~*~ TRANSACTIONS CONNECTICUT. ACADEMY ARTS AND SCIENCES. VOLUME VII. NEW HAVEN: PUBLISHED BY THE ACADEMY. 1885 to 1888. OKKICERS OF YAK ACADEMY, J887-88. ———— PPI President. WILLIAM H. BREWER. Vice-President. CHARLES 8. HASTINGS. Corresponding Secretary. ADDISON VAN NAME. Recording Secretary. RUSSELL H. CHITTENDEN. Librarian. ADDISON VAN NAME. Treasurer. LEWIS E. OSBORN. Publishing Committee. HUBERT A. NEWTON, ELIAS LOOMIS, GEORGE J. BRUSH, ADDISON E. VERRILL, RUSSELL H. CHITTENDEN, EDWARD 8S. DANA, ADDISON VAN NAME. Auditing Committee. ADDISON E. VERRILL, HUBERT A. NEWTON, ADDISON VAN NAME, CONTENTS. PAGE Ligh One ADDITIONS LO THE LIBBARY,..-. .0---2--l:----=2 Vv Arr. I.—On tHe Law or Error In TARGET—-SHOOTING. yah e Ul ne Om nei eo secrete. ea ae ee | II.—Exrensions OF CERTAIN THEOREMS OF CLIFFORD AND OF CAYLEY IN THE GEOMETRY OF 7” DI- UaNsIONey. by) Bio. Moomny dR... 22-2. J) 49 IlJ.—On Kwors, wirh a Crnsus FoR ORvER TEN. Byte Ne Lamrnnny de lategedl—(, os. ke ee ee 27 y ) IV.—Tuer Amytotytic Action oF Drastasre or Matt, AS MODIFIED BY VARIOUS CONDITIONS, STUDIED QUANTITATIVELY. By R. H. CurrrenpEen and ME SAL ON Gi i) TS aS pe aay a a Tet Bead V.— INFLUENCE OF CERTAIN THERAPEUTIC AND Toxic AGENTS ON THE AMYLOLYTIC ACTION OF SALIVA. By R. H. Currrenpen and H. M. Painrer,... 60 VI.—INFLUENCE oF vARIOUS INORGANIC AND ALKALOID Sats oN THE PRoreoLytic ACTION oF PEPSIN- Hyprocutoric Acip. By R. H. Cuirrenpen CASAIG (ats Kegel Daas iA 9) 0px fs) 6 ge eee oe Sen EROORE SBE pOEEEE Ls 84 VIT.—INFLUENCE oF VARIOUS THERAPEUTIC AND Toxic SUBSTANCES ON THE PrRoTEOLYTIC ACTION OF THE Pancreatic Ferment. By R. H. Carr- TENDHN- and GW. (CUMMINS, 22.2. 2225.22. 108 VIUi.—InrLturence or TEMPERATURE ON THE RELATIVE AMYLOLYTIC ACTION OF SALIVA AND THE DtAs- TASE OF Marr. By R. H. Cuirrenprn and AVES SHENG) Li. 2a cf SR a Ane eR Ee gh 125 1X.—INFLvueEnce oF Bink, Brrz Satrs ann Brie Acips oN AMYLOLYTIC AND ProrEotytic Action. By R. H. Cuirrenpren and G. W. Cummnins, ----- 134 X.—ABSORPTION OF ARSENIC BY THE Brain. By R. EH CHrrtin DEN and.ckL, Hi Sir, 222.5225. 149 XIL—INFLUENCE oF Porassium anp Ammonium Bro- MIDES ON Meraporism. By R. H. CairrenpEen Bie alse ULRMRUtemes eee ree seo. Lae 1Voo CONTENTS. XII.—INFLUENCE oF CINCHONIDINE SULPHATE ON MrErTa~- BOLISM. By R. H. CuitrenpEen and H. H. Wanrnnousk, =. -- <2 0.25 Leia dete eee Xi11.—Tne Post-mortem ForMATION OF SUGAR IN THE LivER IN THE PRESENCE OF PEPTONES. By R. H. Cuirrenpen and A. LAMBERT, -__- __.. XIV.—GtLopuLin anp Gropuose Bopirs. By W. Ktune and Rh; HH, CarrrenpEn, )C-)ie) ee XV.—PeEptronges. By W. Kitune and R. H. Carrren- DEN, 242. 02. Stee Pee ee XVI.—On tHe Denypration or GtucosE IN THE Sromacu AND InrEstTines. By R. H. Currren- XVIL—Iveivence or Uranium Sats on THE AMYLOLY- Tic ACTION OF SALIVA AND THE PROTEOLYTIC Action oF Prpsrn anp Trypsin. By R. H. CHITTENDEN and M. T. Hurcurnson, ---- ---- XVUI—Tser Revative Disrrisurion or ANTIMONY IN THE ORGANS AND TISSUES OF THE BODY, UNDER VARYING conpbiTions. By R. H. CurrrenpENn and: JosnPa: A, Baakwen co. fos XIX.—INFLUENCE oF ANTIMONIOUS OxIDE ON MertTa- BoLisM. By R. H. Currrenpren and JosKen A. BRAK, 52'S XX.—Own some Meratiic Compounps oF ALBUMIN AND Myosin. By R. H. Cuirrenpen and Henry H. WoreHouse, . Cra) 6) pie pose eee Dah ees eye) dy ele ie ON 9 Bah 2S 9 2. Ce San ». Care = 2 eer 7 Jhb) MS Tiel OC) Gee Ao See (a) DOG No, >. ora) =) (OC). S =>. eran oe. Corin!) | Date Meal ral ot erat BP or the (unicursal) curve (B) m" in an flat. Cf. Clifford, p. 310. (a) A line A corresponds to the point A. For Q is (1, 0, 0); or u indeterminate, ¢==0; and, therefore, A corresponds to Ne eel g = 0 Os ei oe —). Che m n-fats R,, in R,,4, intersect in R,, a right line. 14 E.. H. Moore, jr.—Theorems of Clifford and Cayley. A point corresponds to the dine OF, viz: the intersection of the full-skew curve O and the line A. y,=0; or w= m, t=0; Xe XS= teense Se = 0, D.C Trane. 5.0 — ae, Se = X= 0: 3. Tangent and osculating flats. On the two-spread let Q be a point and C a curve passing through Q; there are at Q a tangent line R,, and .osculating 2-flat R,, osculating R,... R, to the curve C, determined by 2, 3... (¢+1) consecutive points (Q included) of the curve; the oscu- lating ¢-flat R, meets the curve C in ¢+1 points at P. The tangent plane R, at Q to the two-spread is the locus of tangent lines R, to all curves C through Q; every m-flat R,, through this tangent plane R, meets the spread in a curve having a.double-pt. at Q. Suppose there were an s-flat R, such that every R,, through it met the two-spread in a curve having a (¢+1)ple pt. atQ. Take any curve C through the point P; every R,, through the R, meets the curve C in ¢+1 points at Q, and, therefore, contains the osculating R, to C at Q; hence the R, contains the osculating ¢-flats R to all curves C through Q, or say it meets the two-spread at ¢ consecutive points in every direction from Q. . In the case of this full skew two-spread it will be shown that at every pt. Q there is an osculating R.,., containing the osculating R, to every curve through Q; but probably s=2¢ for the general two-spread in any number of dimensions, R,; (2¢£4, of course). Considerations from the abbildung-system. The general abbildung-system (cf. §1) of curves @” has as base-pts. the (m—1)ple pt. ©, and the m—1 pts. 9; there are m+2 asyzyge- tic curves of the system. Consider those curves of the system which have a (£+1)ple pt. at @, say &” (@‘t’); these correspond to the R,,-sections of the two-spread which have a (¢+1)ple pt. at Q. Such a curve @” (@*') includes the line @@ ¢ times, and, besides, a sup- plementary curve ¢”~ (@), passing through @ and the m—1 pts. 9 and having an (m—t—1)ple pt. at ©. For say the curve &” (@") includes the line ©@ « times, and, therefore, also a supplementary curve &”~ (Q*) with (¢+1—z2)ple pt. at @ and (m—2x#—1)ple point at @; the line ©@ meets the supplementary curves in (m—a—1)+(¢+1—x)=m+t—2x pts. and will be again thrown off, if m—x7 TXy— Xin = 0, TM —Xini0 == (5 2m'(= m) R,, meeting in R,, the line r. The additional R,,, vX,—X,,,,.=0, determines the point 7v on the line r. 16 E. H, Moore, jr—Theorems of Clifford and Cayley. t=1. Kk,, the osculating R,,,_,; along z‘*'-*, the locus of tan- gent planes of points v along the line T. TX, —27X, +X, =0, T Xing 2T Rinses 4 oe To OT Ran =} T” Kins 3 — 21 Meta + hoe Oe TE" Das BT ta 1 = 0, TK DT ee 2(m'—1)=m—2 R,, meeting in R,. The additional R,, vu(z7X,—X,)—(7Xpi2—Xmii3)=0, determines the tangent plane R, at the point tv. And so, in general, the law of formation is clear. The osculating R,,,, along 7‘*', the locus of the oseulating R,, of points v along the line 7. The equations may be written Xe) 0, D Aidinel (eee. Gi laser 1) Xie x)= Ree Xa . X"(7—-XYH4=0, X47 X20; where, after the binomial expansion and multiplication by the exter- nal factor, the exponents of the X are to be exchanged for corres- ponding subscripts; X’ is to be changed to X,. 2(m'—t) =m—2t R,, meeting in R,,,,. The additional R,, (vX’—X”"'’) (r—X')‘=0 (the exponents be- coming subscripts, as above), determines the osculating 2¢-flat R., at the point tv. The osculating R,,, along 7’ lies in the osculating R,,,, along tt; the two R,,, X'(7—X’)'=0, X™t*(r—X’)'=0, with the m—2¢ equas. of the R,,,, written above are easily seen to be equivalent to the m—2(¢—1) equations of the R,_,, X'(r—X')'=0 X”'*(r—X')'=0 Kee x)= 0 xX" **(7—X’)'=0. Thus the equations show that the singly infinite system of osculat-_ ing 2¢-flats R,, at points uv along the generator 7 lie in the oscu- lating (2¢+1)flat R.,,,; along 7‘*’; and, at the same time, form a pencil of R, in the R,,,, having as an avis the osculating (2¢—1) flat R,,_, along 7‘; and the osculating flats of the pencil are homo- graphic* with their points of osculation along the generator T. Compare the well known theorem: Salmon; Geometry of Three * The only equation introducing v is linear in v. E. H. Moore, jr.— Theorems of Clifford and Cayley. iW Dimensions, § 459; “the anharmonic ratio of four tangent planes passing through a generator of a ruled surface is equal to that of their four points of contact.” (6) m odd = 2m" +1. There is one more equation in each case from the first group of coordinates, X, . - Xjw42, than from the second group, Xan. - - X40; the v equation is of exactly the same nature; the conclusions stated above hold equally for m odd or even. As the line 7 generates the two-spread §,,,,,.4, the osculating R.,,, along z*’ generates a (2¢+2)spread of order (¢+1) (m—2t) Sats, ((4-1)(m—2t), m-+1 * Let the m—2t asyzygetic R,, determining Rk,,,, in terms of 7 be Pees 45... :- Agi; the A involve 7 to: the, power ¢-+-1 ; -let Ans» Am—i,0) - + + Asii,s, be what the A become when the coordi- nates of a point, P, in the (m-+1)flat are substituted for the current coordinates. The (2¢+2)spread is met by an (m—2¢—1)flat in say x points P,; any point in the (m—2t—1)flat may be given in terms of m—2t asyzygetic points P,,.. . . Pozi; (ge 7 cet La ya ap 8 é., XA Xs, mt AX, ma SUC G Nog Xr, opt » ete. Substituting the coordinates of P, in the m—2¢ A,, and eliminating from the m—2¢ A,,, the m—2¢ 1 which enter homogeneously in the first degree, we have the determinant of the order m—2t A,,, my ys Nee m—1 9 CEO sae %t+1 =0, JA m9 Bey m—1l) * ° Js is +1 Puoraa: a's Pier 9 oo Aoees, 241 an equation of degree (¢+-1)(m—2t¢) in r; for each value of 1, there is one set of values of the A, one point P,. Hence the order of the (2¢+2)spread, the locus of osculating Ry4,, is (¢+1)(m—2¢), as stated. Through a point P, an osculating R,,, along 7‘t’ may be drawn; this contains the osculating Tas) along zt‘; the R,, joining the point P, with this R,,_, is the osculating R,, at some point v of the line 7, This may be expressed thus; through an R,,_,_, may be passed (¢+1)(m—2t) m-flats R,, which meet the spread in a (t+1)ple line 7; and (¢+1)(m—2t) (m—1)flats R,,_, which meet the spread in a (¢+1)ple point rv. TRANS. Conn. AcAp., Vou. VII. 3 Sept., 1885. 18 EE. H. Moore, jr.—Theorems of Clifford and Cayley. (a) meven=2m'. The simplified abbildung-system; § 1. g”’*" may degenerate into the line 7 taken m’ times, and a line through 4; for this the limiting case t=m’'—1. The (2¢+2)spread, locus of osculating R,,,, along zt! is of order (t-+1)(m—2t). But the (m—-2z¢)spread, locus of osculating R,,», along t”’~ is also of order (m!' —t) (m—2m'-—t—1)=(t+1) (m= 20). For instance, (=0; the two-spread locus of lines R, (i. e., the orig- inal two-spread) is of order m; and, also, the m-spread locus of oscu- lating R,,_, along 7” is of order m. Thus, when m is even, the orders of the (2¢+2)spread for t=#,, t=t,, are equal, if ¢,+-¢,=m’—1. (b) m odd = 2m"-+-1. t=m,; since the curve ¢”"*' in the limiting case degenerates into the line r taken ¢+1—=m"-+1 times. For t=m’" thé order of the (2¢4+2) = 2m" +2 —=(m-+1)spread is (m”+1)(m—2m")=m"+1; i. e@, through every point R,, +41» of Ry may be passed m’-+1 R,, meeting the 2-spread in an (m” + 1)ple line r. - There is no symmetry analogous to that for m even. 4. Curves on the two-spread. (b) m odd = 2m"-+-1. The abbildung-system, @”""' having @ as m’-ple pt. Let us denote the wniqgue curve of order m” in an m’-flat corre- sponding to @® by O; and the right lines of the spread by 7. A curve on the spread of order p, meeting the unique curve O in g points and every line 7 in r points may be written C? (O%7’). A curve on the abbildung plane @°(@') of the order s with a éple point at @ is met by a curve of the system @”""' in s(m”+1j—tm" points and by any line 7 in s—¢ points; therefore, it corresponds to a curve on the spread of order s(m"-+-1)—tm", which meets the unique curve O in ¢ points, and every line z in (s—¢) points, say Cea er Ores: So a curve @* (@‘7°“) transforms into O°™"'t)—™" (Of7**), A curve C? (O°), of order p, meeting the curve O in g pts., must have p>g and p=q (mod. m”+1); say p==s(m" +1) -tin’, qt. s(m"+1)—tm"=p. (s=?%). s=1,t=1,p=1. A liner on X, corresponds to a line 7 on the spread. ss, t=s, p=s. s lines 7 correspond to s lines r. E.. H. Moore, jr.— Theorems of Clifford and Cayley. 19 If s>t, p2=m"+1; which shows that the curve O of order m” is unique, since it is the only curve of so low an order on the two-spread (the lines 7 excepted). Curves on the two-spread and in flats of less than m+1=2im"+2 dimensions are full skew curves. Such a curve is an R,,-intersection or a part of an R,,-intersection ; therefore, its abbildung is a curve 6° (@") ; any m”4+1-s lines 7 belong to the supplementary system, of which the asyzygetic number is m’+2-—s; therefore, m”+42—s asyzygetic R,, meet in an R,,,,,, in which the curve O%"t)—G-Dmttemut lies. A curve C”** in R,,,,,,. is a full skew curve. (I; theorem C.) The general plane curve @’ (¢=0; not through ©) corresponds to a Ot) which does not meet the unique curve. The plane is a full skew 2-spread 8,5 (m=2m"4+1=1, m’=0). All the geometry of plane curves depending upon intersections and tangencies and the order of curves is immediately applicable to the general full skew two-spread of odd order m=2m"-+-1, the curves C*”"’* on the two- spread corresponding completely to the curves @° of the plane. A curve Cw"'t)“"" (O*) meeting the unique curve O ¢ times is a par- ticular case of C%”"t», and in fact plays the same role as a Ot) having a ¢é-ple point. A few examples are given. There is a double infinity of curves C”"*’; two meet in one point; one is determined by two points; they correspond to the lines ¢' of the plane. A line z together with the uniqte curve O is a special case of a curve C”""'. Five points determine a curve Ct); which corresponds to a conic ©? of the plane. Pascal’s theorem becomes : If six points P’.. P® lie on a curve C*"t” the three points of intersection of the two curves Ot’ joining P'P’, P*P®; P’P®, P>P*®; P*P", P’ P*, respectively, lie on another curve O?"", Mo acurve Ctr) (O’r**) there: are .s(s— 1) —2 (¢-+ 1) = (s+-t)(s—t—1) tangent lines 7, and s(s - 1)—¢(¢—1)=(s—t) (s+¢—1) tangent curves C”’'' in a pencil through a point P.* An m-spread of order P meets the 2-spread in a curve C”” meeting the unique curve O m’P times, and every line 7 in P points; ee (ONT a Seay +l), tm’ PP, , To an m-spread of order P in Row,» my: there are mP(P—1) tangent lines lying entirely on the Sy m9, mit Two curves C (O'r*“), C (O’r""), meet in ss’ —¢¢’ points; in particu- lar, two curves C (O'7*”) meet in s’*—@ points; one is determined by * These formule are similar to some given by Chasles, Comptes Rendus, 1861; cf. the following (a). 20 EF. H. Moore, jr.— Theorems of Clifford and Cayley. 44s(s+3)—z¢(¢+1)} points. Hence two curves C (O'r**) through ${s(s+3)—¢(¢+1)}—1 points determine a pencil of such curves through these and ${s(s—8)—¢(¢~-1)}-+-1 additional points. (a) meven=2m’'. The simplified abbildung-system, @”'*’ having @ as m’-ple point and 4 as an ordinary point. Curves @?*' (@,4’) of order p+1 having @ as p-ple pt., through A, correspond to full skew curves of order m'+-p; these are the only curves on the spread in a flat of less than m-+1 dimensions. The proof is like that of (b) for m odd. In particular:—A line y,—Ty,=0 through @ corresponds to a line z on the spread (§ 2); two lines z do not meet. A line v through A corresponds to a full skew curve v of order m’ on the spread ; two curves uv do not meet. Through every point on the spread pass one line 7 and one curve O”’, uv; a line 7 and a curve vu meet in one point. @ corresponds to a curve O of order m’', meeting every line Tt. A corresponds to a line A of order m’, meeting every curve v. The curve O meets the line A in a point OA. (§ 2.) In fact, the curve O, the line A, the point OA in no way differ from an ordinary curve v, line 7, point tu of the spread. Observe that a curve 6’ (@'A") of order s (having O a ¢ple and A an w-ple point), corresponds to a curve C&%™*6™ (7*“*u) of order (s—t)m'+-(s—w) meeting every line 7 in s—¢ points and every curve v in s—w points: it passes s—t—w times through the point OA and meets the line A elsewhere in uw points, the curve O elsewhere in ¢ points; (.°. in all, it meets the line A in s—¢ points, the curve O in 3—w points). A curve G°=6 OE) (OE MA"—E™) with an (s—t—u)}ple point at say @ corresponds to a curve CO™t¢—) (ru) with an (s—t—w)- ple point at Q. Thus the order and character of intersection with the lines 7 and curves v and the (s—¢t—w)ple point are exactly the same. The asyzygetic numbers are equal; as shown by the follow- ing equality (where v=s—t—uw, s’=s+-v=2s—t—u,t’=s—u,w'=s—), (s+1)(s +2)—¢ (¢ +1) —u (uw +1) @' a. =(s'+1)(s'+2)—7’(¢'+ 1)—u'(u' +1)—v(v+1) oO" a Qe The statement above is justified, and it is therefore proper to con- sider only curves which have no especial relation to the point OA; i. e., in the abbildung-plane, only curves @° (©'A") where s=t+u. The spread is ruled with the lines z (§2); and also with the curves v, O™, The curves v correspond to y,—vys=0; the (m'+2)spread E.. H. Moore, jr.— Theorems of Clifford and Cayley. 21 x ? 7 Xnrps ’ X inits 5) ed OO Cet 0 Rl + Ps ’ ee, oA 81k 6) gehen .8 ». +19 Se is cut in the curves v by m’-flats, the intersection of corresponding R,, in the m'+1 projective pencils of m-flats R,,, Meme N= 0, Kg UO Se OR, =O. A curve ¢/*" (@'A") corresponds to a curve Ct! (rv) of order wm' +t meeting each line 7 in w points, each curve v in ¢ points. Since the number of intersections of curves on the two-spread with the lines 7, the curves v and with each other, and all intersection- and tangency-properties, depend only on the abbildung-curves, it is clear that there is a complete correspondence between the curves on a two- spread of even order m=2m', and those on an ordinary hyperboloid or quadric¢ (m'=1); the two systems of ruling curves, the lines 7 and the curves v of order m’, answer to the two systems of generators on the quadric (only, in the latter case, the two systems being of the same order are indistinguishable). Hence many of Chasles’ results (Comptes Rendus, liii, 1861) concerning ‘ Propriétés générales des courbes gauches tracées sur Vhyperboloide” apply in this more general case; for example: A curve O"'*‘(r"v') is determined by ¢u-+-(¢+-~) points. Two curves Ort (r*v'), Ct" (rv), meet in tu’+ut' points. All curves Cv" (r"v') going through tw+(t+u)—1 fixed points form a pencil passing through tu—(¢+u)+1 other fixed points; since any two meet in 2¢w points. To a curve C’’’ (r“v’) 2¢(w—1) lines 7 are tangent. 2u(t—1) curves v are tangent. 2tu curves C”’*} (r'v') of a pencil through a pt. P are tangents. These numbers are easily derived by considering the correspond- ing abbildung-curves. The curve C""'*'(r“v’) corresponds to a curve C*t’(z“v‘)'on an ordinary quadrie; on the quadric there is no distinction between the curves O"+'(z“v'), C“** (z'v"); 1. e., two curves of the same order ¢ +4, which meet the generators of one system T in / points, and those of the other system v in {, points. If, then, there is a theorem about curves of order u,m’+t#, (7=1, 2,.. 8’) meeting the lines 7 in w, pts. and the curves v in ¢, pts., the same theorem will be true about cor- responding curves of order ¢,7'+-u, meeting the lines 7 in ¢, pts. and the curves v in wu, pts. ' A curve OC’ (r“v") must have P=wm' +4. = (0 22 HE. H. Moore, jr.—Theorems of Clifford and Cayley. An m-spread of order P intersects the 2-spread in a curve of order mP=2m' P, meeting the lines 7 in u= P points, and the curves v in == P pointe; sayin a un) = Cs (aren): 2t(u—1)=2m' P(P—1)=mP(P—-1) lines 7 are tangent to this curve. There are mP(P—1) lines 7 lying entirely on the two-spread So, m, m1 and tangent to an m-spread S,, p my of order P.* (m odd or even ; ef. § 4, b). The curves on a full skew two-spread of even order, m=2m', then, have an exact correspondence with those on an ordinary quadric, m=2; those on a full skew two-spread of odd order, m=2m"+1, have almost as exact a correspondence with the curves on a plane, m=1; the unique curve O is a singularity, but curves meeting it in ¢ points play very much the same réle on the two-spread as curves having a ¢-ple pt. at an ordinary point of the spread. These close correspondences with the hyperboloid and plane curves are the marked features of the theory of curves on full skew two-spreads. III. Sereaps or Opp ORDERS ON QUADRICS. The known theorems, that a curve §, of odd order on an ordinary quadricone, cone-Q, ,, passes an odd number of times through the vertex V, and that a general quadric 3-spread Q,, contains no 9-spreads 8, of odd order (ef. Clifford, Mathematical Papers, p. 64), may be extended. Q,,.4: Will denote a general quadric r-spread in R,,,, and cone-Q, ,,, 4 quadric 7-spread in R,,, formed by joining a (general) Q,_i,, to a vertex-point V in R,,,. The section of Q,,,,, made by a tangent r-flat R, is a cone-Q,_,,,. The section of a cone-Q,.,, by an R, through the vertex V is a cone-Q,_,,,; but that by an arbitrary R, is a (general) Q,_,,,. S, will denote an 7-spread. A,. The general Q,,,om ; has on it no spread of odd order unless rm; (III, A). ¢ If an R,, passes through V, it lies completely in the tangent R’o_ at V. ¢ Thus the intersection of an 7, (the projection of an R,,) with the fixed 7r/o,—1 lies completely on the fixed g’; and likewise two rm and the fixed 1’, intersect on the fixed q’. § A quadric m-spread - passing through V would project into an m-flat 7, having a quadric (m—2)spread on q’. E. H. Moore, jr.— Theorems of Clifford and Cayley. 25 (2) m even. Two R,, of opposite systems in general do not intersect, but may intersect in R,, or R;, or. . . or R,_;. Two R,, of the same system in general intersect in a point, but may intersect in an R,, or Ry, or... or R,,5.. This may be expressed (R,= a point, 0 =no intersection), begin- ning with the most infrequent cases: Two R,, of *s =n} system intersect in am Te A in general m even m odd alee aries. OF... | OF kinis: 20 Pee Oe bees sOLt).<... ORS Umea ig (1). A few considerations to be used in the proof are given here. In 7,,, two 7,, meet in a pt. 7; if in another pt., then in a line 7, , if two 7,, have an 7, common, and also another pt., then they have an 7,,, 19 common. Two 7, intersecting on qg’ in r, may intersect in another point ; and thus in 7,,,; but they do not intersect in éwo other asyzygetic points, for then the intersection with the fixed 7’,,,_, would not lie entirely on the g’. (Cf. foot-note {, §1.) If two 7, intersect only in an 7, lying on the q’, the two correspond- ing R,, meet the R,,, joining this common 7, to the fixed point V in two R, (which were both projected into the common 7,) which in the R,,, intersect in an R,,. Asa special case, if two 7, intersect in a point r, on gq’, the two corresponding R,, [intersect the line R, , join- ing 7”, to V, in two points R, and] do not intersect. (2) meven. The7r,, on q' intersect according to (k), m—1 being odd. On q@ ?ma,2 2nd 7, 4,, in general do not intersect, but may in- tersect In 7,7; .... OF Tas. Twor, through them must intersect in a point, at least. Hence, in general, m being even, two R,, of the same system intersect in a point. In the particular cases, if the two 7, mMtersect entirely on g’, the two R,, intersect in R,, R, ... or R,,4; but if the two 7,, intersect also in a point not on q’, the two im intersect in K,, RR, . 2.. or R,3: On q’' 7,,4,, and 7,,,,, intersect in a point 7; but may intersect IN %,%... OF M». Two r, through them would in general in- tersect in no external point; hence, in general, m being even, two R,, of opposite systems do not intersect. In the particular cases, if the two 7,, intersect entirely on gq’, the two R,, intersect in R,, R, ... or R,,; but if the 7, intersect also in a point not on q’, the two R,, intersect in R,, R, ... or R,,_,. Trans. Conn. AcaD., Vou. VII. 4 SEpPt., 1885, 26 E.. H. Moore, jr.— Theorems of Clifford and Cayley. Thus, the (4) holding for 7—1 odd, the (7) hold for m even. (3) m odd. By similar considerations it is shown that, the (?) holding for m even, the (4) hold for m+1 odd. But the (#) hold for m=1 on the quadrie Q,., in R,; (as might be shown immediately by the projection from V on a plane); and, therefore, (4) and (/) are true in general. 3. One m-flat of each system passes through every R,, of the Q,-. The Ry, projects into an vr” having an 7,,_, on g’, through which (suppose) there pass an 7, ,, and an7,,,,,; the original 7,,_, and the 7,,_,,,, intersecting in 7,., determine an 7,, the projection of an R,,, through the R,,_,. This is true on the Q, ,; therefore, in general. ‘ Every pt. in R,,,,,; has a polar R,,, with reference to the Q,,,; the- polar R,,, of every pt. in an R, passes through a certain R,,,_, which is called the polar of the R,. (Clifford.) If the s-flat R, lie on the Q,,,, the polar h,,, of every point in it, i. e., the tangent R,,, at that point, passes through the s-flat itself; hence the polar P,,,_, must include the original s-flat R,. The m-flats R,, are self-polar. The R,,, and R,,, through an R,_, taken together lie in and determine the polar R,,,, of the R,,_,. More generally, two R,, intersecting in an R, lie in and determine the polar R,,,_, of the R,. T11.—On Kwors, with a Census For OrpER TEN. by ONG Lirttx, Lincontn, N rs. 1, Gauss in 1833* called attention to the importance of the study of the ways in which cords might be linked. Nothing, however, appears to have been written upon the subject until in 1847 Listing published his Vorstudien zur Topologie.t In this he briefly but in a masterly way touched upon the subject of knots, established some of the fundamental propositions, and proposed a notation which, as slightly modified by Prof. Tait, furnishes the point of view for the present paper. In a communication} to. Prof. Tait in 1877, Listing points out the fragmentary character of bis own contribution to the . subject, and says that the type-symbol used by him is “nichts weiter als ein derartiger Fingerzeig.” It is to Professor Tait, however, that the greater part of our pres- ent knowledge of the subject is due. He, independently of Listing, obtained the fundamental propositions and found the knots and their forms for orders from three to seven inclusive.§ In 1884 Kirkman || published the forms of knots of orders eight and nine, and imniediately Tait, making use of Kirkman’s work, extended his census of knots to these orders. 4] 2. Professor Tait has shown that any closed plane curve of 7 cross- ings divides its plane into +2 compartments; that these compart- ‘ments are in two groups; that, at the crossings, like compartments are vertically opposite. We shall call these compartments of the plane parts. A part is represented by the number equal to the num- ber of double points on its perimeter. The sum of the numbers representing the parts of either group is 2n, that is, these numbers together constitute a partition of 2n. The partitions for the two groups together make up Listing’s type-symbol. As it can lead to * Hine Hauptaufgabe aus dem Grenzgebiet der Geometria Situs und der Geo- metria Magnitudinis wird die sein, die Umschlingungen zweier geschlossener oder unendlicher Linien zu zihlen.”—Werke. Gottingen. 1867, vol. v, p. 605. + Gottingen Studien, 1847. I have been able to see only Tait’s apparently full abstract in Proc. Roy. Soc. Edin., vol. ix, pp. 306-309. ¢ Proc. Roy. Soc. Edin., vol. ix, p. 316. § On Knots, Trans. Roy. Soc. Edin., xxviii, 145-191, 1876-77 | Trans. Roy. Soc. Edin., xxxii, 281-309, 4] Trans. Roy. Soc. Edin., xxxii, 327-342 28 C. N. Little Knots, with a Census for Order Ten. no ambiguity we shall also call the number representing a compart- ment a part, and either group of compartments a partition. Since every closed plane curve of 7 crossings, having double points only, may be read alternately over and under at the crossings, every such curve which gives parts, none greater than n or less than 2, may be taken as a projection of a reduced knot of » crossings. We call such curves knot-forms or briefly, forms, and regard two forms as distinct if they do not have the same parts similarly arranged. The first part of the problem is to find all the different knot-forms of any order. Since the same knot may be transformed so as to be projected into more than one knot-form, the second part of the problem is, from the complete series of knot-forms of » crossings to find all the different n-fold knots. Knots exist for which the law of over and under does not hold; these are not considered in the present paper. 3. It is unnecessary to do more than allude to two very distinct and very ingenious methods devised and used, the one by Tait and the other by Kirkman, for the solution of the first part of this problem. We may perhaps infer from Professor Tait’s opinion* that “a full study of 10-fold and 11-fold knottiness seems to be relegated to the somewhat distant future,” that they were more laborious than proves to be necessary. 4, A third method, based on Listing’s type-symbol, is thus de- scribed by Professor Tait at page 168 of his first memoir. “Write all the partitions of 22, in which no one shall be greater than » and no one less than 2. Join each of these sets of numbers into a group, so that each number has as many lines terminating in it as it contains units. Then join the middle points of these lines (which must not intersect one another), by a continuous line which intersects itself at these middle points and there only. When this can be done we have the projection of a knot. When more continuous lines than one are required we have the projection of a linkage.” On page 160 of the same memoir, he says, speaking of this method: ‘‘ But we can never be quite sure that we get all possible results by a semi-tentative process of this kind. And we have to try an immensely greater number of partitions than there are knots, as the great majority give links of greater or less complexity.” It seems possible however, with the help of some simple theorems to make the “ Partition Method ” exhaustive, and wholly to do away with the drawing of links. * In 1884. Trans. R. 8S. E., xxxii, 328 EE C. N. Littleh—Knots, with a Census for Order Ten. 29 5. An inspection of form Aa of Plate I will make clear some jerms already introduced and others that we shall now require. Regarding the curve Aq as alone in a plane, it divides it into twelve parts, two 9-gons, two 3-gons and eight 2-gons. The external 3-gon or am- plexum differs in no way from the other parts. Of these twelve parts, two 9-gons and one 2-gon form one group—the leading par- tition; the two 3-gons and seven 2-gons form the other group—the subordinate partition. The terms leading and subordinate are rela- tive merely, but that partition will be taken as leading which has 1 19°2 ! {379"" 6. The double points common to the perimeters of two parts of the same partition will be called bonds of those parts, and the parts are said to be bound by these bonds. It is well known that a type- symbol does not determine a form. For this, it is necessary to know the numbers of bonds between the several parts of either par- tition, together with the arrangement of these parts. In general the parts of a given partition may be bound in more than one way giving forms that may be projections of either links or knots. Each set of numbers of bonds of the several parts of the given partition is a clutch of that partition. The class p of a partition is the number of parts in it. The class of a form is the class of its leading partition. The order of any par- tition is equal to m and is the same as the order of all knot-forms derivable from it. The deficiency x of a partition is its order minus its greatest part. 7. Let the parts of 2n be A, B,C, ... P arranged in order of magnitude, and the numbers of bonds of eack part be respectively a, B,y,... a. Let the number of bonds common to any two parts as A and B be (AB). Then the smaller number of parts. The type-symbol for Aa is GAS PA EAR) Aor cee. = Hae ): GAB) PEt O) oe ey aie (BP)=£ | (AG) BEBO wo ale oe ee y ne (AP) WEBEye ete ee OR ey) or m equations with $7(2—1) unknown quantities which can have only positive integral values. The possible solutions of (@) will evidently give all the clutches for this partition. 30 C. N. Littlh— Knots, with a Census for Order Ten. 8. I, Tororem.—If a part be solely bound to a second part, or if any g parts (¢ “%p—2) be bound mutually in any way and all free bonds of these parts go to a single part, then this portion of the form constitutes a separate knot (unless there be linkage) and the string concerned in it may be drawn tight without affecting the remainder of the knot-form. Such knots are not considered as belonging to order 7. In particular a 2-gon so bound throws out from consideration a clutch. 9. Il. Tororem.—No knot-form of the nth order has as leading par- tition one whose class exceeds + 2. Adding equations (a) above, dividing by two, and subtracting the first and any other, say the second, we find —(AB)+(CD)+(CE)+ ... (CP)+ ... (OP)=n—a—A£, =xu— fi. Therefore, (AB) x 6— x. In a similar way (AC) xy—x (AD) x 6— x (AP) x az— Adding (AB)+(AC)+ ... (AP) xf+y+ ... m—(p—1)x ; Kn+u—(p—1)u XKn— (p—2)x, we have then the two conditions (AB)+(AC)+ ... (AP) xn—(p—2)x () =n— Nn. Now suppose, if possible, p= +3 (AB) + (AC) + pt. (AP)=n=x — i — Kn—("%+1)x Xn—u—H. To the minimum values of (AB), (AC), etce., (that is, to 6—x, VY, -.- ) must be added x in all, and to no one more than x—1, by I. The +2 smallest parts of ~ square are evidently (%—1)* (~—2)«. By adding these ~+2 parts in any way to the mini- mum values, a clutch will be given in which each of four parts will have a single bond not going to A, and each of —2 parts will have two. A part of the latter kind cannot have its two free bonds car- ried to a second part of the same kind, by I. If two parts be joined by a single bond there will be left two free bonds. Ultimately it will be necessary to join two parts of the first kind to a combina- tion of parts having but two free bonds, and I will apply. If any of the x+2 parts of x° be diminished by s then will s parts of 22% be OO es CO. N. Littl—Knots, with a Census for Order Ten. 31 added to those of the first kind, and however the free bonds may be arranged, ultimately the same result as before will be reached. Therefore p cannot equal ~+3 and still give knot-forms. Much less can p be greater than %+3. 10. A given clutch of a leading partition does not uniquely deter- mine a form. The following proposition however holds. IJ. Tatorem.—All or none of the forms determined by any given clutch of a partition are knot-forms of the order considered. For, all forms to be had from any clutch of a given partition may be obtained by taking all the possible different changes (consistent with the given clutch) of relative position of the various parts. But these can all be effected by successive interchanges of the con- nections of two parts, whether such connections are direct (by a single bond), by a 2-gon, by a 3-gon, or are more complicated. We may therefore confine the attention to a definite portion of the knot and keep the remainder fixed. Let A and B, (Fig. 1, Plate I), be two parts connected as shown. ‘Two strings, or two parts of a single string, are involved. If there were more all but two would be closed. Let the ends of these strings, or parts of a single string, leave the portion of the form under consideration at @ and ¢ on the perimeter of A, and 6 and d on that of B. Cut at these points and call the ends a and a’, band 6',c and ¢’,d and d'; a, 6, c and d remain fixed. Now revolve through 180° about the axis AB, and join the free ends. Before the change there may be three cases. The strings may be oe ae or i ee After the change a’ is joined to ¢, and c’ to a; b' tod, and d' to b. He c 4 Heconare ke gee ac’ d'b \" ’ ee bd bd Mee dee i bd'ae wa ac’ b'd Therefore coming up to this part of the knot on any string, we must leave on the same string before and after the change. If then the form was a knot before the change it will be one after, and if a link before it will be a link after. 