 1920 ce: ce. gm. gm, gm. gm. J\yoy AW 0 32 | 0.474) 0.054! 0.008) 0.356 “ 18) 100 | 30 | 0.589) 0.046) 0.008) 0.298 “ 191 51 32 | 0.726] 0.052) 0.015] 0.230 “ 90 85 55 | 1.056] 0.058] 0.051] 0.258 “ 91! 100 | 130 | 1.697 0.056) 0.074| 0.363 TABLE III. Effect of Injection of 0.05 Gm. of Nucleic Acid per Kilo. Urine. Water Date. : Remarks. mae val Total Creati-| Crea-| po ume on nine. | tine. a Rabbit 8. Body weight 2.7 kilos. 1920 | ce. Co gm. gm. gm. | gm. | Mar. 14 0 56 | 0.938) 0.095) 0.013) 0.186 Mar. 14, injection of a “ 15) 80 86 | 0.977) 0.095) 0.009) 0.247) 1 per cent nucleic acid “16 0 | 150 | 1.045 0.096; 0.014) 0.278) solution = 0.0216 gm. Ge 17 100 82 | 0.951) 0.098) 0.020) 0.296) of nitrogen. Rabbit 9. Body weight 2.4 kilos. 0| 60 0.876! 0.062! 0.005| 0.356] Mar. 26, injection of 15 Mar. 25 “ 26) 60 50 | 0.969} 0.070, 0.038) 0.284) cc. of al per cent nu- “ 27; 60| 36 | 1.072| 0.101) 0.029] 0.220) cleie acid solution =— “ 98! 100| 35 | 0.933) ? | ? | 0.304] 0.0192 gm. of nitrogen. 520 40.40 9352) OsO20\8 oe ? | 0.184 the small amount of nitrogen injected in the nucleic acid solution. The creatinine, creatine, and phosphates were unaffected by the injection. F. P. Underhill and M. L. Long 541 In Table V a very marked effect on the urinary nitrogen is shown for Rabbits 3 and 14, after an injection of 0.2 gm. of TABLE IV. Effect of Injection of 0.1 Gm. of Nucleic Acid on Nitrogen Output. Urine. Water Date. : : | Remarks. antake.|“Vol- | Eotal.| Creati-| Crea- ume. | !t™0- | nine. | tine P2Os gen. 7 Rabbit 7. Body weight 1.8 kilos. 1920 ce. ce. gm. gm. gm. gm. Mar. 13, 0} 48 | 0.462) 0.070, 0.015,-0.104) Mar. 14, injection of “ 14 35 | 44 | 0.690, 0.071) 0.010 0.172, 22.3 ce. of al per cent “ 15] 40] 120 | 1.066 0.081) 0.024) 0.207) nucleic acid solution = cle 0 40 | 0.774 0.053, 0.009) 0.167, 0.016 gm. of nitrogen. 0 certa| 100 76 0.558 0.063) 0.028 | TABLE V. Effect of Injection of 0.2 Gm. of Nucleic Acid per Kilo. Urine. 2 Water Date. aifaiee | <2 Total ; . Remarks. Vol- | jitro- | Cteati-| Crea-| pio ume. | “sen. nine. | tine. Ne Rabbit 3. Body weight 1.9 kilos. 1920 cc. ce. gm. gm. gm. gm. Feb. 12} 200 13 | 0.354 0.043) 0.005) 0.045) Feb. 13, injection of 38 13) 55 46 | 0.427| 0.084; 0.005) 0.102) cc. of al per cent nu- 04 0 | 126 | 1.431) 0.093) 0.099) 0.105) cleie acid solution = Tals. 100 140 | 2.196) 0.090) 0.166) 0.390} 0.049 gm. of nitrogen. Set Gla. 25 90 | 1.425) 0.032} 0.155} 0.288] | Rabbit 14. Body weight 1.6 kilos. Apr. 5| 70| 32 | 0.708) 0.060, 0.019] 0.130! Apr. 6, injection of 40 “ 6; 100] 60 | 0.788) 0.049! 0.011| 0.178, ec. of al per cent nu- eA MT 20 | - 134 | 1.120) 0.034) 0.088) 0.258) cleic acid solution = “ 8} 100| 77 | 1.359) 0.046] 0.052] 0.318| 0.051 gm. of nitrogen. « 9! 100] 100 | 1.947] 0.042] 0.105] 0.386 nucleic acid per kilo of body weight. In Rabbit 3 the nitrogen ’ Is increased 1 gm. on the day after injection, and rises still higher on the next day. The creatinine is not changed markedly, but THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLVIII, NO. 2 542 Action of Protein Derivatives. X° the creatine goes up tremendously, indicating a great tissue destruction. It is interesting to note that the rise in phosphates comes on;the 3rd day after injection. Rabbit 14 did not show as striking effects as Rabbit 3. The nitrogen increased 0.5 gm. on the day following injection and continued to rise on the fol- lowing days.’ Creatine is quadrupled, and the phosphates are steadily increased; however, this and the high increase in nitrogen on the last 2 days are probably fasting factors. TABLE VI. Effect on Urinary Nitrogen after Injection of 0.3 Gm. of Nucleic Acid per Kilo. Urine. Water Date. |. z Remarks. intake.) Vol: Hotel Creati-| Crea- | pig ume. | @*TO- | nine. | tine. =e en. Rabbit 5. Body weight 1.76 kilos. 1920 CC: Ge gm. gm. gm. gm. Mar. 8] 200 53 | 1.184) 0.063) 0.028) 0.140} Mar. 9, injection of 65.5 “ 9} 105 | 105 | 1.475) 0.082) 0.063) 0.283) cc. of a1 per cent nu- “ 10; 421] 168 | 1.876) 0.096) 0.086) 0.406) cleic acid solution = “11; 100 | 115 | 1.877| 0.053) 0.101) 0.442} 0.084 gm. of nitrogen. 12} 100] 150 | 1.246] 0.071} 0.197} 0.487 Rabbit 4. Body weight 2.4 kilos. Feb. 16 15 59 | 0.636) 0.095) 0.031| 0.096 Feb. 17, injection of 72 a LY 0 38 | 0.894) 0.102) 0.021) 0.161} ce. of a1 per cent solu- “ 48) ° 30) 105 | 1.189 0.126) 0.078; 0.160 tion of ‘nucleic acid = “19 10 | 56 | 0.650) 0.108 0.000) 0.232) 0.086 gm. of nitrogen. “ 20| 35] 31 | 0.621] 0.092| 0.003] 0.206 Rabbits 4 and 5 were given 0.3 gm. of nucleic acid per kilo. There is a decided rise in both the nitrogen and creatine, as shown in Table VI, but the increase is not as marked as with the 0.2 gm. dose, indicating that larger amounts are no more potent. These experiments show that the intravenous injection of nucleic acid produces a marked rise in the urinary nitrogen and creatine, and that amounts of 0.2 gm. per kilo of body weight give the most striking results. . -F. P. Underhill and M. L. Long 543 The Influence of Intravenous Injection of Nucleic Acid upon the Non-Protein Nitrogen of the Blood. Since the results on urinary nitrogen determinations point toward an increased protein catabolism it is of interest to follow the non-protein nitrogen of the blood, to determine whether there is an accumulation of the catabolic products in the blood. To interpret the results correctly, the hemoglobin curve was likewise followed to note whether there was any blood volume change. TABLE VII. Influence of Intravenous Injection of Nucleic Acid on the Hemoglobin of the Blood. Hemoglobin of the blood. Date. Sn ee Remarks. Hemoglo- Time of drawing blood. ban Rabbit 14. Body weight 1.6 kilos. 1920 per cent Apr. 6 Before injection. 81 Animal fasted 2 days pre- 2hrs. after “ 70 vious to an injection of 40 AS es se 57 ec. of al per cent solution Git st ape 55 of nucleic acid = 0.051 Sie BY wos em. of nitrogen. ce 7 94. “ “cc “ce 61 Of cc “cc ce 61 ce 8 48 (33 ce “ce 65 An increase in volume, represented by a low hemoglobin would mean a decrease in concentration, and if the non-protein nitrogen did not likewise fall in amount, a real increase of the latter might be indicated. ‘There was some question as to whether the frequent bleeding might not have an effect on the volume; consequently hemoglobin determinations only were made on Rabbit 14 after an injection of nucleic acid. Table VII shows that the volume decrease of about 20 per cent is a result of the injection, and not of the bleeding. The blood of Rabbit 15 was analyzed for non-protein nitrogen. The results, as given in Table VIII, show an increase of about 5 per cent in the first 3 hours, and in view of the fact that the injection causes a dilution of the blood, the increase is still greater. 544 Action of Protein Derivatives! 2 Table IX gives a complete picture of the urinary and blood nitrogen after an injection of 0.2 gm. of nucleic acid per kilo in two different rabbits. The urine picture is practically the same as seen in the rabbits previously discussed. The increase in nitrogen on the day following the injection coincides with the accumulation of non-protein nitrogen in the blood within the first 12 hours after injection. In Rabbit 16 the non-protein nitrogen remains practically on the level, but the corresponding decrease in hemoglobin would indicate that there is a marked rise. Rabbit 17 shows a slight increase in non-protein nitrogen, but a decided decrease in hemoglobin, showing a very marked TABLE VIII. Tflunence of Intravenous Injection of Nucleic Acid on the Non-Protein Nitrogen of the Blood. Non-protein nitrogen. Date. Remarks. Time of drawing blood. Per 100 cc. Rabbit 15. Body weight 1.5 kilos. 1920 mg. Apr. 6 Before injection. 55 | Animal fasted 2 days pre- 3 hrs. after “ 61 vious to an injection on Ep ce i 4 59 Apr. 6 of 37 cc. of al per Ores s *e 48 cent solution of nucleic ce ee, oA ae e 56 acid = 0.048 gm. of nitro- Bi a 3 56 gen. “ 8 48 “ “ce “ 57 rise. The marked increase on the last 2 days of the experiment may represent a fasting factor. The animal was very thin and apparently undernourished. These experiments show that the increase of urinary nitrogen is accompanied by an accumulation of protein catabolic products in the blood. . The Influence of Repetition of Nucleic Acid Injections upon Nitrogen Excretion in Rabbits. Rabbit 11 received the same dose of nucleic acid as was injected a week earlier. The increase in nitrogen output is just as marked or more so, as after the first injection, although the rise comes on -_ 545 F. P. Underhill and M. L. Long “UdSO1pIU JO “WS CFO'(O = UOI}N[OS prove oleponu yued Jod I 8 Jo 09 cg jo uorjoolur “QT “Ady |0Z "28 ‘uss0ryIU JO “Ws G/N = UOTY -NjoOs plow d1ojanu 4uUV—0 tod [| & jo "00 €°8G Jo 6 “Ady uo UoTyolUr UG 0} snorAoid skvp Z poysvy [vulUy ‘SYIVUIOYT GE €8 |€°89 » » » O08 96°89 |8°69 » » » “VG C610 |290°0 |8F0'0 |6F6'0 | FOT 0 ERD os O08 8S G G8 ” ”? ”? 6 OGRGoa mae BD mp ta) OS FS |O OOL », LOPE “SIY € F810 |Z00'0 |8F0'0 OFS '0 | 8E 0 OL » PF O&1 ‘uoTzoolUT a1Ojog 9FT 0 |F00'0 \9F0'°0 \2Sc'0 | F8 0OT |6 ‘Ady ‘SOFT FT FySIOM Apog “LT WqGQVY OF 6E |9°98 ”» 9 » OF 00°E |2°S8 Sp 0p » VG GST 0 |820°0 |060°0 | ¢ 68 0 OL .», |98 OF |6°% » 9 » GI vO IP |€ 88 ” ” ” 6 ¥S PE 9°16 ” 9? ” 9 Gl GV G G6 ” ” ”? g 0° O0T », 10978 ‘say ST 6G°8E ZIT | “worjoofur o10jog 0SZ 0 |€S0°0 |Z20°0 |002°0 | Zé og |6 ‘ady "But |quao sad “Wb ‘mo “mb "ub 7) ‘09 O@6T SOIL] €°S FYSIOM Apog “OT FqQqRY Lad wea ‘ued ome) tie] Penson oom, | sor | 8 [ares] 2 | Fen Loner | ag -u0N IO} EM wa *poold “ouIIy) ‘uabouuN poojg pun fiupursQ ay? uo prop orajonyy fo suoyoaluy snouaanyuy fo aruanyfuy “XI GTAViL : 546 Action of Protein Derivatives. X the 2nd day after injection. The phosphates are likewise in- creased, but the amount of creatine remains practically unchanged, leading one to believe that perhaps immunity is established, as the indications are that the tissue catabolism is not so great. Rabbit 10 shows the result of repeating the dose a month later. The effect is quite similar to that shown for Rabbit 11. The nitrogen output is doubled on the 2nd day after injection, but no marked increase is shown in the creatine. TABLE X. Repetition of Nucleic Acid Injection. Urine. Wat Date. Eke! = Total Gee owen SA Remarks. ume. gene nine. | tine. aa Rabbit 11. Body weight 1.8 kilos. 1920 ce. ce. gm. gm. gm. gm. Mar. 25 0) 60 | 0.529) 0.056) 0.006) 0.194) Immunity experiment on ST KO) 35 | 0.603} 0.059] 0.041] 0.232) Rabbit 7, 1 week lat- rere 0 52 | 0.768) 0.063) 0.012) 0.103) er. Mar. 26, injection “ 28) 20'| 110 | 1.245] 0.058) 0.046) 0-410!" of 22°53) ce: of samaper se 29 0 45 | 1.578) 0.039) 0.042] 0.308] cent nucleic acid sol- ution = 0.029 gm. of nitrogen. Rabbit 10. Body weight 2.24 kilos. Mar. 17 0 90 | 1.080, 0.057) 0.017) 0.090) Immunity experiment . = 100 100 2? | 0.066) 0.001) 0.165) on Rabbit 4, 1 month 9) 0 | 125 | 0.945] 0.081] 0.009] 0.258} later. Mar. 18, injec- 20) 0 135 | 1.701] 0.081] 0.024] 0.324) tion of 83.3 cc. of al “ 21) 100} 110 | 2.300] 0.073] 0.062} 0.187; per cent nucleic acid solution = 0.108 gm. of nitrogen. SUMMARY. The experiments reported point toward a marked tissue catabolism, chiefly shown in the urinary nitrogen and creatine. There is likewise a rise in the non-protein nitrogen of the blood, which is not so marked. The fact that non-protein nitrogen is being introduced with the nucleic acid injection must be taken into consideration. This may amount to as much as is already ‘ F. P. Underhill and M. L. Long 547 present in the blood as, for example, is the case with Rabbit 15. According to McQuarrie’s figures on blood volume, a rabbit of 1.5 kilos would have about 100 ce. (6.5 ec. per 100 gm. of body weight), so that the introduction of 48 mg. of nitrogen into the blood stream would double the amount present, which was 55 mg. per 100 ce. of blood. But with normal kidneys, this should be eliminated quickly. The fact that the non-protein nitrogen does not rise very markedly may be explained by the excretion of the catabolic products about as rapidly as they are formed, so that there would be no great accumulation of these products at any one time. We would suppose this to be the case if the kidneys were working efficiently. The dilution of the blood following nucleic acid injection in the rabbit differs from the results obtained by Ringer and Underhill for the dog. In the dog a marked concentration may be present. It would appear in the rabbit experiments that the relatively large volume of fluid introduced is only slowly compensated for in this animal. In the dog com- pensation occurs relatively quickly. CONCLUSION. Injection of nucleic acid into the circulation of the fasting rabbit induces increased tissue catabolism, as indicated by the augmented output of urinary nitrogen and creatine. In this respect, then, the dog and rabbit respond alike to the intravenous introduction of nucleic acid. In the rabbit nucleic acid injection produces dilution of the blood, in the dog concentration. In the rabbit there may be an increased non-protein nitrogen of the blood after nucleic acid injection. STUDIES ON THE PHYSIOLOGICAL ACTION OF SOME PROTEIN DERIVATIVES. XI. THE INFLUENCE OF SOME PROTEIN SPLIT PRODUCTS UPON THE METABOLISM OF FASTING RABBITS.* By FRANK P. UNDERHILL, PHILIP GREENBERG, anp ANTHONY F. ALU. (From the Department of Pharmacology and Toxicology, Yale University, New Haven.) (Received for publication, August 1, 1921.) A previous communication! relative to the influence of various protein split products upon the metabolism of fasting dogs has shown that in general the higher members of hydrolytic change of protein induce increased catabolism as evidenced by a large output of urinary nitrogen, creatine, and phosphates. This is especially true for such substances as Witte peptone, purified proteoses, and Vaughan’s crude soluble poison. In general, how- ever, the dog is peculiarly susceptible to the influence of proteoses and proteose-like substances, and the character of response elicited by the introduction of such substances is sufficiently important in its bearing upon the problems of inflammatory processes to warrant the extension of this type of experiment to a species of animal recognized to be refractory to proteose injec- tion. Such an animal is the rabbit. Methods. In general the methods followed were those outlined in a former paper? hence repetition is unnecessary. * The data are taken from theses presented by Philip Greenberg and Anthony F. Alu in partial fulfillment of the requirements for the degree of Doctor of Medicine, Yale University, 1920. 1 Ringer, M., and Underhill, F. P., J. Biol. Chem., 1921, xlviii, 503. 2 Underhill, F. P., and Long, M. L., J. Biol. Chem., 1921, xlviii, 537. 549 550 Action of Protein Derivatives. XI The Influence of Intravenous Injections of Witte Peptone, and Proteoses upon Nitrogen Excretion. An extensive experience with the influence of fasting upon urinary excretion in the rabbit has demonstrated that this con- Obie ae: a) fa cc. 1 130 2 0 3 0 4 0 5 110 1, 030 Day. or WN Re dition contributes no complicating factor in the interpretation of results obtained after introduction of various solutions.?* Reaction. Peptone on Urinary Nitrogen Output in a Rabbit TABLE I. The Influence of Witte Peptone on Urinary N itrogen Output in a Rabbit of 2.0 Kilos Body Weight. Remarks. Injection of 0.4 gm. of Witte peptone per kilo (0.158 gm. of nitrogen). Volume 50 ce. Bo hen eehan gm. mg. mg. gm. 0.612 0.070 0.662, 33] 9 |0.073 1.068) 106 | 25 |0.138 0.885) 87 8 0.170 0.969) 85 6 0.169 TABLE II. of 1.8 Kilos Body Weight. ro) es See = mM COs 62 |1, 022 65 |1,020 95 |1,021 83 |1,02 80 |1, 021 nS Reaction. Acid. Nitrogen. 0.704 0.978 1.038 1.716 1.603 Creatinine. Creatine. Phosphates. 0.070 0.171 0.152 0.287 0.265 tition of such data, therefore, will be omitted. ® Also from many unpublished data. Remarks. Injection of 0.15 gm. of Witte pep- tone per kilo (0.05 gm. of nitrogen). Volume 50 ee. Repe- Underhill, Greenberg, and Alu 551 Relative to the toxicity for rabbits of Witte peptone and purified proteoses‘ it may be stated that in no instances was there indication that the animals were even mildly ill. However, several animals died without definite symptoms. TABLE III. The Influence of Witte Peptone on Urinary Nitrogen Output in a Rabbit of 2.1 Kilos Body Weight. oO ae g 5 z 2 E . : : E 3 : = 3 < : S Remarks. ees s |e) a Zl Sener ites (ee ce. gm. mg mg . gm 1 40 | 57 |1,040| Acid. |0.663) 100} 21 |0.140 72, 90 | 96 |1,021 “ 0.675) 98 8 |0.199 3 0 | 295 |1,020 “é 1.089} 114 9 0.215) Injection of 0.4 gm. 4 65 | 64 {1,030 Sf 0.813) 98 7 |0.236) of Witte peptone 5 per kilo (0.160 gm. of nitrogen). Volume 100 ce. TABLE IV. The Influence of Deuterocaseose on Urinary Nitrogen Output in a Rabbit of 3.1 Kilos Body Weight. s 2 a 8 g 3) = ; s E 22 = 3 : : 3 Remarks. A iG aylteaes 3 Z Oyalee| | 6s cc cc gm mg. mg gm 1 Specimen lost. 2, Q | 47 Acid. |0.852) 131 8 0.263 3 10 | 65 fe 1.233] 181 | 47 |0.314) Injection of 0.15 4 QO} 62 {1.031 bi 0.978} 123 | 34 (0.226) gm. of deutero- 5) 100 | 46 - 0.834] 104 | 28 |0.194| caseose per kilo (0.074 gm. of ni- trogen). Vol- | ume 50 ce. From the view-point of the influence of Witte peptone and purified proteoses upon nitrogenous metabolism the data given in Tables I to IV show quite clearly that there is an increase in 4The preparation of this substance has already been described. Cf. Underhill, F. P., and Ringer, M., J. Pharmacol. and Exp. Therap., 1921, in press. 552 Action of Protein Derivatives. XI the output of urinary nitrogen. In most cases also there is a definite but smaller augmented excretion of creatine and phos- phates. In spite then of the fact that the rabbit and dog show different responses to the acute effects of the introduction into the circulation of Witte peptone and purified proteoses these substances exert with both types of animal the same effect upon nitrogenous metabolism, a fact which would lead one to the view that the influence upon nitrogenous metabolism is not necessarily closely associated with the influence which produces the toxic symptoms. The Influence of Vaughan’s Crude Soluble Poison and Vaughan’s Non-Toxic Body upon Nitrogenous Metabolism. In Tables V and VI may be found illustrative data selected from many experiments obtained after injection of Vaughan’s crude soluble poison prepared from egg white. An inspection of these tables will make it evident that the intravenous injection of Vaughan’s crude soluble poison (neutralized) calls forth a significant increase in the urinary nitrogen output of the fasting rabbit. The augmented nitrogen excretion is accompanied by a corresponding increase in the phosphate elimination. Creatine and creatinine are not markedly influenced. It will be recalled that in the preparation of Vaughan’s crude soluble poison a portion of the protein employed as the source becomes insoluble in the alkaline alcoholic solution and does not possess toxic properties. Such a product obtained from casein when injected into guinea pigs was harmless. On the other hand, the intravenous introduction of this non-toxic substance into the rabbit induces just as great an increase in urinary nitrogen and phosphorus as comparable doses of Vaughan’s crude soluble poison (see Tables VII and VIII). In other words the non- toxic material is Just as efficacious in accelerating protein cata- bolism as is the poisonous portion. It must therefore be quite apparent that this influence upon protein catabolism is not specific for Vaughan’s crude soluble poison nor is its influence necessarily associated with its toxic properties as evidenced by other symptoms. Underhill, Greenberg, and Alu 553 TABLE V. The Influence of Vaughan’s Crude Soluble Poison upon the Urinary Nitrogen Output in a Rabbit of 2.1 Kilos Body Weight. Urine. & , o ee) 2 ovals 3 3 = + =) r= 2 Remarks. os aq : a a S 3 = (>) pat 43! g os oe A q Fi = ade & S| aa |S of |sd| 22} 8] 3 = = ax > oO on o oO Ss co ° on ow | = = SQ) le les > ea] a a 6) 6) cc cc gm gm mg mg 35 = 0.874,0.064) 88 | 17 | Injection of 105 mg. of 69 Wy 1.552,0.124) 158 | 48 | Preparation I ( = 0.10 50 fs 1.050,0.120/ 160 | 50} gm. of nitrogen) dis- solved in 100 ce. of 0.9 per cent NaCl = 50 mg. per kilo of body weight. Time, 10 minutes. oP WD eH cooos8!: TABLE VI. The Influence of Vaughan’s Crude Soluble Poison upon the Urinary Nitrogen Output in a Rabbit of 1.8 Kilos Body Weight. ’ Urine 2 3 cae | ta s e iB 3 ea Aah & = B a a 3 3 i = S emarks,. fal = > ea Be | Oileo cc. cc gm. gm. mg. mg 1 42 52 | Acid. |0.732)0.215) 82 4 2 30 68 sf 0.762|0.179| 65 10 3 | aes | 3 oes 3s fo | ¢ 3 5 ale | 2.\-e) tee leomins ce cc gm. gm. mg mg 1 0| 54] Acid. |0.600)0.100; 90 8 2 Q0| 42 ge 0.780.0.184| 87 15 3 0 | 122 sf 1.200,0.600| 99 | 53 | Injection of 210 mg. of 4 0| 78 ns 1.398:0.425| 93 | 74 | non-toxic protein ( = 5 OnSO s 2.432|0.425| 88 | 92] 0.23 gm. of nitrogen) dissolved in 0.9 per cent NaCl = 100 mg. per : kilo of body weight. Time, 10 minutes. TABLE VIII. The Influence of Vaughan’s Non-Toxic Protein upon the Urinary Nitrogen Output in a Rabbit of 2.3 Kilos Body Weight. Urine eb Ss a ee g alte ; 2 2 é E E = z : a F Remarks. S 5 5 $8 acl || RO | se = i, Whee a= (28/8 | 8 | 3 Ase We ee Sit | ies Omleo ce. cc gm gm mg mg 1 40 62 | Acid. 0.810/0. 210 69 21 2 0} 45 0.8800.165| 64] 33 3 0| 47 - 1.0140.265| 88 | 42 | Injection of 115 mg. of 4 0} 45 s 0.942/0.238} 72] 55 | non-toxic protein ( = 5 0| 46 “ 1.200/0.240| 70| 70| 0.12 gm. of nitrogen) dissolved in 50 ec. of 0.9. per cent NaCl = 50 mg. per kilo of body weight. Time, 10 minutes. Underhill, Greenberg, and Alu 555 CONCLUSIONS. In spite of the fact that the rabbit is refractory to the acute effects of Witte peptone and proteoses, the intravenous injection of these substances into the fasting animal induces an accelerating influence upon protein catabolism similar to the response obtained in dogs. A similar influence is exerted by the intravenous injection of Vaughan’s crude soluble poison and Vaughan’s non-toxic body. These facts lead to the view that the action exerted upon nitro- genous metabolism is not necessarily related to toxic properties possessed by some of these substances. The influence seen upon nitrogenous metabolism is therefore not specific for a given protein derivative but is probably an indication of the detrimental action incident to the introduction into the circulation of a foreign protein. Z j oe a ith 1 ye oe . % fe } 7 = J rd er hs “a * oe THE INFLUENCE OF THYROPARATHYROIDECTOMY UPON BLOOD SUGAR CONTENT AND ALKALI RESERVE. By FRANK P. UNDERHILL anp CHARLES T. NELLANS. (From the Department of Pharmacology and Toxicology, Yale University, New Haven.) (Received for publication, August 1, 1921.) ~ In a recent communication Hastings and Murray! report their failure to corroborate the work of Underhill and Blatherwick?* who showed that blood sugar content is low after thyropara- thyroidectomy. They state that “their (Underhill and Blather- wick) determinations made with the method of Forschbach and Severin showed extraordinary variations and were not accom- panied by sufficient data to allow for much comment.” In reply it may be stated that the only data essential for the understanding of the problem were the changes in blood sugar content and their correlation to the occurrence of tetany and these were given in full. No mention is made of the experiments of Underhill and Blatherwick? wherein blood sugar estimations were made by the method of Vosburgh and Richards! which involves the actual weighing of cuprous oxide. It is quite fair to admit that results obtained by the Forschbach and Severin method may be subject to criticism because of the small quantity of blood employed in this colorimetric procedure. No such criticism can, however, be applied to the method of Vosburgh and Richards especially since quantities of blood varying from 20 to 60 gm. were employed for sugar estimations. Hastings and Murray employed the MacLean method of sugar estimation and assert that “in our series, at least, there was no marked disturbance in sugar metabolism for the first few days 1 Hastings, A. B., and Murray, H. A., Jr., J. Biol. Chem., 1921, xlvi, 233. 2 Underhill, F. P., and Blatherwick, N. R., J. Biol. Chem., 1914, xviii, 87. § Underhill, F. P., and Blatherwick, N. R., J. Biol. Chem., 1914, xix, 119. 4 Vosburgh, C. H., and Richards, A. N., Am. J. Physiol., 1903, ix, 35. 557 558 Thyroparathyroidectomy on Blood Sugar after operation.” Their protocols agree with their conclusions. The divergence of results appears to demand a reinvestigation of the problem and the results of four experiments are recorded. Methods. Well nourished adult dogs were maintained in a fasting condi- tion throughout the investigation. The operation was performed under morphine-atropine-ether anesthesia, the entire thyroid- parathyroid apparatus being removed. Blood was drawn from an ear vein. Blood sugar estimations were made according to the method of Folin and Wu.’ Carbon dioxide capacity of the plasma was measured by the procedure of Van Slyke. As a rule determinations on the blood were made about 9.30 a.m. and 5 p.m. daily. Does Hypoglycemia Occur After Removal of the Thyroids and Para- thyroids in Dogs? It is quite evident from the data in the table that after com- plete removal of the thyroids and parathyroids there is a variable but distinct fall in the blood sugar content. This usually occurs after the onset of tetany but may be present before signs of tetany are apparent. The reason for the difference in the results of Hastings and Murray and our own is not evident unless indeed it is related to the question of nutrition. In all our work with the exception of a single dog the animals were maintained in a fasting state whereas in the investigation of Hastings and Murray the context of their paper would lead one to the conclusion that food was given (cf. p. 240). That such an explanation is probably inadequate may be inferred from the fact that in Experiment 3 of our first paper? the animal ate food for a period of 12 days after operation, then went into tetany and revealed a condition of hypoglycemia. With the idea in mind that. changes in blood concentration might possibly play a réle in the topic under discussion hemo- globin and total solid values in the blood were followed. These observations indicate perceptible alterations in concentration from time to time but since these values fluctuate in either direc- § Folin, O., and Wu, H., J. Biol. Chem., 1920, xli, 367. F. P. Underhill and C. T. Nellans 559 tion without apparent relationship to tetany or blood sugar content they are without definite significance and hence are omitted here. The work has now been repeated three times employing three different methods for sugar estimation, and whatever may be the reason for the divergent results we see no occasion for modify- ing our former conclusion; namely, that removal of the thyroids and parathyroids in dogs induces a condition of lowered blood sugar content. The Relation of Alkali Reserve to Removal of the Thyroids and Parathyroids. In another section of their communication Hastings and Murray state that after removal of the thyroids and parathyroids there is not the slightest evidence of an alkalosis as indicated by a study of the carbon dioxide capacity of the plasma. ‘This conclusion is directly opposed to the work of Wilson, Stearns, and Thurlow’ published in 1915. These investigators found by deter- mination of Barcroft’s dissociation constant of oxyhemoglobin in venous blood brought into equilibrium with a constant tension of carbon dioxide and measurements of the carbon dioxide tension in the alveolar air evidence of an increasing alkali reserve up to the onset of tetany. McCann’ later published experiments sub- stantiating this. In view of the importance of the -theoretical considerations involved it seemed very desirable to investigate further the problem since the results of Hastings and Murray fail to corroborate the views of Wilson and his colleagues. Advan- tage has therefore been taken of the opportunity afforded by the animals prepared for the previous work on blood sugar. From the data presented in the table it must be concluded that up to the onset of tetany little or no change occurs in the alkali reserve of the blood after removal of the thyroids and para- thyroids. It is true that with Dog 2 the carbon dioxide capacity is greater the day after the operation than previously. The change is slight, however, and probably little significance should be attached to it, since even greater changes may occur in normal 6 Wilson, D. W., Stearns, T., and Thurlow, M. De G., J. Biol. Chem., 1915, xxiii, 89. 7McCann, W. S., J. Biol. Chem., 1918, xxxv, 553. ! 560 Thyroparathyroidectomy on Blood Sugar dogs in a fasting condition. This may be seen from data taken from another investigation in which dogs were subjected to a preliminary period of fasting. The following figures are for simple fasting, no other procedure having been followed with the animals concerned. Daysiotfasting: \cndseeaa meee ceee 1 2 3 COscapseity (Dor )) over ca ee eee 54 68 65 CO, + ear) eee set as i kes air iA hak 51 60 After the onset of tetany in general there may be a decided tendency toward a diminished alkali reserve. CONCLUSIONS. In spite of contrary findings by Hastings and Murray, repetition of previous experiments leads to the reiteration of a former con- clusion; namely, that thyroparathyroidectomy results in a lowered blood sugar content. After this operation there seems to be little or no change in the carbon dioxide capacity of the blood up to the onset of tetany. After this period there may be a decided tendency toward a diminished alkali reserve. The Influence of Thyroparathyroidectomy upon Blood Sugar Content and Alkali Reserve. Plasma Date. Blood sugar per 100 ce. CO2 Remarks. capacity. Dog 1. 1921 mg. vol. per cent Apr. 20 109 a.m. - |70.8 a.m.| Normal. 111 p.m. 68.9 p.m. OI 111 a.m. Normal. A725 93 cs 67.3 oh Removed thy- roids and parathyroids 3 p.m. Apr. 25. ENS 26 149 = Appears normal. 131 p-m. 58.2 .. ae a7 112 & 55.3 Slight tetany. eS 91 a.m. 63.6 78 (4.30 p.m.) 58.2 | Marked tetany. Died in 58 730 a) 44.7 convulsions at 6.30 p.m. The Influence of Thyroparathyroidectomy—Concluded. Plasma Date. Blood sugar per 100 cc. COz Remarks. capacity. Dog 2. 1921 mg. vol. per cent May 25 99 p.m. 53.6 | Normal. ~~ 26 98 a.m. 52.9 : Removed thy- roids and parathyroids at 3 p.m. SF DAG 117 « 60.2 | Appears normal. 119 p.m. 58.3 cos 45 a.m. 30.9 | Severe tetany. 37 p.m. 44.7 fe S Died in convulsions. Dog 3 June 6 103 p.m. 60.3 | Normal. eee 96 a.m. 54.3 8 Removed thy- roids and parathyroids 3 p.m. Sc 8 108 ‘ 57.7 | Runs, normal. 