11. Cotls.—A succession of n 2-gons constitutes an n-coil, which may be open or closed. Since at the 3rd or 2n+ 1st crossing of a coil the strings have the relative position of the first crossing, if the coil be closed by carrying around the ends to the beginning and 32 C. N. Little—Knots, with a Census for Order Ten. joining them so as to preserve the law of over and under one string will be formed. While if from the 27th crossing the strings are car- ried around, that string over at the Ist is under at the 27th and, on joining, there will be two strings. Hence, as is well known, ue is always a knot, while yes always a link. 12. For the purpose of distinguishing between clutches giving knots and those giving links, it follows from Theorem III that we “may take the direct bonds between any two parts together, and these form open coils of the subordinate partition ; and further it is evident from § 11 that any odd open coil (even number of bonds), may be dropped, and any even coil (odd number of bonds) may be replaced by a single bond. If the resulting clutch gives link, so would the original. If the resulting clutch be still too complex for easy recog- nition of its character, the clutch of the subordinate partition of the resulting form may perhaps be still farther reduced in the same way. If the clutches of lower orders were at hand they also could be used for settling the question. 13. We have the following theorems for throwing out clutches unproductive of knot forms. IV. Turorem.—lIf a part be joined to other parts in every case by an even number of bonds, there is linkage. For, the string about this part is closed by Section 12. V. THrorEM.—If two parts are connected by two 2-gons (of the same partition with the parts) there is linking. For they may be put in succession by Theorem III. When this is done there is a 4-gon of the negative partition bound to two other parts in each case by two bonds, and IV applies. VI. Tarorrm.—An odd part joined to one part by an odd number of bonds and to other parts in every case by an even number of bonds may be dropped; for, by Theorem III it becomes a loop with a single crossing, and this can have no effect on the question of linking. In particular a 3-gon joined by one bond to one part and by two bonds to a second part may be dropped. If two odd parts are joined by an odd number of bonds, and are joined to other parts in every case by an even number of bonds there is linkage. In particular two 3-gons so joined throw out the clutch. VIL. Tuzorem.—lIf two 3-gons, C and D, are themselves joined directly and are joined to A and B in each case by a single bond there is linkage. O. N. Little—Knots, with a Census for Order Ten. 33 > For, HCM (fig. 2, Plate I) under we will say at C is over at H and M. LCN is then over at C and under at L and N. LHN is over at L, under at H, and over at N. Its continuation is therefore NML which is over at N, under at M, and over at L. 14. Crass III, OnpER v.—In this class we have (AB) + (AC) + (BC)=n and an unique solution of equations (a), § 9. Therefore (BC)=x, (AB)=6—x and (AC)=y—x. If two or three of these quantities be even, the clutch to which they belong will give a linkage, by Theorem IV. In other cases by § 12 there is a knot. Suppose 2 to be odd and a@ even; then x is odd, and f and y are both odd, or both even. In the first case the clutch gives a link, in the second a knot. Suppose 7 to be odd, and @ odd; then % is even, and of f and y one must be odd and the other even. The clutch gives a link. Suppose 7 to be even and @ even; then x is even and /, y are both odd or both even. In the first case a knot, in the second a link. Suppose z to be even and @ odd; then x is odd, and of f and y one must be odd and one even, and there is a knot. This proves the following : VIII. THrorEem.—In odd orders only partitions of 2 into three even parts, give knots, while in even orders, only these partitions give links. 15. Crass 1V, OrpER n. Here —(AB)+(CD)=x—£f —(AC) +(BD)=x—y —(AD) + (BC)=x—6 (AB) +(AC)+(AD) o> 9, The minimum values of (AB), (AC), (AD), (BC), (BD), (CD), are respectively 6—x, y—u, 0—x,0,0,0. To get every clutch we must add in every possible way to the minimum values of (AB), (AC), (AD), all the partitions of into not more than three parts, none greater than 2—1. But evidently we must increase the minimum values of any quantity of the second set (BC), (BD), (CD), by the same number that we increase the corresponding quantity of the first set. The following scheme which considers in detail every possible case, expresses clearly the propositions for determining whether clutches of partitions of this class furnish knots or links. Let e or o indicate whether a number be even or odd. or Trans, Conn. Acap., Vou. VII. SEPT., 1885, 34 C. N. Little—Knots, with a Census for Order Ten. ns LL Soren OR Props. or n, « | Partition.| Add c= eel Bee eS Form Sunn @ ‘e7/ ee eve eee eee eee eee Link. IV. e€ 00 e€ 0 0 ooe a §12, V eeoo eee e€0oo eoo eee fs IV. e0oo eee ooe ey EVe o0e€0 00° 0e€0 Knot. |§12, VI§11. G4} 101.0) OF ON NOZORO eee 000 000 Link. §12, VIL. oee oee eeo 7; §12, VI. ooee|] 000 eoo oee 000 Knot. |§12, VI, 11. oee 000 ereno ts §12, VI, 11. eoe : eeo eoe Link. Vi. 0 e€| ooee eee oee oee eee a IV. | € 00 000 ooe s §12, V. oeo eeo oe 0 Knot. 812. e|0000/] eee 000 000 ee e ne $12, eoo oee ooe * §12. o|eeece 000 000 eee 000 Link. IV. oee eoo eeo IV. ee0o0| 000 oee eoo 000 : §12, V oee eee eeo Z: LVe eoe ooe eoe Knot. §12. The first line of this scheme says that when 7 is even and % even, and 2 divided into four even parts, the minimum values of (AB), (AC), (AD), will be even, and that if a partition of % into three even parts be added to 6—x, y—u, O—x the numbers constituting the clutch will be even, and the form a link by Prop. IV. One of the propositions proved in this scheme is worthy of separate enunciation. IX. Turorrem.—In even orders partitions of 2” into four even or four odd parts give link-forms only. In odd orders partitions into four even parts give link-forms only. 16. X. THeorem.—In all orders partitions of six even parts give link-forms only. For, an even number may be divided into five parts, of which four, two or none shall be odd. After the application of §12 the only parts to be found in the leading partition will be 4-gons and 2-gons. Every form under consideration will be reduced so as to have as lead- ing partition one of the following, with a clutch of which every number is 1. Ae Ase" AR ee a In the lower orders 4°2°, 472‘, 42°, and 2° have been found to give no knots. If the form given by 4° be drawn it is found in any particular case to consist of four closed curves, and therefore by Theorem III is always a link-form. On drawing 4*2° it also proves to be a linkage. ? Q. N. Littleh—Knots, with a Census for Order Ten. 35 The partition 4°2 with the given clutch can not exist in a plane. This happens in two cases in order 10. On drawing the forms in such cases additional crossings will be found to be necessary. It is therefore true that in all orders, partitions of six even parts give link- forms only. A synthetic proof of this theorem is possible. 17. Instead of continuing the consideration of the general subject we shall now illustrate the method of determining the knot-forms of any order by a particular consideration of order 10. The number of partitions of 20 into parts none greater than 10 or less than 2 is 107.* These, arranged in dictionary order, are given in full in Table I. Of these the single partition of Class H gives a link, §11; the thirty-seven marked II are cut out by Theorem II. In Class III, Theorem VIII throws out 8°4 and 86°; in Class IV, Theorem IX the eight partitions marked IX; and in Class X, Theorem X the three marked X. The partitions remaining in Classes ILI to VI inclusive are alone taken as leading partitions; for, all remaining partitions appear in every possible way as subordinate partitions (§ 2), and can, therefore, furnish no additional knot-forms. We have then to tabulate the clutches of those partitions still remaining in Classes III to VI. We at once write down Tables II and III in which are omitted all clutches that are thrown out by Sections 14 and 15. In Class V, a table with headings as shown in Table IV is used. We take for illustration the first partition, 72°, Here x=3,and 6—x, y—u, O—u, e—x are 4, —1, —1, —1. In this class we must add in every possible way to the minimum values of (AB), (AC), (AD), (AE) the partitions of 2x into four parts, or fewer, none greater than x—1. The only partitions of 6 meeting these conditions are 2° and 2°1°, which may be added in seven different ways to 4, —1, —1, —1, giving the different clutches of the table. Thus adding 1, 2, 2,1, to G—x, y—x, O—u, e+ we have (AB), (AC), (AD), (AE), equal to 5, 1, 1, 0, respectively. Sub- tracting (AB) from {, ete. ='B =(BC) + (BD) + (BE) =2 2C =(BC) + (CD) + (CE) =1 2D =(BD) + (CD)+(DE)=1 2K=(BE) + (CE) + (DE)=2. These quantities are put in the columns headed >B, SC, SD, SE, *See Tait, Trans. Roy. Soc. Edin., vol. xxxii, p. 342. 36 CU, N. Little—Knots, with a Census for Order Ten. and all the possible solutions of this set of equations here as throughout the work are written down by inspection. The rest of this table is made out in the same way. In Table V we must, in every possible way, add the partitions of 3% into four parts or fewer, none greater than x—1. In the general case we add the partitions of (p—3)x into p—1 parts none greater than x—1. In the com- plete Table IV there are 400 clutches, and in Table V 1000. It has not been thought necessary to publish more than a sample of either table. 18. Having completed the tables of clutches we next cast out in Tables IV and V all clutches unproductive of knot-forms. The theorems already established are sufficient for this purpose. The routine followed will be readily understood from considering its use in the samples given of Tables IV and V. Partition 7°2*: in clutch (1) A and B are connected by two 2-gons, and V applies ; (4) becomes (3) by interchanging D and E; (7) becomes (6) by interchanging C and D; and in (2) and (5) we have a 2-gon solely bound to a single part, and I applies. Partition 6? 42°: clutch (1) is thrown out by IV since a 6-gon B is joined to A by four bonds and to C by two; Theorem IV casts out (4) because of C, (14) because of CO, (17) because of B, and (20) be- cause of A; in (2), (3), (8), (9), (11), (12), (15) and (18) a 2-gon is solely joined to a single part, and I applies; in (5) B and C form a combi- nation solely bound to A, and I applies; I throws out also (10) where D and E are bound together and to C, and (13) where the same parts are bound to B; interchanging D and E (7) becomes (6); interchanging A and B (16) becomes (7). Partition 5°32: I throws out (2), (3), (6), (8), (14), (16), (19), (20), (26), (27), (30), (31), (33), (87) and (88) ; by interchanges of parts ()=(4, COD=(5), @=(9), (82)=00), (N=), (08) 8), — (3E( 4), (= (22), (13)=(10), (21)=(10), (28)=(23), (35)=( 1), (15)=(12), (22)=(21), (29)=(28), and (36)=( 9). In (4) D is dropped by VI, and B thus is changed to a 4-gon; then the application of §12 leaves the two 3-gons A and C joined by two 2-gons, E and the still farther reduced B; V, there- fore, shows that the clutch gives links only. In (9), drop C by VI and B becomes a 2-gon and A a 3-gon; the two 3-gons A and D are connected by the two 2-gons E and the reduced B; therefore the clutch gives links, by V. In (11) drop C by VI and the two 3-gons _ A and B are connected by the two 2-gons E and the reduced D; V applies. CO. N. Little—Knots, with a Census for Order Ten. 37 b) e In Table V, clutch No. (1) of 53° is thrown out by I. In (2) the two 3-gons E and F are joined together by a single bond and to A in each case by two bonds, and VI applies; or we might use I. Theorem VI throws out (4), since the two 3-gons B and F are joined to each other by a single bond and to other parts in each case by two bonds. In (5), drop F, by VI; B becomes a 2-gon joining D and K, and they are 3-gons connecting A and C. An obvious exten- sion of Theorem VII throws out the clutch. Since in (7) B and C are joined together and to A, I applies. This process is continued until all clutches which do not give knot-forms have been thrown out, as well as those clutches which are repetitions of clutches previously given. The samples given of Tables IV and V constitute about one-twentieth of the complete tables. 19. We are now ready to draw the forms which the remaining clutches furnish. Derinition.—The number of Circular arrangements of n things is the number of distinct ways in which m things can be arranged in a circle, the order whether direct or retrograde being of no con- sequence. For illustration of the general method we consider in detail the productive clutches of partition 6°42*. In clutch (6) the two 6-gons are connected, by three bonds, by a 2 gon, and by a 4-gon which is connected with one 6-gon by two bonds and with the other by a 2-gon and one bond. There will be as many forms from this clutch as there are circular arrangements of aaa b (cd), where c and d may not be separated. These are aaa b (cd) aaa b (de) aab a (cd) aab a (de). The four forms O’c,, C%c,, C’c,, C*e,, can at once be drawn. See Plate V, Knot LXIV. In (19), the only other clutch of this partition that affords forms, a 6-gon is connected with a 4-gon directly, by a 2-gon, and by a 6-gon which is connected with the 4-gon by a single bond and by a 2-gon. There are two circular arrangements of a b (cd), namely a b (cd) and b a (cd); these furnish the two forms C*d,, C*d, Knot XXX, Plate III. In practice the operations described in this section and in the preceding, are performed simultaneously. 20. In Class VI every productive partition is used as a leading 38 C. N. Littleh—Knots, with a Census for Order Ten. partition. But since the subordinate partitions here belong to the same class, every form, with certain exceptions, will be found twice; this affords a check on the completeness of this part of the work. The exceptions are the amphicheiral knot-forms. These have the same partitions similarly connected both as leading and as sub- ordinate partitions, These therefore appear but once. In the partition 53°, which was given as the shortest possible illustration of Table V, clutch (3) furnishes a single form D’s,, which already had been obtained under 54°32*; clutch (6) gives two which had been obtained, Du, under 64372’ and D*x, under 54°32*. Clutch (8) gives the only new knot-form from this partition, viz: the amphicheiral D*o. From the clutches of Classes III, IV, V and VI 364 ten-fold knot forms are obtained. 21. The Derivation of Knots from Knot-Forms.—Prof. Tait has not described the methods which he used in his derivation of the knots of lower orders from the knot-forms. In 1884 he says :* “ the treatment to which I have subjected Kirkman’s collection of forms, in order to group together mere varieties or transformations of one special form, is undoubtedly still more tentative in its nature; and thus, though I have grouped together many widely different forms, I cannot be absolutely certain that all those groups are essentially dif- ferent from one another.” If a ten-fold knot be placed upon a plane in such a way as to have but ten crossings the eye will project it upon the plane in a form which will be found among the 364 above obtained. If the knot gives more than one form it will be possible to obtain any other of its forms by one or more turnings over of restricted portions of the knot while the remainder is held fixed. Now the string cannot issue from the portion of the knot that is turned at more than four points, for in that case the turning would introduce consecutive overs, and one or more additional crossings; the portion of the knot that is turned must therefore be wholly between two parts of the given knot form and in turning it we untwist two of the strings at one point and twist two at another, the result being simply to change the position of a single bond from one end of the connection to the other. The class of the form is therefore not changed, and all the forms of any knot belong to the same class. 22. Moreover in order 10 and lower orders all the forms coming from any clutch are obtained by changing the position of single * Trans. Roy. Soc. Edin., vol. xxxii, p. 327. — C. N. Little—Knots, with a Census for Order Ten. 39 bonds in the connections of pairs of parts. Therefore in these orders all forms from any clutch are forms of ‘the same knot. The sub- ordinate partitions of these forms are then to be examined and all forms added which are obtained from them by changing the positions of connections of their parts, retaining the given clutch of the subor- dinate partition. These forms in turn are treated in the same way, but it will usually happen that no new form of the knot is obtained and the complete determination of the knot in all of its forms is finished. 23. We take for illustration Knot I of Class IV, shewn on Plate I. Ba, becomes Bu, by twisting about a vertical axis the 2-gon connect- ing the 8-gon and 7-gon. The first crossing below is opened and the strings above are crossed, the rest of the knot remaining fixed. Twisting the 2-gon again Ba, is obtained and nothing new is gotten by further changes of the forms. Since the negative partition in every case consists of two parts joined by three symmetrical connec- tions, which have only one circular arrangement, there are no other forms of Knot I. 24, The knots of Class III, order n, are unique; since three things have but one circular arrangement. 25. The knots of Classes III, [V, V will be found with their forms grouped together in Plates I-V. On Plates V—VII are figured the forms of Class VI grouped as they come from the clutches, except that no form is repeated. The knots of this class will be found in Table VI. Every knot-form is the projection of two knots, one of which is the perverted image of the other, and consequently each group of knot-forms belongs to two knots which are in general different. If a series of knot-forms contains any amphicheiral form then it will also contain the perversion of every form of the series not amphicheiral. The series consists of the forms of one knot and not of two. . 26. In order 10 I find, counting a knot and its irreconcilable perver- sion as two: Class. Forms. Knots. Knots. III, 6 12 6 EV 25 30 15 V, 200 128 64 VI, 133 64 39 Totals, 364 234 124 40 In lower orders Professor Tait has found: Orders. Forms. 3 1 4 1 5 2 6 3 7 10 8 27 9 100 Knots. 82 ] 4 C. N. Little—Knots, with a Census for Order Ten. The fourth column contains the numbers as they are given by Tait, the perversions of knots not being counted. In so long a labor as is involved in making such a census the opportunities for error are many. Any errors or omissions that may be found in the census are to be attributed to the writer rather than to the method, which is simple and direct. TABLE I.—Partitions of 20. | Crass II. Cu. IV.—Contin’d|Cu. V.--Contin’d.| Crass VI. |cz. VIL-Conti’d a IX. 992 (8623 IE 1025 642° Cuass III. 8732 | 85322 II. 9324 11.4 63224 { 1082 IX. 8642 II. 4 8472? II 8424 5295 I eis 863° 84322 a 83223 54324 * \ 1064 8572 834 ( (524 53393 l 105? 8543 ; 7293 II. . 74322 4394 922 xe 848 76322 73592 423298 983 7242 75492 xX. 6224 43492 974 axe 232 75329 65328 359 965 7652 74239 X. 64723 VIII. 8%4 1643 1498 643222 Crass VIIL 875 IX, 1573 624.22 6342 5 VIII. 86? 754? 62329 52423 ee 7°6 IX. 622 65222 523292 i i a 6°53 542322 Scoala IX. 6°42 te 54332 432° 9 3424 10532 65°4 6432 535 10 10422 xan 54 64232 Xe 42° CLass XI. 1043? CLaAss V. 5332 423°2 ( 972? ll { 1042° 52429 4°34 106 oo 9632 > | 10322? 52432 . 3°2 II.-2 9542 9528 5433 Crass VII, (ieee 9532 IL ) 2832 45 Il. 828 aie | 9423 9332 I. 7325 "210 y TABLE II.— Clutches for p=3. re We A! a oh i a) eee ee Partition. =} o 3 Partition. =) 23 Partition, 228 929 811 Aa 974 631 Ac 875 532 Ad 983 ea o} 965 5 41 Ae 7°26 43 3 Af = ee 4] TaBLE III.—Clutches for p=4. C. N. Little—Knots, with a Census for Order Ten. Trans. Conn. Acap., Vou. VII. = 3 od fea] jaa) <9) =: St asta oO 2 = < a = we ae Ss * foe] aoa Ape ki a 3 ae eS Foal s co gam BA. a 3 Set hes ay ae x. = Sue . $ . _ oy 29 + E0fQ Tl ecloaly its | {=| eyany f=» Boo! & al Sm (e) < 5 Sma --Am, - oS (q¥) ls 10 Hieaan 10 on on = _ faa) | (az) TAN a aa4nn _ a = 4 SBH=HANHANR HO HOnARAANeC HR moa] 4 ie (az) ae 4 ef GN Nes a aN aS SN x AdHHANMMHAN MNMAMAMAHMN anel|* 2 (Ox) ee al SWNT igniicn 20) = cx os S| ID DINMMHO1® MHAMOMHM A nmM mM sont m | (dx) In 9 S Met ounGd = os ng = = ed =e a i} i= ¥—3 | | o i eed re fo | * = oy #1 =) y—h je o —) aq4or <7! i —) 4 a —) joa] ¥ ¢) | —¢ le we re o mm fy Aisa ale BI Zn BS gies) ra ag Oo helen an a a & Be a ey on aN 4 A a maa eee ee a ee eet on q a: rs x i = _ aa) : ee - a 2 q eA Ae oon . io ro i) ATHANM AT TAN oF H one es Ia | 3 Leys =| a ZU i NOniost HOw eller hip uataes OS Q 3 erg colesceree at aS taA % $ mSOin1o rFrS O© © 1d OH 10 iP Pet Non el ag KH HHRAN B (OO cy OOOO GO Seis eee naa eee “) oa] Ei san © |pmmamonn Ay II II A Ay 2 2 2 < * a4 Oct., 1886. 42 C. N. Little—Knots, with a Census for Order Ten. TABLE 1V.—Clutches for p—5 (continued). | I Jee ev ve |\amamalanm melon elo Partition. | Add al a lARAA 0°0005 071375 0°2800 25°20 0°0010 071445 0°2944 26°49 0:0020 071242 0°2528 . 22°75 0°0030 0°1252 0°2552 22°96 0 0°1319 0°2684 24°15 Hg(CN), 0°0500 0°1025 0°2084 18°75 In this series of results, it is to be noticed that mercuric bromide is the most energetic in its hindering action; 0°0005 per cent. being even more effective than 0°002 per cent. of the iodide. With the cyanide, however, the first two percentages stimulate or in some way give rise to an increased amylolytic action and even 0°050 per cent. of the salt does not retard the action of the ferment as much as 0°001 per cent. of mercuric bromide. Cupric sulphate. With this salt the following results were obtained: CuS04+5H,20. Wt. Cu in 4. eanoree bodied cena: 0 0°1712 gram. 0°3500 gram. 31°50 per cent. 0:0005 per cent. 0°1445 02944 26°49 0:0020 0°0530 0°1096 9°86 00100 0'0250 00540 4°36 0°0250 0 * Apparently, the double salts so formed act as vigorously as the mercury salt alone could do. 64 Chittenden and Painter—Influence of Therapeutic The hindering action of the copper salt is nearly as pronounced as that of mercuric chloride and even more so than the bromide and iodide of mercury. Lead acetate. With this salt, the smaller percentages experimented with show a slight stimulating action; but the larger percentages fail to retard the amylolytic action of the ferment as the preceding salts. Pb(CgH309)2+38Hy,0. Wt. Cuin 4. reine names. Bes 0 0°1630 gram. 03332 gram. 29°98 per cent. 0°0003 per cent. 071642 0°3356 30°20 0:0005 0°1635 0°3340 30°06 0°0010 071635 0°3340 30°06 0-0020 0°1595 0°3256 29°30 0:0050 071395 0:2840 25°56 0:0100 0°1402 0°2856 25°70 A second series of experiments, with still larger percentages of the lead salt, gave the following results: Total amount Starch _Pb(C2H302)2+8H,0. Wt. Cu in ¥. reducing bodies. converted. 0 0°1742 gram. ' 0°3564 gram. 32°07 per cent. 0°05 per cent. 071735 0°3548 31°93 O10 cI. 0°1720 0:3516 31-64 0°30 0°1657 0°3384 30°45 0°50 ; 0°1555 0°3172 28-54 1:00 4 0°1375 0°2800 25°20 3°00 0°0785 0°1600 14-40 5°00 0:0490 0°1016 9°14 Thus, the presence of even five per cent. of lead acetate fails to completely prevent amylolytic action. Arsenious oxide. Owing to the comparative insolubility of this substance in neutral fluids, small percentages only could be experimented with. With these, the following results were obtained : Total amount Starch As,03. Wt. Cu in ¥4. reducing bodies. converted. 0 0°1475 gram. 0°3004 gram. 27°03 per cent. 0-0003 per cent. —0°1507 0°3072 27°64 0:0005 0°1537 0°3136 28°22 0°0010 0°1475 0°3004 27°03 0°0020 0°1579 0°3204 28°83 0°0050 0°1390 0°2832 25°48 0°0900 01605 0°3276 29°48 Although the results obtained do not wholly accord with each other they still plainly show that arsenious acid, to the extent pres- and Toxic Agents on the Amylolytic Action of Saliva. 65 ent in these experiments, stimulates the amylolytic action of the ferment ; a fact which might be expected, assuming that the acid combines with the proteids of the saliva, for as has been elsewhere* shown, acid-proteids when present in not too large an amount increase the amylolytic action of the salivary ferment. Schafer and Béhmf} state that arsenious acid has no influence whatever on the conversion of starch into sugar by a glycerine ex- tract of the pancreas. Possibly they sought only for retarding action, or it may be that the pancreatic ferment differs in this respect from the ferment of saliva. Arsenic acid. This substance being still more acid than the preceding, might naturally be expected to diminish amylolytic action, when present in quantities which in the preceding would increase the activity of the ferment ; and indeed there is to be seen in the results, a slight in- crease, followed by a rapid decrease of amylolytic action. H3AsOq4. Wt. Cu in ¥%. ean Wot eA eed. 0 01755 gram. 0°3588 gram. 32°29 per cent. 0°0005 per cent. 0°1765 0°3608 32°47 0°0010 0°1635 073340 30°06 0°0030 0°0310 0°0660 5°94 0°0050 0 With 0:005 per cent. of arsenic acid present in the fluid, no reduc- ing bodies were formed in the thirty minutes of the experiment, but the solution did become clear, showing the formation of soluble products. The same fact was observed in the presence of larger percentages of the acid; the starch solution becoming clear, after the addition of saliva, even in the presence of one per cent. of the acid, although, as before, no reducing bodies were formed. Ammonium ursenate. With this salt the following results were obtained: Total amount Starch (NH4)3 A804. Wt. Cu in 4. reducing bodies. converted. 0 0°1527*gram. 0°3112 gram. 28°08 per cent. 0:0005 per cent 0°1620 0°3308 29°17 0:0010 0°1630 0°3340 30°06 0:0050 071675 0°3420 30°78 0°0150 071745 0°3568 ay AIL 0°0250 0-1700 0°3476 31:28 * Chittenden and Smith, Trans. Conn. Acad., vol. vi, p. 343. + Abstract in Jahresbericht fir Thierchemie, 1872, p. 365. Trans. Conn. Acap., Vou. VII. 9 Oct., 1885. 66 Chittenden and Painter—Influence of Therapeutic In a second series, larger percentages were used with the following results : Total amount Starch (NH4)3AsOq. Wt. Cu in 4. reducing bodies. converted. 0 0-1712 gram. 0°3500 gram. 31°50 per cent. 0:05 per cent. 071475 0°3004 27°03 0-10 071147 0°2332 20°98 0°50 0°0165 0:0368 3°31 1:00 The solution became clear but no reducing bodies were formed. With this salt, a very decided stimulation of the ferment is to be observed in the presence of small percentages, while increased amounts of the salt ultimately stop diastatic action. Potassium antimony tartrate. Two series of experiments were tried with this salt, with the fol- lowing results: Total amount Starch K(Sb0O)C4H4Og. Wt. Cu in ¥%. reducing bodies. converted. 0 0:1540 gram. 0°3144 gram. 28°29 per cent. 0:001 per cent. 0°1610 0°3288 29°59 0°005 0°1660 0°3392 30°52 0-010 0-1760 073600 32°40 0°050 01760 0°3600 32°40 0-100 071745 0°3568 31-11 0°200 0°1750 0°3580 32°22 0 0°1545 0°3152 28°36 0°10 0°1850 0:3788 34:09 0°30 0°1640 0°3352 30°16 0°50 0°2565 0°5304 47°73 1:00 0-1570 0°3204 28°83 2:00 071232 0°2504 22°53 5:00 0°0470 00976 8-78 Here we have an illustration, more forcible than with any other salt, of the power possessed by many substances of both increasing ~ and diminishing the action of the ferment. One* of us has for some time held that the addition of very small quantities of hydrochloric acid to neutral saliva tends to increase the amylolytic power of the ferment; that this takes place even when the proteids present are completely saturated with the acid, or in other words when there is present a very small amount of free acid, provided the acid-proteids are not present in too large an amount. It is well known that free hydrochloric acid, when present to the extent of a few thousandths of one per cent. completely stops the action of the ferment. Langley * Chittenden and Smith, Trans. Conn. Acad., vol. vi, p. 360. a and Toxic Agents on the Amylolytic Action of Saliva. 67 and Eves* make this divergence of action of one and the same sub- stance a ground for questioning the accuracy of such a view, for, say they, “since 0°0015 per cent. HCl decreases amylolytic action it seems very unlikely that 0°0005 per cent. should increase it.” The action of many neutral salts here experimented with, where both stimulation and retardation are obtained, plainly show that such a double action, dependent simply on quantity is not an impossible one, Stannous chloride. With this salt very marked results were obtained as follows: Total amount Starch SnClo Wt. Cu in ¥. reducing bodies. converted. 0 0:1475 gram. 0°3004 gram. 27°03 per cent. 0°0003 per cent, 0°1582 0°3232 29°08 0°0010 The solution became clear, but no reduction. 0°0050 The starch was not at all altered in appearance. Here there is stimulation, followed by rapid and complete stopping of amylolytic action. Zine sulphate. ZnSO4+7H20. Wt. Cu in ¥. roifouies Bodies: conentad: 0 0°1495 gram. 0°3048 gram, 27°43 per cert. 0:0003 per cent. 071490 0°3040 27-36 0°0005 0°1510 0°3088 27°79 00010 01475 0°3004 27°03 0°0020 0°1440 0°2936 26°42 0°0050 0°1360 0°2772 24-94 0°0100 0°1260 0°2576 23°18 0 071375 0:2800 25°20 0°05 per cent. 00775 0°1480 13°32 0°10 0-0650 0°1332 ; 11°98 0°30 0°0450 0°0936 8°42 0:44 0 These two series of experiments plainly show a gradually dimin- ished amylolytic action, as the percentage of the zinc salt is in- creased, until with 0-4 per cent. a complete stoppage is effected. Kjeldahl+ found a like retarding action on the addition of zine sulphate to a malt extract. In this connection it is interesting to note that Sternberg} finds zinc sulphate devoid of germicide value, even when used in the proportion of 20 per cent. * Journal of Physiology, vol. iv, No. L. + Jahresbericht fiir Thierchemie, 1879, p. 382. ¢ Amer, Jour. Med. Sciences, April, 1883, p, 330. 68 Chittenden and Painter—Influence of Therapeutic Ferric chloride. With this salt we obtained the following results: FegClg¢.- Wt. Cu in 4. SU HeTB IES, sonvored: 0 0-1740 gram. 0°3560 gram. 32°04 per cent. 0°0005 percent. 0°1597 0°3260 29°34 070020 0°0437 0:0908 817 0°0100 0°0095 0°0236 2°12 0°0250 0 Sternberg states that tincture of ferric chloride is effective as a germicide (upon micrococcus) when present to the extent of 4 per cent. On the unformed ferment of the saliva, it 1s, as the results show, much more active, its hindering action being directly propor- tional to the percentage of iron salt present. ferrous sulphate. With this salt of iron quite different results were obtained; and as we wished simply to compare its action with that of the ferric salt, only very small percentages were experimented with. FeSO4,+7H20. Wt. Cu in ¥. veareitie eiea, convened 0 0°1245 gram. 0°2532 gram. 22°78 per cent. 0:0005 per cent. 071037 0°2108 18°97 0:0020 0°1323 0°2692 24°22 0°0100 0°1365 0°2780 25°02 Here there is decided stimulation with the two larger percentages, while the smallest per cent. shows an apparent decrease of amylolytic action. Kjeldahl* found that this salt exercised a strong hindering action on the amylolytic ferment of malt. Potassium permangunate. Sternberg places this salt next to mercuric chloride in germicide value, it being efficacious in 0°12 per cent. With the unformed fer- ment of the saliva it is likewise active, although no more so than many other salts experimented with. Following are the results: Total amount Starch KoMnogQOg. Wt. Cu in 4. reducing bodies. converted. 0 0°1475 gram. 0°3004 gram. 27°03 per cent. 0°005 per cent, 0-1012 0°2060 18°54 0°025 0 * Jahresbericht fiir Thierchemie, 1879, p. 382, : . and Toxic Agents on the Amylolytic Action of Saliva. 69 Magnesium sulphate. With this salt we obtained the following results: Total amount Starch MgS044+7H20. Wt. Cu in 4%. reducing bodies. converted, 0 071475 gram. 0°3004 gram. 27-03 per cent. 0°025 per cent. 071597 0°3260 29°34 0°500 00510 01056 9°50 Here, there is a slight increase of diastatic action with the smallest percentage, while 0°5 per cent. of the salt greatly retards the action of the ferment. Pfeiffer* has likewise noticed the retarding effect of this salt on salivary digestion. Potassium cyanide. This salt, so powerful as a poison, was found to have a decided effect also on the salivary ferment, causing a rapid decrease in amylolytic action. Total amount Starch KCN. Wt. Cu in 4%. reducing bodies. converted. 0 0°1245 gram. 0°2532 gram. 22°78 per cent. 0-0005 per cent. 0:1080 0-2200 * 19-80 0°0010 0°0896 0°1828 16°45 00030 0°0330 0:0700 6°30 With 1:0 per cent. and even with 5:0 per cent. of potassium cyanide, the starch solutions became clear on the addition of saliva, showing that the ferment was able to effect some change, although in neither case were any reducing bodies formed. It is our intention at some future time, to study the exact nature of the products formed under such conditions. The ferment appears to be peculiarly affected ; for while a very small percentage of a substance like potassium cyanide or borax will completely prevent the formation of reducing bodies, increasing the amount of substance added a hundred-fold, has no effect on the clearing up of the starch solution by the ferment. Some light may be thrown upon the nature of the ferment or its mode of action. Potassium ferrocyanide. A preliminary experiment showed that this salt was less active than the cyanide and therefore larger percentages were used, with the following results: Total amount Starch K,Fe(CN)§+3H20. Wt. Cu in 4. reducing bodies. converted. 0 01417 gram. 0°2884 gram. 25°96 per cent. 0-025 per cent. 0°1497 0°3052 27°46 0°100 071375 0°2800 25°20 0°2084 0°250 0°1025 18°80 * Centralbl, Med. Wiss., 1885, p. 328, abstract. 70 Chittenden and Painter—Influence of Therapeutic Here, unlike the cyanide, there is stimulation of the ferment. Retardation of amylolytic action requires much larger percentages ; thus 1°0 per cent. of ferrocyanide completely prevented the formation of reducing bodies, although soluble starch was apparently formed, as also in the presence of 5-0 per cent. of the salt. Potassium ferricyanide. The action of this salt is almost identical with that of the ferro- cyanide. Total amount Starch KgFeeg(CN) 1 2- Wt. Cu in ¥. reducing bodies. converted. 0 0:1417 gram.. 0°2884 gram. 25°96 per cent. 0°025 per cent. 071515 0°3088 27°79 0°100 0°1295 0°2636 23°72 0°250 0:0975 0°1984 17°85 Like the ferrocyanide, this salt in 1:0 and 5-0 per cent. solutions allows the partial conversion of starch into soluble products, but no reducing bodies are formed. Potassium nitrate and potassium chlorate. Potassium Total amount Starch salt. Wt. Cuin ¥. reducing bodies. converted. 0 0°1513 gram. 0°3080 gram. 27-72 per cent. KNQ3. 0°20 percent. 0°1550 0°3164 28°47 0°50 0°1528 0°3108 27°97 1:00 0°1462 0:2976 26°78 KCIO3. 0°20 071581 0°3228 29°05 0°50 0°1580 0°3228 29°05 1:00 0°1600 0°3268 29°41 With 5-0 per cent. of the salts, the following results were obtained: Potassium Total amount Starch salt. Wt. Cuin 4. reducing bodies. converted. 