101 | p.m. 46.4 29 111 a.m. 52.6 96 p.m. 48.8 | Slight tetany. o510 100 (10 a.m.) 54.0 101 Ge <>) 46.8 | Severe tetany. 67 (10 p.m.) 58.8 eae Bh 74 a.m. 49.7 | Tetany absent. 73 p.m. 47.7 | Dog comatose. ag 85 a.m. 47.7 “e « 67 p.m. 51.4 = 13 107 a.m. 51.4 © | Dog comatose. 93 p.m. 48.3 Dog 4 June 27 111 a.m. 51.7 | Normal. Removal of thy- roids and parathyroids at 3 p.m. “< 28 106 s 49.9 | Seems normal. 100 p.m. 49.9 er 29 83 a.m. 51.7 | Seems normal. 95 p.m. 49.9 £30 81 fs 54.1 | Slight tetany. July 1 104 (9.30 a.m.) 42.5 | Marked tetany. 75 GtESORSS) 34.9 a « 80 (3.00 p.m.) 32.9 | Tetany subsiding. Ome 94 (8.30 a.m.) 46.8 | No tetany. 96 (Noon.) 46.8 561 = THE INFLUENCE OF FOOD INGESTION UPON ENDOGENOUS PURINE METABOLISM. I. By WILLIAM C. ROSE. (From the Laboratory of Biological Chemistry, School of Medicine, University of Texas, Galveston.) (Received for publication, August 24, 1921.) Abundant evidence has been accumulated in recent years to indicate that the original idea of Burian and Schur (1) and Sivén (2), to the effect that the excretion of endogenous purines is con- stant from day to day in the same individual, is not correct. More than 15 years ago Folin (3) found that the change from a diet of milk and eggs to one of starch and cream might be accom- panied by a fall in uric acid elimination of practically 50 per cent, although both diets are purine-free. He stated that the uric acid excretion is reduced whenever the total nitrogen elimination is much diminished, but that the reduction is irregular, and variable for different individuals. Since the work of Folin, numerous investigators, notably Leathes (4), Mendel and Brown (5), Smetanka (6), Taylor and Rose (7), Mendel and Stehle (8), Lewis and Doisy (9), and Hést (10), have corroborated his findings that protein ingestion exerts a marked influence upon urinary uric acid. Smetdnka (6), Mendel and Stehle (8), and Umeda (11) also observed an increase in uric acid elimination, as compared with the fasting output, following the ingestion of carbohydrates. Apparently fats produce the least effect upon purine metabolism of either of the three food- stuffs. The calorific value of the diet is ikewise important in determin- ing the uric acid excretion. According to Graham’ and Poulton (12), diets of protein and fat of insufficient calorie value, cause a fall of 30 to 50 per cent in the output of endogenous uric acid. If most of the fat is replaced by carbohydrate no fall is observed. More recently Hést (10) has affirmed that every increase or 563 564 Endogenous Purine Metabolism. I decrease in the calorific value of the food beyond a certain mini- mum, is accompanied by a like change in uric acid excretion. This effect occurs no matter which foodstuff is responsible for the variation in energy value of the diet, but is greatest with changes due to protein. No unanimity of opinion exists, however, as to the cause of the increases and decreases in endogenous uric acid elimination in- duced by diet. Various factors have been held responsible, and at least six possible explanations have been suggested or advocated in an effort to explain the experimental observations. It is our purpose in this paper to briefly discuss these theories. In the succeeding communication data will be presented which we be- lieve throw additional light on the problem. Alterations in the excretion of endogenous uric acid resulting from variations in the kind and amount of food have been attri- buted to the following factors: (1) Nuclear disintegration in the glands of the alimentary canal occasioned by the work of diges- tion (Mareg (13), Smetdénka (6), Lambling and Dubois (14), Mendel and Stehle (8), Hést (10)). (2) Nuclear disintegration associated with the work of digestion and food storage (Smetdnka, 6). (3) Synthesis of purines from carbohydrates (Graham and Poulton (12), Umeda (11)). (4) Synthesis of purines from arginine and histidine (Ackroyd and Hopkins (15), Harding and Young (16)). (5) Stimulation of the process of elimination -(suggested by Lewis, Dunn, and Doisy (17), but regarded by them as untenable). (6) General stimulation of cellular metabolism by amino-acids or their catabolic derivatives (Lewis, Dunn, and Doisy, 17). The first of these theories was suggested by Mare& (13). This investigator attributed the increase in output of uric acid following the consumption of purine-free food to nuclear disintegration, chiefly in the alimentary glands, incidental to the physiological work of secretion and digestion. Urie acid thus represents, according to Mare’, the wear and tear of these glandular tissues. When the alimentary glands are resting, the output of uric acid is low; when they are actively synthesizing and secreting digestive fluids, wear and tear is increased, and the production of uric acid is accelerated. As further evidence for this mechanism, Mares points to the fact that pilocarpine, which is known to increase secretory activity, likewise augments the excretion of uric acid. W. C. Rose 565 Smetdnka (6), Lambing and Dubois (14), and Hést (10) also regard digestive work as an important factor in the variations in output of endogenous uric acid. Mendel and Stehle (8), on the other hand, state that their experiments “‘offer no obstacle to the assumption that a portion, at least, of the endogenous uric acid may originate from the activity of the alimentary secretory appa- ratus.”’ 'The latter investigators were able to confirm the findings of Mares in regard to the action of pilocarpine, and to make the additional observation that atropine, which diminishes secretory activity, causes a fall in uric acid elimination. The Mares theory seems inadequate to us for several reasons. It does not appear probable that disintegration of the secretory cells during the process of digestion would be sufficiently extensive to account for the increase in uric acid elimination, which in some case is relatively large (cf. Taylor and Rose (7)). It is true that the suggestion of Mendel and Stehle (8) and Hést (10), to the effect that perhaps only a part of the uric acid has its origin in the alimentary cells, obviates this difficulty. Such a suggestion, however, does not afford an explanation of the effect of amino- acids, which require no digestion, upon uric acid elimination. Lewis, Dunn, and Doisy (17) have shown that glycocoll, alanine, and other amino-acids increase the hourly output of uric acid during fasting as much as do proteins. In our own experiments described in the sueceeding paper, the protocols show that even though the subjects lived upon weighed diets, necessitating the daily expenditure of the same amount of physiological labor in the process of digestion, the uric acid output for the individual days of the periods was quite variable. Smetdnka (6), who in the main adheres to the Mares theory, was forced to modify it in view of results obtained by him in experiments somewhat similar in nature to those of Lewis, Dunn, and Doisy. Having observed that the ingestion of honey, a food which like amino-acids requires practically no digestion, causes a marked increase in the output of uric acid, this investigator suggested that in addition to digestive work, the activity involved in glycogenesis may be responsible for a part of the endogenous uric acid. As far as the writer is aware, this is the only sugges- tion in the literature which specifically attributes uric acid forma- tion in part to the process of food storage. While the theory is 566 Endogenous Purine Metabolism. I interesting, and perhaps comes nearer explaining the experimental facts than does the original Mares conception, still it is open to the same ‘criticism as regards the action of amino-acids. If the increased output of uric acid following the ingestion of honey is due to the increased glycogenesis, certainly some other factor must be responsible for the action of glycocoll, alanine, and other compounds which cannot form appreciable quantities of this polysaccharide. In connection with Smetdnka’s observation concerning the effect of honey, the investigations of Graham and Poulton (12) and of Umeda (11) are of interest. These authors believe that part of the endogenous uric acid may arise through synthesis from carbohydrates. They observed that carbohydrate-rich fat- poor diets cause a greater excretion of uric acid than do fat-rich carbohydrate-poor diets, even though the protein content and calorific value of the food are maintained constant. Umeda suggests that uric acid may arise from a condensation of urea with an intermediary product of carbohydrate metabolism, perhaps lactic acid. As evidence for a synthesis of purines from carbohydrates, Graham and Poulton point to the observation of Knoop and Windaus (18) that when glucose is exposed in vitro to the action of sunlight and the strongly dissociated compound, Zn(OH)..4NH3, methyl glyoxal and 5-methyl-imidazole are formed. As interesting as these suggestions are, there exists at the present time no experimental evidence in vivo which justifies the belief that carbohydrates are transformed into purines in the animal organism. The observations of Ackroyd and Hopkins (15) which are discussed below indicate that in the rat, at least, purine synthesis from carbohydrate, if it occurs at all, is not quantitatively sufficient to meet the demands of the growing organism for these nuclear constituents. One of the most important studies of endogenous purine metab- olism of recent years is the paper of Ackroyd and Hopkins (15) alluded to above. These authors found that when young rats were supplied diets deprived of arginine and histidine, but ade- quate in every other respect to meet the demands of growth, erowth ceased and the elimination of allantoin decreased 40 to 50 per cent. When either arginine or histidine was present in the diet, there was no loss of weight, and in some cases growth oc- W. C. Rose 567 curred. The decrease in allantoin excretion was likewise much less than when both diamino-acids were absent from the food. No fall in allantoin elimination occurred when tryptophane was removed from the ration, or as a result of the absence of a vita- mine supply, though nutritional failure in these cases was even greater than when arginine and histidine were withheld. Despite the difficulties which are obviously associated with metabolic studies involving quantitative urine analyses in small animals, the care with which the experiments of Ackroyd and Hopkins are controlled, and the uniformity of their results, justify, in our opinion, their conclusions that arginine and histidine are the most readily available raw materials for purine anabolism in the body. Apparently either of these amino-acids may serve as the substrate for purine formation. A similar conclusion as to the origin of purines in the diamino- acids was recently arrived at by Harding and Young (16). Ac- cording to these investigators, the feeding of placenta, which has a high content of arginine, causes a much greater increase in the output of uric acid and allantoin in young dogs than does the ingestion of an equal quantity of muscle protein. Inasmuch as the diets of their animals were not purine-free, the data of Harding and Young are not as convincing as those of Ackroyd and Hopkins. . On the contrary, Abderhalden and Einbeck (19), Abderhalden, Einbeck, and Schmid (20), and Lewis and Doisy (9) have been unable to show any relationship between the arginine and histidine content of the diet and the uric acid or allantoin output in the urine. Lewis and Doisy compared the effects of diets high and low in arginine and histidine upon the uric acid output in man. Abderhalden and Einbeck studied the effects of adding histidine to the diet upon the allantoin excretion in the dog. In the later experiment of Abderhalden and his coworkers (20), histidine hydrochloride was given in 10 gm. doses to a fasting animal. Neither of the experiments yielded any evidence for an origin of purines in the diamino-acids. We believe the procedures made use of by these investigators were not suitable for studying the relation of amino-acids to purine syntheses. In the experiments of Lewis and Doisy (9), it is quite possible that the ‘‘low”’ histi- dine-arginine diets contained adequate- amounts of purine pre- 568 Endogenous Purine Metabolism. I cursors to support the normal anabolism. Calculation from the authors’ data shows that the ‘‘low”’ diets contained 3.5 to 4.0 gm. of arginine and histidine in each day’s ration. If such amounts are adequate (we have no information as to the quan- tities of diamino-acids required by adult men), one would hardly expect that a more abundant supply would result in an exag- gerated purine synthesis, and an increased uric acid elimination. We shall return to this proposition later. Concerning the experiments of Abderhalden, the question is properly raised by Ackroyd and Hopkins (15) as to whether an abnormal condition like fasting affords the best opportunity for investigating the fate of an amino-acid. They believe! that the « .). ~~. ~synthesis of such essential tissue constituents as the purines continues during starvation, at the expense—as we are entitled to believe— of protein materials liberated by autolysis of the less essential organs. When however an excess of a single amino-acid enters the circulation of a starving animal in a single isolated dose it may well almost completely escape such special utilization. It appears suddenly in excess of current needs, and, because the processes of deamination and direct oxidation are always in action, it will almost certainly survive for but a short period as available material for synthesis.”’ In contrast to the methods of Abderhalden and Lewis and their coworkers, Ackroyd and Hopkins compared the effects of diets free from arginine and histidine, with diets containing ade- quate amounts of the diamino-acids. The importance of this pro- cedure is emphasized by them as follows:? “When an animal is in a state of full nutrition it does not follow that such a process as the synthesis of the purine ring would necessarily be much accelerated or increased by mere increase in the supply of its raw material.”’ 2 9 And again,” “‘An individual amino-acid fed in excess of the immediate current needs of the tissues, as when it is added to an already efficient dietary, will al- most certainly be rapidly broken down on more direct lines, even if it be a normal precursor of the purine (or other) synthesis in the body.’’ 1 Ackroyd and Hopkins (15), pp. 552 and 553. * Ackroyd and Hopkins (15), p. 552. W. C. Rose 569 As important as the investigations of Ackroyd and Hopkins appear to us, we do not believe that they, or other studies of their kind, have an immediate bearing upon the problem of the varia- tions in endogenous uric acid elimination incident to alterations in the kind and amount of purine-free food, when the amino-acids in question are included in the diet. Even if it be admitted, as we are prepared to do, that tissue purines have their ultimate origin in arginine and histidine, this fact, in our opinion, does not war- rant making the assumption that the extent of purine synthesis is proportional to the arginine-histidine supply. On the con- trary, it seems reasonable to suppose that the anabolism of any tissue component is limited quantitatively to the needs of the organism for that particular ingredient. As soon as a diet con- tains sufficient precursors of any given anabolic product, synthesis of that product at the optimum rate probably occurs. It seems unlikely that the optimum would be exceeded however great a redundance of the precursors in question were provided. We believe that this view is entirely in accord with the statements of Ackroyd and Hopkins quoted above, and is completely justified in the case of purine anabolism by the experiments of Abder- halden and Lewis and Doisy. If we are correct, one should no more expect to exaggerate purine anabolism by feeding excessive quantities of purine precursors, than he should anticipate being able to increase the mass of brain substance by feeding unusual amounts of the components of nervous tissue. With the excep- tion of the purely storage forms of foods (glycogen, fats, and to a less extent, amino-acids), the components of the tissues of each species are normally synthesized and retained in remarkably uniform proportions. If conditions were otherwise, tissue com- position would be largely determined by the accident of diet rather than by the expression of the inherent, hereditary tenden- cies and impulses of the organism. It is, therefore, rather sur- prising to us that Harding and Young (16) were able to note differences in purine excretion in pups on diets of placenta (high in diamino-acids) as’ contrasted with diets of muscle (low in diamino-acids), unless the differences were in part due to exogen- ous purines. On the other hand, the fact that in their experi- ments growing animals were used, in which the anabolic reactions are known to predominate, may have been responsible for their unique data. 570 Endogenous Purine Metabolism. I In accordance with these concepts, znstead of there being con- flict between the data of Ackroyd and Hopkins on the one hand, and Lewis and Doisy on the other, we regard them as entirely in accord and mutually supplementary. In the experiments of the former, the decrease in allantoin excretion following the removal of arginine and histidine from the diet is the significant point, rather than the increase, which probably represented the normal purine metabolism, when the diamino-acids were supplied. After removal of arginine and histidine from the diet growth ceased because purine (and perhaps other) anabolism was no longer possible. Because of the deficient anabolism, greater physio- logical enconomy was exercised in catabolism, and the catabolic end-product of purines in the rat (allantoin) decreased in amount. In the experiments of Lewis and Doisy, a high arginine-histidine diet failed to induce a greater elimination of the catabolic end- product of purines in man (uric acid) than did a low arginine- histidine diet, because the latter was adequate to permit the optimum anabolism of purines. The superfluous molecules of arginine and histidine were doubtless oxidized without passing through the purine stage. In other words, the work of Ackroyd and Hopkins, to our mind, renders it very probable that the ultimate sources of tissue purines are arginine and histidine; the investigation of Lewis and Doisy indicates that purine anabolism in the adult is limited in extent to the physiological needs of the organism for purines. Neither investigation, however, permits any conclusions to be drawn as to the cause of variations in purine elimination with diets containing adequate amounts of diamino- acids. The latter problem is more likely one of purine catab- olism or excretion rather than of anabolism. In the course of the exceedingly interesting investigation of Lewis, Dunn,.and Doisy (17) on the influence of diet upon the hourly elimination of uric acid, the possibility occurred to the authors that the inereased uric acid excretion following the in- gestion of a single dose of a protein or of an amino-acid might be due to a stimulation of the processes of excretion under the influence of the food, rather than to increased uric acid formation. They reasoned that if a single dose of an amino-acid produced its effect by bringing about the mobilization and elimination of reserve or stored purines or their precursors, the administration of a a —— To ot W. C. Rose ay ial second dose, after the effect of the first had reached its maximum, should be without further influence. Accordingly, an experiment was made in which successive doses of glycocoll were administered on the same experimental day. The figures show that entirely comparable increases in the hourly output of uric acid occurred after each dose. According to the authors the data* ‘‘clearly demonstrate that the effects of amino-acids on uric acid excretion are not the result of stimulation of excretory processes leading to a removal of preformed uric acid fromthe body.”’? Whileitmight be questioned whether Lewis, Dunn, and Doisy were justified in assuming that the first dose of glycocoll entirely removed all excess or reserve purines from the system, and whether the single experiment reported by them is sufficient to warrant their con- clusion in this regard, data of another sort in the literature, when considered in connection with their work, increase the proba- bility of their contention being well founded. Frequent estima- tions of uric acid in the blood of normal subjects upon widely different purine-free diets led Hést (10) to the conclusion that diet (in the absence of purines) is without influence upon the concentration of uric acid in the blood. Despite the fact that urinary uric acid varies greatly in a given individual as a result of changes in the composition of the food, the proportion in the blood remains constant within the experimental error of the method. Inasmuch as uricolysis is not believed to occur in the human subject, and since the concentration of uric acid in normal blood is invariable under the influence of purine-free food, Hést is of the opinion‘ that ‘‘the endogenous uric acid output becomes a direct expression for the uric acid formation.”’ It must be admitted that rather large quantities of uric acid would have to be retained in the blood in order to alter appreciably the propor- tion present, or that reserve purines in the sense of Lewis, Dunn, and Doisy might be stored in the tissues, and hence not be mani- fested by blood analyses at all. Nevertheless, such evidence as -we have, whether obtained from a study of the urine (Lewis, Dunn, and Doisy), or by means of blood analyses (Hést), indi- cates that the cause of the alterations in output of endogenous ~ 3 Lewis, Dunn, and Doisy (17), p. 17. 4 Host (10), p. 30. 572 Endogenous Purine Metabolism. I uric acid following food consumption is not to be sought in an exaggerated excretion. Having ‘excluded to their own satisfaction the possibility of a stimulation in excretion being the causative factor in the increased output of uric acid following protein ingestion, Lewis, Dunn, and Doisy (17) suggest that the effect may be due to a general stimu- lation of all cellular metabolism by amino-acids. Each of the four amino-acids, glycocoll, alanine, glutaminie acid, and aspartic acid, as well as the closely related asparagine, caused an appre- ciable increase in the hourly fasting output of uric acid. The stimulation caused by the dicarboxylic amino-acids was more marked than that produced by glycocoll and alanine. On the other hand, sarcosine, a substituted amino-acid not readily catabo- lized by the body, and ammonium chloride and urea, were without influence. The authors emphasize the similarity of the effects produced by protein and amino-acid ingestion upon uric acid formation and heat production (specific dynamic action), and point out that the same chemical factors may be responsible for both. We believe that there are no experiments in the literature the results of which invalidate the assumption that general stimu- lation of cellular catabolism, involving both the nuclear purines and the hypoxanthine of muscle tissue, by amino-acids is at least one of the important factors in endogenous purine metabolism. Particularly do the data of Smetdnka (6), Taylor and Rose (7), Mendel and Stehle (8), and Hést (10) lend support to this hy- pothesis. In the succeeding paper we shall present the results of observations which we believe afford additional reasons for ac- cepting this view. BIBLIOGRAPHY. 1. Burian, R., and Schur, H., Arch. ges. Physiol., 1901, Ixxxvii, 239; Burian, R., Z. physiol. Chem., 1904-05, xliii, 532. 2. Sivén, V. O., Skand. Arch. Physiol., 1901, xi, 123. 3. Folin, O., Am. J. Physiol., 1905, xiii, 66. 4, Leathes, J. B., J. Physiol., 1906-07, xxxv, 125. 5. Mendel, L. B., and Brown, E. W., J. Am. Med. Assn., 1907, xlix, 896.. 6. Smetdinka, F., Arch. ges. Physiol., 1911, exxxviii, 217. 7. Taylor, A. E., and Rose, W. C., J. Biol. Chem., 1914, xviii, 519. 8. Mendel, L. B., and Stehle, R. L., J. Biol. Chem., 1915, xxii, 215. W. C. Rose Oe . Lewis, H. B., and Doisy, E. A., J. Biol. Chem., 1918, xxxvi, 1. . Host, H. F., J. Biol. Chem., 1919, xxxviii, 17. . Umeda, N., Biochem. J., 1915, ix, 421. . Graham, G., and Poulton, E. P., Quart. J. Med., 1913-14, vii, 18. . Mares, F., Arch. slav. biol., 1887, 11, 207; Arch. ges. Physiol., 1910, exxxiv, 59. . Lambling, E., and Dubois, F., Compt. rend. Soc. biol., 1914, Ixxvi, 614. . Ackroyd, H., and Hopkins, F. G., Biochem. J., 1916, x, 551. . Harding, V. J., and Young, E. G., J. Biol. Chem., 1919, xl, 227. . Lewis, H. B:, Dunn, M. S., and Doisy, E. A., J. Biol. Chem., 1918, XXXvi, 9. . Knoop, F., and Windaus, A., Beitr. chem. Physiol. u. Path., 1905, vi, 392. . Abderhalden, E., and Einbeck, H., Z. physiol. chem., 1909, xii, 322. . Abderhalden, E., Einbeck, H., and Schmid, J., Z. physiol. Chem., 1910, Ixviii, 395. THE INFLUENCE OF FOOD INGESTION UPON ENDOG- ENOUS PURINE METABOLISM. II. By WILLIAM C. ROSE. Wits THE ASSISTANCE OF J. STERLING Dimmitt AND H. LercH BARTLETT. (From the Laboratory of Biological Chemisiry, School of Medicine, University of Texas, Galveston.) (Received for publication, August 24, 1921.) The investigations reported in this communication have been in progress recurrently for the past 5 years. At first we made numerous studies of the hourly elimination of uric acid in fasting, and following the ingestion of single meals of purine-free foods. The results obtained were, for the most part, similar to those of other investigators who have studied uric acid excretion during short periods, notably, Mendel and Stehle (1), Neuwirth (2), and Lewis, Dunn, and Doisy (3). Invariably, the consumption of food led to an increase in uric acid excretion. But despite every care in the conduct of the experiments, the increases were some- times so slight, and the fluctuations without the influence of food were relatively so large, that we regarded the interpretation of the data as liable to serious error. We finally abandoned hourly studies and adopted 24 hour periods, which yielded more con- sistent results. Methods. The plan of the investigation was similar to that pursued in a study of endogenous creatine-creatinine metabolism, the results of which were reported in a former communication (4). In the earlier experiments, the uric acid and total nitrogen excretion were determined in subjects ingesting diets alternately low and high in protein. Later, two additional series of studies were under- taken. In one series, the nitrogen intake was maintained con- stant throughout, while the calorific value of the diet was altered. In the other, a low protein-low calorific diet was alternated with a high protein-high calorific diet. 575 576 Endogenous Purine Metabolism. II The subjects were four healthy young men, students in the School of Medicine of this University, who were engaged in the usual routine of student life, with practically the same amount of physical activity each day throughout the experiments. By mak- ing use of several individuals, we have excluded the possibility of our findings being accidental, or due to metabolic peculiarities of a single subject. Each student had been subsisting upon a purine-free diet for several days preceding the experimental régime. The diets were prepared, weighed, and ingested in the laboratory, and the meals were served at regular hours three times daily. Urines were preserved with toluene, and were usually TABLE I. Composition of Diets. Articles of diet. Food values.* Novobidiets| ia sete Se a eh Se ee Bread. | Honey.| Butter.| Eggs. | Milk. | Cheese.| Apples. N Calories. gm. gm. gm. gm, ce. gm. gm. gm. 1 400 | 150} 150 6517 | 2a 2 150 655 | 1,500} 50 27.93 | 2,741 7 400 20 200 50 12.91 1, 780 8 400 75 150 100 790 300 12.86 | 3,433 9 300 | 150) 60 [ 300 | 4.75 | 1,978 10 300 90 450 | 1,500; 90 300 27.45 3, 907 * The nitrogen and calorific values here tabulated were calculated from data given by Atwater, W. O., and Bryant, A. P., U. S: Dept. Agric., Office Exp. Stations, Bull. 28 (revised), 1906. analyzed immediately after the end of the 24 hour periods. Total nitrogen was estimated by the Kjeldahl-Gunning method, and uric acid by the procedure of Benedict and Hitchcock (5). Table I indicates the composition, nitrogen content, and calorific value of each diet, all of which were of course practically purine-free.’ 1 White bread, which contains larger traces of purines than any other article of food included in the diets, was kept constant in quantity through- out each experiment with the exception of those recorded in Tables II and III. In these two, more bread was ingested during the low than during the high diet periods. Accordingly, the effect of traces of exogenous pu- rines, if appreciable at all, would be more pronounced during the periods of low diet, and therefore cannot impair the validity of our conclusions. W. C. Rose Si EXPERIMENTAL. The results of the numerous experiments which we have con- ducted are entirely in accord with each other, and therefore need not all be detailed in this communication. The protocols give TABLE II. The Influence of High and Low Protein Diets on Uric Acid Excretion, when the Calorific Intake is Constant. Experiment 8. Subject F. W. D. Body | Urine | Reaction! Total | Uric Date. yaigbt. Palais reencel N. pad. Remarks. 1916 kg. ce. gm. gm. Dee. 7 | 72.5 | 1,310} Acid. | 6.97} 0.40 | Low protein diet, No. 1. Ss fro5on <4 7.38| 0.47 | 6.17 gm. N and 2,770 calo- a> 9 i, HQ) 7.12] 0.41 | ries daily.* ao) 2; 450) se 7.96) 0.44 ro ee scl: 1, 180 ss 6.78] 0.43 PAWERAGCMEN Eee ciis.< 2 x cctce si 24 CORKS Dee. 12 | 71.6 | 1,675) Acid. | 13.00} 0.51 | High protein diet, No. 2. AS alg} 2, 390 & 16.49) 0.49 27.93 gm. N and 2,741 calo- Soe 25780] 20.49} O <6 ries daily. mae LO 2, 440 ee 22.00} 0.51 16 2, 690 ef 22.78) 0.48 PAW ERAGON tte acris cae ia lel oan Ong Dec. 17 | 71.6 | 2,765] Acid. | 15.06] 0.40 | Low protein diet, No. 1. rege he) 15340 |e 9.91) 0.39 | 6.17 gm. N and 2,770 calo- “a 19 20] & 8.74) 0.39 | ries daily. PAVICT AE Cree ence kee a Pl 4 OR 39 1917 Jan. 18 | 71.6 | 1,530) Acid. | 9.03] 0.29 | Starvation level.! * Began eating this diet on Dec. 5. + Subject ate a purine-free diet for 10 days, followed by a fast of 40 hours. Analyses represent urine of last 24 hours of the fast. the data of six experiments, two of each of the three series indi- cated above. In Tables II and III are shown the effects of alternately feeding low protein and high protein diets when the calorific intake is maintained constant. -Although there is some 578 Endogenous Purine Metabolism. II irregularity in the uric acid elimination, as is almost invariably the case with this urine constituent, nevertheless, there is a slight but unmistakable increase in output during the high protein periods. The return to the low protein diet, following the days TABLE III. The Influence of High and Low Prolein Diets on Uric Acid Excretion, when the Calorific Intake is Constant. Experiment 9. Subject H. L. B. ae Reaction i Body | Urine Total | Uric Date. weight. jrobame: eee N. acid. Remarks. 