0 0°1672 gram. 0°3416 gram. 30°74 per cent. KNOs (5:0 pr. ct.) 071559 0°3176 28°58 KClO0; (5:0 pr. ct.) 0°1351 02752 24°76 With these two salts it is very obvious that small fractions of one per cent. decidedly increase amylolytic action and that potassium chlorate is the more energetic of the two in this respect. With one per cent. of the salts, potassium chlorate still shows increased action, while the nitrate causes a decrease of amylolytic activity; in the presence of 5 per cent. of the salts, on the other hand, potassium chlorate causes the greatest decrease in ferment action. Of these two oxidizing agents, potassium chlorate does not appear to have been hitherto experimented with, but with potassium nitrate —— ‘ a and Toxic Agents on the Amylolytic Action of Saliva. 71 O. Nasse* found increased amylolytic action with human saliva in the presence of 4:0 per cent. of the salt. Possibly this difference in our results is dependent in part upon difference in the relative amount of salt and ferment. Sodium tetraborate [Na,B,O,+10H,O]. With this salt experiments were tried with quantities varying from 0°050 to 3°0 per cent. and in each instance the starch was dissolved, but no reducing bodies whatever were formed. Dumast has pre- viously noted a like retarding effect on the diastatic action of emul- sin, diastase and other like ferments. Sternberg states that this salt is without germicide value, even though used in a saturated solution ; its antiseptic power, 1. e. its capacity for preventing the multiplica- tion of bacterial organisms, is, however, considerable. Potassium bromide and potassium iodide. These two common therapeutic agents gave the following results: Total amount Starch Salt used. Wt. Cuin 4. reducing bodies. converted. 0 ’ 01483 gram. 0°3020 gram. 27:18 per cent. KBr. 0°5 per cent. 0°1566 0°3192 28°72 30 0°1450 0°2956 26°60 5:0 071314 0°2668 23°51 KI. 0°5 0°1550 0°3164 28°47 3°0 0°1557 0°3172 28°54 50 0°1467 0°2984 26°85 Both of these salts show a stimulating action which is more per- sistent in the case of the iodide than with the bromide; 5:0 per cent. of the bromide causes a marked diminution of amylolytic action. Sodium chloride. Previous experiments have been tried with this salt by several investigators, notably by O. Nasse{ and E. Pfeiffer. The former found that the presence of 4:0 per cent. of the salt [the only percent- age experimented with] caused an increase in the ferment action of saliva [128:100]; the latter experimenter likewise found that the * Pfliger’s Archiv. fiir Physiologie, vol. xi, p. 150. + Berichte der deutsch. Chem. Gesell., vol. v, p. 826. ¢ Pfliger’s Archiv fiir Physiologie, vol. xi, p. 155. § Centralbl. med. Wiss., 1885, p. 329, 72 Chittenden and Painter—Influence of Therapeutic presence of the salt, in concentrations up to 2 per cent., greatly in- creased the amylolytic action of saliva. Our results with different percentages are as follows: NaCl. Wt. Cuin 4. roaeIne TOE: canvercelis 0 0°1660 gram. 0°3392 gram. 30°52 per cent. 0°3 per cent. 01765 0°3608 32°47 0°5 0°1750 0°3580 32°22 10 01715 0°3504 31°53 2°0 071715 0°3504 31°53 3°0 01770 0°3620 32°58 50 071630 0°3332 29°98 These accord with the results mentioned above and show, more- over, that with 5:0 per cent. of the salt, hindering action just com- mences. Increasing the amount of salt beyond this point, however, only slowly diminishes the action of the ferment; thus, in the pres- ence of 10:0 per cent. of the salt, 22°78 per cent. of starch was con- verted into sugar, while without it 25°20 per cent. of starch was converted. Morphine sulphate. With this alkaloid O. Nasse* has experimented, using, however, the acetate. He found that the presence of 0:1 per cent. of the salt caused a slight increase in the diastatic action of saliva (109: 100). Our results with the sulphate of morphine are as follows: Total amount Starch Alkaloid salt. Wt. Cuin ¥%. reducing bodies. converted. 0 0°1245 gram. 0°2532 gram. 22°78 per cent. 0°05 per cent. 071415 0°2880 25°92 0°50 071605 0°3276 : 29°48> 2°00 071428 02908 Zou The stimulating action of the alkaloid salt up to 2°0 per cent. is very apparent. Quinine sulphate. With the acetate of this alkaloid, Nasse found, by the use of 0:1 per cent., an increase in the starch-converting power of the saliva (115: 100). With the sulphate we obtained the following results : Total amount Starch Alkaloid salt. Wt. Cuin 4. reducing bodies. converted. 0 0°1358 gram. 0°2768 gram. 24°91 per cent. 0:05 percent. 0°1475 0°3004 27°03 0°50 071355 0°2760 24°84 2°00 0:0981 071996 17:96 nnn ee EEE EE EIEIEEEIESESEIE NEESER * Pfliger’s Archiv, vol. xi, p. 161. _s a se On, OO and Toxic Agents on the Amylolytic Action of Saliva. fe, In accord with Nasse’s result we see that 0°05 per cent. increases the amylolytic action of the ferment. This we verified by an addi- tional experiment which led to a like result, although not showing so great a difference as the preceding one; thus, while the saliva alone converted 23°72 per cent. starch into reducing bodies, the presence of 0°05 per cent. of quinine sulphate led to the conversion of 24°58 per cent. of starch. Voit, as quoted by v. Boeck,* has stated that qui- nine is without influence on the ferment of saliva. Cinchonine sulphate. With this alkaloid, previous experiments have not to our knowl- edge been tried. Our results are as follows: Alkaloid salt. Wt. Cuin 4. rohaehin bedisek ghee acted: 0 0°1358 gram. 02768 gram. 24°91 per cent. 0°05 per cent. 071452 0°2960 26°64 0°50 0°1455 0°2964 26°67 2°00 071440 0°2936 26°42 Cinchonidine sulphate. This alkaloid, like the cinchonine, shows a steady accelerating action on the ferment. Total amount Starch Alkaloid salt. Wt. Cu in 4. reducing bodies. converted. 0 0°1358 gram. 0°2768 gram. 24°91 per cent. 0:05 per cent. 0°1505 0°3068 a 2ugor 0°50 0°1460 0°2976 26°78 1-75 071498 0°3056 27°50 The cinchona group of alkaloids thus show throughout an acceler- ating influence on amylolytic action, most pronounced in the case of cinchonidine. These alkaloids have long been known to prevent putrefaction and to check alcoholic fermentation and Binzt has demonstrated that this antiseptic action, in the case of quinine at least, is due to the poisonous influence exerted by the latter upon the fungi which are the immediate cause of the putrefactive changes. Couzen, moreover, has shown that the action of cinchonine on infusoria and on fermentation is similar to that of quinine, but weaker. Hence there is no similarity of action whatever, on the two kinds of fer- ments. * Zeitschrift fiir Biologie, vol. vii, p. 428. + Virchow’s Archiv, vol. xlvi, 1869, p. 68. TRANS. Conn. AcaAD., Vou. VII. 10 Oct., 1885. 74 Chittenden and Painter—Influence of Therapeutic Atropine sulphate. With this alkaloid we obtained the following results: Alkaloid salt. Wt. Cu in 4. PCIE DEICR, Cohveie 0 071485 gram. 0°3028 gram. 27°25 per cent. 0°025 per cent. 0°1460 0:2976 26°78 07050 0°1410 0°2872 25°62 0°200 0°1530 0°3124 28°11 0°500 0°1407 0°2864 25°77 1-000 0°1475 0°3004 27°03 2°000 0°1245 0°2532 22°78 The main action of the smaller percentages of this alkaloid seems to be a slightly hindering one, although there are one or two irregu- larities in the results which are not readily explainable. In the presence of 2°0 per cent. of atropine sulphate there is a decided diminution in amylolytic action. In connection with this alkaloid we have to note some recent ex- periments of Stolnikow* of St. Petersburg. This investigator, pro- ducing artificial fever in dogs by the injection of putrid matter into the blood found, first, that the salivary and pancreatic secretions were for a time increased in amount and then rapidly diminished and finally entirely ceased. This latter action of the septic poison, Stolnikow found to be very persistent and he moreover states that in physiological action the septic poison resembles atropine. Fur- thermore that artificial fever, produced as described, exercises a decided influence on the content of ferments in the pancreatic gland; that in fevers of short duration (2-10 hours) the ex- tract of this gland has a more energetic ferment action than the normal extract, while in fevers of long duration the corresponding extract has a much weaker action. Overlooking now the physiologi- cal explanation suggested for these facts we come to the chemical one, viz: that the septic poison possibly exerts either a destructive or hindering influence on the ferment or its action. In support of this view, Stolnikow found that large quantities of the poison did weaken the amylolytic and proteolytic action of extracts from the pancreatic gland, although small quantities of the septic ferment were without action. Likewise, Stolnikow states that small quanti- ties of atropine sulphate are without action on a glycerine extract of the pancreas, but by adding to 10 c. c. of a glycerine extract, 5 ¢.¢. of a 3°0 per cent. atropine sulphate solution and allowing the mixture to stand at the ordinary temperature for 10 hours, then on * Beitrage zur Lehre von der Function des Pancreas im Fieber. Virchow’s Archiv, vol, xe, p. 389, 1882, and Toxie Agents on the Amylolytic Action of Saliva. 75 testing the amylolytic power of the ferment its action was found to be much weaker than the control. From this fact, Stolnikow consi- ders that the septic poison acts upon the ferment outside the body in a manner similar to atropine. Now it is obvious, in view of the extreme susceptibility of the ferments of the saliva and pancreas to the action of acids and alka- lies, that the atropine solution must be perfectly neutral. Several specimens of atropine sulphate that we have examined, have had a slight acid reaction. In view of the apparent identity of the amylolytic ferments of the salivary and pancreatic secretions we have repeated in principle Stolnikow’s experiment with human saliva, using perfectly neutral atropine sulphate. To 10 ¢.c¢. of the dilute, neutral, saliva hitherto used, 0°3 gram of pure atropine sulphate was added (=3-0 per cent. of the alkaloid salt, while Stolnikow’s mixture contained but 1:0 per cent.) and the solution allowed to stand for 18 hours at the Laboratory tempera- ture. On now being added to the starch solution, diluted up to 100 c.c. [0°3 per cent. atropine sulphate] and placed at 40° C. for 30 minutes, the starch paste quickly became clear and it was found on examination that 29:16 per cent. of starch had been converted, while the control, in the presence of 0:2 per cent. of atropine sul- phate, showed a conversion of 28°11 per cent. of the starch. Hence there had been no destruction of the salivary ferment by even 3-0 per cent. of pure atropine sulphate, although as our previous experi- ments show very much smaller percentages may, by their presence, hinder the auction of the ferment. Strychnine sulphate and brucine sulphate. O. Nasse has previously studied the influence of 0-1 per cent. strychnine acetate on the diastatic action of saliva and has noted a slight increase in amylolytic action in the presence of the strychnine [109:100]. Our results with the two alkaloids are as follows: Total amount Starch Alkaloid. Wt. Cu in 4%. reducing bodies. converted. 0 0°1485 gram. 0°3028 gram. 27-25 per cent. Strychnine sulphate. 0-050 per cent. 01444 0°2936 26°42 ("250 0°1448 0°2936 26°42 0500 071462 02976 26°78 Brucine sulphate. 0:050 per cent. 0°1503 0°3060 27-54 0°500 071524 0°3100 27°90 1000 0°1505 03060 27°54 76 Chittenden and Painter—Influence of Therapeutic With the brucine salt a slightly increased action is noticed in all three of the experiments; while with strychnine a constant diminu- tion in amylolytic action is to be seen. In this connection it is to be remembered, that a trace of free acid in the alkaloid salts would introduce an appreciable error into the results, and therefore all of the alkaloid salts experimented with, were especially purified for this purpose, any adhering acid being removed by repeated crystallization, etc. The following table shows the relative acceleration and retardation of the various salts (the percentages more generally used) compared with their controls expressed as 100. Table showing relative amylolytic action. | | | s Aaa 0°0005 | 0°001 | 0:002 | 07005 | 0°010 | 0:025 | 0°05 O1 05 1°0 2°0 p.c. | p.c.| p.c.| p.c.| p.c. | p.c. | p.c. | p.c.| p.c.| p.c. | pie | | ; HipGls SY Jk. Soot ees 94-81 5078] (38:2 Gr (0) | 2.22) Ghee ese ele eee a$i3 EBs GSo4-4--+2=2-e- SSIES) aya) 7 (53 | Slee Cet) See eee eee 2 ee pls 222255224 = =-- ae oh a en Se EC) 7G eA al | Ral | De Se ea AE | os! ce he a (CN) RY sesh O2 = See 2 EAOG 2 B39) bere eee ae TTG6le 225.) 33. eee eee CuSOne: HeWOk se se 8s So 2° 84:0) oS SB ee 2s) ba Oe Se he eee | eee Pb(C2H302)2+3H O _..|100-7|100°3]100°3) 97-7) 85-2) 85-7) .-. | 99°5| 98°6 88°9|} 78°B} .2-- Wes Os Shee Cle LER 02-2) 104-4:1.00:0))1 0676) 9422) cece) eosai eck - Ape ee eee AEG ates a este 11005) 93:0) 22-2" (O02) s202).coe2) 2.25) 22 oe (NH,)3;AsO4 a aes ae 106-2 107°0| -..-/109°6) =.=. |/111"4! 85°8) 66°G) 10:5) ‘Ory Seee K(SbO)C,H40¢ ie Wek See eis _..|104°5| _..-|107°9}114°5] __-_|114°5}120°2)168°3]101-6) 79°4 SO lara eta eee NOS ests] Oli eee eae eee eee ....) ceed) ese eee eee WnSOne UelgO be ae ee 99°7|101°3| 98°5| 96°3} 90°9) 84-5] _.. | 52:8) 47°5) O | -2.-] ..-. Heal, eeseee ae see ieee = L OUD) ceneet DOB ect GED) 2 Obi sss ae op cree ee MaSOpa TiisO oo 2k oes Sock 8322) 22221063) Seer MOO yee Sy | ere rere ree eee Sen K,.Mn.O3 Se oe ey ee | =---| ---- --=-| 68'D 2 0 mone] seen] - eee] ccce} = see MgSO,+7H.0 EAN eee oe Vk aah et ee Ciea| £2 cole 508 Oi, | ene 3571 eee IKON oc ow eet case Se BGO eT 22a eee eee ofl lel es Ss) See eee K,Fe(CN),+3H20 SS ete lami --- ----| ----| ---- ---- -|105°7 as<= 970) 22 ae eee K,Fe.(CN)s2 Zee ood bo eae joeee| cmne | ----| ---- See LOMO ee 91y3| ee eee = KEN Os eee Fe ee ee ees eae eee Fee) Pes set Petes Sees Pose oo 100°9| 96:6)/28e2 EOL OE eae ee a eee nel teria ieee ate Porm) i SSIs acy eta 3 -2oci104:8/106'0) See ROBT ae ick bee ee See Nee peice) se on) t= se rel cis ero ae 2._|05*G)\ eee ee TEE Be em PN TE RN | 2 eS) | ses ee ed eco feast econ ea oe a Se Oss, NaG@lee 22 on SSUES LE EP Ee 2 eit) Octal) SER] P= eta, eet hi Sie Reiti= 105°5/103°3/103"4 Wanb.One OH. Oe ssa == | pee— | --- | ----| -- SESS Sal esso 0° 64a) 3252) See eee (Mo). . H ASOat Dele Ota eee Pee ese Seyegs | ames Saori ere MAG) | = Se 129-4 wees “\114-9 (Q)e- H.S0,+7Hs Orage Se5 SEG | Geel ooe. | oost bas se 108°5| .-._|| 99:4) 22a aie (Ci)s. H,SO,+2H,O ---] ----| ----] ----| ----] ----] ----| ---- 106°9] _-_.|107°0} _._-]106°6 (Ci dine).. H.S0O,+3H,O| ----| ----|] ----| ----] ----| ----| ---- 110:8|°2. 2.110% "bese see (At)e. HG SOp eso. See eeeee eal Se hess | ==! vom are _.--| 98.2] 94:0] _...] 94°56] 9971] 83:5 : (Sr)o. H.S0, +6H,0 ada] sane] ----] enes|)----] see <| S-es] meee 96:9//e22- 98:2) SA > (Br), . H.SO,+ H,O ---.] ----|] ----] ----] -+--| ----| ----1 ---- 101°0| ~-_-|102°4|101-0] -.--| 2 and Toxic Agents on the Amylolytic Action of Saliva, ~T ~vT Influence of gases on the amylolytic action of saliva. The well known analysis by Pfliiger* of the gases of the submax- illary saliva have shown the presence of both oxygen and carbonic acid in this secretion; oxygen to the extent of 0°6 vol.-per cent. and carbonic acid, by pump extraction, 22°5 vol.-per cent. It is, moreover, a well known fact that as the saliva flows into the mouth and becomes mixed with the food during mastication much air is ab- sorbed. Do these three gases exert any influence on the amylolytic action of the ferment with which they are so constantly in contact ? Again, the amylolytic ferment of the pancreatic secretion, so near yakin, it not identical with the salivary ferment, is subjected to the influence of the reducing gases of the intestinal canal, among which hydrogen may be present to the extent of 220+ vol.-per cent. and hydrogen sulphide in traces. What likewise is the effect of these two gases on amylolytic action ? The experiments were conducted as follows: 90 ¢. c. of diluted starch paste were placed in small, partially stoppered flasks and a stream of the gas allowed to pass through, until the fluid was thought to be saturated, then 10 c.¢. of dilute saliva were added and the gas allowed to bubble through the solution for 30 minutes when the mixtures were boiled and the reducing bodies determined. Follow- ing are the results: Total amount Starch. Gases. Wt. Cuin 4. reducing bodies. converted. 0 071319 gram. 0 2684 gram. 24°15 per cent. ATP CR Stee eu svdde Shs 0°13€5 0°2780 25:02 Oxyorenetss5%. ceckos = 071511 0°3080 20-72 Carbonic acid...-.--.- 0°1537 0°3136 28°22 Hydrogen sulphide ----0°1377 0°2804 — 25.23 iEydropeniss2 2---+=: 0°1248 0°2540 22°86 It is interesting to see that air, oxygen and carbonic acid all stimu- late and approximately in proportion to the extent in which they are present in the natural secretion, while of the sees gases hydro- gen retards and hydrogen sulphide stimulates. The following table shows the relative acceleration and retardation of the several gases, compared with the control, expressed as 100. AUT We Oe Wet Je ek ts ee ees 103°6 Oxygenias2 85 2s ul Seer ee eee ss 114-7 Carbonic /aeila=2 52.2.5 Serer a eo 116°8 Hydrogen sulphide.) 2 2eosoe. - ee ce 104.4 la OO RN oe RO eee ees Saeaee eee 94°6 * Physiologische Chemie, Hoppe-Seyler, p. 192. + Maly in Hermann's Handbuch der Physiologie, vol, v, p. 25, 78 Chittenden and Painter—Influence of Therapeutic In accord with our results, Detmer* has found that the presence of carbonic acid invariably increases the amylolytic action of the diastase of malt. The same fact was previously observed by Bas- witz.t O. Nasse,{ however, has stated that the activity of ptyaline in human mixed saliva is not materially affected by oxygen, hydro- gen or air. With carbonic acid, however, he noticed acceleration in amylolytic action. Nature of the action of the metallic and other salts. In what manner do the metallic and other salts act when they, by their presence, retard or completely stop the amylolytic action of saliva? Is it a process of gradual or sudden destruction of the fer- ment, or does the metallic salt combine with the ferment, forming a compound incapable of ferment action? or again, is the ferment mechanically thrown down with the precipitate of albumin or globu- lin produced by the addition of the metallic salt to saliva, or lastly does the salt by its mere presence introduce a condition unfavorable to the action of the ferment? All of these questions are interesting ones, and possibly all of them might be answered in the affirmative and be correct for some one or more of the substances experimented with. . It is obvious that the presence of 10 or 20 per cent. of such a salt as sodium chloride or potassium nitrate in a digestive mixture might retard the action of the ferment, since solutions so saturated, even with the products of digestion, do not admit of vigorous ferment action. But the larger number of metallic salts decidedly retard amylolytic action when present to the extent of only a few thou- sandths of one per cent., consequently their action must be of an entirely different nature. A number of these salts, such as mercuric chloride, are well known precipitants of albumin, but the saliva being so greatly diluted, in great part for this very reason, cannot yield sufficient precipitate with the mercury salt to mechanically precipi- tate the ferment. As a matter of fact, when the mercuric chloride solution is added to the diluted saliva, a very faint turbidity only, is produced. If now, some of the small percentages of mercuric chloride are added to the starch solution and then larger quantities of saliva, thus giving a larger amount of ferment together with a larger amount of accompanying albumin and globulin, what would * Zeitschrift fir physiol. Chemie, vol. vii, p. 3. + Berichte d. deutsch. chem. Gesell., vol. xi, p. 1443, f Pfliiger’s Archiv, vol, xv, p. 471-481, and Toxic Agents on the Amylolytic Action of Saliva. 79 be the effect on the amylolytic action of the ferment? Might we not expect, knowing that albumin and mercuric chloride readily combine, that the proteid matter present in the saliva, would serve as a shield to protect the ferment from the action of the mercury or other similar metallic salt? At the same time it might be supposed that, the fer- ment being left intact, any mercury-albumin compound formed might retard or destroy the ferment, though less energetically than the metallic salt alone. In an attempt to throw some light upon these points the following experiments were tried: Action of mercuric chloride in the presence of larger amounts of Jerment and proteid matter. a. with 10 e.¢. of original saliva. HegClo. Wt. Cu in 4. solani bodies: éconorted. 0 0-1772 gram. 0°3624 gram. 32°61 per cent. 0°0005 per cent. 071735 0°3548 31°93 0°0010 01695 0°3464 31°16 b. with 5 c. ec. of original saliva. 0 071720 gram. 0°3516 gram. 31°64 per cent. 0°0005 per cent. 01340 0°2728 24°55 Comparing these results with those previously obtained with the same percentages of mercuric chloride, but with 2 c.c. of original saliva, we have: HegClo. 2¢.¢. Saliva. 5c. c. Saliva, 10 ¢.c. saliva. 0 44°28 per cent. 31°64 per cent. 32°61 per cent. 9:0005 per cent. 23°40 24°55 31°93 0-001 16°92 has oe 31°16 The intensity of action of the mercuric chloride, say 0°0005 per cent. in the three cases, varies greatly; thus with 2 c.c. of saliva the difference in the percentage of starch converted, between the control and the 0°0005 per cent. is 20°88, while with 5 ¢.c. of saliva the differ- ence is 7°09 and with 10 c.c. of saliva only 0°68. Obviously then, the action of a given percentage of mercuric chloride can be considered as constant only for a given mixture or under definite conditions. Moreover, it would appear (in the 10 ¢.c.) that either the albuminous matter of the saliva has combined with all of the mercury, leaving the ferment free to act in a normal manner, except so far as it is impeded by the mercury-albumin compound, or else that only a small proportion of the ferment has been chemically precipitated, leaving an amount sufficient for energetic amylolytic action, since, as is well 80 Chittenden and Painter—Influence of Therapeutic known, increase or decrease in the amount of ferment is not always followed by a proportionate change in the amount of reducing bodies formed. Of these two views the former is by far the most probable. Certainly the ferment is not mechanically precipitated by the formation of a mercury-albumin prcipitate; if such were the case with 10 c.c. of saliva and 0-001 per cent. of mercuric chloride, decided retardation ought to have been observed. Action of cupric sulphate in the presence of larger amounts of CusO4 +5H» O. 0 0°0005 per cent. 0 0°0005 per cent. 0 0:0005 per cent. Wt. Cuin 4. Serment and proteid matter. a. witn 10 ¢c.c. saliva. Total amount reducing bodies. 0°1830 gram. 03748 gram, 01775 0°3628 Difference, b. with 5 c.e. saliva. 0°1745 gram. 0°3568 gram. 0°1640 0°3352 Difference, c. with 2 c.c¢. saliva. 0:1645 gram. 0°3360 gram. 0:1140 0°2320 Difference, Starch conyerted. 33°73 per cent. 32°65 1:08 32°11 per cent. 30°16 1°95 30°24 per cent. 20°88 9°36 Action of zinc sulphate in the presence of larger amounts of ferment ZuSO4 +7H,0. 0 0°05 per cent. 0 0°05 per cent. 0 0°05 per cent. Wt. Cuin 4. and proteid matter. a. with 10 ¢.c¢. saliva. Total amount reducing bodies. 0°3748 gram. 0°3552 0°1830 gram. O-1737 Difference, b. with 5 c.c. saliva. 0°1745 gram. 0°3568 gram. 01610 3288 Difference, c. with 2c. ¢. saliva. 0°1645 gram. 0°3360 gram. 0:1320 0°2688 Difference, Starch converted. 33°73 per cent. 31°96 Lr rf 32°11 per cent. 29°59 2°52 30°24 per cent. 24.19 6°05 and Toxie Agents on the Amylolytic Action of Saliva. 81 { yoouy ) Glancing at the differences in these two series of experiments, we see that they accord with what was observed in the case of mercuric chloride, viz: that a given percentage of the metallic salt will pro- duce a constant result only under definite conditions; increasing the proportion of albuminous matter diminishes, as in the case of the mercury salt, although not so greatly, the retarding action of the salt. Evidently, the metallic salts do not act upon the ferment by their mere presence, for if such were the case the mere combination of the salt with the albumin present, would not so materially affect the result. If, on the other hand, they do act by combining with the ferment, forming it may be an insoluble compound or one incapable of ferment action, it is fair to presume that the combination would take place immediately upon mixing the two or very soon thereafter, and thus we should expect that the length of time the two stood in contact after the first few minutes, would have no effect on the amyl- olytic power of the mixture, while a gradual destructive action would be manifested by a gradual decrease of amylolytic power. With a view to testing this point we have tried the following experiment. Three mixtures were prepared as follows: A. B. G Sallivaerarae earns Ne cee. yon: 2, G28 YOKE TEU (Octet se eet de eee 8 7 8 Hig WlS sols SISce St Suse 0 1 2 10 10 10 Bercent HeCly esse cece 0 0°005 07010 These were placed in a bath and warmed at 40° C. for 18 hours, after which 1 ¢.c. of the same mercuric chloride solution was added to A and then starch and water added to all three, making the volume in each case up to 100 ¢.c. The mixtures were then warmed at 40° C. for thirty minutes to test the activity of the ferment; A containing now 0°0005 per cent. mercuric chloride, 6 the same per- centage and C’ 0-001 per cent.. In A, 19°8 per cent. of the starch was converted into reducing bodies, while in 6 and C there was no amylolytic action whatever. Thus by the previous action, for this length of time, of 0:005 per cent. mercuric chloride, the ferment was rendered incapable, on subsequent dilution, of exerting any diastatic action whatever. Again, in a similar manner it was found that by warming the , saliva for thirty minutes at 40° C. with 0°005 per cent. mercuric chloride and then adding starch paste and diluting to 100 ¢. ¢. so TRANS. Conn. AcApD., Vou. VII. 11 Oot., 1885, 82 Chittenden and Painter—Influence of Therapeutic that the percentage of mercuric chloride was 0:0005, only 2°0 per cent. of the starch was converted, while the same quantity of saliva, in the presence of the same amount of mercury salt (0°0005 per cent.) converted 19°69 per cent. of starch. Working with larger amounts of saliva, the following results were obtained : Ar B. C. OV eee we eee 10 c.c. 10 ¢. ¢. 10. cc. [nL O Sut 5 us 8 ie apd ee 16 15 15 Hic Ci soln ee ose ss 0 1 1 26 26 26 Percent, ie Clas == 0 0°002 0-002 B was warmed at 40° C, for 15 minutes and C for 30 minutes; then 1 c. c. of the mercuric chloride solution was added to A, and all three diluted and mixed with starch paste. The three solutions were now exactly alike; all contained the same percentage of mercury salt (0°0005 per cent.) but B and C had been previously warmed with the salt for 15 and 30 minutes respectively. A, converted 31°32 per cent. of the starch, B 29°48 per cent. and C 27:97 per cent. Here we have what appears to be a gradual decrease in amylolytic power, but it does not seem sufficiently pronounced to account for the action of the mercury salt. It would appear rather, in this instance, as if the mercuric chloride exercised a selective action, combining with the proteid matter of the saliva, leaving the ferment free; but the mer- cury-proteid compound, being apparently possessed of some destruc- tive action, exerts its influence, and thus the gradual decrease of amylolytic power noticed in B and C. In the previous experiments, on the other hand, where /ree mer- curic chloride is present, there not being sufficient albumin to com- bine with all of the mercury, there is apparently destructive action. Experiments of like nature as the preceding, tried with cupric sulphate, gave the following results: A. B. C D. Saliva ose eee os = 2 G.C, 2 Cle 22GNG, 2ac¢ HOM Sse ee ore 18 17°8 17°8 17°8 CuSO.s0l- sess: 0 072 0-2 0:2 20 20°0 20°0 20°0 Per cent. CuSO,--. 0 0-0005 0:0005 0°0005 B was warmed at 40° C. for 15 minutes, C for 30 minutes and D for 1 hour; 0:2 ¢.c. of the cupric sulphate solution was then added to A and lastly starch paste and water to 100 c.c. The amylolytic ee i i i and Toxic Agents on the Amylolytic Action of Saliva. 83 power of the four mixtures, expressed in the percentage of starch converted, was as follows: A. B. Cc. D. 28°72 22°35 23°04 20°23 With zinc sulphate, somewhat similar results were obtained: A. B. C. SENT 2 SO nee ee 2¢c.¢. PONG: 2¢.¢. IG OMe ea tise te ee 18 16 16 PNSOT SON Se sap Seacue 0 2 2 20 20 20 Revicenta/Z0 SO, ease = = 0 0°05 0°05 B was warmed at 40° ©. for 30 minutes and C for 1 hour; then 2 c.c. of the zinc sulphate solution were added to A, and all three ~ mixed with starch paste and water to 100 ¢.c. Each now contained 0°01 per cent. zine sulphate and all three were then warmed at 40° C. for 30 minutes, to determine the activity of the ferment. A, converted 22°24 per cent. of the starch, 6 11°88 per cent. and C 10°98 per cent. These experiments would therefore indicate, on the part of the metallic salts experimented with, a destructive action towards the ferment, though loss of amylolytic power under the conditions of the experiments might also be due to more complete precipitation of the ferment in the more concentrated solution and under longer exposure to a temperature of 40° C. At the same time it is to be noticed, that any metallic-proteid compound formed with the above salts, has a far less destructive or retarding action than the free salt. Of these, the destructive action of mercuric chloride is most pronounced. Potassium permanganate acts, doubtless, by direct destruction of the ferment through oxidation, while many of the alkali and alkali- earth salts produce their retarding effects by simple clogging of the digestive fluid; but the fact that 0°5 per cent. of one salt, as potassium antimony tartrate, for example, increases the amount of starch con- verted 68 per cent., and 0°5 per cent. of another salt, as magnesium sulphate, diminishes the amount of starch converted by 65 per cent., plainly indicates that there is something in the presence of these salts, dependent upon chemical constitution, that controls the action of the ferment. VI.—INFivence or Various Inorganic AND ALKALOID SALTS ON THE PRotTEOLyTIC AcTION oF PEpsIN-HypROcHLoRIC AcIp. By R.-H. Currrenpen anpv S. E. Aten. ALTHOUGH many experiments have been tried to ascertain the influence of various salts on ferment action since 1870, when Liebig * recorded the statement that the fermentative power of yeast is somewhat increased by a little potassium or sodium chloride, few systematic experiments, with a large variety of salts, have been made with the ferment of the gastric juice. Alex. Schmidt + in 1876 studied the influence of sodium chloride on the digestive action of pepsin and hydrochloric acid. Wolberg { in 1880 studied, with the same ferment, the action of ammonium, potassium and sodium salts of nitric, hydrochloric and sulphuric acids and also the action of several alkaloids. _Wernitz§ and also Petit|| have studied the action of several metallic salts. Still later, Pfeiffer] has examined the influence of several alkali and alkali-earth salts on the digestive action of pepsin as well as of other ferments. Isolated experiments with single salts have likewise been recorded ; these will be noticed later on. It is thus seen that almost all work in this direction has been done with salts of the alkali and alkali-earth metals. No systematic attempt has been made to ascertain the influence on gastric diges- tion of the large number of metallic salts, in common use as poisons or therapeutic agents. With the exception of a few isolated cases, no accurate data are recorded bearing on this question. Observation has led to the belief that certain metallic salts interfere with diges- tion in the stomach, but few quantitative results are recorded to show the truth of such a belief. * Ueber Gahrung, Quelle der Muskelkraft und Ernabrung. Separatabdruck aus den Annalen der Chemie u. Pharmacie, 1870, p. 61. + Pfliiger’s Archiv, vol. xiii, p. 97. Ueber die Beziehung des Kochsalzes zu einigen thierischen Fermentationsprocessen. ¢ Pfliiger’s Archiv, vol. xxii, p. 291. Ueber den Hinfluss einiger Salze und Alka- loiden auf die Verdauung. § Quoted by Brunton. Pharmacology, p. 85-86. || Etudes sur les ferments digestifs. Abstract in Jahresbericht fiir Thierchemie, 1880, p. 309. ; “| Ueber den Einfluss einige Salze auf verschiedene kiinstliche Verdauungsvorgange, Abstract in Centralbl. med. Wiss., 1885, p. 328. on the Proteolytic Action of Pepsin-hydrochloric Acid. 85 @ Our aim has been, therefore, to study more particularly the com- parative influence on gastric digestion of various percentages of those salts, well known as poisons or therapeutic agents, which have hitherto been overlooked or but imperfectly studied. At the same time in order to make the work more complete, we have studied somewhat, the action of the alkali salts, experimented with by other observers. Method employed. The experiments were conducted in series, in which one of each series served as a control for comparison. The artificial gastric juice employed, was made from 0:2 per cent. hydrochloric acid and a glycerine extract of pepsin, in the proportion of 10 ¢.c. of the latter to 1 litre of the former. The volume of each digestive mixture was 50 c.c.; made up of 25 c.c. of the above-mentioned gastric juice and 25 ¢.c. of 0°2 per cent. hydrochloric acid, containing the salt to be experimented with. The material to be digested, consisted of puri- fied and dried blood-fibrin, prepared by thorough washing with water, extraction with cold and boiling alcohol and lastly with ether. It was then ground to a coarse powder and dried at 100-110°C. 1 gram of the fibrin was used in each experiment. The digestive mix- tures were warmed at 40° C. for two hours, then filtered upon weighed filters by the aid of pumps, the residue washed thoroughly with water, lastly with alcohol, and finally dried at 100-110° C. until of constant weight (48 hours). The amount of fibrin digested or dis- solved, is a measure of the proteolytic action. Cupric sulphate. With this salt two series of experiments were made; one to ascer- tain the influence of small quantities, the other to show the effects of larger amounts of the substance. 7 Undigested Fibrin Relative proteo- CuS04+5H20. residue. digested. lytic action. 0 0°2854 gram. 71°46 per cent. 100°0 0°001 per cent. 0°2508 74:92 104°8 0°005 0°2650 73°50 102°8 07010 0°3067 69°33 970 0:025 0°3845 61°55 86°] 0°050 0°3877 61°23 85°6 0 0°2352 76°48 100°0 0'1 0°5315 46°85 61°2 0°3 0°7585 24°15 31°5 0°5 0-7976 20°24 26°4 0'8 0°8214 17°86 23°3 15 0°8480 15°20 19°8 86 Chittenden and Allen—Influence of various Salts The action of the salt is very marked; with even 0°010 per cent. there is a diminution in proteolytic action amounting to 3:0 per cent., while in the presence of 0°5 per cent. of the salt, there is retardation to the amount of nearly 75 per cent. The copper salt prevents almost entirely the swelling of the fibrin and doubtless its retarding action is due in part to this fact. Lead acetate. In view of the frequent cases of chronic poisoning with lead salts, the influence of the acetate on gastric digestion, seems especially interesting. The results, moreover, show decided action on the part of the salt; with small fractions of a per cent. pronounced increase in proteolytic action is to be noticed, while beyond 0°5 per cent. there is sudden and almost complete cessation of ferment action. In this respect, the salt acts very differently from the copper salt, with which a more gradual diminution is observed. The two largest percentages of the lead salt prevented entirely the swelling of the fibrin. Undigested Fibrin Relative proteo- Pb(C2H302)2+3H20. residue. digested. lytic action. 