1916 kg. (Fre gm. gm. Dec. 7 | 68.6 | 400) Acid. | 6.73) 0.45 | Low protein diet, No. 1. 8 460). “ 6.96) 0.51 | 6.17 gm. N and 2,770 calo- or 9 450) “ 6.62) 0.54 ries daily.* 0 | 730 ie 7.26) 0.56 a. 520)" 6.42| 0.54 Dec. 12 | =| 980; Acid. | 12.45) 0.66 | High protein diet, No. 2. A. als 960) “ 16.33] 0.56 | 27.93 gm. N and 2,741 calo- sage al! 980} “ 19.02} 0.52 | ries daily. ae ay 1160) ss 21.74) 0.54 Sse G 1,100) “ 22.18] 0.55 PAV OTAGC cts ie pe Va one 18.35] 0.57 Dec. 17 | 69.1 | 635| Acid. | 12.89] 0.47 | Low protein diet, No. 1. Fas 460) se 8.86] 0.48 | 6.17 gm. N and 2,770 calo- «34S 450) “ 8.21; 0.46 | ries daily. AN CTAGC Se. ioc ole nomen p erie ae 9.99) 0.47 1917 | Jan. 18 | 69.5 | 710) Acid. | 9.36] 0.43 | Starvation level.! * Began eating this diet on Dec. 5. { Subject ate a purine-free diet for 10 days, followed by a fast of 40 hours. Analyses represent urine of last 24 hours of the fast. of high protein ingestion, is in each case accompanied by a fall in uric acid excretion. At the bottom of each table is recorded the subject’s fasting output of uric acid. As indicated in the protocols, purine-free diets were ingested for 10 days preceding W. C. Rose 579 the 40 hour fasts. The urines for the last 24 hours of the absti- nence periods were used for the analyses. In Experiment 8, the starvation uric acid excretion dropped to 0.29 gm., and in Experi- ment 9, to 0.43 gm. per day. TABLE IV. The Influence on Uric Acid Excretion of Diets High and Low in Calories, when the Protein Intake ts Constant. Experiment 15. Subject J. 8. D. Te Reaction : es Body | Urine Total | Uric : oe weight.| volume. 2 N. acid. Remarks. litmus. 1917 Dec. “cc kg. cc. gm, gm. 62.2 | 685) Acid. | 11.63] 0.41 | Low calorific diet, No. 7. ‘¢ 13.33) 0.47 | 12.91 gm. N and 1,780 calo- | “ce 7 8 9 100017 iG 12.35} 0.49 | ries daily.* ae #10 | 860K beats 11.66 0.47 1 2 il Gia 2 12.56) 0.47 oo hl G20) ss 12.20] 0.54 PAR STL CNR aiscrsic sis, 00-3 I 12.29) 0.48 Dec. 13 | 61.4 625| Acid. | 11.15] 0.50 | High calorific diet, No. 8. «4A Ne esas) ee PAZ 0258 12.86 gm. N and 3,433 calo- ae be 1,490) “ 11.17] 0.59 | ries daily. er 16 800) “ 9.16) 0.55 AASV ELEN 2 eee eR en Naas a 10.67) 0.56 Dec. 17 | 61.5 | 650, Acid. | 10.84] 0.55 | Low calorific diet, No. 7. Ly ale 890) “ 12.30) 0.56 | plus 300 gm. apples.’ 13.10 419 995). 47 12.35| 0.57 | gm. N and 1,971 ealories fe 20 ol) ence 11.93} 0.57 daily. PAN CLIAU.C eye tea arate rtcece mote ns 11.86) 0.56 * Began eating this diet on Dec. 3. { The apples were added because of their laxative properties. Tables IV and V show the effects of alternately feeding diets low and high in calories when the protein intake is maintained practically constant. Throughout each experiment the nitrogen consumption was approximately 13.0 gm. (12.86 to 13.10 gm.) daily. In the first and third periods the energy values of the diets were 1,780 and 1,971 calories respectively. In the second 580 Endogenous Purine Metabolism. II or high calorific period, an increase to 3,433 calories daily was made by the liberal addition of carbohydrates and fats. The data indicate that in each experiment the change to the ration of greater calorific value was accompanied by a rise in uric acid elimination. In Experiment 15, the increase was from an average TABLE V. The Influence on Uric Acid Excretion of Diets High and Low in Calories, when the Protein Intake is Constant. Experiment 16. Subject J. B. F. Body | Urine | Reaction) Total | Uri Date. eight. olive: niches N. Bid: Remarks. 1917 kg. ce. gm. gm. Dec. 7 | 65.2 975| Acid. | 11.81) 0.42 | Low calorific diet, No. 7. Py SS 840; “ 11.48) 0.45 | 12.91 gm. N and 1, 780 calo- ee 880) “ 12.96) 0.44 | ries daily.* ‘10 LE O70le 12.60) 0.48 joe 1] 74 0) 13.00 0.58 satan | 1,040; “ 12.26) 0.52 FASVCTAL Ceo a. ais, dncrteee oe eee 12.35} 0.48 Dee. 13 | 64.3 720) Acid. | 11.51] 0.57 | High calorific diet, No. 8. i,” ode {a0 © tes 10.21) 0.59 | 12.86 gm. N and 3,433 calo- of GES 1; ASb | 10.69, 0.64 | ries daily. in 6 17 (4 | en 9.26) 0.55 AMOETAGC® occctocent cle bless oe ale LOS42 | ROE Dee. 17 | 65.5 | 1,360| Acid. | 10.67| 0.59 | Low calorific diet, No. 7, sales | 1,040] “ 10.96] 0.52 | plus 300 gm. apples.‘ 13.10 e AS 920} “ 11.68} 0.59 | gm. N and 1,971 calories = 20 | 850} “ 11.92) 0.62 | daily. ASV CTAL Cons sacan she eee 11.31) 0.58 * Began eating this diet on Dec. 3. { The apples were added because of their laxative properties. of 0.48 gm. daily in the low period, to an average of 0.56 gm.in the high period. In Experiment 16, the increase was quantita- tively similar. In each experiment the rise in uric acid elimina- tion amounted to 17 to 18 per cent. A third investigation upon another subject gave results entirely comparable in every particu- lar to the two here reported. W. C. Rose 581 In each experiment of this type, as contrasted with those in which the energy value of the food was maintained constant while the protein intake was varied (Tables II and III), there was no decrease in uric acid elimination during the 4 days of . the period inwhich a return was made to the low calorific diet. Uric acid continued to be excreted at the higher level established upon the diet of greater energy value. Perhaps the storage of carbohydrates and fats upon the high calorie diet was sufficiently large to supply an abundance of energy-yielding food material during the after period, and thus temporarily prevent the effects of a return to the low ration becoming apparent in the uric acid excretion. We did not determine how long this condition would persist before the output would again decrease to the low diet ‘level. Experiments 19 and 20 are illustrative of the effects brought about by alternately feeding low protein-low calorific and high protein-high calorific diets. Each of these experiments was con- tinued over four periods—two upon the low and two upon the high ration—so that each increase and decrease in uric acid elimination is reproduced a second time. The data are given in Tables VI and VII. The food consumption was identical in both experiments. The low ration consisted of 4.75 gm. of nitrogen and 1,978 calories per day; the high, of 27.45 gm. of nitrogen and 3,907 calories per day. As shown in the tables, the change from a ration low in protein and calories to one having a high protein and calorific content is in every instance attended by an increase in uric acid excretion. On certain days this is relatively quite large, as for example on the 15th, 16th, and 27th in each experiment. On the latter date the urines of both sub- jects contained rather large quantities of uric acid crystals. Like- wise, the change from a high to a low diet is associated with a prompt and distinct drop in uric acid elimination, as for instance on the 22nd in each experiment. The averages for the periods manifest an unmistakable relation- ship between uric acid excretion and the character of the food, but we believe the figures for the individual days are more instruc- tive than are the averages. A study of the data indicates that the most decided alterations in uric acid output associated with changes in the quantity of protein, occur almost invariably on 582 TABLE VI. The Influence of a High Protein-High Calorific Diet and a Low Protein-Low Calorific Diet on Uric Acid Excretion. Experiment 19. Subject H. L. B. Endogenous Purine Metabolism. II ; Tr Reaction : Body | Urine Total U Date. ae A ne Ged N- Enid Remarks. 1918 | kq. cc. gm. gm, Nov. 11 | 66.4 | 680, Acid. 5.08 0.44 | Low protein-low calorific Reo wt De [s GZOlh Pay 4.37; 0.46 | diet, No.9. 4.75 gm. N and ale: G70 4 5.06) 0.43 1,978 calories daily.* Sees 680) se 4.96, 0.438 AVETACE: jee oe em eee 4.87| 0.44 Nov. 15 | 65.9 | 960! Acid. | 12.56] 0.55 | High protein-high calorific Succ LG 1, 160 sf 12.92) 0.58 diet, No. 10. 27.45 gm. N gare ly | 1, 420 " 18.17; 0.49 | and 3,907 calories daily. ALS | 1, 610) a 19.08) 0.51 < glS 1, 400 sf 20.08} 0.51 20 1, 230 ae 20.79) 0.54 eae 1, 470) ne 21.34) 0.56 AN CTAGE 2 isis le. ice. RE LTO RODS Nov. 22 | 68.2 | 1,490. Acid. | 12.56) 0.42 | Low. protein-low calorific «93 s4o| « | g.g210.43| diet, No. 9. 4.75 gm. N «og | 820“ 7.22| 0.42 | and 1,978 calories daily. ao S800 - 7.69) 0.49 26 | AO) aes 7.14] 0.58 AV CTA PCR I Ao. sc:t acters 8.69} 0.47 ~ Noy. 27) 66.4! 1,030) Acid. | 15.96] 0.714] High protein-high calorific “« 98 | 1,220; “ | 18.54| 0.53] diet No. 10. 27.45 gm. N i 20 | IP 240) |e 18.73) 0.49 | and 3,907 calories daily. Ai, | | 1, 360) M 25.18} 0.49 ALVETA DO hs xs nhe ee ereNSeee 19.60) 0.56 * Began eating this diet on Nov. 4. + Urine contained a deposit of uric acid crystals on this day. the first day after the inauguration of the dietary changes. After the initial maximum variations, there appears to be a general tendeney to gradual recovery from the effects of the sudden This is particularly noticeable in the alteration in type of food. W. C. Rose 583 TABLE VII. The Influence of a High Protein-High Calorific Diet, and a Low Protein- Low Calorific Diet on Uric Acid Excretion. Subject J. 8S. D. Bod Urine Reaction T 1 Uri y Date. Sere: Ealaie: ee we aia Remarks. 1918 kg cee gm gm 4 Nov. 11 | 60.2 | 1,070) Acid. | 4.75] 0.42 | Low protein-low calorific Samer 600 4.41] 0.41 | diet, No. 9. 4.75 gm. N haa |: 1,080 s 5:42) 0.49 | and 1,978 calories daily.* “14 700 es 4.12) 0.39 Average....... RR Tee ae 4.68] 0.48 Nov. 15 | 60.0 | 1,380) Acid. | 13.23) 0.67 | High protein-high calorific ro LG 1, 830 se 16.21 0.59 diet, No. 10. 27.45 gm. N a 26 ( |e 16.52) 0.50 | and 3,907 calories daily. els 1,200) ‘ 17.28} 0.47 SMe ke) 1,190) <“ 19.07| 0.51 0) 1,310 fs 21.13) 0.52 “91 1,470} “ | 21.67] 0.56 MAVCT AGEs... cancerceveside ee bases 17.87| 0.55 Nov. 22 | 61.6 | 1,595) Acid. | 12.19} 0.37 | Low protein-low calorific neo 500) “ 7.48| 0.34 | diet, No. 9. 4.75 gm. N re24. 7D) 7.48) 0.33 | and 1,978 calories daily. en 590) “* 6.34/ 0.39 eeee26 998} “ 6.85} 0.44 PACT ELE Chi lcvah steel eral Maa set soeves 8.07) 0.37 Nov. 27 | 59.8 810) Acid. | 12.01) 0.667; High protein-high calorific Bo 2S TOON. ise 18.09] 0.55 | diet, No. 10. 27.45 gm. N OF Wi 2Q 1,740) - * 17.44) 0.52 | and 3,907 calories daily. FS 2, 020 oe 21.49) 0.46 PA ViCTALC Way set Wate ots Re 17.26 0.55 * Began eating this diet on Nov. 4. 7 Urine contained a deposit of uric acid crystals on this day. last periods of Experiments 19 and 20. In Experiment 19, the uric acid figures for the 4 days of the final period are 0.71, 0.53, 0.49, and 0.49 gm. respectively, showing that the effect upon uric acid became less pronounced the longer the diet was ingested. 584 Endogenous Purine Metabolism. IT Experiment 20 manifests a similar behavior. In this subject, the figures for uric acid for the last 4 days of high diet are 0.66, 0.55, 0.52,.and 0.46 gm. respectively. Furthermore, the changes in urie acid excretion are more marked during the latter half than during the first half of Experi- ments 19 and 20. Apparently the influence became exaggerated in these experiments in proportion to the frequency with which the alterations in diet were instituted. Thus the increase from the average output of the first period to the first day of the second period, is not so pronounced as is the increase from the average elimination of the third period to the first day of the fourth period. The latter amounts to 51 and 78 per cent respectively in the two experiments. While the actual quantities of “extra” uric acid are not large in any of the experiments, yet in proportion to the normal out- put, the changes are appreciable, and in our opinion, leave no room for doubt as to the influence of food upon endogenous purine metabolism. In none of the experiments is there any proportionality between the volume of urine and the quantity of uric acid excreted. DISCUSSION. The interpretation of our results is fraught with difficulty because of unavoidable irregularities, as noted above, in the uric acid data. The following features, however, warrant special comment. (a) An increase in the consumption of purine-free food, either in the form of protein or as non-nitrogenous articles of diet, leads to a small but distinct increase in the daily output of uric acid. This is in accord with the findings of other investi- gators, notably Mares (6), Leathes (7), Smetdnka (8), Taylor and Rose (9), Mendel and Stehle (1), Lewis and Doisy (10), and Host (11). (6) Under the conditions of our experiments, the maximum effect upon uric acid excretion produced by an increase in protein consumption, usually manifests itself upon the first day after the inauguration of the dietary change, and in some cases is followed by a tendency to return to a lower level of elimination with continued use of the high protein ration. (c) Increases or decreases in endogenous uric acid excretion result- ing from abrupt alterations in food consumption, seemingly be- W.. C. Rose 585 come more pronounced the more frequently the changes in diet are instituted (cf. Tables VI and VII). The above results are in some respects quite different from what we had expected to obtain. In the single experiment reported several years ago by Taylor and Rose (9), the uric acid elimination steadily increased upon a high protein diet until it amounted to 0.82 gm. per day, as contrasted with an average of 0.29 gm. for the low protein period. At no time did the uric acid excretion manifest a tendency to decrease, but on the con- trary progressively increased upon the high protein ration. We therefore anticipated that similar effects would be observed in the experiments described in the present communication. Prob- ably the differences are to be accounted for in the quantity of food ingested. In the experiment of Taylor and Rose, an accurate record of food consumption was not attempted, but the subject, for a period of 4 days, ingested as heavily of white of egg as possible. The nitrogen consumption each day amounted to over 40 gem. Furthermore, in the experiment of Taylor and Rose, the diet of the fore period consisted of purified starch and cane- sugar, and hence -was nitrogen-free. The abrupt change from such a diet to one containing an excess of 40 gm. of nitrogen per day, was a far more radical dietary alteration than any which our subjects experienced in the present investigation. In attempting to explain the effects of protein upon uric acid excretion, we are forced to a conclusion similar to that of Lewis, Dunn, and Doisy (3); namely, that at least one of the factors involved is a general stimulation of cellular metabolism by amino- acids. For reasons stated in the preceding paper, we do not believe that this stimulating effect is limited to the digestive glands, or that the increases in uric acid are due to digestive work. If physiological labor is to be held responsible for the effect of diet upon uric acid, it would seem more reasonable to include as contributing factors all of the activities involved in digesting, storing, and metabolizing the foods, rather than to single out one group of activities, as is done in the Mares theory, and attach the responsibility solely to them. We are convinced, however, that physiological activity per se is not the important factor. The work involved in metabolizing the comparatively small doses of amino-acids fed in the experiments of Lewis, Dunn, 586 Endogenous Purine Metabolism. II and Doisy would scarcely have necessitated sufficient cellular wear and tear to account for the increases in uric acid elimination observed by these authors. We believe that a more logical explanation is to be sought in a general accelerating action of amino-acids upon the metabolic processes. Why in our experi- ments this should have apparently become more pronounced the more frequently the changes in diet were instituted, is not evident. Lewis and his collaborators (3) call attention to the fact that probably the causal agents for the cellular stimulation are ‘‘either the amino-acids or their non-nitrogenous rest, a-ketoniec or hydroxy acids.’ Certain facts in our experiments may be of interest in this connection. In spite of irregularities, the data in Tables Vi and VII evidence a general tendency for the uric acid output to be greatest when the temporary retention of nitrogen is most pronounced, and to fall to lower levels when the nitrogen of the food is promptly excreted. In other words, during a condition of plus-balance, uric acid excretion is usually increased. When a state of minus-balance pertains, as for a few days following the change from a high protein to a low protein ration, the elimination of uric acid tends to decrease. We know from the work of Mendel and R. C. Lewis (12) and others, that urea nitrogen, in normal individuals, is promptly excreted. On the other hand, Folin and Denis (13),and Van Slyke and Meyer (14) have shown that amino-acids may be temporarily stored unchanged in the tissues. Folin and Denis (13) make the fol- lowing interesting comment concerning their important discovery :* “The muscles and other tissues as well evidently serve as a storehouse for such reserve materials [amino-acids]. The existence of such a res- ervoir must be taken into account in our theories of protein metabolism. . . . The peculiar lag extending over several days in the establishment of a constant level of nitrogen elimination when extreme changes are made in the nitrogen intake is probably due to a filling or depletion as the case may be of the reservoir.’’ It would thus seem reasonable to assume that in our experi- ments retained nitrogen was in the form of amino-acids, which were being used to fill the “reservoir.” But during the process ? Lewis, Dunn, and Doisy (3), p. 25. 3 Folin and Denis (13), pp. 94 and 95. — W. C. Rose 587 of “filling,” uric acid excretion was at a higher level than during the course of “‘depletion.”” May this not indicate that unchanged amino-acids, rather than their disintegration products, are respon- sible for the stimulating effect upon cellular metabolism? In Chart 1 are shown curves of the uric acid excretion and the state! of nitrogen equilibrium in Experiment 19, Table VI. The curve of nitrogen balance is not strictly correct since we have no data upon alimentary nitrogen loss. Despite this fact, and the irregularities in the uric acid figures, a general interrelationship is manifested. It must be recalled that uric acid is probably the most difficult of the urinary ingredients for the kidneys to excrete, and that accordingly, irregularities in elimination and temporary retentions are more likely to occur in the case of it than of any other metabolic product. Hence a close agreement between the curves would hardly be expected. Experiments 8, 9, and 20 (Tables II, III, and VII) show a similar general pro- portionality between the uric acid output and the nitrogen balance. In regard to the increased excretion of uric acid in Experiments 15 and 16 (Tables IV and V), where the protein consumption was constant, but the calorific values of the diets varied, the interpretation is more difficult. Similar effects of increases in the caloric value of the food have been observed by Hést (11). The protein-sparing action of carbohydrates is indicated in the second period of each of our experiments by a fall in the out- put, of total nitrogen. Doubtless the retained amino-acids were partly responsible for the rise in uric acid, through their stimu- lating action upon cellular catabolism. It is also quite possible that metabolism may be accelerated by intermediary products derived from carbohydrates and fats. We know that these food- stuffs do manifest a stimulating effect upon heat production, but to a more limited extent than do proteins. Another factor which we believe should be taken into con- sideration in a discussion of endogenous purine metabolism, is the possibility of a reutilization of the purines liberated in catabolism. We are in the habit of thinking of these purines as being trans- formed into uric acid and with more or less promptness eliminated. On the other hand, in starvation and in other conditions of physio- logical stress, unusual economy may be exercised in metabolism. ‘aT FL wolqng ‘6, quoumrodxy *T LUVH/ bZ LT Car £7 1% b/ Li SI €! IAN 4$iH usin eouvlog— ross ojo Ol+ a, ‘wh -w§ PD YT on N W. C. .Rose 589 May it not be possible, when there is a deficiency of purine precur- sors (arginine and histidine?; cf. Ackroyd and Hopkins (15), and Harding and Young (16)) in the diet, that a reutilization of purines liberated in tissue wear and tear may occur for synthetic pur- poses? Such an assumption would account for the remarkably low figures for uric acid excretion observed by Raiziss, Dubin, and Ringer (17) in individuals living upon starch-cream diets. Re- utilization might also have been a contributing factor in pro- ducing the low values which we obtained during brief fasts (Tables II and III). Under such conditions urinary uric acid would represent a balance between the formation and conserva- tion of purines. A consideration of all the known facts concerning purine metab- olism, both those discussed in the preceding paper, as well as the experimental data presented in this communication, appears to indicate that endogenous purines have their ultimate origin in arginine and histidine, but that the extent of their synthesis is limited quantitatively to the anabolic needs of the organism. Superfluous molecules of arginine and histidine, which are not required for anabolism, are probably in the adult at least oxidized without preliminary transformation into purines. Under con- ditions of constant diet and nitrogen equilibrium, purine metab- olism, as measured by the uric acid output, proceeds at a fairly constant rate, but this rate may be altered by changes in the character or quantity of food ingested. Amino-acids and prob- ably digestive (or metabolic) products of carbohydrates and fats, exert a general stimulating action upon cellular catabolism, which is manifested by a rise in uric acid elimination following marked increases in food consumption. Moreover, indirect evidence indicates that perhaps in the case of the amino-acids, they them- selves, rather than their nitrogen-free derivatives, are the stimu- lating agents. It is suggested that when the organism is deprived of purine precursors, an additional factor leading to variations in uric acid excretion, may be a reutilization for anabolic purposes of part of the purines liberated in catabolism. Such a working hypothesis, while wholly tentative, serves for the present to explain many apparent contradictions in the literature. We expect to test the possibility of a reutilization of purines in subsequent studies. 590 Endogenous Purine Metabolism. II oo bo BIBLIOGRAPHY. _ Mendel, L. B., and Stehle, R. L., J. Biol. Chem., 1915, xxii, 215. . Neuwirth, I., J. Biol. Chem., 1917, xxix, 477. . Lewis, H. B., Dunn, M. 8., and Doisy, E. A., J. Biol. Chem., 1918, XXXvi, 9. . Rose, W. C., Dimmitt, J. S., and Bartlett, H. L., J. Biol. Chem., 1918, xxxiv, 601. . Benedict, S. R., and Hitchcock, E. H., J. Biol. Chem., 1915, xx, 619. ». Mares, F., Arch. ges. Physiol., 1910, exxxiv, 59. . Leathes, J. B., J. Physiol., 1906-07, xxxv, 125. . Smetanka, F., Arch. ges. Physiol., 1911, exxxviii, 217. . Taylor, A. E., and Rose, W. C., J. Biol. Chem., 1914, xviii, 519. . Lewis, H. B., and Doisy, E. A., J. Biol. Chem., 1918, xxxvi, 1. . Host, H. F., J. Biol. Chem., 1919, xxxviii, 17. 2. Mendel, L. B., and Lewis, R. C., J. Biol. Chem., 1913-14, xvi, 55. 3. Folin, O., and Denis, W., J. Biol. Chem., 1912, xi, 87. . Van Slyke, D. D., and Meyer, G. M., J. Biol. Chem., 1913-14, xvi, 197. . Ackroyd, H., and Hopkins, F. G., Biochem. J.5 1916, x, 551. . Harding, V. J., and Young, E. G., J. Biol. Chem., 1919, xl, 227. . Raiziss, G. W., Dubin, H., and Ringer, A. I., J. Biol. Chem., 1914, Rix, Alo: » INDEX TO VOLUME XLVIII. ACER saccharinum, maple seed, * acerin, the globulin of, 23 Acerin, the globulin of the maple seed (Acer saccharinum), 23 Acetone and diacetic acid, estima- tion of creatinine in the pres- ence, 105 ——, excretion from the lungs, 413 Acid, animal nucleic, preparation and analysis, 177 — -base balance of the blood, normal and abnormal vari- ations in, 153 ——, citric, content of, in milk and milk products, 453 ——, diacetic, and acetone, estima- tion of creatinine in the pres- ence, 105 ——, hippuric, in urine, rapid method for the determination of, 13 —— neutralization, mechanism of, in the animal organism, the bearing of ammonia content of blood on, 463 ——, picric, and tungstic acid methods of deproteinization, comparison, 127 ——, plant nucleic, possible bearing of thymus nucleic acid on the structure, 119 ——, thymus nucleic, structure of, and its possible bearing on the structure of plant nucleic acid, 119 —, tungstic, and picric acid methods of deproteinization, comparison, 127 Acidosis, studies of, 153 591 Acids, amino-, of feeds, quantita- tive determination, 249 —, aminomalic, (para- and anti- hydroxyaspartic acids), synthe- sis of inactive, 273 —, monoamino-, in the hydro- lytic cleavage products of lac- talbumin, determination, 347 ——, nucleic, influence on the me- tabolism of fasting dogs, 523 : - on the metabolism of the fasting rabbit, 537 —, para- and anti-hydroxyas- partic, (aminomalic acids), syn- thesis of inactive, 273 —, sugar, numerical values of the optical retations in, 197 Air analysis, a simplified form of apparatus for, 369 ——, expired, for analysis, a gas receiver of convenient and prac- tical form for sampling, 373 Alkali reserve and blood sugar content, influence of thyro- parathyroidectomy upon, 557 experimental shock, 533 Atu, ANTHONY F. See UNDERHILL, GREENBERG, and ALv, 549 Amino-acids of feeds, quantitative determination, 249 Aminomalic acids (para- and anti- hydroxyaspartic acids), synthe-— sis of inactive, 273 Ammonia content of blood and its bearing on the mechanism of acid neutralization in the ani- mal organism, 463 Analysis, air, a simplified form of apparatus for, 365 592 Analysis, expired air for, a gas re- ceiver of convenient and prac- tical form for sampling, 373 — and preparation of animal nucleic acid, 177 ANDERSON, J. A. See FRED, PETER- son, and ANDERSON, 385 ANDERSON, R. J. Acerin. The globulin of the maple seed (Acer saccharinum), 23 Animal nucleic acid, preparation and analysis, 177 —— organism, mechanism of acid neutralization in, bearing of ammonia content of blood on, 463 Antiscorbutic vitamine, effect of heating, in the presence of invertase, 323 Apparatus for air analysis, a simpli- fied form, 365 ——,, thyroid, studies of, 148 Arabinose and xylose, character- istics of certain pentose-de- stroying bacteria especially as concerns their action on, 385 BACTERIA as a source of the water-soluble B vitamine, 379 — and certain yeasts, vitamine requirements, 437 ——, pentose-destroying, character- istics of certain, especially as concerns their action on ara- binose and xylose, 385 Base, acid-, balance of the blood, normal and abnormal ations in, 153 Bavupiscu, Oskar. The mechan- ism of reduction of nitrates and nitrites in processes of assimi- lation, 489 Beuuis, B. BE.LLIs, 453 Bence-Jones protein, note on a possible source of error in testing for, 21 See SuprpLeEE and vari- - Index Benepict, STanuey R., and OstTER- BERG, Emin. A method for the determination of sugar in nor- mal urine, 51 —. See Nasu and Brenepict, 463 Buiavu, NatHan F. The estimation of creatinine in the presence of acetone and diacetiec acid, 105 Blood, ammonia content of, and its bearing on the mechanism of acid neutralization in the animal organism, 463 ——., catalase content of, effect of subcutaneous injections of solu- tions of potassium cyanide on, 445 —, direct quantitative determi- nation of sodium, potassium, calcium, and magnesium in small amounts, 223 —, normal and abnormal vari- ations in the acid-base balance, 153 . —— some pathological human, potassium content, 83 —— phosphate, inorganic, studies in, 293 —— sugar content and alkali re- serve, influence of thyropara- thyroidectomy upon, 557 —— ——,, studies on, 313 —., total amount of circulating sugar in, in diabetes mellitus and other conditions, 313 Bock, ARIE V. See Firz and Bock, 313 Bopansky, Meyer. The zine and copper content of the human brain, 361 Bouiman, J. L. See Wetker and BoLiMAN, 445 Brain, human, zine and copper content of, 361 Briaes, A. P., and SHaArreR, PHILIP A. The excretion of acetone from the lungs, 413 Index (CABBAGE, orange juice, cod liver oil, and green and dried plant tissue on calcium assimilation, comparative influence, 33 Calcium assimilation, comparative influence of green and dried plant tissue, cabbage, orange juice, and cod liver oil on, 33 , dietary factors influenc- ing, 33 —, magnesium, sodium, and potassium in small amounts of blood, direct quantitative de- termination, 223 —, ; , and potassium in urine and stools, methods for the direct quantitative deter- mination, 1 Carbohydrate content of the king salmon tissues during the spawning migration, 429 Catalase content of blood, effect of subcutaneous injections of potassium cyanide on, 445 Chemical development of the ova- ries of the king salmon during the spawning migration, 59 — study of the California sardine (Sardinia cerulea), 93 — certain Pacifie Coast fishes, 73 Citric acid content of milk and milk products, 453 Cod liver oil, green and dried plant tissue, cabbage, and orange juice on calcium assimilation, comparative influence, 33 Collodion membranes, preparation and standardization, 203 Copper and zine content of the human brain, 361 Creatinine and creatine in muscle extracts, 127, 133 in the presence of acetone and diacetic acid, estimation, 105 Creatinine-creatine balance in incu- bated extracts of muscle tissue 593 of the albino rat, influence of parathyroid and thyroid tissue on, 143 a incubated extracts of muscle tissue of the albino rat, influence of the reaction of the medium on, 133 Creatine, creatinine-, balance in incubated extracts on muscle tissue of the albino rat, influ- ence of parathyroid and thyroid tissue on, 143 —_, — , in