0 0°1936 gram. 80°64 per cent. 100°0 0-001 per cent. 0°1592 84-08 104°2 0°005 071892 81°08 100°5 0-010 01781 82°19 101°9 0:025 01691 83°09 103°0 0 0°2140 78°60 100°0 O01 072310 76:90 97°8 0°3 0°4523 - 54°77 69°6 0°5 0°7419 25.81 32°8 0°8 0-9779 2:21 2°8 1°5 079938 0°62 07 Mereuric chloride. This salt, which showed such a marked action on the amylolytic ferment of the saliva, causes a like diminution of proteolytic action in the case of pepsin; even with 0°001 per cent. there is retardation to the extent of over 6 per cent., calling the action of the control 100. Petit* very erroneously states that mercuric chloride up to 04 per cent. does not hinder the action of pepsin. * Btudes sur les ferments digestifs. Abstract in Jahresbericht fir Thierchemie, 1880, p. 309. ‘ean on the Proteolytic Action of Pepsin-hydrochloric Acid. 87 Undigested Fibrin Relative HgClg. residue. digested. proteolytic action. 0 0°3759 gram. 62°41 per cent. 100°0 0-001 per cent. 04140 58°60 93°8 0-005 0°4210 57°90 92:7 0 0°1307 86°93 100°0 0-1 04765 52°35 60°2 0°5 0:9007 SR 11°4 10 1:0495 ; 0 0 M. Marle* has previously experimented with mercuric chloride and has likewise found that small quantities of the salt exercise a retarding action upon gastric digestion; that as the percentage of corrosive sublimate is increased, the retarding action is correspond- ingly increased, although this effect is diminished up to a certain point, by increasing the strength of the digestive mixture. Marle considers that this action of mercuric chloride does not depend upon decompo- sition of the ferment nor upon a contraction of the albuminous matter, but rather that the salt in an acid solution enters into a chemical combination with the proteid matter and the latter is thus rendered impervious to the digestive action of the ferment. In support of this view we offer the fact that fibrin introduced into an acid solution of pepsin in the presence of 1 per cent. of mercuric chloride, increases in weight; in the experiment given above to the extent of 49:5 milligrams. This would clearly indicate a combina- tion of the two. Moreover, that mercuric chloride does not act by destroying the ferment we have ample proof, as the following experiment shows : : A. B. G H,0 sol. glycerine pepsin _--- 5c.¢. Sec: biexe: UCI. (0:2: per cent.) 2-222 - 20 20 0 EUG Ul npeyens be Ace Sere Meee ao 0 07025 gram. 07025 gram. 155 (0), ie a Ra a hs 0 0 20 ¢ ¢: 25 25 ¢. ©. 25 Rercent, tieCl, 20es oo ses 0 0-1 01 These three mixtures were warmed at 40° C. for 24 hours; then to A was added 0:025 gram HgCl, dissolved in 25 ¢. c. 0°2 per cent. HCl, to B 25 c.c. 0:2 per cent. HCl and to C 25 c.c. 0-4 per cent. HCl. The three solutions were now exactly alike; in 6, however, the ferment had been exposed to the action of 071 per cent. HgCl, in an acid solution for 24 hours, in C’ to the action of the same percent- age of the mercury salt in an aqueous solution, while A served as * Abstract in Jahresbericht fir Thierchemie, 1875, p. 168. 88 Chittenden and Allen—Influence of various Salts a control. 1 gram of fibrin was added to each of the mixtures, which were then placed at 40° C. for 2 hours. Band C digested the same amount of fibrin as A, consequently the mercuric chloride could have exerted no destructive action whatever on the ferment. Wassilieff,* in Hoppe-Seyler’s laboratory, found by comparative experiments that mercurous chloride (calomel) has no effect on the proteolytic action of pepsin. Mereuric bromide, Mercuric iodide and Mercurie cyanide. These three salts of mercury were experimented with, only so far as to compare the action of small quantities, with the action of like quantities of mercuric chloride. In using the bromide and iodide it was necessary, on account of their insolubility, to dissolve them with the aid of an equal weight of sodium chloride, consequently these two salts of mercury were doubtless present in the digestive mix- tures, in part at least, as double salts. Marle, however, found that the action of mercuric chloride with small quantities of sodium chloride was not different from that of mercuric chloride alone, and doubtless the same is true of the iodide and bromide of mercury. Following are the results we obtained: Mercury Undigested Fibrin Relative proteo- salt. residue. digested. lytic action. 0 0°3590 gram. 64°10 per cent. 100°0 HegBr. 0-005 per cent. 0°3731 62°69 97°8 0°025 073980 60°20 95°9 Hel, 0-005 03114 68:86 107-4. 0°025 0°3904 60°96 95:1 He(CN). 0°005 i 0°3105 68°95 107°5 0°025 0°3985 60°15 93°8 0-100 0°3183 68°17 106°3 From these it is evident that mercuric bromide is less vigorous in its hindering action than mercuric chloride ; the iodide still less so, while mercuric cyanide, in similar percentages, appears to cause an increase in proteolytic action. The iodide, likewise, in the smallest percentage experimented with, causes increased proteolytic action. None of these salts then, approach mercuric chloride in the intensity — of its hindering action on gastric digestion. * Ueber die Wirkung des Calomel auf Gihrungsprozesse und das Leben von Mikro- organismen. Zeitschrift f. Physiologische Chemie, vol. vi, 113. > a 2 on the Proteolytic Action of Pepsin-hydrochloric Acid. 89 Stannous chloride. This salt shows marked action in retarding gastric digestion; its retarding effect increasing directly with the amount of stannous chloride added. Undigested Fibrin Relative proteo- SnCly. residue. digested. lytic action. 0 0°2576 gram, 74-24 per cent. 100°0 0-025 per cent. 0:2728 72°72 97°5 0-1 04826 51°74 69°6 0°5 0°7332 26°68 35°9 1-0 0°8155 18°45 24°8 2°0 0°9010 9:90 15 %3: Arsenious oxide. This substance might naturally be expected, in view of its well known antiseptic properties, to hinder proteolytic action, more or less. It is known to hinder putrefaction and to prevent also the fermentative action of yeast. Contrary to our expectations, however, the action of arsenious oxide, so far as it is to be seen, is an acceler- ating one, causing increased proteolytic action. The following results were obtained : 5 Undigested Fibrin Relative proteo- A8s9Q3. residue. digested. lytic action. 0 0:2111 gram. 78°89 per cent. 100°0 a 0-05 per cent. 01872 81-28 103-0 O01 0:2160 78°40 99°3 0-2 0°1900 81:00 102°6 0°5 ~ 0:1707 82°93 105°1 The stimulating action is slight, still it is plainly recognizable. Drs. Schiifer and Béhm* have previously studied the action of arseni- ous acid on the digestion of albumin by artificial gastric juice, and they came to the conclusion, using 0°02 and 0:04 gram As,O, respec- tively, in 34 c. c. of fluid containing egg-albumin, that arsenious oxide is without influence on the decomposition of albumin by the gastric juice ferment. Our results, though not so large in number as theirs, would indicate a slight accelerating action. Arsenic is known, when administered in small, repeated doses, to act as a tonic; the history of arsenic-eating, indicates that the sub- stance has some positive tonic influence over nutrition, and Dr. * Jahresbericht fiir Theirchemie, 1872, p. 363. Ueber den Hinfluss des Arsens auf die Wirkung der ungeformten Fermente. Trans. Conn. Acap., Vou. VII. 12 Oot., 1885. 90 Chittenden and Allen—Influence of various Salts Wood* states, “ there is much reason for believing that it acts largely as a direct stimulant to nutrition.” The results obtained in our ex- periments certainly accord with this statement. Arsenic acid. The experiments tried with arsenic acid, tend to confirm the accelerating action noticed with arsenious oxide. In the first series of experiments the following results were obtained: Undigested Fibrin Relative proteo- H3As04. residue. digested. lytic action. 0 0°2696 gram. 73°04 per cent. 100°0 0-2 per cent. 02614 73°86 LOTS" 0°5 01514 84°86 1161 2°0 0°2583 7417 101°5 5-0 0°3915 60°85 83°3 \ The accelerating action is here so very pronounced, that a second series of experiments was undertaken by way of confirmation. These — give in a general way the same results, although with 0°5 per cent. the stimulating action is not so pronounced as in the first experiment. These two series of experiments illustrate another point, which it is well to mention here, namely: that definite percentages of any par- ticular substance do not invariably give precisely the same result, even when compared with their respective controls. They do, however, generally point in the same direction, and although not always giving exactly the same numerical expression, they show clearly the nature and extent of the action. : Undigested Fibrin Relative proteo- H3AsOq. residue. digested. lytic action. 0 0°2490 gram. 75°10 per cent. 100°0 _ 0°2 per cent. 0°2401 75°99 101°2 0°5 0°2367 76°33 -101°6 1:0 0°2335 76°65 102°0 2°0 0°2622 73°78 98°2 5°0 0°3176 68°24 90°8 0 071493 85°07 100°0 10:0 0°4207 57°93 681 Plainly then, arsenic acid in small percentages does accelerate the proteolytic action of pepsin-hydrochloric acid, while in large percent-_ ages (5-10) it causes a diminution in the action of the ferment. Arsenic acid tends to make the fibrin become very gelatinous. * Therapeutics, Materia Medica and Toxicology, p. 390. on the Proteolytic Action of Pepsin-hydrochloric Acid. 91 Zine sulphate. With this salt, no experiments appear to have been hitherto made. Our results show a decided diminution in proteolytic action, even in the presence of 0°01 per cent. of the salt, while with a few thou- sandths of one per cent. the figures indicate a slight accelerating action. Three distinct experiments were made as follows: Undigested Fibrin Relative proteo- ZnSO4+%7H 20. residue. digested. lytic action. 0 0°1744 gram. 82°56 per cent. 100°0 0-001 per cent. 0°1609 83°91 101°6 0°005 01617 83°83 101°5 0°010 0°2053 79°46 96°2 0°025 0°2573 74:27 89-9 3°000 0°8400 16°00 EOS 0 0°1630 83°20 100-0 0-1 0°4848 51°52 61°9 0°3 0°7133 28°67 34°4 0°5 0°7382 26°18 31-4 0°8 O:'T671 23°29 27:9 1:5 0°8202 17-98 21°6 0 071493 85:07 100:0 1:0 0:7683 23°17 27°2 A glance at these results, shows plainly a gradual decrease in pro- teolytic activity. It is to be noticed that in the presence of the larger percentages of these metallic salts, the fibrin does not swell up in the 0:2 per cent. acid. Manganous chloride. In small fractions of one per cent. this salt gave such irregular results that it is doubtful if they can be relied upon as expressing any particular action. With 0°3 per cent. the retarding action of the manganese salt commences to be very pronounced. Following are the results: Undigested Fibrin Relative proteo- MnCly. residue. digested, lytic action 0 01923 gram. 80°77 per cent. 100°0 0-001 per cent. 0°2022 79°78 98°7 0:010 0°1815 81°85 101°3 0°025 0°2066 79°34. 98-2 0:050 071855 81°45 100°8 0 0°1880 81°20 100°0 0°3 0°3687 63°13 SoS 08 0°6438 35°62 43°8 15 0°6612 33°88 41:7 3-0 0°7400 26°00 32°0 92 Chittenden and Allen—Influence of various Salts Ferrous sulphate and Ferric chloride. The salts of iron, doubtless on account of their physiological importance and their great therapeutic value, have been experimented with by several observers. It has been a prevalent opinion that iron salts tend to produce disturbances in gastric digestion. Petit,* how- ever, states as a result of experiment, that preparations of iron, in small quantities, do not hinder the action of pepsin, but in large quan- tities they retard the action of the ferment, doing so according to Petit, by the hydrochloric acid of the gastric juice displacing the acid of the iron salt, thus forcing the pepsin to act with a less energetic acid. Diisterhoff,t dealing with the same question, came to the con- clusion that iron salts of the organic acids, exercise the greatest retarding effect on pepsin digestion, and moreover, that ferrous salts are better adapted to the organism than ferric salts. Diister- hoff also concludes that while the retarding action of iron salts is doubtless due, in part, to the setting-free of the acid of the iron salt by the acid of the gastric juice, there is in addition a specific action of the iron preparation of an unknown nature, prejudicial to digestion. Lastly, Bubnow { found that moist ferric hydroxide in small quanti- ties (not weighed) causes a scarcely recognizable diminution in pro- teolytic action, while the presence of 1 per cent. of ferrous chloride and ferrous sulphate causes marked retardation, as does also an excess of ferric hydroxide. The most intense action was observed on the addition of 5 per cent. of ferrous sulphate. No quantitative results, that is, percentages of albumin digested were, however, obtained. Our experiments were made only with crystallized ferrous sulphate and ferric chloride. It appears superfluous to try the action of ferric hydroxide, which must necessarily, if in sufficient quantity, neutralize the acid of the gastric juice and thus prevent digestion by with- drawal of the free acid. * Quoted by Bubnow in Zeitschrift fiir physiologische Chemie, vol. vii, p. 316; also abstract by Herter in Jahresbericht fiir Thierchemie, 1880, p. 309. + Ueber den Einfluss von Kisenpraparaten auf die Magenverdauung. Jahresbericht fiir Thierchemie, 1882, p. 257. ¢ Ueber den Einfluss des Hisenoxyhydrats und der Hisenoxydulsalze auf kiinstliche Magenverdauung und Faulniss mit Pancreas. Zeitschrift fiir Physiologische Chemie, vol. vii, p. 315. ‘on the Proteolytic Action of Pepsin-hydrochloric Acid. 93 Undigested Fibrin Relative proteo- FeS04+7H20. residue. digested. lytic action. 0 0°1835 gram. 81°65 100°0 0-001 per cent. 0'1916 80°84. 99°0 0°005 0°2241 UT(3a8) 95:0 0°010 071895 81°05 99°2 0:025 0°2573 74:27 90°9 0:050 02773 72°27 88°5 0 0°1935 80°65 100°0 0-1 0°3467 65°33 81°0 0:3 0°7274. 27°26 33°8 0°8 0°8080 19°20 23°8 1°5 0°8447 15°53 19:2 Here, with the ferrous salt, we find pronounced diminution of pro- teolytic action, commencing even with 0:001 per cent. With ferric chloride, the following results were obtained. Undigested . Fibrin Relative proteo- Fe2Clg. residue. digested. lytic action. 0 0°1842 gram. 81°58 per cent. 100°0 0-001 per cent. 0°2111 78°89 96°7 0°005 0°2059 79°41 Sige 0-010 0°2165 78°35 96°0 0050 0°2332 76°68 93°9 0 01961 80°39 100°G 0°3 0°6526 34°74. 432 0°5 0°8035 19°65 24-4 0'8 08794 12:06 15:0 30 0°9582 4°18 5:2 A comparison of the two series of results, shows no pronounced and constant difference in the amount of action between the two iron salts; both retard proteolytic action about equally; although with the larger amounts, as with 0°5 per cent. and beyond, ferrie chloride appears the most injurious. Comparing the results with those obtained with the manganese salt, which of late has been recom- mended as a therapeutic agent where iron cannot be taken, we see that the manganese is throughout, far less injurious than the two salts of iron. As to the manner in which the iron salts produce their retarding effect on proteolytic action, it is evident that it cannot be due to a simple displacement of the acid of the iron salt, by which the pepsin is made to act with a less compatible acid, since ferric chloride acts similarly to the sulphate, in which case there could be no such injurious replacement, 94 Chittenden and Allen—Influence of various Salts Magnesium sulphate. Pfeiffer * alone appears to have studied the influence of magnesium sulphate on gastric digestion. He found that retarding action com- menced in the presence of 0°24 per cent. of the salt and was very great in the presence of 4:0 per cent. Our results show decided retarding action, even in the presence of 0°005 per cent. of the crys- tallized salt. At the same time, it is to be remembered throughout, that probably differences in the strength of gastric juice, would cause some variation in the amount of retardation, produced by any given percentage. ' Undigested Fibrin Relative proteo- MgS04+7H20. residue. digested. lytic action. 0 0°1081 gram. 89°19 per cent. 100°0 0005 per cent. 01910 80°90 90°7 0°010 0:2330 76°70 86°0 0°050 0°3260 67:40 755 0°100 0°4428 55712 62°3 0 0°2605 73°95 100°0 0°3 07551 24°49 33°1 05 0-7886 21°14 28°5 0°8 0°8250 17°50 23 T 15 0°8891 11°09 15:0 3°0 0°8894 11:06 14°9 It is noticeable here, that while the retarding influence of the salt, becomes more and more pronounced as the percentage is increased, there comes a point (1°5 per cent.) when further addition does not materially influence the action of the ferment. Potassium permanganate. This salt, as with the amylolytic ferment of the saliva, shows very energetic action. Its influence is, without doubt, due to rapid oxida- tion and consequent destruction of the ferment ; indeed, the (at first) bright red color of the solution became almost immediately bleached out and the solution, at the same time, completely deprived of pro- teolytic power. The following results testify to its extreme activity. Undigested Fibrin Relative proteo- KoMnoOg. residue. digested. lytic action. 0 0-1951 gram. 80°48 per cent. 100°0 0°005 per cent. 08278 17°22 21°3 0°010 0°9949 0°51 0°6 It is thus more active in preventing proteolytic action, in gastric juice of the strength used, than in hindering the development of * Abstract in Centralbl. med. Wiss., 1885, p. 328. on the Proteolytic Action of Pepsin-hydrochloric Acid. 95 bacteria; although doubtless, the action of any one percentage is de- pendent in part, upon the amount of organic matter present. Marcus and Pinet* found that the permanganate in 0-1 per cent. would pre- vent the development of bacteria and in 1°5 per cent. would kill the fully developed organisms. Potassium dichromate. A single experiment with this salt gave the following results; showing a decided retarding action on gastric digestion. - Undigested Fibrin Relative proteo- KeCr207. residue. digested. lytic action. 0 0°2028 gram. 79°72 per cent. 100-0 0-01 per cent. 0°2476 75°24 94-4 0-10 0°6383 36°17 45°3 Potassium cyanide. Potassium cyanide we found very active in diminishing the diges- tive power of pepsin; due in great part doubtless, to decomposition of the cyanide by the hydrochloric acid of the gastric juice with conseqent formation of pepsin-hydrocyanic acid. Our first results were as follows: Undigested Fibrin Relative proteo- KCN. residue. digested. lytic action. 0 0°3255 gram. 67°45 per cent. 100°0 0°25 per cent. 09687 3:13 4°6 0°50 079912 0°88 163} Here, the fibrin did not swell at all, indicating the probable ab- sence of free hydrochloric acid, although of course the potassium cyanide might, per se, prevent swelling. With very much smaller percentages of cyanide, we obtained the following results: KCN. “residue. digested. Maytie action. 0 0°3098 gram. 69°02 per cent. 100°0 0°'005 per cent. 04376 56°24 81°5 0°025 0°3750 62°50 90°5 Potassium ferrocyanide. With this salt, the results are practically the same as with potas- sium cyanide; almost complete stopping of proteolytic action, even in the presence of small fractions of one per cent. * Action de quelques substances sur les bactéries de la putréfaction, Compt. rend. Soc. de Biolog., 1882, p.718. Abstract in Jahresbericht fiir Thierchemie, 1882, p. 515. 96 Chittenden and Allen—Influence of various Salts Undigested Fibrin Relative proteo- K4Fe(CN) ¢+3H20. residue. digested, lytic action. 0 0°3717 gram. 62°83 per cent. 100°0 0°05 per cent. 0:5969 40°31 64:1 0-10 0°7922 20°78 33°0 0°25 0°9585 4°15 6°6 0-5 10 0 0 0 0°3098 69°02 100°0 0-005 0°3562 64°38 93°93 0°025 0°3471 65°29 94°5 Potassium chlorate and Potassium nitrate. These two oxidizing agents produce almost exactly the same effect on pepsin-hydrochloric acid digestion; a retarding action directly proportional to the amount of salt present. Undigested Fibrin Relative proteo- KCl1O3. residue. digested. lytic action. 0 0°2683 gram. 73°17 per cent. 100-0 0°3 per cent. 0°4565 54°35 74°3 0°8 07019 29°81 40% © 15 0°8173 18:27 25°0 30 0°87T07 12°93 17°6 KNO3. 0 0:2870 gram. 71:30 per cent. 100°0 0°3 per cent. 0°5370 46°30 64°9 0°5 0°6247 37°53 52°6 0°8 0-7158 28°42 39°8 15 0°8148 18°52 25°9 3°0 0°8907 10°93 15°3 With potassium chlorate no experiments have been previously tried; with potassium nitrate, however, Wolberg* experimenting with quantities varying from 0°5 to 8:0 per cent. found in every instance, diminution in the proteolytic action of his pepsin solution. This, however, amounted to but little, except in the presence of 8 grams (8 per cent.) of the nitrate, where there was a diminution in the amount of fibrin digested, equal to 490 per cent. Even with 6 per cent. of the salt, Wolberg found after 24 hours, only a diminution of 6°8 per cent. in the fibrin digested. In quantity, therefore, our results do not accord at all with Wol- berg’s, since as the table shows, even 0°3 per cent. of potassium nitrate caused a diminution in the quantity of fibrin digested, amounting to 35°1 per cent., when compared with the control (100); while the pres- * Pfliiger’s Archiv, vol. xxii, p. 300. Ueber den Winfluss einiger Salze und Alka- loiden auf die Verdauung. : on the Proteolytic Action of Pepsin-hydrochloric Acid. 97 ence of 3 per cent. of the salt caused a diminution in proteolytic action amounting, in the quantity of fibrin digested, to nearly 85 per cent. The only apparent explanation of this difference in the results [unless due to difference in the amount of ferment] is in the length of time the mixtures were warmed at 40° C.; in Wolberg’s 24 hours, in ours 2 hours. This, if the true reason of the difference, would imply on the part of the ferment, ability to gradually overcome the influence of small amounts of the substance and thus eventually to digest an equal quantity of proteid matter. This, however, would in turn imply that the object sought for, viz: the influence of different quan- tities or percentages of a substance on the action of the ferment is lost sight of. The length of time best adapted to the experiment, is naturally that which will bring out most clearly and decisively all differences of action. Sodium tetraborate (Borax) and Boracic acid. Sternberg’s experiments* with both of these substances, have shown that, although possessed of no germicide value, they prevent the multiplication of bacterial organisms and are thus valuable anti- septics. Wolberg, in experiments made with artificial gastric juice, found that in a 24 hours digestion, 0°5 gram (0°5 per cent.) of borax caused a slight acceleration in proteolytic action (0°4 per cent.), while with 1 per cent. of the salt, retardation occurred to the extent of 23°3 per cent., and in the presence of 4:0 per cent. almost complete stopping of proteolytic action. Our results, however, fail to show any stimu- lating action on the part of the borate, although retardation is very pronounced. Undigested Fibrin Relative proteo- Na2B407+10H20. residue, digested lytic action. 0 0°3610 gram. 63°90 per cent. 100°0 0°05 per cent. 0°3852 61°48 96°2 0°20 04080 59°20 92°6 0°65 0-7710 22°90 35°8 1:0 0°9899 101 15 Doubtless, the retarding action of this salt is due wholly to the liberation of boracie acid and the consequent neutralization of the hydrochloric acid of the gastric juice. Boracic acid itself, offers no obstacle to the proteolytic action of pepsin-hydrochloric acid; on the contrary it increases it, but pepsin-boracic acid has little digestive * Amer. Jour. Med. Sciences, April, 1883, p. 335. Trans. Conn. Acav., Vou. VII. 13 OcT., 1885. 98 Chittenden and Allen—Influence of various Salts power. The influence of boracic acid on pepsin-hydrochlori¢ acid is — seen from the following experiments: : - : d Undigested Fibrin. Relative proteo- H; BOs. residue, digested. lytic action. 0 0 2395 gram. 76°05 per cent. 100°0 01 per cent. 0°2252 71768 1021 0 0°2049 (Gin! 100-0 0°5 0°1875 81°25 102-2 3°0 01729 82°71 1042 . 60 01445 85°55 107°6 Evidently then, the action of borax consists simply in withdrawing from the pepsin the hydrochloric acid of the gastric juice. The low digestive power of pepsin and boracic acid is shown by the following experiment : A, B. Cc. D. H.0O sol. pepsin ----- 50 ce 50 c.¢. 50 cc. 50 ¢. ¢. HCl 0:2 per cent..... 50, 0 0 0 HeBOpeese ees ae eee 0 0-2 gram. 0°3 gram. 05 gram. HORS zeetesteis oe 0 50 ec. 50 ee. 50 ¢.¢. ’ 100 100 100 100 01¢HCl 024HsBO; 034H,BO, 05%H;BO, | To each, was added 1 gram of purified fibrin, after which the mix- tures were warmed at 40° C. for 2 hours. Following are the results. A, B. C. D. Wt. of undigested residue ---- -- 2 071180 09615 0°9705 0°9620 HBerjicent; digested 2222 255-22+222 88:20 3°85 2°95 3°80 Ammonium oxalate. With this salt the following results were obtained : ey Undigested. Fibrin Relative proteo- (NH4)262044+2H20. residue. digested. lytic action. 0 0°3254 gram. 67°46 per cent. 100-0 0°610 per cent. 0°3579 64°20 95:1 0:025 0°3627 63°73 94°6 0-1 0°3920 60°80 901 0°5 0:9049 9°51 14:1 0 0°3098 69°02 100°0 1:0 0°9958 0 42 6°6 As to the cause of this retarding action, it is probable, that, as in the case of borax, the oxalic acid of the salt is displaced and the ferment compelled to act with the acid thus liberated. But if we compare the results of the present series with those of the preceding, on the Proteolytic Action of Pepsin-hydrochloric Acid. 99 we find that, with like percentages of the two salts, ammonium oxal- ate has far the greater retarding power, and yet it is well known that the ferment acts well when combined with oxalic acid. More- over, our experiments show further on, that ammonium chloride has no greater retarding power than sodium chloride. Hence, there must be some reason, other than the one mentioned above, to account for all of the retarding action manifested by the oxalate. Petit* states, that the maximum digestive action of oxalic acid, with 0°2—0-4 per cent. of his pepsin, is attained with 0°5—4-0 per cent. of the acid, according to the amount of pepsin. The comparative digestive power of pepsin-oxalic acid and pepsin- hydrochloric acid is shown by the following experiments :f A. B. 0. HO sol. pepsin --.-- 50 ce. 50 c.c. 50 c.c. 0°2 per cent. HOl.-.. 50 0 0 Okina Ope te te a 0 0°5 gram. 1:0 gram. JB ly, O) 8 Sas ee 0 50 c.c, 50 ce. 100 100 100 01% HCl 0°5 % CoH2O. 1:0 4 C.H20, Warmed at 40°C. for 2 hours with 1 gram of pure, dry fibrin, the following results were obtained : A. B. C. Wt. of undigested residue --... 0°2170 04450 0°4371 Per cent. digested .-.-.2------ 78°30 55°45 56:29 A second series, with larger amounts of oxalic acid, gave the fol- lowing results: A. B. C. H,0 sol. pepsin--- 50c.c. : 50 ec. e. 50 ¢c. ¢. 0-2 per cent. HCl. 50 0 0 eta Og ese. 2 0 1°5 grams. 2:0 grams. ib (0) ee eee 0 50 c.¢. 50 ¢. ¢. 100 100 100 0-1 per cent. HCl 1°5 per cent. C.H.0,4 2°0 per cent. Cp H.04 Undigested residue 0-1085 gram. 03090 gram. 0:3640 gram. Per cent. digested 89°15 69°10 63°60 These four results being placed on the same basis, i. e. compared with their respective controls (100) show as follows: * Jahresbericht fiir Thierchemie, 1880, p. 309. + Made in this laboratory by Mr. R. W. Pinney. 100 Chittenden and Allen—Influence of various Salts Relative proteo- Per cent. of acid. lytic action 071 HCl 100°0 0°5 CoH.O,4 70-7 1:0 (er 15 he 2°0 (files This shows;maximum action, with our amount of pepsin, in the presence of 1°5 per cent. of oxalic acid and shows, moreover, that the retarding influence of ammonium oxalate on proteolytic action, is not fully explained by the suggested neutralization of the hydrochloric acid of the gastric juice, unless it be that the ammonium chloride, formed by the reaction, effects pepsin-oxalic acid differently than it does pepsin-hydrochloric acid. Sodium chloride. With this salt we obtained, by our method, the following results : Undigested Fibrin Relative proteo- NaCl. residue. digested. lytic action. 0 0°2936 gram. 70°64 per cent. 100-0 0°005 per cent. 0°2824 : 71°76 101°6 0-010 02896 ; 71:04 100°5 0°025 03441 65°59 92°8 0°050 O35)15s 64°89 91°8 0°100 0°3744 62°56 88°5 0 0°2669 {ersil 100°0 0°3 0°4953 50°47 68°8 0°5 06175 38°25 52°1 0:8 0°6898 31°02 42°3 15 0°7825 ARTs 29°6 3°0 0°8249 L751 23°8 Alex. Schmidt* has recorded the retarding effect of sodium chlo- ride on the proteolytic action of gastric juice; Petitt likewise, has stated that small quantities of sodium chloride interrupt the action of the ferment, and Wolberg{ has recorded the same result with varying percentages of the salt; noting in addition, that very small quantities cause a slight acceleration of proteolytic action, amounting in his experiment to 2°5 per cent. With 0-005 per cent. of the salt, we found as the results show, acceleration amounting to 1°6 per cent., larger amounts causing a gradual and proportional diminution in proteolytic action. * Pfliiger’s Archiv, xiii, p. 98. + Jahresbericht fiir Thierchemie, 1880, p. 309. ¢ Pfliiger’s Archiv, xxii, p. 298. | ; | | on the Proteolytic Action of Pepsin-hydrochloric Acid. 101 Pfeiffer* has also called attention to the retarding action of this substance. Potassium chloride. With this salt our results are as follows: Undigested Fibrin Relative proteo- KCl. residue. digested. lytic action. 0 0°1552 gram. 84-48 per cent. 100-0 0:005 per cent. O-1817 81°83 96°8 0°025 0°1550 84°50 100-0 0°050 0°2565 74°35 88:0 0-100 0°2097 79°03 93°5 0 0°1930 80-70 100-0 0°3 0°3997 60°02 74:3 15 0°6815 31°85 39°4 3°0 0:°7282 27°18 33°6 The only noticeable difference between the action of this salt and the preceding, is the absence of any acceleration on the part of the potassium chloride and with larger percentages, a less vigorous retard- ing action. Ammonium chloride. For the sake of comparison a few experiments were tried with this salt, with the following results : Undigested Fibrin Relative proteo- (NH4)Cl. residue. digested. lytic action. 0 0°1880 gram. 81:20 per cent. 100°0 0-3 per cent. — 0°4649 53°51 65°9 0'8 0°6615 33°85 41‘7 3°0 0°6970 30°30 37:3 By looking at the table of comparisons, we see there is little con- stant difference in the amount of retardation caused by the ammo- nium, potassium and sodium salts of hydrochloric acid. This result, however, is quite different from that obtained by Wolberg, who found that ammonium chloride influenced proteolytic action but very little, while both potassium and sodium chloride caused great retard- ation. his difference in result, may be due to difference in strength of gastric juice or to difference in length of time the mixtures were warmed at 40° C. ; certainly in our experiments, with pure anhydrous salts and 2 hours digestion, very little difference in digestive action is noticeable ; throughout, sodium chloride causes a little less proteolytic action than the corresponding potassium salt, while of the ammonium salt, little is to be said except that it causes equal retardation. * Centralbl. med, Wiss., 1885, p. 328. 102 Chittenden and Allen—Influence of various Salts Wolberg in speaking of the relative retarding action of the three sodium salts of hydrochloric, nitric and sulphuric acids, states that the sulphate retards the most, then the nitrate and lastly the chloride. We have noticed the same fact in our work and take it as additional evidence of the liberation of the acid of the salt added, the retarding influence of the salt being dependent, in part, on the digestive power of the pepsin-acid formed. The following experiments,* showing the relative digestive power of the several acids in conjunction with pepsin, testify to the proba- bility of this view. All of the solutions contained 50 c. c. of an aqueous solution of glycerine-pepsin, each having a total volume of 100 c. c. and differing from each other only in the nature and percentage of the acid present. In testing the digestive power of the solution, 1 gram of pure fibrin was added and the mixtures warmed at 40° C. for two hours, after which the amount digested was determined in the usual manner. Percentage of fibrin digested in the presence of the different per- centages of acids. Control. 0°05% O-1% 0:2% O38 G O4% 05% 0:°1% HCl HNOst-.-. 9°20 48°75 73°65 67°20 46:00 87°65 SO. 19°50 24°70 25°80 22°55 24°95 88°05 CeHuOsas-- 3°70 2°30 87°50 Comparing these results with their respective controls, figured as 100, we have the following values for sulphuric and nitrie acids, ex- pressive of their relative proteolytic power when combined with pepsin, as compared with 01 per cent. hydrochloric acid under simi- lar conditions. Per cent. acid. HNO3. H2S804. 0°05 10°5 Atta § 01 55:5 22°1 0°2 84:0 28°0 03 767 29:1 04 52°5 25°6 0°5 eee 28°2 From this it is evident, that nitric acid is most active, with our amount of pepsin, in a 0°2 per cent. solution, while sulphuric acid attains its maximum action in 0°3 per cent.; moreover, nitric acid is * Made in this laboratory by Mr. R. W. Pinney. + The acids are calculated as pure HNOs, H.SO,, ete. on the Proteolytic Action of Pepsin-hydrochloric Acid. 163 more than four-fifths as active as 0°1 per cent. hydrochloric acid, while sulphuric acid is only a little more than one-fourth as active as the hydrochloric acid; acetic acid being practically worthless. Henee it is obvious, that, the base being the same, acetates, borates and other salts, the acids of which are not capable of working with pepsin, will most readily retard gastric digestion; then of the other salts experimented with, sulphates and lastly nitrates. A glance at the table of comparisons, shows here and there, results which manifestly accord with this view, notably lead acetate, cupric sulphate, zine sulphate,* magnesium sulphate and potassium nitrate. That this withdrawal of free hydrochloric from the pepsin, is only a partial explanation of the mode of action of neutral salts is plain from the fact that chlorides exert very pronounced retarding actions moreover the action of these latter as well as of the others cannot be due merely to mechanical causes, viz: the semi-saturation of the digestive fluid, since 3-0 per cent. of boracic acid stimulates instead of retarding, and even the presence of 10 per cent. of arsenic acid causes retardation amounting to only 32 per cent. According to Petit,t moreover, saccharose to the extent of 16 per cent. does not interrupt gastric digestion. Again, that the base entering into the composition of the salt has much to do, in many cases, with its re- tarding proteolytic action, is apparent when we compare the action of ferric chloride, manganous chloride, sodium chloride, mercuric chloride and other metallic salts. That this action is due in part to capability of combining with the proteid matter, thus rendering it non-digestible, is unquestionable. Acceleration, produced by neutral salts has been noticed by other observers, but no explanation of the cause has been offered. It seems probable, however, that in the case of pepsin-hydrochloric acid, one plausible reason, at least, may be suggested. If, as has been mentioned, many of the neutral salts are decomposed by the acid of the gastric juice, it would follow that the addition of very small amounts of the salts would diminish slightly the percentage of free hydrochloric acid, while the acid liberated from the salt, would be entirely without deleterious action. Nearly every experimenter in this direction has employed in the preparation of gastric juice 0°2 per cent. hydrochloric acid, which is well adapted for the purpose, but the action of the acid is dependent in part on the amount of ferment. With our solution of pepsin, the following results, expressed in the percentage of fibrin * Compare Petit, Jahresbericht fiir Thierchemie, 1880, p. 309. { Jahresbericht fur Thierchemie, 1880, p. 309. 104 Chittenden and Allen—Influence of various Salts digested, were obtained with different percentages of hydrochloric acid; the amount of pepsin being the same in each case. Per cent, MG sg see _ O05 Ol O°2 O38 O-4 Per cent. fibrin digested 73°8 89°3 84-0 81°7 63°8 This shows plainly, that, with the amount of pepsin employed in our experiments, acceleration of proteolytic action on the addition of neutral salts, might in some instances be due to slight diminution of free hydrochloric acid. That this is not the sole cause, however, is plain from the fact that closely related salts do not act alike. Potassium bromide and Potassium iodide. With these two potassium salts, the following results were ob- tained : Undigested Fibrin Relative proteo- KBr. residue. digested. lytic action. 0 0°3837 gram. 61°63 per cent. 100°0 0-005 per cent. 0°3205 67-95 110:2 07025 0°3035 69°65 113°0 0710 0°4204 57:96 94-0 0°50 075690 43°10 70:0 1:00 0°6203 3797 61:6 Kl. 0 0°2572 gram. 74°28 per cent. 100-0 0005 per cent. 0:2755 72°45 97-5 0025 073421 65°78 88°6 0°10 0°2841 TONES) 96°4 0°50 0°6367 36°33 48'9 1:00 0°7586 24°14 32°5 By comparison of these two series of results, we see that there is a very decided difference in the action of the two salts; potassium bromide in very small quantity, causes a decided acceleration in pro- teolytic action, which is wholly lacking with potassium iodide. Again, there is a very great difference in the amount of retardation produced by the two salts ; thus by comparison with their respective controls we see, that while 1 per cent. of potassium bromide causes retardation in digestive action, amounting to 38-4 per cent., the same percentage of iodide causes retardation amounting to 67°5 per cent. It is to be supposed, naturally, that in the presence of large amounts of the salts, the ferment must act wholly in combination with hydrobromic and hydriodic acid respectively; that is to say, the hydrochloric acid of the gastric juice must have replaced these two acids in the ° potassium salts, and thus new pepsin-compounds formed, of which pepsin-hydrobromic acid is the more active digestive agent. ig SE ny on the Proteolytic Action of Pepsin-hydrochloric Acid. 105 Putzeys,* indeed, found that he obtained practically the same proteolytic action [retarding] with pepsin and hydriodic acid, as with potassium iodide and pepsin-hydrochloric acid. ‘The following results, showing the relative digestive action of pepsin with hydro- bromic and hydriodic acid respectively, were obtained by Putzeys. Digestive pro- HI. HBr. ducts formed. 0°625 gram. Beas 15°86 per cent. 0:937 ae SillFais) 3°310 Sets : 2°33 ahaa 0°882 gram. 26°40 Ae 2°200 45°60 3°309 46°60 It is thus evident, that both hydrobromic and hydriodic acid can, to a certain extent, replace the hydrochloric acid of the gastric juice, although they are much less active than the latter acid. Moreover, hydrobromic acid is much more efficient than hydriodic in connec- tion with the ferment, for in comparatively large doses the latter acid will completely stop proteolytic action. As a practical result, Putzeys suggests, that for therapeutic purposes, potassium bromide and iodide should be given $ to 1 hour before meals. Our results are plainly in accord with Putzeys’ observations. The following table shows the relative influence on the proteolytic action of pepsin, of the various inorganic salts experimented with, compared with the controls, expressed as 100. * Jahresbericht fiir Thierchemie, 1877, 279. De l’influence de l’iodure et du bro- mure de potassium sur la digestion stomacale. TRANS. Conn. AcAD., VoL. VII. 14 Oot., 1885. Chittenden and Allen—Influence of various Salts 106 1-89 |8-06 GLE 8-6 G-G& 9-19 "402900 aughjoa},0Nd aaunjas Humoys 21907, LTP &:6P 8-6€ L-0F “ond £0-0 Es ss wee O° HOT + ‘O'a*eN rae One Oro CEN) up aes O*HE + °(NO)9a' eT rane a pe oe a Tt ces, §o°unN sy Sane ae a 0°HL + OSs 1B: (es Wes < gia ame se 100 O:GGal\ a eee O°HL + OSaT LOGRIR GE Cet eee °10°0a ONON Faas see O°HL + 'OSUz zeus | ect cla here ae ’OSV°H POT Sa arit Sine anaes aie 219ug Z-FOL- ~~ OFHS +%(20"H"%0)4d GimOe ses cae aa O*HS +*osng Soe mals as ers? ae ae) AE Alkaloid salts. | o— m Pa o oH Se ° =| ° om ~~ o oS o ao ~ ~ Q =] ial i o ~ =| = Y 2) =| fe) FS mM nS o re Ss v4 —_ lo] Shy oe _ o Ay ° a) on S orm oS in (2) Oo < Wolberg, however, found that morphine chloride, in acid solution. 1e8 Il quantit in very sma d ine, use ine, quin ine sulphate and narcot strychn al OD op o.8 as TEs ro o & 2s 3 § § Pichia “> & 3 te mo ee feb) o = eS ae = 2 3S nrc ap ket 5 Sy on 19 .E Semper on the Proteolytic Action of Pepsin-hydrochloric acid. 107 per cent.; with quinine sulphate, slight acceleration was obtained. Strychnine and narcotine, in very small quantity, gave slight accel- eration. Dr. H. v. Boeck* states, that quinine is without influence on the activity of pepsin. In our experiments, using larger amounts of the alkaloid salts, retardation is very pronounced, doubtless due in part to replacement of the sulphuric acid of the salts by the acid of the gastric juice. Undigested Fibrin Relative proteo- Alkaloid salt. resiaue. digested. lytic action. 0 0°3555 gram. 64°45 per cent. 100°0 (Sr)p . H,SO, +6H,0. 0°5 per cent. 0°6825 31°75 49°2 1-0 0°8213 17°87 27-7 (Br), . H,SO, +H,0. 0°5 05685 43°15 66°9 10 0°7700 23°00 35°6 (At). . H,S0,. 0°5 0°6412 35°88 55°6 (Q)..H,SO, +7H,0. 0°5 0°6606 33°94 §2°6 (Ci)p + H,SO, +2H,0. ~—05 ‘ 0°6885 31°15 48°3 (Mo),. H,SO, +5H,0. 0:5 0°6225 27-15 58°5 (Na), . H,SO, + H,0. 0°5 0°6365 36°35 56°4 Percentage retardation of the alkaloid salts (0°5 per cent.) MURV CHIMING nae easels ons) ros heen eae etal eae 50°8 BEG Cees hoe ee ee She a ee ane Fre ft eem Shee 2 ea ee Soul Atropine _ shee Ms. Se Seale Ses SaaS 44°4 Outlines Sze ssee - Su. ss ci seide ee Cele ates 47-4. Cinghonme ts... Se ssee soso Se aetna ee ee ae 517 Morphine -...-- Pee Se ee sae sets eos ae wie iets 41°5 INARCOMMOM Ste ieee Soke er Mone SP ee hes Se 43°6 * Zeitschrift fiir Biologie, vol. vii, p. 428. VIT.—InFivuence or Various THERAPEUTIC AND Toxic Sup. STANCES ON THE Proreotytic ACTION OF THE PANCREATIC FER- MENT. By R. H. Cuirrenpen anv Gro. W. Cummins, Pu.B. Wir the proteolytic ferment of the pancreatic juice, few sys- tematic experiments have been tried to ascertain the influence of those substances the frequency of whose use, as therapeutic or toxic agents, renders their action on this ferment a matter of no small con- sequence. The well known experiments of Heidenhain, Kiihne and Schmidt have been confined to the action of sodium carbonate, sodium chloride, and kindred salts of physiological importance. Pfeiffer* has indeed, in addition, experimented with a few of the sulphates of the alkalies and alkali-earths, but of the large number of metallic and other salts in common use, few have been tried, while the action of alkaloids and the gases occurring in the intestinal canal, has not been studied at all. Method employed. It is a characteristic of the proteolytic ferment of the pancreatic juice, that it exercises considerable digestive power in a neutral solution.t In view therefore of the fact that the ferment, while in the intestinal canal, may be obliged to act in an acid-reacting medium (due to acid-proteids) as frequently as in an alkaline or neutral one, it seemed advisable in the experiments, to employ a neutral solution of trypsin. Moreover, it becomes necessary to use such a solution, if salts of the heavy metals are to be experimented with, since in alkaline solutions, carbonates or oxides of the metals would be formed and thus affect the results; still, again, the action of all substances can be best studied in neutral solution, since under such conditions, no replacements or decompositions of any kind take place to compli- cate the action of the substance, other than the direct action on the ferment or the proteid matter. Hence, a neutral solution has in every case been employed as being the most satisfactory, while only such substances have been experimented with, as contained no free acid ~ or alkali, the destructive action of which is well known. * Centralbl. med. Wiss., 1885, p. 328. + See Chittenden and Cummins, Amer. Chem. Jour., vol. vii, p. 46. Also Trans. Conn, Acad., vol. vii. Chittenden and Cummins— Action of the Pancreatic Ferment. 109 The solution of trypsin was prepared according to Kiihne’s* method from dried ox pancreas freed from fat; 40 grams dry pan- creas in 500 ¢. ¢. 0°1 per cent. salicylic acid, neutralized and diluted to 2 litres.t In each digestion experiment, 25 ¢. c. of this neutral trypsin solution were used, to which was added 25 c. c. of water containing the substance to be experimented with, or in the control 25 ¢. ¢. of water alone, making the volume of the digestive mixture in each case 50 c.c. In testing the proteolytic action of the different solutions, 1 gram of pure dry, pulverized fibrin, described in the preceding article, was added and the mixtures warmed at 40° C. for six hours, after which the undigested fibrin was filtered on weighed filters, washed thoroughly and dried at 100-110° C. until of constant weight. In this work as in the study of the salivary ptyalin and pepsin, it has been the effort mainly to ascertain the relative action of small quantities of the various salts, rather than the percentages necessary to completely stop the action of the ferment, this to our mind being much the more important. Mercurie chloride. With small percentages of this salt, the following results were obtained : Undigested Fibrin Relative proteo- HgCly. residue. digested. lytic action. 0 0'4495 gram. 55°05 per cent. 100°0 0:002 per cent. 0°4465 55°35 100°5 07003 0-44.05 55°95 101°6 0°005 0°4562 54°38 98°7 0°025 05076 49°24 89°4 0-100 0°7753 22°47 40°8 As seen from the table, 0-1 per cent. mercuric chloride diminishes the proteolytic action of the ferment more than one-half. Its action in this percentage is more energetic than on pepsin, although the smaller quantities are proportionally far less active; in one case (0003 per cent.) even causing acceleration. Wassilieff { has studied the influence of mercurous chloride on pan- creatic digestion and finds that calomel does not affect the action of the proteolytic ferment, while it does prevent the formation of putrefaction products, viz: indol, phenol, ete. * Untersuchungen aus der physiolog. Inst. d. Universitat Heidelberg, vol. i, p. 222. + The solution was kept from putrefaction by the use of a little 20 per cent. alcoholic solution of thymol; enough of which remained dissolved in the fluid to pre- vent putrefaction also during the six hours digestion at 40° C. } Zeitschrift fir physiol. Chemie, vol. vi, p. 112. 110 Chittenden and Cummins—Influence of various Substances ‘ Mercuric iodide and Mercurie bromide. These salts dissolved in sodium chloride in such proportion that the ultimate solutions contained like percentages of both salts, gave the following results: HglIo. 0 0°005 per cent. 0°025 0°100 0°200 HgBro. 0 0°005 per cent 0°025 0-100 0°200 Undigested residue. 0-4707 gram. 0°4780 0°5085 05994 0°6580 0°4707 gram. 0°4400 0°4840 05721 0°6548 Fibrin digested. 52°93 per cent. 52:20 49°15 40°06 34°20 52°93 per cent. 56°00 51°60 42°79 34°52 Relative proteo- lytic action. 160°0 98°6 92°8 75°6 64°6 100°0 105°& 974 80°8 65°2 Aside from a slight acceleration, noticed with 0°005 per cent. of bromide, the two salts act very much alike, causing retardation of proteolytic action; less pronounced however, than that caused by mercuric chloride. Mercurie cyanide. The action of this salt is somewhat peculiar, causing at first when in small quantity, noticeable retardation followed in the presence of larger percentages by increased proteolytic action, though still below the action of the normal solution of trypsin. In a general way, its action on this ferment accords very nearly with its action on the amylolytic ferment of saliva and the proteo- lytic ferment of the gastric juice. Undigested Fibrin Relative proteo- Hg(CN) 9. residue. digested. lytic action. 0 0°2668 gram. 73°32 per cent. 100°0 0°005 per cent. 0°3192 68°08 92°8 0:025 0°3209 67:91 92°6 0:050 0°3244 67°56 9251 07100 0:3308 66°92 91°2 0 0°3675 gram. 63°25 per cent. 100°0 0°3 per cent. 0°4094. 59°06 93°3 0°5 0°3880 61°20 96°7 1°5 0°3932 60°68 95°9 Its ation is to be attributed mainly to the hydrocyanic acid — radical, judging from the action of potassium cyanide on the one hand and the action of mercury salts on the other. There is, how- ever, without doubt a close connection between the action of these salts and their power of combining with proteid matter in general. on the Proteolytic Action of the Pancreatic Ferment. 111 Cupric sulphate. With this salt a more energetic retarding action is to be noticed, than in the case of the proteolytic ferment of gastric juice; 0°1 per cent. of the salt causing a retardation in proteolytic action of 65°7 per cent. as compared with a retardation of 38°8 per cent. in the case of pepsin. Undigested Fibrin Relative proteo- CuSO4+5H20. residue. digested. lytic action. 0 0°5035 gram. 49°65 per cent. 100°0 0°005 per cent. 0°5027 49°73 10071 07025 075320 46°80 94:2 0°050 0°5856 41°44 83°4. 0°100 0°8295 17°05 34°3 Lead acetate. With this salt the following results were obtained, agreeing essen- tially in the smaller percentages, with those obtained with cupric sulphate. In the presence of 0°1 per cent., however, retardation is far less than with the same percentage of cupric sulphate; 0°5 per cent. completely stops proteolytic action. Undigested Fibrin Relative proteo- Pb(CyH302)9+3H20. residue. digested. lytic action. 0 0°4562 gram. 54°38 per cent. 100°0 0-005 per cent. 0°4655 53°45 98:2 0:025 0°5391 46°09 84°7 0°050 0°5660 43°40 79°8 07100 0°6410 35 90 66°0 0°500 1:0 0 0 Stannous chloride. This salt, in conformity with its action on the amylolytic ferment of saliva, causes very decided retardation in the proteolytic action of trypsin, requiring but a very small amount of the salt to com- pletely stop the action of the ferment. Undigested Fibrin Relative proteo- SnClg. residue. digested. lytic action. 0 0°3875 gram. 61°25 per cent. 100°0 0°0005 per cent. 0°4495 55:05 89°8 0°005 0°5370 46°30 15:5 0°025 1:0 0 0 Arsenious oxide. Schiifer and Béhm have experimented with arsenious acid, study- ing its influence on the proteolytic action of a glycerine infusion of * Abstract in Jahresbericht fiir Thierchemie, 1872, p. 363, 112 Chittenden and Cummins—Influence of various Substances pancreas. They find that arsenious acid is without influence on the action of the ferment. We do not know whether they added arsen- ious acid to a neutral or alkaline solution of the ferment, as we have’ seen only an abstract of their paper, but we find that the addition of very small amounts of arsenious oxide to a neutral solution of trypsin, does produce a slight retardation in the proteolytic action — of the ferment. Owing to the comparative insolubility of arsenious — oxide in water, only small percentages could be employed, but these — show, as might be expected from the acid character of the substance, — a decrease in proteolytic action. Undigested Fibrin Relative proteo- As203, residue. digested. lytic action. 0 0°4598 gram. 54°02 per cent. 100°0 0-001 per cent. 0-4630 53-70 99-4 0-005 04513 5487 101°5 0°025 0:4710 52°90 97-9 0°050 0°4723 52°17 97°6 4 Arsenic acid. This substance, in conformity with its more decided acid character, causes greater retardation than arsenious acid; which fact tends, by — analogy, to make the retarding action of the latter still more proba- ble. The results are as follows: Undigested Fibrin Relative proteo- H3AsOq. residue. - digested. lytic action. 0 0°4598 gram. 54:02 per cent. 100°0 0°005 per cent. 0°4563 54°37 100°6 0°025 0°4887 51°13 94°6 0°050 075264 47°36 87:4 0°100 0°6340 36°60 67:7 Ammonium arsenate. This salt in small amount, appears to increase the proteolytic action of the ferment, which fact would indicate that the retarding action of the two preceding arsenic compounds is due more to their acid — character than to the arsenic contained in them. Undigested Fibrin Relative proteo- (NH4)3A804q4. residue, digested. lytic action. 0 0°4598 gram. 54°02 per cent. 100°0 0°050 per cent. 0°4620 53°80 99°6 0°100 0°4442 55°58 102°8 0 0°3675 63°25 100°0 0°5 0°4071 59°29 931 on the Proteolytic Action of the Pancreatic Ferment. 113 Potassium antimony tartrate. This compound of antimony fails to produce with trypsin the marked acceleration, noticed with the amylolytic ferment studied. This difference in action, since both ferments were in neutral solu- tion, indicates a specific difference in the nature of the two ferments. Following are the results obtained : Undigested Fibrin Relative proteo- K(SbO)C4H40¢. residue. digested. lytic action. 0 0°3978 gram. 60°22 per cent. 100-0 0-2 per cent. 9°4105 58°95 97°8 0°5 0°4129 58°71 97-4 1:0 0°4258 57-42 95°3 15 0°5406 45°94. 76°2 Ferric chloride and ferrous sulphate. With these two salts of iron the following results were obtained : Undigested Fibrin . Relative proteo- Fe2Clg. residue. digested. lytic action. 0 0°5215 gram. 47°85 per cent. 100-0 0°005 per cent. 0°5431 45°69 95°4 0°025 0°6243 Sa 78°5 0°050 0°7457 25°43 53:1 0°100 10 ‘ 0 0 FeSO4+7H20. 0 0-4075 gram. 59°25 per cent. 100°0 0°005 per cent. 0°4548 54°52 92-0 0°05 0°6225 SIs 63°71 0°10 O71T7 28°23 47°6 0°25 0°7104 28°96 48°8 0°50 0°7219 27°81 46°9 1:00 0-7675 23°25 39-2 1°50 0-7861 21°39 361 Ferric chloride is seen to be far more energetic in its hindering action on the proteolytic ferment, than the ferrous salt. A like result was obtained with the amylolytic ferment of the saliva and to a lesser extent with pepsin-hydrochloric acid. Bubnow* has tried experiments with both of these salts and found, as might be expected, that when present to the extent of 5 per cent. they prevented the appearance of putrefaction products (indol, phe- nol, etc.) but did not interfere with the action of the unorganized ferment. As no quantitative results were obtained, we can make no direct comparisons. In our experiments, it is to be remembered that putrefaction is prevented by thymol. . * Zeitschrift fiir physiol. Chemie., vol. vii, p. 327. TRANS. Conn. ACAD., Vou. VII. fs} te OctT., 1886, 114 Chittenden and Cummins—Influence of various Substances Manyanous chloride. Following are the results obtained with this salt : Undigested Fibrin Relative proteo- MnCly,. residue. digested. lytic action. 0 0°5215 gram. 47°85 per cent. 100-0 0-005 per cent. 0°5201 47-99 100°3 0°050 0°5139 48°61 101°6 0°100 0°5483 45°17 94:4 0:500 0°6807 3193 66°7 Comparing the influence of this salt on the proteolytic action of trypsin, with the results obtained with the closely related iron salts, it is seen that the manganese salt has a far less retarding influ- ence than the latter; a fact which agrees with what was observed in studying its action on pepsin-hydrochloric acid. Consequently, manganese salts, in so far as they are adapted to replace iron salts as therapeutic agents, are apparently less liable to interfere with normal digestive action. Zine sulphate. Undigested Fibrin Relative proteo- ZnS04+7H20. residue. digested. lytic action. 0 _ 0°5035 gram. 49°65 per cent. 100°0 0°005 per cent. 05175 48°25 97°2 0°025 0°53805 41°95 84°5 0°050 0°6802 31°98 64°4 0100 0°8301 16°99 34:2 The action of this salt on trypsin is, both in character and extent, similar to its action on the amylolytic ferment of saliva. Com- pared with pepsin-hydrochloric acid, however, the action of the salt is seen to be more energetic on the pancreatic ferment. Barium chloride. The following results were obtained : Undigested Fibrin Relative proteo- BaCl,.+2H,0. residue. digested. lytic action. 0 0°3710 gram. 62°90 per cent. 100°0 0°05 per cent. 0°3515 64°85 103°1 0°5 0°3885 61°15 97-2 3°0 0°4986 50°14 19°7 With this salt a slight acceleration in proteolytic action is to be seen with 0°05 per cent., while the larger amounts cause noticeable retardation. | Magnesium sulphate. Undigested Fibrin Relative proteo- MgS0O,4+7H20. residue. digested. lytie action. 0 0°3495 gram. 65°05 per cent. 100°0 0°05 per cent. 0°3541 64°59 99°3 0°5 03810 61°90 951 ~ 3°0 04706 52°94 813 oo on the Proteolytic Action of the Pancreatic Ferment. 115 Here retarding action is similar in extent to the action of barium chloride. Compared with ziné sulphate, the difference in action on trypsin is about the same in extent as the difference found in the action of the two salts on the amylolytic ferment of saliva. The retarding action of this salt on pancreatic digestion has been previously noticed by Pfeiffer.* Potassium permanganate. The retarding action of potassium permanganate 1s very pro- nounced. There is, however, a noticeable difference between the action of the salt on trypsin and its action on pepsin and ptyalin, the two latter being much more readily affected by the permanganate ; that is, by much smaller percentages. Undigested Fibrin Relative proteo- KoMn2O0g. residue. digested. lytic action. 0 0°4562 gram. 54°38 per cent. 100°0 0-001 per cent. 0°4570 54°30 99°8 0:003 0:4608 53°92 SEAL 0-010 0°4833 51°67 95°0 0 03675 "63°25 100-0 01 0:4487 55:13 87-1 0°5 07769 22°31 35°2 10 1:0 0 0 Potassium dichromate. Undigested Fibrin Relative proteo- KoCr207- residue. digested. lytic action. 0 0°3978 gram. 60°22 per cent. 100-0 0°05 per cent. 0°4032 59°68 fer 0:2 0°4245 57°55 95°5 0°5 04693 53°07 88:1 1:0 0°5525 44°75 74:3 15 0°6321 36°79 61°0 The retarding action of this salt is proportionally greater than that of the other potassium salts, indicating that the acid has a specific action of its own. Moreover, being an acid salt, the acid radi- cal is present in larger amount than in the neutral potassium salts experimented with. Potassium cyanide. The very pronounced acceleration in proteolytic action caused by the larger percentages of this salt is very interesting, especially when we recall the fact, that the retarding action of the same salt on the * Centralbl. med. Wiss., 1885, p. 328. 116 Chittenden and Cummins—Influence of various Substances amylolytic ferment of saliva and on the proteolytic ferment of gastric juice was equally decided, even with very small pecentages. Undigested Fibrin Relative proteo- KCN. residue. digested. lytic action. 0 0°2668 gram. 73°32 per cent. 100°0 0°005 per cent. 0°2790 72°10 98°3 0°025 0°2840 71°60 97°6 0°050 0°2678 73°22 99°8 0°100 0°2600 74:00 100°9 ; 0 0°3675 63°25 . 1000 0°3 0:2076 79°24 125°2 0°5 0°1985 80°15 126°7 10 0:2697* 73°03 115°4 115) 0°3315* 66°85 105°6 Potassium ferrocyanide and Potassium ferricyanide. With these two salts the following results were obtained : Undigested Fibrin Relative proteo K4Fe(CN) 5+3H, 0. residue. digested. lytic action. 0 0°3045 gram. 69°55 per cent. 100°0 0°005 per cent. 0°3363 66°37 9574 0°50 0°3293 67:07 96°4 2°00 0°3753 62°47 89°8 K,¢Fe(CN))¢ 0°005 per cent. 0°3268 67°32 96°8 0°05 0°3370 66°30 95°3 2°00 0°3912 69°88 875 Here, unlike the cyanide, there is no acceleration in the proteolytic action of the ferment, neither on the other hand is there very pro- nounced retardation; less indeed than is produced by a like per- centage (2°0) of sodium chloride. Sodium tetraborate. With this salt we obtained the following results: j Undigested Fibrin Relative proteo- NaeB407+10H20. residue. digested. lytic action 0 0:4116 gram. 58°84 per cent. 10070 0:05 per cent. 0°3729 62°71 106°5 0°2 0°3260 67°40 114°5 0° 0°2332 76°68 130°3 1:0 071860 81°40 138°3 2°0 0°1663 83°37 141.7 3°0 0°2276 77°24 13i°2 5°0 0°3141 68°59 116°5 * Tt was impossible to wash these completely, consequently the weights are undoubtedly too high. iv, on the Proteolytic Action of the Pancreatic Ferment. 117 Here, unlike the action of the salt on the two preceding ferments studied, there is to be seen a gradual increase in proteolytic action, which is very pronounced in the presence of 2 per cent. of the salt, then gradually diminishes ; although even with 5 per cent. of the crystallized salt, the action of the ferment continues to be increased far above that of the normal juice. Sodium sulphate. Following are the results obtained : Undigested Fibrin Relative proteo- NagS04+10H20. residue, digested. lytic action. 0 0°3495 gram. 65°05 per cent. 100°0 0°05 per cent. 0°3602 63°98 98°3 0°5 073558 64°42 ; 99°0 2-0 0°3938 60°62 93°1 5-0 0°4360 56°40 86°7 The retarding action of this salt is quite pronounced, although it is noticeable that its retarding action is not equal to that of mag- nesium sulphate nor indeed to that of sodium chloride. Pfeiffer* has also noticed the retarding action of this salt. Weiss,+ however, has stated that sodium sulphate in very small quantity increases the proteolytic activity of a pancreas extract. As we have not seen the original article we do not know whether definite per- centages were employed or not; with 0°05 per cent. of the crys- tallized salt, under the conditions of our experiment, there was certainly no acceleration in proteolytic action. Potassium chlorate and Potassium nitrate. Undigested Fibrin Relative proteo- KC103. residue. digested. lytic action. 0 0°3662 gram. 63°38 per cent. 100°0 0°05 per cent. 0°3991 60°09 94:8 0°50 0°3743 62°57 98°7 2°00 04101 58°99 93°0 KNO3. 0 0°3710 gram. 62°90 per cent. 100-0 0°05 per cent. 0°3751 62°49 99°3 0°5 0°3804 61°96 98°5 2°0 04080 59°20 941 5°0 0:44.73 55°27 87°8 * Loe. cit. + Jahresbericht fiir Thierchemie, 1876,%p. 177. 118 Chittenden and Cummins—Influence of various Substances The effects of these two salts on the proteolytic action of the fer ment are very similar, with the exception that potassium chlorate appears to be a little more energetic in action than the nitrate. Weiss* has stated that a very small quantity of potassium nitrate added to a pancreas infusion, causes slight increase in the proteolytic — activity of the ferment. This, however, is not observable in our ex-— periment with 0°05 per cent., although retardation is very slight. Potassium chloride. This salt, unlike sodium chloride, appears to cause retardation of proteolytic action, and is without any accelerating influence what- ever. The extent of its retardation, however, is not so great as in — the case of sodium chloride. Following are the results obtained. Undigested Fibrin Relative proteo- KCl. residue. digested. lytic action. 0 0°3662 gram. 63°38 per cent. 100-0 0°05 per cent. 0°3740 62°60 98°7 0°5 0°3672 63°27 99°8 2°0 0°4331 56°69 89°4 5°0 0°4723 52°77 83°2 Sodium chloride. With this salt many previous experiments have been tried; Heid- enhain,+ after noting the accelerating action of sodium carbonate, tried the influence of sodium chloride and found that it increased | the proteolytic power of the ferment. . Pfeiffer, however, states — that he found sodium chloride to exert a retarding influence on the proteolytic action of the pancreatic ferment. Pfeiffer[ moreover states that he found sodium carbonate to exert its accelerating action only when present in 0°5 per cent., not in 0°24 per cent. as found by Heidenhain. Recent results§ obtained by us in another connection, however, fully substantiate Heidenhain’s statements on this point. Lastly, Lindberger|| has found that sodium chloride, which in a neu- tral or alkaline solution of trypsin accelerates its proteolytic action, causes in an acid solution (0°018 per cent. HCl, presumably as acid- proteid) marked retardation of ferment action, without, however, — * Loe. cit. } Beitrage zur Kenntniss des Pancreas, Pfliiger’s Archiv, vol. x, p. 578, t Loe. cit. § Chittenden and Cummins, Amer, Chem. Jour., vol. vii, p. 46-47. Also Trans. Conn. Acad., vol. vii. || Jahresbericht fiir Thierchemie, 1883, p. 281. on the Proteolytic Action of the Pancreatic Ferment. 119 ~~ Ra causing destruction of the ferment, as after dialysis the ferment was -again active. Our results accord in the main with Heidenhain’s; acceleration, _ followed by retardation of proteolytic action as seen from the follow- ing figures. Undigested Fibrin Relative proteo- NaCl. residue. digested. lytic action. 0 0°2993 gram. 70°07 per cent. 100°0 0-05 per cent. 0°2635 73°65 1051 0°2 0°3001 69°99 99°8 0°5 0°3372 66°28 94°5 10 j 0°3923 60°77 86°7 2°0 074352 56°48 80°6 3°0 0°436] 56°39 80°4 5°0 04740 52°60 75°0 Potassium bromide and potassium iodide. The action of these two salts is wholly an accelerating one; more pronounced, however, in the case of the bromide than in the iodide. Following are the results of the experiments : Undigested Fibrin Relative proteo- KBr. residue. digested. lytic action. 0 0°3881 gram. 61°19 per cent. 100:0 0°05 per cent. 0°3773 62°20 101°6 05 0°3681 63°19 103°2 20 0°3795 62:05 101-4 5-0 0°3278 67°22 109°8 KI. 0 0°3881 gram. 61°19 per cent. 100°0 0°05 per cent. 0°3575 64°25 105°0 0°5 0°3915 60°85 99:4 : 3°0 0°3897 61:03 99°7 Influence of gases on the proteolytic action of trypsin. Podolinski* in Heidenhain’s laboratory, has shown that oxygen "gas has the power of converting the zymogen of pancreas. into the active ferment and that carbonic acid is without such power; more- over, that carbonic acid added to a sodium carbonate solution of -pancreatin (trypsin) retards the proteolytic action of the ferment, because the normal carbonate is thus changed into acid carbon- ate which does not favor the action of the ferment so well as the simple salt. Hydrogen was found to be without such action. Podo- linski further found that while oxygen favored the conversion of _ zymogen into the ferment, it did not influence the proteolytic action * Pfliiger’s Archiv, vol, xiii, p. 426. 120 Chittenden and Cummins—Influence of various Substances the ferment. Hydrogen was also found to be without marked action on the ferment, but carbonic acid retarded decidedly the proteolytic action of the ferment in an alkaline solution. We have tried the influence of three gases, such as the ferment would naturally be brought in contact with in the intestinal canal, and find that they exert a marked influence on the activity of the ferment. We employed, as in the preceding experiments, a neutral solution of trypsin, and kept the solutions saturated with the gas during the experiment, by passing a constant current through the fluid contained in a small. flask. As a control, air was passed through one digestive mixture, which served to keep the powdered fibrin in an equal state of agitation and thus make accurate com- parison possible. Following are the results : Undigested Fibrin Relative proteo- ‘ residue. digested. * lytic action. DN ae eee a ee 0°4218 gram. 57°82 per cent. 100-0 Hydrogent(El)ieeaessee 0°3670 63°30 109°1 Carbonic acid (CO,) ----- 0°5665 43°35 14:9 Hydrogen sulphide (HS). 0°4884 51°16 88°5 From this it is seen that hydrogen gas causes a slight acceleration in the proteolytic action of the ferment, while carbonic acid causes marked retardation, which fact agrees with Podolinski’s results, and shows moreover, that the action of the gas is the same in neutral and alkaline solutions. Hydrogen sulphide also causes retardation, although not so marked as carbonic acid. Influence of alkaloid salts on the proteolytic action of trypsin. So far as we know, no previous experiments have been tried on this subject. Our results show, so far as our experiments extend, a greater susceptibility on the part of trypsin to the action of alkaloids than the amylolytic ferment of saliva. Moreover, with trypsin, the alka- loids in only one case cause acceleration of proteolytic action. Com- pared with pepsin-hydrochloric acid, however, we find that neutral solutions of trypsin are not so readily affected as the former ferment, except by one alkaloid, viz: narcotine, where retarding action is very pronounced. All of the alkaloids experimented with were in the form of pure sulphates. Morphine sulphate. This salt exerts but little retarding action. The results are as follows: ee Le Lae tree Snr en ae ee eee Oe ee ee ee a es siabhinitiastainriintaienameeibastemiiaaaatn daa. cee th ea on the Proteolytic Action of the Pancreatic Ferment. 121 (Mo) .H2S04+5H 90. 0 0°05 per cent. 0-5 2°0 Undigested residue. 0°3875 gram. 0°3952 0°4105 0°4542 Fibrin digested. 61°25 per cent. 60°48 58°95 54°58 Atropine sulphate. Relative proteo- lytic action. 100°0 98.7 96-2 89-1 This alkaloid is very similar to morphine in its action. (At): . HgSO4. 0 0°05 per cent. 0-5 2°0 Undigested residue. 0°3875 gram. 0°3909 0°4078 0°4619 Fibrin digested. 61°25 per cent. 60°91 59°22 53°81 Relative proteo- lytic action. 100°0 99°4 96°6 87°8 Stolnikow* has stated that a very small amount of atropine sul- phate is without influence on the ferment power of a glycerine ex- tract of pancreas, but that large amounts of the alkaloid salt, con- siderably diminish the proteolytic action of the ferment. Strychnine sulphate and Brucine sulphate. These two alkaloids produce more marked effects on the ferment, than the preceding. Of the two, as the results show, strychnine is more powerful in preventing proteolytic action. Undigested Fibrin Relative proteo- Alkaloid salt. residue. digested. lytic action. | 0 0°4611 gram. 54°89 per cent. 100°0 (Sr), . H,SO, +6H,0. 0:05 per cent. 0°4760 52°40 95°4 0°5 0°5792 42:08 76°6 2°0 0°6882 31°18 56°8 (Br), . H,SO,+H,0. 0:05 0°4915 50°85 92°6 0°5 0°5480 45:20 82°3 2°0 0°5452 45°48 82°8 Narcotine sulphate. Fibrin Relative proteo- Undigested (Na)».H2SO4+H20. residue. digested. lytic action. 0 04511 gram. 54°89 per cent. 100°0 0°05 per cent. 0°5205 47°95 87°3 0°5 0°9943 0°57 1:0 2°0 1:0 0 0 This alkaloid causes a complete stopping of proteolytic action; nearly so, even when present to the extent of only 0°5 per cent. * Virchow’s Archiv, vol. xe, p. 435. TRANS. CoNN. ACAD., Vou. VII. 16 Oct., 1885, i= 122 Chittenden and Cummins—Influence of various Substances Quinine sulphate, Cinchonine sulphate and Cinchonidine sulphate. The following table of results shows the action of these three salts: ‘ Undigested Fibrin Relative ‘proteo- Alkaloid salt. residue. digested. lytic action. 0 04679 gram. 53°21 per cent. 100°0 (Q).. Ho80,+ 7H,0. 0:05 per cent. 0°4811 51°89 97°5 0°5 0°6310 36°90 69°3 2°0 0°7600 24°00 45°] (Ci)... H.SO, + 2H,0. 0°05 0°4868 51°32 96°4 0°5 0°6409 35°91 67°4 2°0 0°8327 16°73 314 (Cidine), . H»SO,+3H,0. 0:05 0°4467 54°33 104°0 0°5 05977 40°23 75°6 2°0 0-7 707 22°93 43°0 Cinchonidine, in the smallest percentage, causes a slight accelera- tion in proteolytic action; retardation is also less marked with 0°5 per cent, than in the case of the other two alkaloids. Otherwise the results are much alike. On the opposite page is a table showing relative action of the various salts on the proteolytic power of the ferment, compared with the action of the controls expressed as i00. O. Nasse,* by a study of the influence of various salts (inorganic) on fermentation, particularly their influence on the amylolytic action of the salivary ferment, pancreatic ferment, and the invert ferment of yeast, came to the conclusion that there is a manifest and import- ant dependence on the part of the ferments in question, in their action, on the presence of salt molecules, and moreover, that this dependence is specific for each individual ferment; that the quantity, as well as the quality of a salt, exercises a specific influence upon each ferment. Our own results, with still different ferments, and with a larger number of substances, both related and more varied in character, all testify to the truth of this statement, viz: that the unorganized fer- ments are much influenced by the presence of salts, and moreover, that there is no distinct relationship among the ferments in question in their behavior towards the various salts experimented with, as a study of the three tables of comparisons show. Thus one and the same salt may affect two ferments in quite a different manner, as seen * Pfliiger’s Archiv, vol. xi, p. 157. 