incubated ex- tracts of muscle tissue of the albino rat, influence of the reaction of the medium on, 133 — and creatinine in muscle extracts, 127, 133 Cyanide, potassium, solutions of, effect of subcutaneous injec- tions of, on the catalase content of blood, 445 AKIN, H. D. The synthesis of inactive para- and anti- hydroxyaspartic acids (amino- malic acids), 273 Damon, SAMUEL R. Bacteria as a source of the water-soluble B vitamine, 379. Deproteinization, comparison of the picric acid and tungstic acid methods, 127 Determination, direct quantitative, of sodium, potassium, calcium, and magnesium in_ small amounts of blood, 223 ——, — — of sodium, potas- sium, calcium, and magnesium _in urine and stools, methods for, 1 —— of creatinine in the presence of acetone and diacetic acid, 105 —— —— hippuric acid in urine, rapid method for, 13 594 Index Determination of the monoamino- acids in the hydrolytic cleav- age products of lactalbumin, 347 sugar in normal urine, method for, 51 ——, quantitative, of amino-acids of feeds, 249 Diabetes mellitus, total amount of circulating sugar in the blood in, 313 Diacetie acid and acetone, estima- tion of creatinine in the pres- ence, 105 Dietary factors influencing calcium assimilation, 33 Ditut, D. B. A chemical study of the California sardine (Sardinia cerulea), 93 —. A chemical study of certain Pacific Coast fishes, 73 Dupin, Harry E. See Funk and Dustin, 437 J{GGERTH, Arnotp H. The preparation and standardi- zation of collodion mem- branes, 203 Esters, phosphoric, of some sub- stituted glucoses and their rate of hydrolysis, 233 Excretion of acetone from the lungs, 413 Extracts, incubated, of muscle tis= sue of the albino rat, influence of parathyroid and _ thyroid tissue on the creatinine- creatine balance in, 143 ,— muscle tissue of the albino rat, influence of the reaction of the medium on the creatinine-creatine balance in, 133 —, muscle, creatinine and crea- tine in, 127, 133 FASTING dogs, metabolism of, in- fluence of nucleic acids on, 523 Fasting dogs, metabolism of, influ- ence of various protein split products on, 503 — rabbit, metabolism of, influ- ence of nucleic acids on, 537 —— ——,, —— of, influence of some protein split products upon, 549 Feeds, amino-acids of, quantita- tive determination, 249 Fishes, certain Pacifie Coast, chemi- cal study, 73 Fitz, ReGInALp, and Bock, ARLIE V. Studies on blood sugar. The total amount of circulating sugar in the blood in diabetes mellitus and other conditions, 313 Food ingestion upon endogenous purine metabolism, influence, 563, 575 Frep, E. B., Prererson, W. H., and ANDERSON, J. A. Char- acteristics of certain pentose- destroying bacteria especially as concerns their action on arabinose and xylose, 385 Funk, Casimir, and Dustin, Harry E. Vitamine requirements of certain yeasts and bacteria, 437 AS receiver of convenient and practical form for sampling expired air for analysis, 373 Globulin of the maple seed (Acer saccharinum) acerin, 23 Glucoses, phosphoric esters of some substituted, and their rate of hydrolysis, 233 GREENBERG, Pattie. See UNDER- HILL, GREENBERG, and ALU, 549 GREENE, CHARLES W. Carbohy- drate content of the king salmon tissues during the spawning migration, 429 —. Chemical development of the ovaries of the king salmon dur- ing the spawning migration, 59 tie on lh Index GRINDLEY, H. 8S. See HaAmiLton, NEVENS, and GRINDLEY, 249 GUTHRIE, CHARLES CLAUDE. A gas receiver of convenient and prac- tical form for sampling expired air for analysis, 373 A simplified form of appa- ratus for air analysis, 365 H4MILTON, T. S., NEvEns, W. B., and Grinpuey, H. S. The quantitative determina- tion of amino-acids of feeds, 249 “Hammett, FREDERICK S. Crea- tinine and creatine in muscle extracts.. I. A comparison of the picric acid and the tungstic acid methods of deproteini- zation, 127. II. The influence of the reaction of the medium on the creatinine-creatine bal- ance in incubated extracts of musele tissue of the albino rat, 133 Studies of the thyroid appa- ratus. IV. The influence of parathyroid and thyroid tissue on the creatinine-creatine bal- ance in incubated extracts of muscle tissue of the albino rat, 143 Hart, E. B., and Humpurey, G. C. Can “home grown rations” supply proteins of adequate quality and quantity for high milk production? III, 305 ——, STEENBOCK, H., and Hoprert, C. A. Dietary factors influ- encing calcium assimilation. I. The comparative influence of green and dried plant tissue, cabbage, orange juice, and cod liver oil on calcium assimila- tion, 33 Heating the antiscorbutic vitamine in the presence of invertase, 323 595 Hippuric acid in urine, determina- tion of, rapid method for, 13 Hoprert, C. A. See Hart, STEEN- BocK, and Hopprrt, 33 Humpurey, G. C. See Hart and Humpurey, 305 Hydrolysis, rate of phosphoric esters of some substituted glu- coses, 233 Hydrolytic cleavage products of lactalbumin, determination of the monoamino-acids in, 347 Hydroxyaspartie acids, para- and anti-, (aminomalic acids), syn- thesis of inactive, 273 [ NGESTION, food, influence upon endogenous purine metabolism, 563, 575 Injections, subcutaneous, of solu- tions of potassium cyanide, effect of, on the catalase content of blood, 445 Inorganic blood phosphate, studies in, 293 Invertase activity of yeast, effect of certain stimulating sub- stances on, 329 ——.,, effect of heating the antiscor- butic vitamine in the presence, 328 JOHNS, Cart O. See Jones and JOHNS, 347 JoNnEs, D. BreEse, and JOHNS, Cart O. Determination of the monoamino-acids in the hydro- lytic cleavage products of lact- albumin, 347 KINGSBURY, F. B., and Swan- son, W. W. A rapid method for the determination of hip- puric acid in urine, 13 KRAMER, BENJAMIN, and TISDALL, FREDERICK F, The direct quantitative determination of 596 calcium, small sodium, potassium, and magnesium in amounts of blood, 223 —. See TispALu and KRaAm_Er, 1 LACTALBUMIN, monoamino- acids in the hydrolytic cleav- age products of, determination, 347 Lecithin, liver, 185 Lepman, Epwin P. Studies in inorganic blood phosphate, 293 LEvENE, P. A. On the numerical values of the optical rotations in the sugar acids, 197 On the structure of thymus nucleic acid and on its possible bearing on the structure of plant nucleic acid, 119 Preparation and analysis of animal nucleic acid, 177 — and Meyer, G. M. Phos- phorie esters of some substi- tuted glucoses and their rate of hydrolysis, 233 — and Simms, H. S. The liver lecthin, 185 Liver lecithin, 185 Lone, Mary Lovisa. HILL and Lone, 537 Lungs, excretion of acetone from, 413 See UNDER- MAGNESIUM, | sodium, _potas- sium, and calcium in small amounts of blood, direct quan- titative determination, 223 —., , ——, and calcium in urine and stools, methods for the direct quantitative deter- mination, 1 Maple seed (Acer saccharinum), acerin, the globulin of, 23 Mepes, Grace. See SmitH and MeEpEs, 323 Medium, influence of the reaction of, on the creatinine-creatine Index balance in incubated extracts of muscle tissue of the albino rat, 133 Membranes, collodion, preparation and standardization, 203 Metabolism, endogenous purine, influence of food ingestion upon, 563, 575 — of fasting dogs, influence of nucleic acids on, 523 dogs, influence of various protein split products on, 503 —— —— the fasting rabbit, influ- ence of nucleic acids on, 537 —— —— fasting rabbits, influence of some protein split products upon, 549 Method for the determination of sugar in normal urine, 51 —, rapid, for the determination of hippuric acid in urine, 13 Methods for the direct quantita- tive determination of. sodium, potassium, calcium, and mag- nesium in urine and stools, 1 — of deproteinization, compari- son of picric and tungstie acid, 127 Meyer, G. M. MEYER, 233 Milk and milk products, acid content, 453 — production, high, can home grown rations supply proteins of adequate quality and quan- tity for, 305 —— products and milk, citric acid content, 453 MitueER, C. W., and Sweet, J. E. Note on a possible source of error in testing for Bence-Jones protein, 21 Miter, EvizasetH W. The effect of certain stimulating sub- stances on the _ invertase activity of yeast, 529 See LEvENE and citric Index 597 Monoamino-acids in the hydrolytic cleavage products of lactalbu- min, 347 Muscle extracts, creatinine and creatine in, 127, 133 —— tissue of the albino rat, incu- bated extracts of, influence of parathyroid and thyroid tissue on the creatinine-creatine bal- ance in, 143 —— —— —— the albino rat, incu- bated extracts of, influence of the reaction of the medium on the creatinine-creatine balance in, 133 Myers, Victor C., and Snort, JAMES J. The potassium con- tent of normal and some patho- logical human bloods, 83 NASH, THomas P., Jr. and BENEDICT, STANLEY R. The ammonia content of the blood and its bearing on the mecha- nism of acid neutralization in the animal organism, 463 NELLANS, CHARLES T. See UNDER- HILL and NELLANS, 557 Nevens, W. B. See Hamitton, NEVENs,.and GRINDLEY, 249 Nitrates and nitrites in process of assimilation, mechanism of reduction, 489 Nitrites and nitrates in process of assimilation, mechanism of reduction, 489 Nucleic acid, animal, preparation and analysis, 177 —— -——, plant, possible bearing of thymus nucleic acid on the structure, 119 , thymus, structure of, and its possible bearing on the structure of plant nucleic acid, 119 — acids, influence on the meta- bolism of fasting dogs, 525 Nucleic acids, influence on the metabolism of the fasting rab- bit, 537 QL, cod liver, green and dried plant tissue, cabbage, and orange juice on calcium assimi- lation, comparative influence, 33 Orange juice, cod liver oil, green and dried plant tissue, and cabbage on calcium assimila- tion, comparative influence, 33 Organism, animal, mechanism of acid neutralization in, bearing of ammonia content of blood on, 463 OsTERBERG, Emin. See BENEDICT and OSTERBERG, 51 Ovaries of the king salmon during the spawning migration, chemi- cal development, 59 PARATHYROID and thyroid tis- sue on the creatinine-creatine balance in incubated extracts of muscle tissue of the albino rat, 143 Pentose-destroying bacteria, char- acteristics of, especially as concerns their action on ara- binose and xylose, 385 Peterson, W. H. See FRED, PETER- son, and ANDERSON, 385 Phosphate, inorganic blood, studies in, 293 Phosphoric esters of some _ sub- stituted glucoses and their rate of hydrolysis, 233 Physiological action of some protein derivatives, studies on, 503, " 523, 533, 537, 549 Picric acid and tungstic acid meth- ods of deproteinization, com- parison, 127 598 Index Plant nucleic acid, possible bearing of thymus nucleic acid on the structure, 119 —— tissue, green and dried, cab- bage, orange juice, and cod liver oil on calcium assimila- tion, comparative influence, 33 Potassium, calcium, magnesium, and sodium in small amounts of blood, direct quantitative de- termination, 223 ; ‘1 , and sodium in urine and stools, methods for the direct quantitative determina- tion, 1 — content of normal and some pathological human bloods, 83 — cyanide, solutions of, effect of subcutaneous Injections of, on the catalase content of blood, 445 Protein, Bence-Jones, note on a possible source of error in testing for, 21 — derivatives, physiological ac- tion of some, studies on, 503, 523, 533, 537, 549 —— split products, influence upon the metabolism of fasting dogs, 503 —— —— ——.,, influence upon the metabolism of fasting rabbits, 549 Proteins of adequate quality and quantity for high milk produc- tion, can home grown rations supply, 305 Purine metabolism, endogenous, influence of food ingestion upon, 563, 575 RESERVE, alkali, and blood sugar content, influence of thyro- parathyroidectomy upon, 557 ; experimental shock, 533 Rincer, Micwarn, and UNDER- HILL, FRANK P. Studies on the physiological action of some protein derivatives. VII. The influence of various protein split products on the metabolism of fasting dogs, 503. VIII. The influence of nucleic acids on the metabolism of fasting dogs, 523 —. See UNDERHILL and RINGER, 533 Rose, Witi1am C. Influence of food ingestion upon endogenous purine metabolism. I, 563. II, 575 SARDINIA crerulea, California sardine, chemical study, 93 Seed, maple (Acer saccharinum), acerin, the globulin of, 23 Shock, experimental, and alkali reserve, 533 SHort, JAMes J. See Myers and SHort, 83 Stmms, H. S. See Levene and Srums, 185 SmirH, Erma, and Mrepss, GRACE. Effect of heating the antiscor- butic vitamine in the presence of invertase, 323 Sodium, potassium, calcium, and magnesium in small amounts of blood, direct quantitative determination, 223 —, —, —, and magnesium in urine and stools, methods for the direct quantitative determination, 1 Solutions of potassium cyanide, effect of subcutaneous injec- tions of, on the catalase con- tent of blood, 445 Standardization and preparation of collodion membranes, 203 — Index SrrEnsock, H. See Hart, STEEN- BocK, and HoppeErt, 33 Stimulating substances on the in- vertase activity of yeast, effect of certain, 329 Stools and urine, sodium, potas- sium, calcium, and magnesium in, methods for the direct quantitative determination in, 1 Subcutaneous injections of solu- tions of potassium cyanide, effect of, on the catalase con- tent of blood, 445 Sugar acids, numerical values of the optical rotations in, 197 ——,, blood, studies on, 313 — content, blood, and alkali reserve, influence of thyro- parathyroidectomy upon, 557 — in the blood in diabetes mel- litus and other conditions, total amount of circulating, 313 —— —— normal urine, method for the determination, 51 SuppLee, G. C., and Bettis, B. Citric acid content of milk and milk products, 453 Swanson, W. W. See KINGSBURY and Swanson, 13 Sweet, J. E. See Minuer and SWEET, 21 ‘TESTING for Bence-Jones: pro- tein, note on a possible source of error, 21 Thymus nucleic acid, structure of, and its possible bearing on the structure of plant nucleic acid, 119 Thyroid apparatus, studies of, 143 — and parathyroid tissue on the creatinine-creatine balance in incubated extracts of muscle tissue of the albino rat, influ- ence, 143 599 Thyroparathyroidectomy, influence upon blood sugar content and alkali reserve, 557 TIsDALL, FREDERICK F., and Kramer, BensamMin. Methods for the direct quantitative determination of sodium, potas- sium, calcium, and magnesium in urine and stools, 1 See Kramer and TISDALL, 223 Tissue, incubated — extracts of muscle, of the albino rat, influence of parathyroid and thyroid tissue on the creatinine balance in, 143 —, muscle, of the albino rat, incubated extracts of, influence of the reaction of the medium on the creatinine-creatine bal- ance in, 133 —., parathyroid and thyroid, on the creatinine-creatine balance in incubated extracts of muscle tissue of the albino rat, influ- ence, 143 ——, plant, green and dried, cab- bage, orange juice, and cod liver oil on calcium assimila- tion, comparative influence, 33 Tissues, king salmon, carbohydrate content of, during the spawning migration, 429 Tungstic acid method of deprotein- ization, comparison of picric acid method with, 127 UNDERHILL, Frank P., and Lone, Mary Louisa. Stud- ies on the physiological action of some protein derivatives. X. The influence of nucleic acid on the metabolism of the fast- ing rabbit, 537 —— and Newuans, CHaries T. The influence of thyropara- 600 thyroidectomy upon blood sugar content and alkali reserve, 557 UNDERHILL, FRANK P., and RINGER, MicuareL, Studies