125 of the Pancreatic Ferment. ton O on the Proteolytic Act 8-601 0-9) G-&8 8-18 1-98 G91 8-Z8 8-99 0.89 PE LSP eral) ee orks 1-68 1-66 “--" Ie TOL F-08 9.08 “7"* 17.68 1-F6 0-86 1-€6 L-IPT 9-18 8-68 } ---- eeienes | ie | 6-16 6-001 0-66 V-96 9-001 G-101 G-G) 6-86 1-001 8-66 9-86 8-S0T 010-0 | 00-0 1-86 |9-TOT/S-00T £00-0 | 600-0 ase PROSE (g) Tap toLyaystqay OLA(Arap)) Es . O*°H9 + *08"H * *(18) === Sel Oe + OS": S(eulpi)) SRaS Seen SO SEC +. OSH “O) Tinks eae ae eae ee -ONHE WOS es “(Op Bo ae Ge tan cae SE OCC hase END Ties aay Ree aoe ae ae CTD ee eW a} Cee TD ge Osher Os He (On 7 ee ee a ee Sarees - Sees So NOL itr wee OR ee i. vas ONT Se aa ene esa ee ye 0) (5 foal Re tase, eee er) PEA te eg SELEY IOS on Amylolytic and Proteolytic Action. 145 shown that the addition of 0°1 per cent. hydrochloric acid to an aqueous extract of the pancreas stops its action, OC. A. Ewald* how- ever, found that while pepsin-hydrochloric acid destroyed trypsin, trypsin could digest fibrin in the presence of 0°3 per cent. hydrochlo- ric acid. Mayst likewise found that trypsin digestion could take place in the presence of 0:3 per cent. hydrochloric acid, but only when a relatively large proportion of fibrin was present, and in cor- roboration of Kiihne’s statement, he showed that trypsin could be destroyed both by pepsin and dilute hydrochloric acid. Engesser{ found that a pancreatic juice did not lose its tryptic power by two hours warming with a gastric juice containing 0°5 per cent. hydro- chloric acid. Langley,§ on the contrary, has shown that a glycerine extract of the pancreas loses a very appreciable amount of trypsin when warmed for two and a half hours with 0°05 per cent. hydrochloric acid. Lindberger,|| working with an alcohol precipitate from a glyc- erine extract of ox pancreas, in which there would naturally be present but a small amount of proteid matter in addition to the tryp- sin, found that in the presence of 071 per cent. hydrochloric acid the ferment was entirely without action, and even in the presence of 0°012 per cent. hydrochloric acid, fibrin was much more slowly dis- solved than by a neutral trypsin solution. Lindberger, moreover, found that weaker acids, as acetic and lactic, had a much different effect than the stronger hydrochloric acid; thus with dilute acetic acid, digestion of the fibrin was almost as rapid as with a neutral solution of trypsin, while with small amounts of lactic acid, fer- ment action was even more energetic than in a neutral solution. There is, however, no guarantee that in these experiments free acid was present. We have found that free acids, even when present in small per- centages, completely stop the proteolytic action of trypsin, and that when considerable albuminous matter is present, the action of tryp- sin is much hindered by the addition of acid to a neutral solution, even before the proteid matters present are saturated with acid. 0°1 per cent. free salicylic acid, in the presence of proteids already satu - * Jahresbericht fiir Thierchemie, 1880, p. 297. + Untersuchungen a. d. physiolog. Inst. d. Univ. Heidelberg, vol. iii, p. 378, 1880. { Jahresbericht fiir Thierchemie, 1880, p. 297. § Journal of Physiology, vol. iii, No. 3. || Jahresbericht fiir Thierchemie, 1883, p. 281. S| We have seen only the abstract of Lindberger’s paper, so cannot speak positively on this point. TRANS. Conn. Acap., VOL. VII. 19 Oot., 1885. 146 Chittenden and Cummins—Influence of Bile rated with the acid, allows no proteolytic action whatewer. Further- more, a sufficient amount of proteid matter just saturated with hydrochloric acid, no free acid being present, will almost completely stop the action of trypsin. Proteid matter, however, only partially saturated with acid has a much smaller retarding action. This, doubtless, was the condition of the mixtures in Mays’ and Enges- ser’s experiments above referred to, for, as Mays states, the ferment could act in the presence of 0°3 per cent. hydrochloric acid only when a relatively large proportion of fibrin was present. A pancreatic juice prepared from 20 grams of dried pancreas by warming it at 40° C. with 200 ¢.c. 0°71 per cent. salicylic acid, etc., was finally made exactly neutral and diluted to 500 c.c.; 25 ¢.c. of this solution required 7°5 ¢.c. of a 2°0 per cent. solution of salicylic acid to completely saturate the proteids present,* the excess of free acid necessary to give the tropzolin reaction being deducted. Three digestive mixtures were made as follows : 1. 25 ¢.c. of the neutral pancreatic solution + 50 ¢.c. water. 2. 25 cc. of the same pancreatic solution+7°5 c.c. 2°0 per cent. salicylic acid solution +17°5 cc. water. The mixture was acid to test papers, but gave no reaction with tropzolin 00. It therefore contained no free acid, but 0°3 per cent. of combined acid. 3. The same as No. 2, but 2°5 ¢.c. more of 2°0 per cent. salicylic acid, so that the solution contained, in addition to the acid proteids, 0-1 per cent. free salicylic acid. One gram of fibrin was added to each of these and the mixtures warmed at 40° C. for 8 hours and 40 minutes. No. 1 digested 88°34 per cent. of the fibrin, No. 2, 13°44 per cent., while No. 3 had no action whatever. Much smaller percentages of combined salicylic acid cause an equally diminished proteolytic action; thus, in the case of a carefully dialyzed juice where the proteid matter was much diminished, the digestive mixture, with its proteids wholly saturated, contained but 0°060 per cent. of combined salicylic acid; yet this mixture, in 15 hours at 40° C. digested but 17°10 per cent. of fibrin, while the same amount of the neutral trypsin solution digested 57°80 per cent. * Tested by tropzolin 00 according to the method of Danilewsky (Centralbl. med. Wiss., 1880). One drop of a solution containing 0-028 per cent. free salicylic acid gives a reddish-violet color, which is, however, not permanent as in the case of hydro- chloric acid, but transient. With hydrochloric acid, one drop of a 0:003 per cent. solution will give the reaction. on Amylolytic and Proteolytic Action. 147 Combined hydrochloric acid has a greater hindering action than salicylic acid, as the following results show : Fibrin digested Pancreatic solution of trypsin. in 18 hours. neutral 57°80 per cent. 0-034 per cent. combined HCl+no free HCl 3°90 O;034,5~ 4 ss HC1l+0°'C05 per cent. free HCl 2°31 0°034 aS # HCl+ 0-010 ti Ls 0°87 It is thus evident that in an ordinary digestive mixture, or even where albuminous matter is present only in limited quantity, the addition of hydrochloric or salicylic acid to a neutral solution of trypsin reduces its proteolytic action to a minimum before any free acid is present. 4.— Influence of Bile, Bile Salts and Bile Acids on the Proteolytic Action of Trypsin. The addition of bile to a neutral pancreatic juice causes but little change in its proteolytic action, as is seen from the following results obtained with ox bile containing 8°3 per cent. solid matter : Weight of Fibrin Bile. undigested residue. digested, 0 per cent. f 0°4118 gram. 59°82 per cent. 1:0 0°3907 60°93 10°0 0°3938 60°62 A slightly increased action is the only effect produced on the trypsin.* The addition of bile to an alkaline pancreatic juice does not produce any very different results. The following were obtained with a pancreatic juice containing 0°3 per cent. sodium carbonate and fresh ox bile containing 10-02 per cent. solid matter: Weight of Fibrin Bile. undigested residue. digested. 0 per cent. 0°3056 gram. 69°44 per cent. 0°25 03074 69°26 0°50 0°3488 65°12 1:00 0°3633 63°67 5°00 0°3278 67°22 10:00 0°3603 63°97 Here there is no increased proteolytic action, neither is there any very great retarding effect produced. Pure sodium glycocholate and taurocholate produce results similar to bile, as the following table shows. The pancreatic juice contained 0°3 per cent. sodium carbonate : * Compare Heidenhain, Pfliiger’s Archiv., vol. x, p. 579, 148 Chittenden and Cummins—Influence of Bile Sodium ‘Weight of taurocholate. indivested vesldne: enn 0 per cent. 0°2308 gram. 76°92 per cent. 0°05 0°2566 74°34 O° Ones 073048 69°52 1:00 0°2832 71°68 sodium glycocholate. 0°10 0°2576 74°24 0°20 0°3154 68°46 The presence of 3°0 per cent. crystallized ox bile caused a some- what different result, increasing the proteolytic action slightly; thus, while the control, containing 0°3 per cent. sodium carbonate, digested 88°69 per cent. of fibrin, the same trypsin solution plus 3 per cent. of crystallized bile digested in the same time 89°73 per cent. of fibrin. While bile or bile salts have but little influence on the proteolytic action of trypsin, the bile acids, even small percentages, have a much more marked effect. The following results, obtained by the addition of the bile acids to a neutral pancreatic juice, show the extent of the action : Weight of Fibrin Bile acids. undigested residue. digested. 0 0°2516 gram. 74°84 per cent. Glycocholic, 0°03 per cent. 0°1993 80°07 Taurocholic, 0°10 073455 65°45 0°20 04332 56°68 0°50 0:4170 58°30 Here the retarding influence of taurocholic’ acid is very manifest, while, on the other hand, the small percentage of glycocholic acid appears to increase the action of the ferment. - In view of the possible acid-reacting character of the contents of the small intestines, it becomes an interesting point to ascertain the influence of bile on the action of trypsin in the presence of more or less combined acid. With a pancreatic juice in which the proteids were partially saturated with salicylic acid, 0°1 per cent. combined acid being present, the following results were obtained : Weight of Fibrin Bile. undigested residue. digested. 0 per cent. 0°4822 gram. 51°78 per cent. 1:0 0°4858 51°42 10°0 0-4091 59°09 This increased action in the presence of 10 per cent. of bile accords with Lindberger’s results, this experimenter having found that bile in the presence of small percentages of (combined?) acetic and lactic acids tends to diminish the retarding effect produced by the acids alone. In the presence of combined hydrochloric acid, the bile salts pro- duced no effects whatever; the trypsin was entirely without action. X.—ABSORPTION OF ARSENIC BY THE Brain. By R. H. Cuirren- DEN AND Hersert E. Smita, M.D. Some time since one of us* advanced the view that the amount of arsenic present in the brain, in cases of arsenical poisoning, is an in- dex to the form in which the poison was taken, viz: whether in a readily soluble and diffusible form, such as sodium arsenite, or in a comparatively insoluble form, as arsenious oxide or aceto-arsenite of copper. The original experiments of Scolosuboff+ on animals, with sodium arsenite, plainly showed the capability of nerve tissue for the absorption of arsenic ; yet the recorded observations of toxicologists tend to show, as a rule, the presence of but traces of this metal in cases of arsenic poisoning, either acute or chronic. Scolosuboff’s re- sults are, however, undoubtedly correct ; arsenic when taken in a very soluble and diffusible form without doubt does accumulate in the brain, but in our opinion only when in that condition, and thus in the more common forms of poisoning with the white oxide or other insoluble forms of arsenic, but a trace of the poison is to be found in the brain at any one time. With a firm belief in the truth of the above statement, founded on personal experience and the recorded results of other workers in this field, it was maintained by one of us in a previous paper{ that the presence of weighable amounts of absorbed arsenic in the brain may be taken as an indication of the administration of a soluble form of the poison. Experiments on animals tend to show the correctness of the theory and the results of toxical investigations, so far as our knowledge extends, contain nothing contrary to this view. If true, we ought never to find under any circumstances an accumulation of arsenic in the brain, after the administration of an insoluble form of the poison. Hence, the study of arsenic cases, where the form of poison is known, is of great importance in this connection. We have had a recent opportunity of adding two more cases to * Chittenden, Amer. Chem. Jour., vol. v, p. 8; and Medico-Legal Journal, vol. ii, p. 237. + Bulletin de la Société Chimique de Paris, vol, xxiv, p. 125, ¢ Chittenden, loc. cit. 150 Chittenden and Smith—Absorption of Arsenic by the Brain. the list of those which bear testimony to the truth of the above theory ; two fatal cases of arsenic poisoning, one of which was caused by the white oxide, the other presumably by Paris green or aceto- arsenite of copper. Case A.—L. K., a middle-aged laboring man, ate for his dinner at noon a quantity of bean soup. Almost immediately after, he was seized with vomiting and purging, cramps in the legs, and all the ordinary symptoms of acute arsenic poisoning. There were no marked cerebral symptoms. At 9 Pp. m. of the same day the patient died in a condition of collapse, having thus lived nine hours after | eating the poisoned soup. An autopsy made the following day by Dr. M. C. White of the Yale Medical School, to whose courtesy we are indebted for a description of the case, and also for the organs for analysis, revealed the following points of interest: “The mucous membrane of the stomach was very much inflamed, especially around the cardiac orifice. The duodenum was likewise much inflamed, also the lower part of the rectum, showing here as a red mottled conges- tion. The remaining portions of the intestines were normal. The brain showed marked congestion. The kidneys’ were normal in ap- pearance, the urinary bladder was nearly empty, and the mucous lining somewhat reddened. The lungs were normal, except the lower half of the right one, which was a little congested. The heart normal; small fibrinous clot in the right ventricle.” In order to draw deductions of any value from the distribution of arsenic in the body of the deceased we must know positively as to the form in which the poison was taken. Fortunately, we were able to obtain the residue of the soup eaten by the deceased. Microscopic examina- tion of the sediment plainly showed octahedral crystals of arsen- ious oxide, and we were able to separate from a small portion of the solid residue 24 milligrams of the oxide. Plainly the soup was poisoned by simply mixing with it arsenious oxide in substance. As to the quantity of arsenic present in the soup we have the follow- ing data: 125 ¢. ¢., oxidized with hydrochloric acid and potassium chlorate, yielded 314°6 milligrams of arsenious sulphide, equal to 253°2 milligrams of arsenious oxide. As to the amount taken by the deceased we have no knowledge; we infer, however, since the soup constituted the main portion of his dinner, that a large quantity was eaten, which view we think is substantiated by the intensity of the vomiting and purging so characteristic of large doses of the poison. Here then, we have an unquestionable case of poisoning with arsenious oxide, and under conditions most suitable for rapid absorp- ahs AL ana RTRs PF ALT {Every tel Chittenden and Smith—Absorption of Arsenic by the Brain, 151 tion ; a probable empty condition of the stomach, together with a large amount of the poison, a considerable portion of which must probably have been dissolved in the soup. Added to this, nine hours intervened between the taking of the poison and death. Certainly then everything favored an absorption of poison by the brain, if such is characteristic of this form of arsenic. Naturally the vomit- ing and purging would remove much of the poison, still the relative proportion of absorbed arsenic would not be materially altered, and thus if Scolosuboff’s results with soluble arsenites are applicable to arsenious oxide, we ought to find in this case, in conformity with his results, a larger percentage of arsenic in the brain than in the liver or kidneys. Following are the results actually found:* Liver (1259 grams) contained 76:0 milligrams As,O;. Kidney and bladder (332 grams) contained 0°6 milligram As2Os. Brain (=328 grams dry) contained simply a recognizable trace. Case B.—J. G., a young woman, age unknown. Regarding the details of this case we have less definite knowledge. She was last seen alive on Friday night, at which time she threatened to poison herself. The following Monday morning she was found dead, and near her an open package of Paris Green. She had evidently been dead some time, and both the condition of her room and person gave evidence of excessive purging and vomiting. An autopsy by Dr. White showed an entire absence of inflammation of the alimentary tract, and also a lack of any abnormal condition sufficient to account for death. The verdict was therefore, death by poisoning with Paris green or aceto-arsenite of copper. Through the kindness of Dr. White we were able to obtain por- tions of the body for analysis. The contents of the stomach were entirely free from arsenic, the poison having been wholly removed by the purging and vomiting; the trace found therefore, was the amount absorbed by the muscle tissue of the stomach. Following are the amounts of arsenic found in the parts analyzed : Liver (2984 grams) contained 12‘78 milligrams As.O3. Kidneys and bladder (515 grams) contained 3°40 milligrams As.Os. Muscle of thigh (735 grams) contained 0°97 milligram As,Q3. Stomach (425 grams) contained a trace. Brain (1179 grams) contained a trace. * The method of analysis consisted in the oxidation of the tissue with nitric and sulphuric acids, the arsenic being weighed as metallic arsenic. See Amer. Chem, Journal, vol, ii, p. 235, 152 Chittenden and Smith—Absorption of Arsenic by the Brain. The trace of arsenic in both the muscle tissue of the stomach and in the brain was very small; the entire brain could not have con- tained more than 0:2 of a milligram of arsenic. These results plainly substantiate the views set forth above and lend favor to the belief that Scolosuboff’s results with sodium arsen- ite are applicable only to that form of poison, and not to the more insoluble compounds of arsenic. These two cases, therefore, are additional evidence that in poisoning with arsenic the presence of an appreciable amount of poison in the brain, is an indication amount- ing almost to proof positive of the administration of a soluble and diffusible form of arsenic. XI.—InFLUENCE oF PorassittumM anp Ammontum BromiIpEs oN Merasorisms By R. H. Cuirrenpren anp W. L. CuLpert, Pu.B. As a question in the physiology of nutrition, it is very desirable to be able to state something definite regarding the influence of bro- mides upon the metabolism of the body; particularly their influence upon the metabolism of proteid matter, as shown in the excretion of urea and uric acid, and in view of their special application as therapeutic agents in diseases of the nervous system, their influence also on the decomposition of nerve substance, as shown in the excre- tion of phosphoric acid. Two complete investigations appear to have been made upon this subject; one in 1868 by Dr. J. H. Bill,* and one in 1883, by Dr. B. Schulze,} the results of which are more or less in direct opposition to each other. We have also seen a refer- ence to two other investigations, quoted by Dr. Wood,{ in which Dr. Rabuteau found the daily excretion of urea slightly lessened under the influence of bromide, as did also Dr. Bartholow. Dr. Bill’s investigation, which was a very thorough one, had for one of its objects to ascertain whether bromides reduce the amount of phosphoric acid excreted, like such known hypnotics as morphine ; at the same time careful examination was made of the variations in urea and uric acid under the different conditions of the experiments. The experiments were all conducted on one person with a body weight of 160 pounds, which remained fairly constant throughout. The experiments were made in series under known conditions with uniformity of habits, diet, etc. Unfortunately, however, no data are given regarding the nature of the diet, the closeness with which it was adhered to, or whether the body was kept ina state of nitro- genous equilibrium. Each series of experiments, moreover, covers at the most, but six days; three days without bromide and three days with, consequently slight variations might easily be absorbed in an average of three results. The urea in Dr. Bill’s work was deter- * Amer. Jour. med. Sciences, July, 1868. Experimental Researches into the action and Therapeutic value of Bromide of Potassium. + Zeitschrift fir Biologie, vol. xix, p. 301. Einfluss des Bromkalium auf den Stoff- wechsel. } Therapeutics, p. 337. Trans. Conn. Acapd., Vou. VII. 20 Noy., 1885. mined with a “mercury solution,’ phosphoric acid with uranium solution and uric acid by precipitation and weighing as such. The results obtained were as follows: With moderate doses of potassium bromide (3°0-8°0 grams per day) urea was not affected, phosphoric acid was slightly increased, uric acid likewise, though much more decidedly; with larger doses of bromide (10°0-14:°0 grams per day, continued for 2-3 days) phosphoric acid was diminished in amount, but this Dr. Bill intimates could not be attributed to regular hypnotic action, since the other urinary constituents were likewise diminished, notably the urea. Both large and small doses of bro- mide increased the quantity of urine passed in the twenty-four hours. This Dr. Bill asserts was not due to the increased drinking of water, for no thirst, not even with the largest doses, was ever present. Dr. Schulze, experimenting on his own person, obtained results quite different from these. This investigator lived on a fixed diet of the following composition : 154 Chittenden and Culbert Influence of Potassium 220 grams fresh meat =17'13 grams N. 50 grams air-dried wheat bread=0'92 grams N. 30 grams cocoa powder =1'14 grams N. 30 grams butter. 30 grams sugar. 5 grams salt. 1500 grams water. 9:19 grams N. The average amount of nitrogen excreted daily was about 11 grams. Taking this amount of food daily, with uniform habits of sleep, exercise, etc., Dr. Schulze states that he soon reached a point where the daily excretion of nitrogen, sulphur and phosphorus remained fairly constant. Potassium bromide was then taken three days, in divided doses of 10 grams each day. Diuretic action was very noticeable. Phosphorus was diminished, sulphur very much in- creased, and nitrogen (on two days) apparently slightly increased under the influence of the bromide. As, however, the increased excretion of sulphur was not accompanied with a corresponding increase in the excretion of nitrogen, Schultze considers that the increase in sulphur cannot be due to increased metabolism of sim- ple albuminous matter, and seeks to show that the potassium bro- mide must have decomposed, to a slight extent, some nitrogenous phosphorized principle or principles, such as lecithin (glycerine-phos- phoric acid) and nuclein, so abundant in the brain and nerve sub- stance in general. Schulze therefore concludes that under the influ- Ce el, v thee meres ae and Ammonium Bromides on Metabolism. 155 ence of potassium bromide there is probably a decided diminution of metabolic activity in the nervous system, accompanied by decreased nervous irritability. By determining the nitrogen of the feces, Schulze concluded that the bromide exercised no particular influence on the digestibility of the food. In our experiments great care was taken first, to insure body equi- librium and then to obtain sufficient data by analysis, to be sure of the requisite constancy in the composition of the daily excretions. The experiments were tried throughout on the person of one of us (W. L. C.), of good physique and vigorous constitution. The diet was weighed out each day with scrupulous care and was as follows: regi mest Pieeths iis eeprisl bi oe es yee 142°0 grams. IPO COSIs) SBS Ree om sek Es ee ee ae ee eee, Sic nl ene Wine abe RGa Gest saceiee 2 ee Ae ees Skary Ta 8 gre ad hoa 2560 ee (Opie Tae se Sees ia eS baa a es Tae ee DOsOmmne TEU IST es eae tar to aa hee sated i as dl Sahn ld ap SR green SO nies SUPEBee sense ee Se cies Sa Aerio Me OMten sen Phere) | ta ORI erase APSO fe OW Led BOS Ge eae beg SL ERE BOS OB 2 es mee Oxtw as WIN ce Benet ae oe hres Seen ty wee ete ee iy fe ae AE re ee O0;0me ce AWE G3 ot es ee aS ee ede See ee ee See ee ere 34D: ae This diet was commenced on the third day of April and continued for nine days before any attempt was made to ascertain the daily amounts of urea, etc., excreted. Then the urine was analyzed for nine successive days, after which doses of potassium bromide were taken. The above daily amount of food was divided into three por- tions and taken at the same time each day; at 7:30 a. m., 1p. m., and 6 p.m. Exercise was taken regularly and in stated amounts; consisting of a walk each morning before breakfast and exercise with dumbbells just before retiring at 11 p.m. Care was taken not to exercise so freely as to induce perspiration. Throughout the day routine duties allowed of regular habits. It was thus found possi- ble to keep even the minor conditions of the experiment constant throughout. The urine was collected from 7:30 a. m. of one day to 7:30 a.m. of the next, and was at once analyzed. Urea was deter- mined by Pfliiger’s* modification of Liebig’s method, with a standard solution of mercuric nitrate. All the precautions so carefully worked out by Pfliiger; preparation of a mercuric nitrate solution of the proper specific gravity, a standard solution of sodium carbonate to 156 Chittenden and Culbert—Influence of Potassium chlorine and removal of the same, before precipitating the urea; were carried out with very satisfactory results. Uric acid was determined according to the older method of Heintz* with the modi- fication of Zablins, and being conducted each time under exactly the same conditions and with a urine of approximately the same composition, the results are to be considered as strictly comparable. Chlorine and bromine were determined in the usual manner with a standard solution of silver nitrate; the results, however, are not given as they are of value only as essential to the urea determinations. Total phosphoric acid was determined by means of a standard solu- tion of uranium nitrate.t Phosphoric acid in combination with alkali- earths was determined by precipitation with ammonium hydroxide, allowing the mixture to stand 24 hours, filtermg the precipitated phosphates, washing thoroughly with diluted ammonia, then dissolv- ing in a definite amount of dilute acetic acid and titrating with uranium solution. Total amount of solid matter contained in the 24 hours’ urine, was calculated by the use of Christison’s formula. The diet specified, was commenced on the 3d day of April; on the 12th the urine was collected for analysis, the body weight taken and the investigation then carried forward without interruption. Table No. I. gives the results of the analysis of the urine for nine con- secutive days, and shows the average amount of variation to be expected under the conditions of the experiment. On the 21st of April, 60 grains of potassium bromide were cake in divided doses as seen in Table No. Il. The bromide was taken about midway between the hours of eating, so that it might not affect digestion. On the 22d the dose was increased to 100 grains and then to 150, the latter amount being taken daily for three consecutive days. Table No. IL. shows the effects of the bromide on the system, for the six days it was taken. On the first day, the only apparent influence of the bromide is to cause a diminution in the amount of phosphoric acid excreted; seen both in the total P,O, and in the P,O, in combination with alkali- earths. On the second day, the diuretic action of the salt is apparent, accompanied with an increase in specific gravity, and a decided increase in the amount of urea excreted, together with a slight in- crease in the amount of uric acid. Phosphoric acid was still dimin- ished in amount. On the third day, the body weight commenced to diminish and continued to do so throughout the pe ; diuretic * Die Lehre vom Harn, ceo und Leube, p. 94-95. + Die Lehre vom Harn, p, 184. and Ammonium Bromides on Metabolism. 157 action was still apparent as was also increased elimination of urea, and to a slight extent of uric acid likewise. Indeed, the most notice- able effect of the bromide, next to its diuretic action is its decided influénce on proteid metabolism as shown by the increased elimina- tion of urea. As to phosphoric acid the results are not so striking, although an average of the two series shows a diminished excretion, both of total P,O, and of P,O, in combination with alkali-earths. The average difference in the two series of results is clearly shown by the following table: Average of Table No. I, without KBr. No. Il, with KBr. Motaliquantityy on urine 2.2 5ees. eee 926 ¢. ¢. HOMOFexe: Syd. (CRSA pS AEs Sh A Se ere eee ee 1025, 8 1026, 3 Rotalesolid: matters 2250 4_ 2. se seek 56°7329 grams. 63°6252 crams. Metal Ope Ss Sos ole sce OM 2°7540 2°5426 *. P.O; in combination with Ca'and Mg. —-0-6022 0°5452 (Ofte: Exerc |e eee pe des pee Begs eee 0°6752 0°6858 TiccyE Da Rae rn a 34-8681 35-9454 Our results therefore plainly indicate, that under the influence of potassium bromide, nitrogenous metabolism is increased while the excretion of phosphoric acid is slightly diminished in amount; not however, to any such extent as would be expected by an active hypnotic agent. The bromide taken, the largest doses about 10 grams per day, produced its usual physiological effects; such as drow- Siness, diminution of the circulation with accompanying coldness and paleness of the skfn. Constipation was not noticed while taking the bromide, but later on it became somewhat troublesome, once or twice alternating with a slight diarrhcea. In accord with Dr. Bill, we no- ticed an increase in the acidity of the urine while taking the bromide, as also a deepening of the color. With bromide of potassium therefore, our results agree with those of Dr. Schulze in showing an increased excretion of nitrogen (urea and uric acid), although far more pronounced than he found in his experiments, while the diminution in phosphorus is less pronounced than found by Schulze. With Dr. Bill’s experiments our results agree in so far as the diminution of phosphoric acid is concerned, but are entirely different as regards the urea. Since, however, Dr. bill retained uniformity in diet only during the days of the experiments, it is quite possible that lack of nitrogenous equilibrium may have had some influence on his results. The increased elimination of urea noticed in our experi- ments is certainly indicative of increase! metabolic activity ; it is, 158 Chittenden and Culbert—Influence of Potassium however, suggestive that potassium bromide has been recently found to exercise a very decided accelerating influence on the proteolytic action of both pepsin-hydrochloric acid* and trypsin ;+ while, there- fore, this fact may have something to do with the increased -elim- ination of nitrogen, particularly as the diet used is quite rich in pro- teid matter, it seems more probable to suppose that the above changes in the excretion of nitrogen are due rather to changes in the tissue proteids ; still it would have been interesting if the nitrogen in the feces had been determined each day. Although the last dose of potassium bromide was taken on the 26th of April, the same diet was still continued and the urine carefully examined daily, until the 8th of May, at which time the amount of bromine in the urine was reduced to a minimum. Dr. Bill states that bromine usually disappears entirely from the urine in ten days aie the last dose of bromide. The results of the twelve days analyses are shown in Table No. III. In examining this table it is interesting to note how quickly the elimination of urea is changed on stopping the doses of bromide. On the 26th, the last day the bromide was taken, the excretion of urea amounted to 37°5 grams; on the 27th it fell to 31°8 grams, far below what it had been any time before the bromide was taken. It would thus appear that after withdrawal of the bromide, nutrition which had been accelerated, rebounded in proportion to the preceding acceleration. Uric acid, moreover, which had likewise been increased in amount by the bromide, was now also correspondingly diminished. Furthermore, the diuretic action of the bromide was at once stopped, and the specific gravity fell to 125°5. In the case of phosphoric acid, however, the action of the bromide appears to be continued for a day or two after its withdrawal, and indeed it is noticeable through- out, that the diminution in phosphoric acid excreted, is not at all proportional to the amount of bromide taken. In fact phosphoric acid, both total P,O, and alkali-earth P,O,, appears to be more deci- dedly diminished on those days when the amount of bromide in the blood was the smallest, notably on the 21st, 24th and 27th of April. By the 3d day after withdrawal of the bromide, the excretion of urea had gone nearly back to the daily amount, prior to taking the bromide; still it is to be seen in Table No. IJ, that the average ex- cretion of urea, uric acid and phosphoric acid is below the average excretion recorded in Table No. I. In fact after the continued doses * Chittenden and Allen. Trans. Conn. Acad., vol. vii, } Chittenden and Cummins, Ibid. and Ammonium Bromides on Metabolism. 159 _ of potassium bromide, the metabolism of the body did not fall back to its original height; but being temporarily accelerated during the exhibition of potassium bromide, it [the nitrogenous metabolism | fell back on withdrawal of the same, to a far lower level, and al- though later somewhat increased in amount, its average was still lower than recorded in Table No. I. Nutrition had evidently been disturbed; the body weight showed gradual diminution, and on the 4th and 6th of May there was slight diarrhcea accompanied with a decided decrease in the amount of urea and uric acid excreted. Dr. Bill appears to have experimented somewhat with sodium bro- mide, although we find no results recorded, aside from the fact that this salt, like potassium bromide, caused an increased excretion of uric acid, and the» general statement that ‘when taken by the mouth, bromide of sodium does not produce the same effects as bro- mide of potassium.” In view of the increased excretion of urea, noticed under the influence of the potassium salt, we were interested in seeing whether ammonium bromide would have a like influence, especially in view of the fact that v. Schreeder* has shown that ammo- nium carbonate is directly convertible into urea by passage through the liver. The physiological action of ammonium bromide is stated to re- semble in many points that of potassium bromide, while in other points it differs essentially.t As to its influence on metabolism no experiments whatever appear to have been made. On the 9th of May, 75 grains of ammonium bromide were taken, in divided doses, as shown in Table No. IV. In all, 425 grains of the salt were taken in four consecutive days. The action of the salt on the system was not as pleasant as that of potassium bromide; caus- ing a general weakness and indisposition, a slight diminution in the pulse, an occasional cold perspiration, more marked lividity of the countenance and a parched, dry taste in the mouth. An habitual eruption of the skin was moreover much increased and accompanied with acne on the back and shoulders. Undoubtedly these disagree- able symptoms were much augmented by the temporary lassitude which was beginning to be apparent; doubtless due to the approach _ of warm weather together with lack of the accustomed vigorous exercise and the long continued use of the somewhat monotonous’ diet. * Archiv f. exp. Pathol., vol. xv, p. 364. Also Report on Progress in Physiological Chemistry in Amer. Chem. Jour., vol. v, p. 219. + Wood, Therapeuties, p. 341. asstum 160 Chittenden and Culbert—Influence of Pot COCE- FS 69LG-0 9FLS-0 G00L-6 L88¢-1¢ 9C0T ‘poy 0&8 OOTEL 06 096F-98 T¢¢9-0 6689-0 1886-8 LPTP-09 90. = “PHY 816 QOTSL 61 O9ES-FE 966¢-0 69S¢.0 6069-6 GCL Log ¢.9G0T | ‘PDV 088 OOFGL 8T GLES-ZE LOFL«0 9768-0 5 8189-2 orgL-9¢ | &-¢0T ‘poy G16 OOP | LE LOGP-GE 9FTL-0 8819-0 6CEL-S PLOP-SS CoOT | ‘ploVv 866 O08SL 9T LVGE- SE 6969-0 L8S¢-0 8E0L-6 8666-7S CoOL | ‘prPeVv 066 00061 cT 6698-P& P6990 0909-0 6761-6 LEOP-09 GoOT | ‘pov SLOL 00062 tL 1900-28 1069-0 BE19-0 9608-2 06S%-L6 9801 ‘poy 0g6 00TSL el a ae oe i ee vary) ‘plow og eee "0%d TOL | pros oy | 82 U8 | ‘woHovex eae bes qudy ‘aqdImoug LAOHLIM—'T AIAV 161 and Ammonium Bromides on Metabolism. ‘md Of = g ‘ud g 0¢ LIIG-LE | F86L-0 | 9609-0 | 9099-2 | L8T9-69 960 ‘UL “B OT 72 OC ‘urd #OT og ‘ua ‘dg 0g SLLP-98 | O&L9-0 | 9609-0 | 8106-3 | 6808-T& | -90T "UL "® QT 98 0g res ot | Boor-ge 9 918e-@ | 2090-99 ‘UB OT 9B QC : SIT9-0 | 208F-0 186: . 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As to its influence on metabolism, a study of Table No. IV, and comparison with No. III, plainly shows a decided accelerating influence on the excretion of urea; the average of the results, moreover, shows a very slight diminution in the excre- tion of total phosphoric acid, at the same time it would appear, in accord with what was observed with potassium bromide, that the diminution was greatest with the smallest amounts of bromide, as on May 9th and 10th and on the 13th and 14th after withdrawal of the bromide. With the largest amount of ammonium bromide, on the other hand, phosphoric acid appeared to be increased in amount, thus according with what Dr. Bill observed with like quantities of potassium bromide. The average difference in the two series of results is shown by the following table: 164 Chittenden and Culbert—Influence of Potassium : : Average of Table No. III No. IV without (NH4)Br. with (NH4)Br. Total quantity of urine -....----..-- 915 c. c. 1072 ¢. ¢. Splert. ete Wek bo Eee pete 1024, 8 1024, 4 Total solidtmatterss-.. -=.-2-.-- 4.-- 54'3970 grams. 62°3608 grams. Total sPrOpeeeesneeant eee eee ees 2°5643 2°5130 P.O; in combination with Ca and Mg. 074972 0°5749 Wriclacid\ 2-6 ooo chee goes toe 0°6599 06751 Wrea lio cee ce pe meant 32°8579 34°6505 It is thus seen that diuretic action is even greater with the ammo- nium salt than with potassium bromide; likewise the excretion of both urea and uric acid is greater under the influence of ammonium bromide than in the case of the potassium salt; as to phosphoric acid the table of averages shows practically nothing, but as before ob- served a study of the individual results does indicate some action of the salt, although diminution in the excretion of phosphoric acid under the influence of ammonium bromide cannot be so surely claimed as with the potassium salt. After withdrawal of the ammonium bromide, the urine was exam- ined for two days more; the results showing the same, or even greater drop in the excretion of urea, observed under like conditions with the potassium salt (Table No. V.). Hence, so far as our experiments ex- tend, the’ influence of the two salts on the metabolism of the body is very much alike, differing only in extent of action; the ammonium 2 and Ammonium Bromides on Metabolism. 165 salt, as might be expected, causing the largest excretion of urea, not, however, necessarily from any greater influence on proteid metabolism, but merely as furnishing a certain amount of ammonia to be excreted as urea. Finally, our results, with both salts, fail to show that dimi- nution in the excretion of phosphoric acid to be expected from active hypnotic agents, and in this respect, therefore, our results show noth- ing antagonistic to Dr. Bill’s conclusion “that bromide of potassium in its legitimate action, is an anesthetic to the nerves of the mucous membranes and a depressor of their action. Its hypnotic effects are secondary.” XIT.—INFivence or CrncnonipInr SunpHATE on METABOLISM. By R. H. Currrenpen anp Henry H. Wurrenovuse, Pu.B. Wu8ILE much attention has been paid to the physiological study of the cinchona alkaloids; quinine particularly having been experimented with by several observers, to ascertain its influence on the metabol- ism of the body ; we have not been able to find any recorded state- ments bearing on the action of the closely related alkaloid, cinchoni- dine. In physiological action, quinine is taken as a type of the group ; cinchonine is stated* to be similar to quinine but less power- ful, and that its history in the organism is parallel with that of qui- nine; cinchonidine is likewise stated to be weaker than quinine and in pugniolseacal action, apparently its equivalent when taken in doses one-third larger. Presumably, therefore, its action on the metabol- ism of the body is similar to that of quinine, although apparently no attempt has been made to determine this point. In view of the spe- cial action of quinine on nitrogenous metabolism, we have devoted our attention mainly to a study of the influence of cinchonidine on the excretion of urea, uric acid, phosphoric acid and chlorine. The experiments were conducted wholly on the person of one of us (H. H. W.) under uniform conditions of diet, exercise, etc. The diet, weighed out accurately each day, was composed of 255 grams meat (beef). 255 grams wheat bread. 149 grams potatoes. 50 grams oat meal. 35 grams butter. 21 grams sugar. 570 grams milk. 350 grams water. This diet, divided into three definite portions each day, was taken for some time previous to the experiments, so that the system might become accustomed to it and the metabolism of the body brought to a constant point. The body weight was determined each morning; the urine collected from nine a. m. of one day to nine a. m. of the next, making the 24 hours’ urine, the analysis being made the same * H. C. Wood, Therapeutics, p. 81. Chittenden and Whitehouse—Sulphate on Metabolism. 167 day. Urea was determined by Pfliiger’s* modification of Liebig’s method, chlorine being removed by a standard solution of silver nitrate. Chlorine was determined by evaporating 10 ¢.¢. of the urine with a weighed amount of potassium nitrate in a platinum cru- cible, igniting until the organic matter was completely removed, dis- solving in water, acidifying with nitric acid, neutralizing with cal- cium carbonate and then titrating with silver nitrate solution. Uric acid was determined by Heintz’s method as modified by Zablinst and phosphoric acid with a standard solution of uranium nitrate.[ The amount of solid matter was calculated by the use of Christison’s formula. After taking the above diet for some time, the urine was analyzed for seven consecutive days prior to the exhibition of cinchonidine. The results, seen in Table No. I, show a very close agreement in the daily excretions. On the 11th of May the first dose of cinchonidine sulphate was taken. The alkaloid salt was a finely crystallized preparation ob- tained from Powers and Weightman. The daily dose was usually divided into three portions and taken in tiny gelatine capsules, about five hours apart. In view of the fact that cinchonidine is a weaker alkaloid than quinine, it was not deemed necessary to try the influ- ence of very small doses; on the first day, therefore, 15 grains of the salt were taken; on the second day 21-8 grains; on the third day 35°1 grains; and on the fourth 50 grains, making a total of 121°9 grains of cinchonidine sulphate in four consecutive days.§ The re- sults of the analyses of the four days’ urine, as well as those of the three following days, on which no cinchonidine was taken, are shown in Table No. IL. Comparing these results with those in Table No. I, and in Table No. ITI, it is seen that cinchonidine exercises a very decided influ- ence on the nitrogenous metabolism of the body. Urea is at once affected : its excretion on the first day even, is diminished 6 per cent., while on the second day it is diminished 10 per cent., and on the fourth day when the largest dose of cinchonidine was taken, the excretion of urea was 16 per cent. less than in the normal urine. The influence * Pfliiger’s Archiv, vol.‘ xxi, p. 248. + Die Lehre vom Harn, Salkowski and Leube, p. 94-95. } Die Lehre vom Harn, p. 184. § While taking the larger doses of cinchonidine, an intense ringing in the ears was temporarily experienced (cinchonism) together with partial deafness and slight dizziness. On one or two occasions a slight nausea was felt. 168 i) BS 's ‘=> Ss S < S Ss s > js) O 2 is) S > al @ s < S 5 rs Re g 8 § S ~S S $ s 5 ¢86-1F 79L-0 0F0-€ 6FL-9 188-99 9801 OF9-TF 6FL-0 6188 991-9 198-99 ¢.980T POPP 89L-0 0182 POE-9 896-89 @- 9601 666-2F 618-0 OTL@ O19 696-29 LeOL LL8- TP TeL-0 B0L-8 G6F-S TLP-89 LeOr $89-CF 161-0 740-8 gge.g eT L-89 8801 meae: |. ig: | oe te | ag | ver = | “ploeong §| “Ota OL | “ouMopyQ | mo Nen ap ‘dg ‘ANIUQ) TVNUON—'T IAVI, PHOS [THIOL ‘PPV ‘ploy ‘PPV ‘PPV ‘PPV ‘PPV ‘PPV “MOTIOVOY oSOT ccor LLOT 066 £06 ‘086 0 °D GL6 ‘oul, 4yyuenb eyo, 0096S 0096S 0096S 0096S 0066S 00F6S 00F6E “‘SuUIvIs ‘yqs10m Lpog OL 169 Sulphate on Metabolism. 0 998-66 TTL-0 | T08-6 98S-¢ 161-79 ¢- L601 ‘PIV CL6 0088S ui! 0 686-9& 999-0 068-6 860-9 616-09 ¢-960T ‘ploy 0&6 OOF6S 9T 0 I8P-Cé 869-0 G6L-T Col-9 LOP-9S E601 ‘PPV O&0T 00809 CT Og CTI: Sé CLg-0 861-6 Cg8-¢ G6S-8G voOL ‘PrPy GGOT 00009 las T-G& 6OE-8E 661-0 666-6 | CSP: h 86F-89 PoOT ‘PPV C6LT 00009 &1 8-16 196-LE 6LL-0 669-6 988-¢ L6L-19 ¢-960T “PPV cL6 00669 6I Se CTL: 66 STL-0 9LL-G vP0-G OFP-6S LoOT ‘plOV |'9 *O 066 0096¢ Ir “SUIGIS *SUBIS ues SUTRAS “*SUIRIO *SUIBLO “‘suvad Toyey ca ‘ si aqyeqdyns . 2 . 4u3 are supiioy, YN | PePoEA | “ota mmo, | “enwoIg SME | ap dg | snonovoy | SMA A | AMIE | soy “aD 4. y , ‘HNIGINOHONIO AO SLOMAA AHL YNIMOHG-—"J] ATAV Nov., 1885. 22 TRANS. 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On the fourth day it is nearly back to the normal amount [see Table No. II.], and on the fifth and sixth days the excretion of urea rises somewhat above the average. Uric acid does not appear to be correspondingly affected. It is only under the influence of the largest doses, or rather under the long continued action of the alkaloid that the ex- cretion of uric acid is diminished; thus on May 14th, when the final dose of 50 grains of cinchonidine sulphate was taken, the excretion of uric acid was for the first time diminished ; its diminution, how- ever, was very perceptible and it continued for the two succeeding days, after withdrawal of the alkaloid salt. Phosphoric acid was greatly diminished in amount under the influence of cinchonidine; the diminution commencing to show, as in the case of urea, with the first dose of the alkaloid salt, then gradually increasing in amount with increase in the dose of cinchonidine until maximum diminution was reached on the day after the final dose of the alkaloid. The alkaloid salt, moreover, appears to have had a slight diuretic action. On the 21st of May, cinchonidine sulphate was again taken, the results of the analyses of the 19th and 20th showing that the urine had returned to its normal composition. Accordingly, 95°8 grains of the salt were taken on the 21st and 22d, producing the same results (Table No. IIL.) as noticed in the first series, viz: diminution in the amount of urea and uric acid excreted, a like diminution in the amount of phosphoric acid and an increase in the total amount of fluid. Table No. [V. shows the average results under the different condi- tions of the experiments. | It is evident then, that cinchonidine has the power of lessening very materially the elimination of nitrogen, i. e. the consumption of tissue. Ranke* first pointed out the retarding influence of quinine on the elimination of uric acid. Zuntz found by experiment upon himself that 25 grains of quinine reduced his elimination of urea nearly 40 per cent. A like diminution in the excretion of urea, under the in- fluence of quinine, was noticed by Rabuteau in experiments upon dogs and also by Hermann von Boeck.t{ The most interesting ex- periments, however, with quinine are those carried out by Dr. G. * Quoted from Dr: H. C. Wood, Therapeutics, p. 74, + Zeitschrift fiir Biologie, vol. vii, p. 422. ——s —?P . eet Seed te te ee oe tT Wyte apne cee oe Sulphate on Metabolism. - 178 Kerner,* and more recently by Dr. Priorft and Dr. Sassetzky.{ In Dr. Prior’s article isto be found a very complete account of the liter- ature of the subject. Dr. Kerner found that taking 9°3 grains of quinine hydrochloride per day in divided doses, for 3 days, making a total of 27-9 grains, caused a diminution in the excretion of urea, amounting on an average for the three days to 12 per cent., while the excretion of uric acid under like conditions was diminished 54 per cent. Phosphoric acid, too, was diminished somewhat; on an aver- age about 4 per cent. per day. Diuretic action was very slight. With larger doses of quinine; 77°5 grains of the hydrochloride in divided doses during three days; diuretic action was quite pro- nounced, the average increase in volume, amounting to 200 c.e. Moreover, urea was diminished on an average 23 per cent. per day, uric acid 82 per cent. and phosphoric acid 15 per cent. Oppenheim,§ however, by a dose of 30°8 grains of quinine found an increase in the daily excretion of urea amounting to 4 grams, and he considers that Kerner’s results are due to diminution of digestive action. It is cer- tain that both quinine and cinchonidine do interfere with the pro- teolytic action of the gastric and pancreatic juices,|| but this retarding action can hardly be taken as explaining in full, the results obtained by Kerner or those obtained by us with cinchonidine. Prior, moreover, by very carefully conducted experiments with quinine, has completely corroborated Kerner’s results and has shown in addition, by a daily determination of nitrogen in the feces, that diminution of urea and uric acid is not due to lack of digestive action, as suggested by Oppenheim. Prior’s results show on an aver- age, without reference to the size of the dose, the following effects of the quinine.4 Quantity of Uric Sodium Sulphuric Phosphoric urine. Urea. acid, chloride. acid. acid. increase decrease decrease _ decrease decrease decrease 10°65 per cent. 19°60 % 12°29 % 9°06 % 33°70 % 23°38 % Sassetzky’s results with fever patients, also corroborate Kerner’s statements. * Pfliiger’s Archiv, vol. iii, p. 104. + Ueber den Einfluss des Chinin auf den Stoffwechsel des gesunden Organismus. Pfliiger’s Archiv, vol. xxxiv, p: 237. { Ueber den Einfluss fieberhafte Zusténde und Antipyretischer Behandlung auf den Umsatz der stickstoffhaltigen Substanzen und die Assimilation stickstoff-haltiger Bestandtheile der Milch. Virchow’s Archiv, vol. xciv, p. 485. § Pfliiger’s Archiv, vol. xxiii, p. 476-477. | Chittenden and Allen; Chittenden and Cummins. Trans. Conn. Acad., vol. vii. §] Pfliger’s Archiv, vol. xxxiv, p. 263, 174 Chittenden and Whitehouse—Influence of Cinchonidine v. Boeck considers that quinine owes its retarding influence on proteid metabolism to a direct action of the alkaloid upon the cells and their activity, although the alkaloid doubtless does unite with albu- min or alter its constitution so as to render it less readily decompos- able. Metallic salts, as lead and mercury, certainly form compounds with albumin difficultly decomposable, as also does arsenic, and vy. Boeck suggests that these metallic poisons unite with the proteid matter of the various organs of the body, while quinine unites sim- ply with the circulating albumin, explaining in this manner the ready elimination of quinine as compared with the slow excretion of mer- cury or arsenic, the latter of which v. Boeck* has shown has little if any influence on proteid metabolism. Prior, moreover, states that diminution in the urine of the end-products of hitrogenous metabol- ism is due, not to hindering of their excretion, but to actual hindering of their formation. Comparing now Kerner’s results, with the results obtained by us with cinchonidine, we see great similarity of action but decided dif- ference in extent, particularly so far as the excretion of uric acid is concerned, With cinchonidine, the greatest average daily diminu- tion in uric acid amounts to but 15 per cent., and this after taking about 121 grains of the alkaloid during four consecutive days. Se- lecting the lowest single result, that obtained on the day 50 grains of cinchonidine were taken and comparing the diminution then, with the average normal excretion, it is seen to amount to but 26 per cent. In the case of urea and phosphoric acid, the divergence is not so great; thus for urea the average daily diminution was 11 per cent. for the three days following the last dose of cinchonidine, while the greatest diminution noticed in any one day was 16 per cent.; with phosphoric acid the average diminution for the same period amounted to 19 per cent., while the greatest diminution noticed any one day was 38 per cent.; a diminution which at no time was reached in Kerner’s experiments with quinine. Thus in drawing a comparison between Kerner’s results with 7775 grains of quinine distributed through three days, and our results with 121 grains of cinchonidine extended over four days, we see two strik- ing points of difference; with quinine there is a diminution in the amount of uric acid excreted of 82 per cent., with cinchonidine an average diminution of but-15 per cent. ; with quinine there is a dimin- ution of phosphoric acid amounting to 15 per cent., with cinchoni- dine, on the other hand, a diminution of 19 per cent. Hence it is to * Zeitschrift fir Biologie, vol. vii, p. 430, Sulphate on Metabolism. 175 be seen that cinchonidine has a far less pronounced specific action on the excretion of uric acid than quinine, while on the other hand, diminution of phosphoric acid is much more pronounced with cincho- nidine than with quinine. In Prior’s experiments, however, with qui- nine, diminution of phosphoric acid is more pronounced. Is this diminution in the excretion of phosphoric acid under the influence of cinchonidine to be attributed simply to decrease of pro- teid metabolism, or is it in part due to a special action of cinchoni- dine on the metabolism of some phosphorized principles, presumably those of nerve tissue? If due to general decrease of proteid meta- bolism, we might expect to find that the addition of any non-nitro- genous principle to our fixed diet, whereby the decomposition of albuminous matter would be diminished, would cause a corresponding decrease in the excretion of phosphoric acid, or in other words that diminution of urea and phosphoric acid excreted, would be in the same ratio as noticed under the influence of cinchonidine. This question we have endeavored to answer by a study of the influence of pure glucose on the elimination of urea, uric acid, and phosphoric acid, under the same conditions of diet etc., as observed in the experiments with cinchonidine. The influence of carbohydrate food on proteid metabolism has been illustrated in many ways by various investigators, but so far as we know, no experiments with pure glucose have ever been tried. Through the courtesy of Dr. Arno Behr, of Chicago, we have been supplied with an abundance of chemically pure anhydrous glucose, which we have used in the following experiment. Before taking the sugar, the urine was analyzed for ten consecutive days, to insure an accurate average of the normal excretion under the conditions of the experiment. The results are shown in Table No. V. 200 grams of glucose were then taken daily in addition to the fixed diet, for nine consecutive days. The effect on the excretion of urea, etc., is shown in Table No. VI. At no time was sugar to be detected in the urine by Trommer’s test. A comparison of the two tables shows the usual effects of carbohydrate matter on the excre- tion of nitrogen, viz: a diminution in the amount of both urea and uric acid. The volume of the fluid excreted, appears to be consider- ably lessened by taking the glucose. The excretion of phosphoric acid is likewise diminished. Chittenden and Whitehouse—Influence of Cinchonidine 176 ’ ———s ‘PPV Ss L6G-2 816-9 CF0-29 8e0r OL6 006g OT 006-9F 126-0 CFO-8 TPL-8 eh9-0L G-L80T ‘poy SLOT 0076S 6 GOT: LF CTL-0 POE-E O8LeL CCP OL LOI ‘poy 060T 0086¢ 8 000:FF BBL-0 129-2 028-9 POF-29 9201 ‘poy POOT 0086¢ A 8L0-FF 708-0 629-2 aah C1919 @-880T ‘ply 066 0086g 9 19T-&F 86-0 988-2 868-9 P8E-F9 8e0T ‘py 096 00009 g OL SF 106-0 LOT’ 060-9 OSF- Sh 6201 ‘pov SPOT 006g P 109-9F 308-0 e8E-£ F619 TOL TL SCOT ‘poy OLOT 00F6S g es 890-1 oO: 080-4 C68-2L @-800T ‘poy 0901 00968 g rma od, | see, | ome, | mes, | cm | eer fomome | gyag | roel ‘ple og «| “Od yelog, | “ommoryo ae “19 “dg WOHOrOT |e aaunb pog| M8 Spoa Bie ‘ANIUQ. TVWHON—'A FIAVI, 0 COP: PP 168-0 696-6 OSE-9 969-99 9601 “PPV 9g0T 0006S &@ eee Tae a 0 6&F-CP 106-0 80L-S | 686-9 618-99 960T “PPV 9LOT 00009 6G es 0 CE8-66 608-0 ¢99-6 6¢¢.9 C60-89 | 9601 “POV FIO 0096S 1@ ‘ 0 OFL- TP 998-0 P&6-6 3 066-4 089-99 9601 “PlV CLOT 00609 06 7 ] é 006 COL-8E 789-0 2 LEG-G | OF9-S 691-19 £-9601 7 Ppley £96 00009 61 : 006 008-66 18L-0 ~ F69-6 90T-9 ¢go-g9 8c0T sPIOY OL6 0086S 8I 39 ; d S 006 188-05 6F9-0 _ 688-6 PLO-9 &TL-&9 8G0T ‘PV 0&6 0096S AL s 006 GOG-6P 08-0 O&T-& - OGT-L 616-04 8201 PHY LVOT 00009 ot : s 006 OLO-0F G62:0 area 5 ee 676-99 ; ¢-860T ‘PPV OL6 0066S cT ie - Z Tp eee ae ree 006 600-09 699-0 Wes z 698-7 OLE-LG » $8601 ‘POV OF8 0086S ia! 3 . 006 SLL: OF €69-0 T&L-@ | 860-9 &FT-09 6C0T PPV g98 O06S &I : ie ee eS A i uS Cte Paice SI ES 4 006 PIL-GP ToL-0 T8L-6 | LOL-D L6L-F9 O&0T “PPV 006 00S6¢ él é a ee eee a ae ‘aden "Bod ‘ploe og | “O%q [10 “eutIO[YO, Ripe tid PASE D awe CAC eal eet % “Ty "dg “WOOBOY eet) | youuy | | a alli si pros. reHon sen Tea _Spog, jo Leg 178 Chittenden and Whitehouse—Sulphate on Metabolism. These points of difference are all shown more clearly in the follow- ing table of averages : Average of Normal Urine under the urine. influence of glucose. Total quantity urine--.----../.-.==- 1030 ¢. ©. Rien (On [sere ~-= oes at ga ee ee eae SHAG, OOSGs0) bse “==> 7a wigan Peper oe AG EenCsh LP EGra B mes A “wu 0). O° 9 “TUBIS ? "ONM + HOW 2 emsseig | ‘J, Fic ? aa UJI Torsny : J 8 | 19yje OQ N ‘punoj N 9) 09 H “‘HSOTOAAOTNOLOUd dO SISATVNY O $ A tN H @) 00-2 8&-1¢ 00T6-0 O¢s-0 fc FE1S-0 mn LET6-0 nae. C9LS-0 oe 9EPL-0 Be: STEP-0 ee 868S-0 OLOE-0 966F-0° TTGP-0 L02L9-0 [oven Ro} ‘werd PaO “pasn OH eouRysqng Kiihne and Chittenden— Globulin and Globulose Bodies. 215 simply by concentration of the solution freed from chlorine, pre- cipitation with alcohol and washing with ether. The ash (1°17 per cent.) consisted only of calcium phosphate and a trace of sulphate. Leteroglobulose. This body was obtained from the gummy precipitate which sepa- rated, during dialysis, from the solution of the first precipitate thrown down from the neutralized digestive fluid by salt alone. The sticky mass was separated from the sides of the parchment tubes, dissolved in sodium chloride of from 3 to 5 per cent., repre- cipitated by saturation of the solution with salt, the precipitate again dissolved in dilute salt solution and the substance finally separated by long continued dialysis in running water. After thorough wash- ing with water, alcohol and ether it appeared as a light, white powder, not unlike heteroalbumouse in general behavior and _ reac- tions. After each precipitation and treatment with dilute sodium chloride, heteroglobulose left a residue, which like dysalbumose was readily soluble only in dilute acids. From the following analysis ‘it is to be seen that the preparation, in spite of its long continued and repeated dialysis, contained 2°03 per cent. of ash, which con- sisted mainly of calcium carbonate with a small amount of phos- phate and sulphate. The composition of the three globulose bodies shows the same slight differences as noticed in the case of the various albumose bodies (from fibrin). Unlike the latter, however, the content of car- bon in the globulose bodies never falls below 51 per cent., and fur- thermore it is always higher than that of the globulin from which the globulose was derived. The percentage of nitrogen, which in the albumose bodies was found a little higher than in fibrin, exceeds that of the globulin more yet, in some cases by more than 1 per cent., and the same holds true of the percentage of sulphur. In contrast to the albumose bodies, the percentage composition of the globulose bodies gives no grounds whatever for the assumption that they arise from the digested globulin by simple hydration. It must not be for- gotten, however, that the digestion of globulin by gastric juice is a process quite different from that of fibrin digestion and one hitherto much less clearly understood, since besides the globulose bodies there is formed a large quantity of a substance which is separated by boiling and which resembles ordinary coagulated albumin. Some- thing similar, indeed, has been known ever since Briicke’s study of fibrin digestion, but it has long been accepted that the coagulum vés. 16 Kiihne and Chittenden— Globulin and Globulose Bod 2 00-001 Gh bE 98-T %6-ST G6-9 6G-1G ‘ISC1OA VW TL-T 66-ST 96-9 9¢-1¢ ‘asojnqojbouajnaq aauf-yso fo uorwrsodwos abnjuadsag 61-1 LLO0:0= 1 = Sak ee ae Sere) yas plage te om OTT 9400-0 | ~~~" as pcs i os Te. alee alee ea ea ~_ San = Ser 6680-0 lo ot ir a cs ea: as a Se nook 6690-0 Sais ‘ues nie peisaiads| | teas eutce se BH | 6-1 9£90-0 Snel, ate Ed | easier pe Pros eee jeri. 9L-ST | OF-9GL 7-61 10-69 | ~~" eS at eas ae GL-ST | OT-99L | 9-61 | F6-SL | ~~~ ese Ter Bae oc 2 Be Sa i 7 iS) = 198-08. || 1Srs-0 as ee oi ‘ae 9 mi | al 86-08 | 0628-0 "MBS ; : : ae p Sr Reig ‘ONY + HOM 2 kone heres leecamcas pane} Yysy "SV 5 Joye 'Ogug N ‘punoz N 9 09 ¥6-9 87-1 OMmARO 6F16-0 0166-0 “mes “punoy O*H “ASOTOAZOTNONNLAAG AO SISATVYNY LYP9-0 OFe9-0 GLPI-0 PE9IS-0 9199-0 901-0 G0FS-0 89FE-0 TTLP-0 “WIR ‘posn eouRysqng ON 217 Kithne and Chittenden— Globulin and Globulose Bodies. ‘aSOTASCOTNOUELER AO SISA TVNY ‘ 00-001 89°66 aja: eS amas aes ated ee Te) IG S16 06-6 anay a aS es mp ie SS 80-91 Bs aes TT-9T c0-9T E Se: pee NT 86-9 ayes oa ae Sar Sie 60-4 c6-9 H OL-6¢ Be ty ak i a ree C0-6¢ cree 0 OSRI9A W ‘asopnqopbouwajzazy aatf-ysp fo uovpsodwuoo abnyualad 00-6 SON Ose rece | ce tenn te ste ale Sa Sg Pi wall Saseaee eh ane ae aah pe L68F-0 90-6 OGRORUS SI) see har ee eric ae pepe NK ie Hela ares ean aa See eae east TT8&-0 ite ee irae soll 80-6 9160-0 teil rays PO Ae ae lly Ne | a Z att C709-0 ee REE fe C16 6680-0 Maes oe en BA, lle Peaee ea ee sch aa mae ee igs ea” ei at LEGG-0 Fy ag Sea ee $k, Cele ee ee 6L-GT | BE POL — |.0-OL = | LORBP | ee | Dies 'C; | The analysis of the product is shown in the accompanying table. Antipeptone (D). The behavior of the preceding preparation while being dried, nat- urally suggested the suspicion that the substance was either decom- posable at 100° C. or less in the air, or else that it contained some decomposable admixture. We therefore attempted a further parifi- cation of antipeptone and at the same time a more cautious method of drying. For this purpose another preparation of antipeptone was made in the following manner. 230 grams of commercial dry pancreas, somewhat less active than that employed in the preceding preparation, were warmed at 40° C. for three hours with 1200 ¢. ¢ of 0:1 per cent. salicylic acid, after which the mixture was neutralized with sodium carbonate and to it was added directly 1920 grams of boiled, moist fibrin, 82 grams of dry sodium carbonate and 32 grams of thymol. This mixture was warmed at 40° C. for seven days, at the end of which time the residue of the pancreas, the antialbumid prodused, and considerable sepa- rated tyrosin, formed a noticeable sediment, which was filtered off and pressed, after the residue had been thoroughly washed with water warmed at 40° C. The filtrate was made slightly acid, heated to boiling, and as this produced only aslight precipitate it was imme- diately concentrated to about three litres. On cooling, an abundant slate-colored precipitate separated, composed almost entirely of tyro- sin. After removing this by filtration, the solution was saturated — with ammonium sulphate and the resultant filtrate treated as in the 253 Ss SF At) a ae | OS 10-0 et SEO Rg = eet el aoe I ~ S s S = = wie . Weis % age yse ayy ay} wos] mod1y S rogeg _00-001. 17.86 E40 8-91 Eke9 08-41 *ODVIOAW 68-9T F8-91 oL-9 1é-LV ‘aounjsqns aauf-ysv ayy fo uoyrsodiuos abpyua1ag FL-9 08-L7 OAZRO 18-0 $8-0 01-0 19-0 0680-0 8960-0 6&-G 86-¢ 0160-0 8160-0 Dy “ysv jo % § Suyonpep| § 1OYFE S “UIRLS *punoy "Ose qsV TURAS “punoy Ysy ae Ses eer gee gre eee =" | @pT9-0 | XI Aigner Seo er cass? “hae SIS ooo aoa ee eave ee THA wage D- ge Noee“ | 228 Ghee eaters i ead ae I rage Re aes dlrs [oS pee ler gee We aac a ee EEO ‘TA gait er Spe cee Wee alee eee eae ee ee CCRT GO:01 cOCOL 1 G:y Opener Peet eee Tego eU el Al 96-01 | 0-894 | 0-9) 9674 | <2 | cee | oo | oo | tan@o | 1 sre Joos free] =2°" | 196%] OLL6-0 | GLO | STFE-O | 6F62-0 | II “v7 [owes [oro] ===" | og.pp | g0g6-0 | 88-9 | eaee-0 | 7829-0 | I et sun ; : “wes £ bamat "(oe penne | Me | man oP oa ‘punoj NS 00 a -“qug (2) ANOLATIILIN VW Nov., 1886. TRANS. Conn. AcAD., Von. VII. 234 Kihne and Chittenden—Peptones. previous case; that is, a barium peptone compound was formed, puri- fied with aleohol and this exactly decomposed with sulphuric acid. In order to separate the free peptone, the solution was concentrated at a gentle heat with the addition of a little ammonia, precipitated and boiled with alcohol, the almost liquid’ precipitate dissolved in water, the solution acidified with acetic acid, concentrated again, pre- cipitated and extracted hot with alvohol, repeatedly washed and kneaded with ether, kept for a long time under ether and then slowly dissolved in as small an amount of cold water as possible. When filtered, a small residue of tyrosin appeared in the preparation. The new solution was concentrated at a gentle heat, precipitated again with alcohol, the peptone boiled and washed with alcohol, allowed to stand for some time with a large quantity of absolute alcohol, again treated with ether as before, and as it had now become friable, it was immediately dried in vacuo over sulphuric acid. On attempt- _ ing todry it at 100° C. the melting mass foamed so much that it had to be dried in the air. When this had been done with frequent stirring on the water bath for several days, the mass was finally dried com- pletely im vacuo, first at 100° C., then at 105° C. A few weeks of this drying were needed to bring the substance to a constant weight, and in order to prepare the various quantities for analysis they had to remain in vacuo over sulphuric acid and at 105° C. for some time. Probably in consequence of the thorough treatment with ether, the preparation when warmed gave much less odor than the former one, Composition of Antipeptone (D). I. 0°5061 gram substance gave 0°2875 gram H,O = 6°31 per cent. H and 0°7937 gram OO, = 42°76 per cent. C. II. 0:4449 gram substance gave 0°2527 gram H,O = 6°31 per cent. H and 0°7024 gram CO, = 43°05 per cent. C. III. 0°5010 gram substance gave 0'2870 gram H,O = 6°36 per cent. H and 0°7885 gram CO, = 42°91 per cent. C. IV. 0:4414 gram substance gave 56°0 c.c. N at 21°0° C. and 759°2 mm. pressure = 14°91 per cent. N. V. 0°5870 gram substance gave 76:5 c.c. N at 216° C. and 758'0 mm. pressure = 15°13 per cent. N. VI. 06930 gram substance gave 0°0694 gram ash = 10°01 per cent. VII. 05017 gram substance gave 00504 gram ash= 10:04 per cent. VIII. The ash from 05017 gram substance gave 0°0325 gram BaSO, =0'89 per cent. S calculated on the original substance. Kiithne and Chittenden— Peptones. 235 On account of the large percentage of ash and the large amount of sulphates, the sulphur of the organic matter was not determined. Percentage composition of the ash-free substance. Average. Cie ee 47°52 47°83 47°69 are seat 47°68 Neier stn 2 701 7-01 7-07 Ee aes 7:03 IN ee a Baer eee amare 16°57 16°80 16°68 Antipeptone (E). In order to obtain a still purer preparation and especially to free it from the large percentage of ash, a portion of antipeptone (D) was dissolved in boiling water after the first treatment with ether, then when cold the solution was acidified with sulphuric acid to such an extent that it contained 6 per cent. of acid and precipitated with a large excess of phosphotungstic acid. The precipitate, after the manner already described under amphopeptone (B), was washed thor- oughly with dilute sulphuric acid and then with water, finally de- composed with baryta, the barium-peptone precipitated with alcohol, washed, the alcohol driven off by heat, the aqueous solution of the compound exactly decomposed with sulphuric acid, the solution con- centrated after the addition of a few drops of ammonia, precipitated with alcohol, dissolved again in water, concentrated with the addi- tion of a little acetic acid, again precipitated with alcohol, and the product so obtained treated thoroughly with alcohol and ether in the same manner as preparation D, and finally dried in the same manner as that. The substance so prepared, was lighter colored than the preceding, not quite so hygroscopic, and in drying gave scarcely any odor. The analysis of the product is shown in the accompanying table. Antipeptone (F). (Gland peptone.) This peptone was obtained as a bye product in a preparation of trypsin from 1,000 grams of dry pancreas and was formed wholly from the albuminous bodies of the gland substance, after extraction with alcohol and ether. It is not probable that the peptone con- tained, in any considerable quantity, any products from the digestion of elastin, since the elastic tissue could have been but little altered under the conditions in which the self-digestion of the gland took place during the preparation of the infusion, and furthermore there would have ben needed for solution in the latter, a finer subdivision Kiihine and Chittenden— Peptones. 236 00-001 hike kG 49-0 86-81 69-9 69-97 ‘OBVIOAV 99-0 Go-81 18-81 86-81 19:9 CP-9F ‘aounjsqns aatf-ysn ayz fo uovwrsoduod abnyuasagd TL-9 LL: OP OMARO v yse oly WOIy S 00TO-0 maaden ta} “yse ol} woiz |g Suyonpop ‘osra c9.0 29-0 b ‘se Jo Joye g 92-0 bL-0 0080-0 0060-0 89:6 19-8 V-1G F-1G 8-66 “meBIs ‘punoyz rosea UsV TURLS *punoz ysV > N TU eans -Solg ‘Do i 69-96 0-061 89-69 ‘punoy N ‘(q) ANOLMUdI INV 8616-1 8EFC-0 0616-0 689-0 961-0 6LE9-0 006L-0 1166-0 Baten Rs} “panoy °00 Kiihne and Chittenden—Peptones. 237 and long continued action. However this may be, the albumins of the pancreas naturally cannot be classified with the substances ordi- narily used in digestion experiments, such for example as fibrin, without further investigation, for although there may be substances in the gland cells like serum-albumin, globulin and myosin, there are also many bodies quite different from these, as, for example, the leu- coid precipitated by excess of acetic acid, zymogen and trypsinogen, all of which are decomposed by self-digestion and yield amido acids and peptones. So long as trypsin digestions are not ordinarily con- ducted with pure trypsin, it is of especial interest to find out the composition of the gland peptones, which, as a rule, have invariably been mixed in greater or less quantity with the antipeptones hitherto investigated. Preparation.