on the physi- ological action of some protein derivatives. IX. Alkalireserve and experimental shock, 533 ——, GREENBERG, PHILIP, and ALU, AntTHony F. Studies on the physiological action ‘of some protein derivatives. XI. The influence of some protein split products upon the metabolism of fasting rabbits, 549 —. See RincerR and UNDER- HILL, 503, 523, Urine, hippuric acid in, rapid method for the determination of, 13 —, normal sugar in, method for the determination, 51 and stools, sodium, potassium, calcium, and magnesium in, methods for the direct quanti- tative determination in, 1 VAN SLYKE, Donatp D. Studies of acidosis. XVII. The nor- mal and abnormal variations in the acid-base balance of the blood, 153 ; Index Vitamine, antiscorbutic, effect of heating, in the presence of invertase, 323 —— requirements of certain yeasts and bacteria, 437 , water-soluble B, bacteria as a source, 379 WATER-SOLUBLE B vitamine, bacteria as a source, 379 WELKER, WiLuIAm H., and Bot- MAN, J. L. The effect of sub- cutaneous injections of solutions of potassium cyanide on the catalase content of the blood, 445 XYLOSE and arabinose, char- acteristics of certain pentose- destroying bacteria especially as concerns their action on, 385 YEAST, invertase activity of, effect of certain stimulating substances, 329 Yeasts, certain, and bacteria, vita- mine requirements, 437 ZINC and copper content of the human brain, 361 AN OUTFIT FOR Basal Metabolism Estimations AS DESIGNED BY Dr. Cameron Vernon Bailey of the Laboratory of Pathological Chemistry New York Post Graduate Medical School and Hospital . Vie ™ SINAMADANAAS ANALY SEERA TES BO = GiGe Gees = AAAAURAAARARANUA DAREN For detailed description see September issue |Z Journal of Laboratory and Clinical Medicine Wh \\\\\\\ SSS \\\ \ \ RTT] REPRESENT QUALITY OF HIGHEST STANDARD “MANUFACTURED BY C. M. SORENSEN CO., Inc. DEPT. K 177 East 87th Street New York or In buying e } RARE SUGARS Specify the STANDARD OF PFANSTIEHL The highest possible purity, containing no sulphates, chlorides, Al, Ca, or heavy metals and a minimum of can and water Neutral to methyl red. Solution perfectly C.P. SPECIAL Strength and Pliability are Found in Armour’s lodized Sheep Gut Ligatures which are made from lamb’s gut selected in our abattoirs especially for surgical purposes. The Armour Iodized Ligatures possess full tensile strength and their pli- ability, prevents breakage at the knot. They are iodized to the core and are absolutely sterile. Regular lengths, sizes 00 to number 4 at $2.50 per dozen. We also offer Plain and Chromic Ligatures, sizes 000 to number 4 regular lengths 2. 50 per dozen, emergency lengths, £1.50 per dozen (nothing but the smooth side of the intestine is used in the manu- facture of the Armour Ligatures). Suprarenalin Solution, 1:1000 is stable, uni- form and free from preservatives. Pituitary Liquid is physiologically standardized and is ready for hypodermatic use—4 c. c. ampoules = for obstetrical and 1 c.c. ampoules for surgical use. Literature upon the ARMOUR LABORATORY PRODUCTS for the medical profession only. ARMOUR sxc COMPANY CHICAGO Headquarters for the erdocrines and other organotherapeutic products. LaMotte Standards GUARANTEED Section 1.—Standardized Indicator dyes. Covering a wide range of H-ion con- centration. Supplied in dry form and in sterile stock solutions. Each indicator is standardized in strict accordance with the specifications of Clark & Lubs. (Jr. Bact., Vol. II, Nos. 1, 2, 3, 1917.) Common Name Color Change Pu Value Thymol Blue (acid range) red-yellow 1.2-2:8 Methyl! Orange red-yellow 2.9-4.0 Bromphenol Blue yellow-blue 3.04.6 Resorcin Blue pink-blue 4.0-7.2 Methyl Red red-yellow 4.4-6.0 Bromcresol Purple yellow-purple 5.2-6.8 Litmus (special) red-blue 5.5-8.9 Bromthymol Blue yellow-blue 6.0-7.6 Phenol-Red yellow-red 6.8-8.4 Cresol Red yellow-red 7.2-8.8 Thymol Blue (alkaline range) yellow-blue 8.0-9.6 Cresol-phthalein colorless-red 8.2-9.8 Phenol-phthalein colorless-red 8.4-9.2 Section 2-B.—Specially prepared and standardized Buffer salts and solutions. Buffer mixtures may be obtained in series covering any particular range of H-ion concentration from Px 1.0 to 10.0. Standardized Buffer Solutions (M/5) Potassium Phosphate Potassium Phthalate Sodium Hydroxide (CO: free) Potassium Chloride Hydrochloric Acid Di-Sodium Phosphate, 2H:0 A large number of general synthetic and purified compounds are manufactured by us and information concerning them may be obtained by addressing LA MOTTE CHEMICAL PRODUCTS CO. *‘Standards Department”’ 13 W. Saratoga St. Baltimore, Md. INTERNATIONAL CENTRIFUGES Holds Any Standard Razor Blade Inexpensiv Convenience for Laboratories Will Razor Blade Holder For histological, botanical and chemical laboratories. Indispensable for taking tissue sections in histological work; plant and drug sections in botanical and pharmacognostic examinations; tissue work in clinical laboratories; of great assistance in food and drug examinations. Made of nickel plated steel. Blade Lat est model, self-balancing cen- easily inserted and removed. trifuge with brake and brush re- lease, and removable top adapted Admirable individual equipment for students. to use with perforated basket. PRICE 60c Discount in quantities. International Equipment Co. pie mal Corporation 352 Western Avenue (Brighton) WwW - CHEMICHLS LABORATORY APPARATUS Boston 35, Mass. ROCHESTER . N.Y. Catalogue Cy sent on Request ~I The Journal of General Physiology Edited by JACQUES LOEB, New York, N. Y. W. J. V. OSTERHOUT, Cambridge, Mass. The Journal of General Physiology is devoted to the explanation of life phenomena on the basis of the physical and chemical consti- tution of living matter. , The Journal of General Physiology is issued bimonthly, one volume of about 600 pages appearing in a year. Contributions should be sent to the editors of The Journal of General Physiology, The Rockefeller Institute for Medical Research, Avenue A and 66th Street, New York, N. Y., or 60 Buckingham Street, Cam- bridge, Mass. The papers should be limited preferably to twenty printed pages, not counting the space occupied by illustrations. Authors receive 100 reprints of their papers free of charge; addi- tional copies may be obtained at cost. The subscription price per year (one volume) is $5.00, payable in advance. Single copies cost $1.00. Remittances should be made by draft or check on New York, or by postal money order, payable to The Journal of General Physi- ology, Mount Royal and Guilford Avenues, Baltimore, Md., or Avenue A and 66th Street, New York, N. Y. CONTENTS OF VOL. IV, No. 1, SEPTEMBER 20, 1921 OsrerHout, W. J. V. Conductivity and permeability. Maxwett, 8. S. Stereotropic reactions of the shovel-nosed ray, IRhinobatus productus. | Maxwett, 8.8. The stereotropism of the dogfish (Mustelus cal/fornicus) and its reversal through change of intensity of the timulus. Moore, A. R. Chemical stimulation of the nerve cord of Lumbricus terrestris. Cuampers, Rorert. The formation of the aster in artificial parthenogenesis. CuaMBers, RoserT. Studies on the organization of the starfish egg. MircHetyt, Pure H., and Witson, J. Water. The selective absorption of potassium by animal cells. I. Conditions controlling absorption and reten- tion of potassium. Norrurop, Joun H. Comparative hydrolysis of gelatin by pepsin, trypsin, acid, and alkali. Logs, Jacques. Donnan equilibrium and the physical properties of proteins. IV. Viscosity—continued. Lors, Jacaurs. The reciprocal relation between the osmotic pressure and the viscosity of gelatin solutions. Published by The Rockefeller Institute for Medical Research Avenue A and 66th Street, New York, N. Y. | S PHYSIOLOGICAL ABSTRACTS Monthly, price 5s. net. Annual Subscription, post free, 40s. This Journal is issued by the PHYSIOLOGICAL SOCIETY, acting in cooperation with numer- ous physiological organisations in Great Britain, America, and other countries. The Editor, Professor Hattisurron, is assisted by a staff of competent abstractors both at home and abroad. The Journal aims at issuing promptly abstracts of the papers published throughout the world, in physiological and allied sciences (including plant physiology). A number is published at the beginning of each month. Volume VI begins with the April (1921) issue. Most of the numbers of preceding volumes can still be ob- tained from the Publishers at the price of 4s. per No. A few numbers which are now scarce are 8s. per No. Subscriptions are only taken for April to March of the next year, and are payable in advance. PUBLISHED BY H. K. LEWIS & CO., Ltd. 28 Gower Place LONDON, W.C. 1 For Chemists, Metallurgists, Biologists, and Bacteriologists The Determination of Hydrogen Ions ~ W. Mansfield Clark, Ph.D. This treatise on the hydrogen electrode, indicator, and supplementary methods of determining hydrogen ion concentrations, with an indexed bib- liography of eleven hundred references on applications, is an important con- tribution to the literature in this field. Are You Interested in analytical methods, in the acidity of your garden soil, in enzymes, in blood and the determination of acidosis, in modern methods of handling protein solutions, in the cultivation and study of bacteria, yeasts, molds and tissue, in autolysis, hydrogen ion catalyses, colloids, the digestive system, filtration, plant distribution, natural waters, the hydrogen ion concentration of biolog- ical fluids? If so, you will need the methods and the references found in The Determination of Hydrogen Ions Limited Edition 318 pages NUMEROUS ILLUSTRATIONS INDICATOR CHART IN COLORS WILLIAMS & WILKINS COMPANY, Publishers WiiuiaMs AND WILKINS Company, Mount Royal and Guilford Avenues, Baltimore, U.S. A. Please enter an order for.......... copy(s) of The Determination of Hydrogen Ions by W. Mansfield Clark, Ph.D. Remittance for $5.00, United States, Mexico, Cuba; $5.25. Canada; $5.50, other countries, is enclosed to cover. (or) Remittance will be made on receipt of your statement. (or) Remittance will be made EMIS ae ea lete ste! corms alsin o'ale sie ,clcie lsie Sates seco nic 1921. THE AMERICAN JOURNAL TROPICAL MEDICINE OFFICIAL ORGAN OF THE AMERICAN SOCIETY OF TROPICAL MEDICINE Editor-in-Chief H. J. NICHOLS Medical Corps, U. S. Army Army Medical School, Washington, D. C. Advisory Editorial Board B. K. ASHFORD G. W. McCoy Medical Corps, U. S. Army, San Juan, Porto Rico Director, Hygienic Laboratory, U.S. P. H.S., C. C. Bass Washington, D.C. Tulane University, New Orleans, La. K. F. MEYER ; M. F. Bovp University of California, San Francisco, Calif. University of Texas, Galveston, Texas B. H. Ransom C. F. Craic Department of Agriculture, Washington, D. C. Medical Corps, U. S. Army, Army Medical School, R. P. STRONG Washington, D. C. Harvard University, Cambridge, Mass. GEoRGE Dock Washington University, St. Louis, Mo. A. J. SMITH 3 A 5 Suck ieee University of Pennsylvania, Philadelphia, Pa. Rockefeller Institute, New York Citv E. R. StrTT F Wirtiam Krauss Surgeon General U.S. Navy, Washington, D. C. Memphis, Tenn. W. S. THAYER : : W. D. McCaw Johns Hopkins University, Baltimore, Md. Asst. Surgeon General, U.S. Army, Army Medical E. J. Woop School, Washington, D. C. Wilmington, N. C. Ex Officio Advisory Editorial Board The American Society of Tropical Medicine J. M. Swan, President GEORGE Dock, Councillor K. F. Meyer, /st Vice-President C. L. FursusH, Councillor V. G. HEIsEr, 2nd Vice-President J. F. SIrer, Councillor S. K. Smuon, Sec’y and Treasurer J. H. Waite, Councillor A. J. Smita, Asst. Sec’y and Treasurer C. S. BuTLER, Councillor Issued Bimonthly; six issues a year, 85 pages, approximately, an issue; one volume a year is planned at present. Price, net postpaid: $5.00, United States and dependencies, Mexico, Cuba; $5.25, Canada; $5.50, other countries. Order from WILLIAMS & WILKINS COMPANY Publishers of Scientific Journals and Books BALTIMORE WaSaA. HE PRESENTATION of essential data concerning vitamines to succeeding groups of students has become increasingly difficult with the development of research in this field. The literature itself has assumed a bulk that precludes sending the student to original sources except in those instances when they are them- selves to become investigators. The demand on the part of the layman for concise information about the new food factors is increasing and worthy of attention. For all of these reasons it has seemed worth while to collate the existing | data and put it in a form which would be avail- ° able for both student and layman. Such is the purpose of The Vitamine Manual. Li e Ai [It HAS been called a manual since the arrange- ment aims to provide the student with working material and suggestions for investiga- tion as well as information. The bibliography, CODY 00 the data in the chapter on vitamine testing, the tables and the subdivision of subject matter have all been arranged to aid the laboratory workers and it is the hope that this plan may that answers the demand make the manual of especial value to the stu- . A ; ‘ dent investigator. The details necessary to t tH € \ laboratory investigation are separated from the or concise In ormation more purely historical aspects of the subject, an arrangement that will be appreciated by the about the new i lay reader as well as the student. N° APOLOGIES are made for data which food factors on publication shall be found obsolete. The whole subject is in too active a state of in- | vestigation to permit of more than a record of events and their apparent bearing. (From the preface of THE VITAMINE MANUAL) | THE VITAMINE MANUAL BY WALTER H. EDDY ASSOCIATE PROFESSOR PHYSIOLOGICAL CHEMISTRY Teachers College, Columbia University The Vitamine Manual will prove useful to Medical Men, Public Health Officers, Dietitians, Nutrition Experts, Chemists, and to all individuals interested in promot- ing correct habits of living and correct habits of diet. The Vitamine Manual will serve as a valuable text for classroom purposes. WILLIAMS & WILKINS COMPANY Publishers of Scientific Journals and Books Wittrams & Wirkins Company, Publishers of Scientific Journals and Books, 3altimore. Maryland, U. S. A. I (or) We enclose S......for......copy (copies) of THE VITAMINE MANUAL. Price, $2.50 2 copy, net postpaid 5S Copics - - - - 10% discount 25 Copies - - - - 15% discount 10 Copies - - - - 12% discount 50 Copies - - - - 20% discount. CSiptied ym N ree Se ii e : eC BP sere Address — BINDING “~~ T, MAY 2 4 1966 QP The Journal of biological 501 chemistry Atads (I fa) ) Biological & Medical Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY mye Saye ee ~~ tere” pa ee te te eee Th tena aed Paes erera are? oe take: Shee rete ee eS th eye: i sbi Hoey AST IG mo. Sern Pr reeeiien ty: - yds Met oe . 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Aarpceheseies tite Satsts ong) mck ; . pie ye abbietecs 534i, tirtereh ss oe meted Shei tetas dhe pe) thsi , G) aby! . ates nt Pe ia age hD yt eh He 4 ESTE RIY = JF tr hot Lernyet meee vepeetap aru hete) mite “ z 33 287 Set Ot = Fapbenseden vs z z 7 0 Eee be oe fans ere ary ene faf ec aren ee bekee nets paneer ewereneee-b—9-0 1005 O Sr sey op -2°8 + 759-54) 277 t oe AIRF Ap gt A rhe Letter Partin 0° $= P28 Serie sty Py ature oe aoe Seite, rer sae os mmeke 47h = c mage HIE Taber ciar ges rate 2 ten : Sane hat FA a a ss CTY ope i yrs get ey : ve Tet ” hes orks ot Sa