—1,000 grams of dry pancreas were warmed at 40° C, for twelve hours with five litres of 0:1 per cent. salicylic acid and 0:25 per cent. of thymol, filtered through muslin, the residue warmed another twelve hours with two litres of 0°25 per cent. sodium carbonate and 0°5 per cent. of thymol, again filtered and pressed, the two fluids united, brought up to an alkalinity of 0°25 per cent. of sodium carbonate and then warmed at 40° C. for three days. After filtering through paper, the whole solution was slightly acidi- fied with acetic acid and then saturated with five kilos. of ammo- nium sulphate, by which means there was precipitated a little albu- ' mose and all of the trypsin, the further treatment of which is of no interest here, while the gland peptone remained in solution. It is to be noticed in the separation of this peptone that it was treated exactly like preparation (C), excepting that the second purification with ether could be omitted. The preparation, after drying for some time, left a small residue of tyrosin when dissolved in cold water. It was therefore precipitated from this solution with alcohol, then freed from alcohol by boiling with water, dried directly over a water-bath and finally im vacuo at 106° C. until a constant weight was obtained. The analysis of the product is seen in the following table. Antipeptone (G). (Gland peptone.) This preparation was obtained from the preceding product by the following process; the solution of the peptone was acidified with 6 per cent. of sulphuric acid, precipitated with a large excess of phos- photungstic acid, the precipitate carefully washed, then decomposed with barium hydroxide, the latter exactly removed with dilute sul- Kiihne and Chittenden—Peptones. 238 00-00L 88-08 0-0 90-41 AT -k S147 ‘OSRIOAW 0g-0 60-LT CTL a a ‘aounjsqns aat{-ysn ayz fo worjzisodwuos abnzuaowag . 61 OVP ST-0 8610-0 62-0 09-0 9F-0 LP-0 w-oe- 0160-0 6860-0 1980-0 6f&0-0 GT-91 80-91 0-F7E2 |9-8T 0-694 IP-8T “TRIS “yse oy) WOT "OSta % ‘YS jog | % suyonpep | § Jaye g ‘TUB1S *punoy *OS*d Usy “ues *panoyz UsV N “UU ‘O.ins -s01q Do no ‘0 °O % “punoy NV 0&8¢-0 oC9l-T “TRIS “‘punoj FOO | “ON ‘(quojdadpun])) (4) ANOLIAdIINY Kithne and Chittenden—Peptones. 239 phuric acid, the solution concentrated, the peptone precipitated and washed with alcohol and finally boiled with alcohol. The partially dry product was then dissolved in cold water, leaving a small amor- phous residue which gave no reaction for tyrosin. The solution was then concentrated on the water-bath and again precipitated with alcohol, after which it was dried finally, over sulphuric acid in vacuo and at 106° C. in vacuo, until of constant weight. The following table shows the results of the analysis. Antipeptone (H). (Gland peptone.) This product was prepared and purified in exactly the same manner as the former one (G), but was made from another trypsin prepara- tion, in which a smaller amount of dry pancreas was used. When dissolved for the last time in cold water, some little insoluble matter remained (from which it was freed) which, however gave no reaction for tyrosin. : The following table shows the results of the analysis. GENERAL PROPERTIES OF THE PEPTONES. We should have liked to study more accurately the physical be- havior of the different samples of peptones, especially their optical properties as determined by specific rotary power. It was easy to show that they were all laevo-rotary, but we have not yet succeeded in making any quantitative determinations of sufficient accuracy to be of value. The decidedly brown color of the solutions prevented the use of a sufficiently long tube, or a solution of the proper concen- tration, necessary to determine specific rotation. It would be of still greater importance to investigate the rate of diffusion of peptones, it being more necessary from the fact, that pre- vious observations on the diffusion of the products of digestion can have but little reference to pure peptones, but rather to the albumose bodies so long overlooked. We have, however, not as yet begun these investigations owing to lack of material. All of the peptones obtained by us in the dry state, showed considerable rise of temperature when moistened with, or dissolved in water. It is worthy of notice, physiologically, that according to observations made in the physiological Institute at Heidelberg, by Dr. Pollitzer of New York, no one of the peptones would hasten or retard coagu- lation of the blood, either when injected into the veins or added to the shed blood, such action being due wholly to certain of the albumose bodies. Kiihne and Chittenden—Peptones. 240 60-0 00-001 49-TE T&-0 O8-4T 96-4 96-07 ‘OGBIOAY 08-0 os ae ag T6-AT. OLA “eS ah gst ian P+) 86-0F ‘2oUDISQns dauf-yso ayy fo WoYpsodUod abnpUdILaT OoDZARO a 18-0 OF-0 oa 66-0 86-0 4800-0 a ea e--- ---- e<-- 8LL0-0 6760-0 OA. GAN eT ORO a aes cae a el ae oe TGh a yCevOs0 dp aes) oes alae ele en) 782 | heiat | 8-60L; Pear] 9-69 | -7": ---- |) se-- | gent | @.89L OLE 876 | 77° arse) Soeeeerie eg aeng| sey eee ROTA "yse olf} wody iS) WUBI % “Yyse “yse jog % ayy Woy | Suyonpep g "Osta IOIFE S “TURAL “punoy FOsed UsV “TUL aanen fol 4 ‘9ans *punoy N -Solg see ‘punoy N Oa ar di Owe 8666-0 0092-0 “mes “punoy *00 -2-- THIP-0 G9TE-0 6909-0 9¢88-0 0898-1 GEL9-0 8689-0 9cLP-0 1889-0 CLP9-0 F16P-0 “mes “punoj O°H “mRs “posn gourys “Qug ‘(auojdadpun})) (9) UNOLAAdIIN VY 241 00.001 48:66 nay ma, ero ee O 49-0 LG-0 lees og eS 16-40 isa ¥6-L1 a = N GT+h coe ps TTk 06-2 H % ADT eae eye 9¢-FP 68-FF 90 ~ IBVIOA Y s Q ‘aoupysqns aatf-ysn ay, fo uorprsoduos abnyuaolad < Eee s er zoe | as i ee cis le ee, [RE | ete ee wate ia ar ae s&s 9¢-0 99-0 | 0F60-0 ‘ 6967-0 |A Ss = OT-0 | 8800-0 ae aa" noe Seas =o Fe Wie eel pee | ae | pe = me eS Ee Oe er Ss ios mn rey a oe ser se Oe rieGOLOnO: le caer eas” ee eee |e Se es =r oie (PAO SAE ta is. ww pa aioe Si Fa re “""~ | 8G-LT | 8-094 |9-8T 87-88 | ~~ ~ =1Es. | OFGE-O Tit S 8 ms or — eG) on ae Eat ge aoe vroo fosees foes") =""" | 59.8% | 0888-0 | 96-9 | She8-0 | PATS-0 |IT a Lee S ae = ee 7 perenne ee aeons sors jocere fomse} mao" | 7F.8F | SPS9-0 | 80-4 | 2096-0 | LOTF-0 |T x | ums | 4 a ee oe | a ee awa | el ‘yse oj] se =| cysegjog | % | oot Bh Re ge fees Be gent See le catia |e ten eet | a eer Oa MOIf | OY} WoIZ | Suonpop g yee ‘US V aie N seid ) ak H pe) e0uR4s oN . 8 rosea 1ojJV § . “‘puno} N iii) ‘(auojdadpun2,p) (H) INOLIAdIINY Noy., 1886, 31 Trans. Conn. Acap., Vou. VII. 249 Kihne and Chittenden—Peptones. A few observations on the taste of peptones are of interest. While the genuine albumins and the albumose bodies excite practically no sense of taste, the less so the purer they are, it appears as if peptones belong to the most offensively tasting bodies. In order to see what an important change the taste of an albuminous body undergoes on digestion, and at the same time what the taste of peptones is, warm 50 ¢.c. of fresh milk to 40° C. and then add to it a small fragment of soluble trypsin (prepared from ox pancreas), which excites no taste of itself. The milk at first coagulates, tnen regains its former appearance by solution of the coagulum, but tastes no better than gall. Never- theless we believe that the especially objectionable taste proceeds not from peptones, but from certain compounds heretofore only acci- dentally separated from them. For, among our preparations, which as a whole tasted something like roast meat, as if burnt, but above all nauseatingly bitter and astringent even in a 2 per cent. solution, we found one that in a 10 per cent. solution was free from this dis- agreeable peculiarity and had a pleasant, sweet taste like meat. It is to be noticed that this was the preparation of antipeptone (F) which had not been purified by phosphotungstic acid. Only by taking a large swallow was there noticed a not unpleasant taste, peculiar to certain cheeses after becoming alkaline, The most important property for the separation and treatment of peptones is for the present, the lack of precipitation by a great number of reagents, which precipitate more or less perfectly albumins and albumose bodies, especially sodium chloride, either alone, or with the’addition of acid. We have repeatedly confirmed the observations made by Wenz that even sodium chloride and acetic acid, sodium chloride and nitric acid or metaphosphoric acid do not completely precipitate the albumose bodies. In whatever proportion these addi- tions may be made, there always remains at the end a solution which gives with alcohol a precipitate of salt, in which albumose is still to be found, or from which albumose may be separated by dialysing and concentrating, according to the method already used. The only per- fect precipitant of these substances is ammonium sulphate. It is how- ever an error to attribute to this salt the same action on peptones. Wherever peptones occur, they will always be found in the filtrate from a solution saturated with ammonium sulphate, and we must conclude from some opposed statements that in the experiments on which they are based, albumose bodies instead of peptones were pres- ent, since we are certain that by means of our new method, it can be generally shown what an unexpected difference exists between the ap- Kiihne and Chittenden—Peptones. 243 parently vigorous action of a poorly prepared gastric juice, or com- mercial preparation of pepsin and the action of solutions actually rich in pepsin. Even where the fibrin almost instantly disappears, the amount of ferment may still be quite insufficient to produce noticeable traces of peptones. Therefore care must be taken not to conclude immediately from a speedy solution, that digestion has been com- plete, since this is to be determined only by the disappearance of the primary cleavage products of digestion, that is, the change of albumose into peptones. It was especially interesting to ascertain whether peptones iso- lated according to the methods already described, were likewise pre- cipitated by ammonium sulphate or by other reagents that precipi- tate the albumose bodies, a question which was interesting considering the oft-asserted formation of albumin or albumose from peptones. To our surprise we noticed in the beginning that both antipep- tone and amphopeptone after complete purification, under certain circumstances gave rise to a turbidity or even a resinous precipitate, not only with ammonium sulphate, but also when their solutions were saturated with salt or when treated with acetic acid, nitric acid or metaphosphoric acid, just as if albumose had been formed or the albumose not completely removed by the previous treatment. Even if these precipitates concerned only a small part of the material in solution, their appearance would need explanation. So far as we can now determine, the occasion of this behavior is a circumstance con- cerning which we do not care to decide whether it really depends on the formation of albumose from peptone or not. It is to be ob- served that if the purification of the peptone by sulphuric acid is conducted incautiously, either on decomposing the barium-peptone or on acidifying before precipitation with phosphotungstic acid, the appearance afterward of albumose is avoided provided the solutions, when warm, are never exposed to an excess of acid. That a resinous precipitate appears, while boiling the solution saturated with ammo- nium sulphate at 110° C. has already been mentioned, but this can- not cause any impurity of the peptone remaining in solution, any more than the well known precipitation of antialbumid during trypsin digestion can occasion an impurity of the antipeptone. Since the fact is proved that peptones are not precipitated by am- monium sulphate, these bodies are then characterized more than ever by the property long attributed to them of being rendered turbid by very few reagents and completely precipitated by a still more lim- ited number. A list of the latter reagents includes only tannin 244 Kiihne and Chittenden— Peptones. and mercuric iodide in potassium iodide, while imperfect precipitants of peptones are phosphotungstic acid or phosphomolybdic acid and picric acid. The following list shows the further action of various reagents. Reactions of Peptones free from albumose and purified by phos- photungstic acid. In 5 per cent. solution, after being made noticeably alkaline with a trace of sodium carbonate. Fibrin antipeptone. Fibrin amphopeptone. Acetic acid and potassium|At first perfectly clear, later trace/The same. ferrocyanide. of opalescence. Neutral lead acetate. First drop, 0; more, turbidity. The same, but much weaker. Basic lead acetate. Turbidity | immediately “more, strong turbidity. The same, but weaker. Mercurie chloride. First drop, 0; more, strong tur- bidity. Turbidity immediately, growing stronger. 5 per cent. cupric sulphate. | At first clear ; more, slight turbid- Nothing. ity disappearing with great ex- cess. | 5 per cent. platinum chloride./Only excess, strong turbidity. IN othing. Chromic acid. Nothing. Nothing. Ferric chloride. A trace gives turbidity vanishing) Nothing. with the least excess. Glacial acetic acid and conc.|Brownish red. |The same. sulphuric acid. Nitric acid. The color changing yellow in the The same. cold. Boiling with cone. hydro- The color becomes slightly darker./The same. chloric acid. Millon’s reaction. At first a heavy white precipitate ; on warming, dirty yellow or reddish. The same, then beauti- ful red color. Without desiring to claim especial value in general for these reac- tions and for smaller differences between the two peptones, some of them, however, may be more closely examined. The slight intensification of color by boiling with concentrated hydrochloric acid is striking, for we have not ordinarily been able to obtain it, even with peptone in substance or even on the addition of Kihne and Chittenden—Peptones. 245 concentrated sulphuric acid. Likewise, the reaction with sulphuric acid and glacial acetic must be described as almost unsuccessful. As this, however, is nothing other than Pettenkofer’s test for bile acids, for which its discoverer has recommended concentrated acetic acid as a substitute for sugar, we have also tried the reaction with sugar, without however obtaining any better result, particularly not the beautiful violet-red which albumin and the albumose bodies give. Finally, the poor result of Millon’s reaction with antipeptone, in con- trast to the brilliant red obtained with amphopeptone, is also to be remarked. To this reaction we shall return later. It has already been observed by many investigators that among the products of the digestion of albumin, bodies are not infrequently met with, which give little or no lead sulphide on boiling with sodium hydroxide and lead acetate. This is not at all strange, since the pep- tones prepared by us show on analysis less than one per cent. of sul- phur, in striking contrast to the albumins and albumose bodies, all of which contain much larger percentages. In the gland peptones (F, G, H,) the sulphur amounted to only 0°50, 0°31 and 0°57 per cent. respectively. The peculiarity of peptones in giving up a part of their sulphur when warmed with alkali, apparently stands in no direct connection with the percentage amount of sulphur. Indeed, solutions of the antipeptones G and H, of which the first possessed the lowest percentage of sulphur found, showed no browning with this test, and only a trace of it when solid particles of the peptone were heated with a concentrated solution of alkali containing lead. But the gland peptone (F) not purified by phosphotungstic acid and with only 0°15 per cent. of sulphur became slightly darkened in solution. On the other hand, the purest amphopeptone (B) with 0°77 per cent. of sul- phur gave the reaction very faintly, while antipeptone C with 0°73 per cent. of sulphur gave it very plainly. Probably the reaction is not to be attributed to the peptones themselves, but proceeds from contamination with an easily decomposed substance containing sul- phur, whose removal still depends on chance. It may be assumed as completely proved that the rose or violet coloration, which the products of pancreatic digestion assume with bromine or chlorine water, is due to some special body and not to antipeptone. We had previously shown this to be the case with the antipeptone obtained by the action of trypsin from antialbumid, and have now also found in all antipeptones purified with phospho- tungstic acid, the absence of all color on addition of bromine water, either in large or small quantity. 246 Kiihne and Chittenden—Peptones. Finally, not to overlook the most striking reaction of peptones, we mention further the brilliant so-called biuret reaction which is to be seen, if possible, still more intense in the purer peptones, what- ever their origin, than in those formerly used. Composition of the Peptones. For the sake of comparison we present in the following table the - percentage composition of the various samples of peptones, calculated on the ash-free substance. The ash found, consisting in every case of calcium, a little sodium, potassium, traces of barium and iron, car- bonic acid, phosphoric acid and sulphuric acid, is placed at the foot of the columns. Ampho- (pepsin) peptone from || Anti- (trypsin) peptone, A. B. | b. C. D. E. HS G. H. Prepared from fibrin. Glandpeptone. Certain a ae pepsin|| — 7 = 3 Ee = : an | 3 ‘ing mucin- SRAM niaGa: ae Purified al brane Purified with eptone. i more wi B- osphotungstie me avid: ether. | Photungs- y Pacid. = tic acid. | | 47°30} 47°68 46:59 | 4445] 42°96 44-47 44:53 48°75 48°47 649 721 7:02 6:69 ot 7:26 7-15 2 I ow —T i=) ioe) C H N | 16°73 16°26 16°86 | 1683. 16°68 18°28 | 17°06 | 17°80 17°94 S | Ou2 | Ost By | (ei eee 0°67 | 050| 031 | O57 ©) | 31:53 HO) res We 28°41 | Pa Deel } Ash iil yuS 224) W216 M6254 10°02 3°67 | 5:54] 193 2°07 The nature of the substance, which at present affords but little proof that we have to deal with a simple body and not a mix- ture of chemical bodies, places the greatest restrictions on the use of the above values, and only under such reserve is to be understood what is hereafter added. As regards the analysis of the single sub- stances, we are inclined to believe that only in amphopeptone B, is there a difference in the percentage of carbon greater than the ordi- nary differences naturally to be expected in amorphous materials so difficult to prepare. Amphopeptone A, with the lowest percentage of carbon, has already been described as a mixture of fibrin-peptone and mucin- peptone. If we have succeeded in removing the latter, by using Kiihne and Chittenden—Peptones. 247 purified pepsin, then the figures obtained from B and } may be con- sidered as representing the first gastric peptone prepared free from albumose. The correspondence between the two, in spite of the fact that by continued purification of b we succeeded in reducing the ash by more than 1 per cent., we think may be considered as grounds for this assumption. In opposition therefore to the majority of pre- vious statements, including our own, which as now easily understood referred to mixtures of albumose and peptones, there is to be noticed in pure amphopeptone about 1 per cent. lower content of carbon, about as much higher a percentage of nitrogen and 0-3-0-4 per cent. lower content of sulphur. With antipeptone, the variation from the previous results was less expected, for we are not inclined to believe that in our former long and thorough typsin digestions, any appreciable quantity of albumose remained and in the more recent ones, the precipitate pro- duced by ammonium sulphate was never abundant if the matter ‘separated by boiling in a slightly acid solution was previously rémoved. The differences found, however, might be readily explained by the fact that the purification of the peptone had this time been more complete, owing partly doubtless to the formation of the barium compound, and partly also to the precipitation with phospho- tungstic acid. The content of carbon is seen to be about | per cent. lower, the content of sulphur likewise lower and the percentage of nitrogen decidedly higher, in one case as much as 4 per cent. more than before. The real reason for this difference in composition appears to us to lie in the use of much larger quantities of trypsin, which formerly was only possible by using large quantities of the gland substance, so that the fibrin-antipeptone would naturally be obtained mixed with the gland peptone. It is well for the future that we know the composition of these gland peptones, for they differ essentially, in the lower content of carbon (in one case 42:96 per cent.), from all other peptones hitherto investigated. These bodies might be pronounced troublesome intruders with the same right as the mucin-peptone arising from gastric digestion, although we found in it, aside from the percentage composition, no reaction and no property which would serve to distinguish it from the other peptones. After these considerations, little stress can be laid on the differences between the composition of pepsin and trypsin peptones. We have found, however, another difference which we will examine more closely. 248 Kiihne and Chittenden— Peptones. Cleavage of the peptones. As already mentioned, Millon’s reaction appears very brilliant with amphopeptone, but more or less imperfectly with antipeptone. As the reaction is sure only under certain circumstances we have not neglected to perform it in every possible way, either by using the same concentration of solutions of the two peptones under exactly the same conditions, or by trying the most successful variations of the experiment for antipeptone. Thus it was found that the preparations C, D, E,and F, treated in a suitable way, gave an appreciable reaction, but in no case so that more than a dirty, generally orange red pre- cipitate was obtained. On the contrary, all trials with gland pep- tone purified with phosphotungstic acid, failed to give more than a simple yellow color. Since Millon’s reaction for albumin corresponds with the so-called Hoffmann’s test for tyrosin, and since with albu- min it probably depends on the separation of tyrosin by boiling with the acid solution of murcuric nitrate, if not by the formation of _ further decomposition products of tyrosin (hydroparacumarice acids) which likewise redden with the test, it might be presumed that anti- peptone, in contrast to the amphopeptone of pepsin digestion, forms no tyrosin by cleavage. So far as the action of trypsin is concerned, this was already known, since the real difference between gastric and pancreatic peptones consists in the fact that only the former, when treated with trypsin, yield tyrosin together with leucin and other decomposition products—in our opinion because they contain hemi- peptone capable of further cleavage (together with antipeptone). It was also known to us, however, that antipeptone during cleavage with boiling sulphuric acid yields the amido acids, and among them also tyrosin. Renewed investigations on this subject appeared called for now, since we thought ourselves in possession of much purer pre- parations of antipeptone. First, we established the possibility of decomposing with trypsin, amphopeptone entirely free from albumose. A few hours’ digestion in a small test tube, of 1 gram of peptone in 10 ¢. ¢. of water, contain- ing 0°25 per cent. of sodium carbonate with a little thymol and a fragment of purified pepsin, sufficed for this purpose. By concen- trating the neutralized solution and boiling the residue with alcohol, a decided residue was obtained in which balls of leucin and bundles of tyrosin were to be seen under the microscope without further preparation. The residue was also colored a beautiful violet with bromine water. We also sacrificed a large quantity of ampho- peptone to the same experiment and obtained the tyrosin pure (free a Kiihne and Chittenden— Peptones. 249 from peptone) and tested it, both by Millon’s reaction and Piria’s test. Further, a few grams of the same preparation were heated for several days with six times its weight of sulphuric acid (2 : 3 water), and after removing the acid with barium hydroxide, tyrosin and leucin were found among the decomposition products. Antipeptone was now likewise submitted to the above treatment. The method of conducting this experiment with albuminous bodies which we have used successfully, even with 2-3 grams of substance, is as follows. The substance is placed in a small, strong flask stand- ing on an asbestos plate, and five or six times the weight of sulphuric acid of the above mentioned strength is added and the mixture kept boiling as long as desired. The loss of water by evaporation is pre- vented by connecting to the neck of the flask a glass tube 1 metre long and 1 centimetre in diameter, so that the lower end cannot be closed by a drop of water. The upper end of the tube is drawn out to a capillary point and as it does not become warm at the top dur- ing the boiling of the fluid, all loss of water is prevented. To be as sure as possible, we have treated the antipeptone in this . manner for 48 hours. The contents of the flask were then much diluted with water, filtered from the sticky residue always present, made alkaline with a concentrated solution of barium hydroxide, the barium separated from the filtrate by sulphuric acid, after which the clear fluid was concentrated and allowed to crystallize. It is not advisable to remove the sulphuric acid with barium carbonate, since the latter is unavoidably used in excess, for it is very undesirable to have the barium precipitate unnecessarily increased in bulk, as the cleay- age products of albumin are difficult to remove even with hot water. Further, the mixure is made alkaline by barium carbonate and soluble barium compounds always appear, which must be removed by sulphuric acid in every case. In the strongly concentrated solution finally obtained, in case leucin and tyrosin have really been formed, as with most albuminous bodies, their presence may be readily shown microscopically and after suitable separation of the mother liquor, or if necessary, after re-crystallization from dilute alcohol, their chemical reactions may likewise be obtained. We have succeeded after this manner in showing the presence of leucin always in antipeptone ; tyrosin, however, only in a few cases, and even then only after repeated crystallization. When- ever tyrosin occurred it was in exceedingly small quantities. From the antipeptones (gland peptones H and G) with which Millon’s test had hitherto failed, tyrosin could not be obtained at all, and that TRANS. Conn. AcAD., Vou. VII. 32 Nov., 1886. 250 Kiihne and Chittenden—Peptones, tyrosin was really absent in this case was shown by the final exam- ination of the whole united residues by Hoffman’s test, which was | absolutely negative, while it appeared plainly with the products of the remaining antipeptones. Although we do not wish to consider the behavior of the gland peptones as a criterion for antipeptone in general, still this result, united with the small amount of tyrosin obtained from the latter, seemed to call for further investigations concerning the decomposi- tion of those primary cleavage products of albumin related to the antipeptones, especially antialbumid. We began this extension of our work because during our treatment of the subject an article of Maly’s* appeared, in which he described a very interesting cleavage and oxidation product, obtained by treating albumin with potassium permanganate, which product possesses the essential properties of the albumins, and yet on further decomposition does not yield tyrosin. It is questionable, therefore, whether this property is not the one directly distinguishing the bodies of the anti- group from the pri- mary cleavage products of the albumins. A few preliminary experiments were made with samples of anti- albumid prepared by the action of boiling dilute sulphuric acid, both on fibrin and Thiry’s neutralization precipitate from egg-albumin, also with the antialbumid remaining from the digestion of fibrin with trypsin, and finally with a small neutralization precipitate of so-called parapeptone from an incomplete pepsin digestion of fibrin, which we regarded as antialbumose. After these experiments as a whole, had resulted contrary to our expectations, in that a moderate amount of tyrosin appeared after boiling the substance for a long time with sulphuric acid, we submitted to decomposition a prepara- tion from which we thought we could expect a decisive result. This preparation was an antialbumid from egg albumin, made in one of our former investigations as follows: White of egg freed from mem- brane, was coagulated by heat in an acid solution, the coagulum fil- tered, thoroughly washed and then heated for a long time at 100° C, with dilute sulphuric acid, the residue filtered, washed thoroughly with water, dissolved in sodium carbonate, precipitated by neutraliz- ation, the precipitate dissolved in 0-2 per cent. hydrochloric acid and the antialbumid freed from all other other albuminous bodies by long continued digestion with pepsin. The antialbumid was then separated {rom the solution by neutralization, washed, dissolved in 0:5 per * Wiener Acad. Sitzungsber., xci, Abth. 5, February, 1885. Se Kiihne and Chittenden— Peptones. 251 cent. sodium carbonate, warmed with trypsin at 40° C., and after the antialbumid had partially separated as a jelly-like mass, it was filtered and washed with water, finally with alcohol and ether. This product could not possibly contain any known albumin, albumose or peptone and undoubtedly formed the purest sample of antialbumid yet prepared. By decomposing this body with sulphuric acid, a residue was finally obtained which to our surprise gave but the slightest reaction for tyrosin with Millon’s and Hoffmann’s test, with Piria’s test no reaction whatever, and in spite of endeavors continued for weeks not a single crystal of tyrosin could be detected. Leucin was found in very small quantity and in addition there were seen large lustrous balls of crystals of some nitrogenous substance, too small in quantity to be identified. Heidelberg, ) G 5 New Haven, § December, 1885. XVI.—ON THE DEHYDRATION OF GLUCOSE IN THE STOMACH AND IntTesTINEsS. By R. H. CuHirrenpen. In a series of interesting communications* “ On the physiology of the carbohydrates in the animal system,” Dr. F. W. Pavy has brought forward evidence to show that glucose, generally considered as the final product of amylolytic action, can be converted within the animal body into a product of less cupric oxide-reducing power ; that there exists particularly in the stomach and intestines of rabbits, a ferment which has a dehydrating action upon glucose or dextrose, transforming it into a body akin to maltose in reducing power. ‘Hitherto, it has been generally supposed that the transformations which carbohydrates undergo in the animal system are in the nature of gradual hydration changes, in which each step forward toward the final product is attended with the formation of bodies of in- creased cupric oxide-reducing power. Dr. Pavy’s results, however, would tend to show that transformations in the opposite direction do occur and this notably in the stomach and intestines of rabbits. Dr. Pavy’s conclusions concerning this dehydration of glucose in the animal system, are based upon changes in the cupric oxide-reduc- ing power of the carbohydrate, after contact with portions of the stomach and intestines for short periods of time at 48°8° C. Itisa well known fact that the reducing power of pure glucose is not affected by boiling with dilute sulphuric acid, while under like treat- ment, maltose and similar bodies are readily converted into glucose or into a body of like cupric oxide-reducing power. Dr. Pavy finds, as the result of a large number of experiments, that a solution of glucose or grape sugar, by mere contact with the stomach and intestines of a rabbit at 48°8° C. is changed into a body of less cupric-oxide reducing power, and that by boiling with dilute sulphuric acid this product is carried back again into glucose. Thus, in one experiment, 0°138 gram of glucose in contact with strips of stomach from a rabbit for one hour and a half at 48°8° C. showed, after removal of the dissolved albumin by boiling with sodium sul- phate, a reducing power calculated to the entire amount equivalent to only 0:080 gram of glucose; while after boiling with dilute sul- * Chemical News, 1884, vol. xlix, pages 128, 140, 155, 162, 172 and 183. a R. H. Chittenden—Dehydration of Glucose. 958 phuric acid (the solution containing two per cent. H,SO,) the cupric _oxide-reducing power was increased to the equivalent of 0°134 gram of glucose, or nearly equal to the amount started with. It is to be noticed in the experiment just quoted, that the cupric oxide-reducing power, before and after treatment with sulphuric acid, stand to each other in the proportion of 58 : 100, or in about the rela- tion of maltose (61) to glucose (100). In some experiments, how- ever, the reducing power before boiling with dilute acid, was so low as to warrant the belief that dextrins were also formed. This result is a type of many similar ones obtained by Pavy with the stomach and intestines from various animals and in no instance, in the case of rabbits at least, so far as reported, were negative results obtained. The discovery of such a dehydrating ferment, hitherto unsus- pected, appeared to be a matter of so much importance that experi- ments have been tried in this laboratory from time to time during the past two years, with the view of confirming in part at least some of Dr. Pavy’s. results. To our ‘surprise, however, in no case, have we been able to obtain results corresponding to those of Pavy’s, although the animals experimented with (rabbits and cats) were taken in various stages of digestion. We therefore record here, some of the results simply in the hope that some light may be thrown upon the cause of this discrepancy ; or if, as may be, the ferment is not invariably present, some reason may be found for its constant absence in the tissues of the animals experimented with, and thus light be thrown upon the conditions which control its presence. The glucose used in the following experiments was a sample of crystallized anhydrous glucose presented to the laboratory by Dr. Arno Behr. The sugar was quite pure, as was ascertained by testing both its reducing power and specific rotary power, and more import- ant still, was not at all affected by boiling with dilute sulphuric acid. Thus 50 c¢.¢. of a one per cent. solution of the glucose, mixed with sufficient 10 per cent. sulphuric acid to have the mixture contain two per cent. of H,SO,, was boiled for one and one-half hours, the flask being connected with an inverted Liebig’s condenser to prevent con- centration. The solution was then neutralized, diluted to 100 «. ¢. and tested with Fehling’s solution according to the method of Allihn. 25 c.c. yielded 0°2414 gram of metallic copper, corresponding to 0°1246 gram of glucose, whereas the 25 ¢. c. of solution should have contained 0°1250 gram of sugar. Evidently then, the reducing power of the sugar is not affected by treatment with dilute acid. First experiment.—A rabbit in full digestion was killed, the stom- 954 R. H. Chittenden—Dehydration of Glucose. ach emptied of its contents and then divided into two longitudinal halves along the curvatures. One-half, after being cleansed, was finely divided and placed in a small beaker with 70 c. ec. of water con- taining 0°200 gram of glucose. An equivalent amount of the small intestine, similarly cleaned and divided, was placed in a second beaker in contact with 70 c¢.¢. of water containing 0°150 gram of glucose. In both cases the entire walls, including muscularis and mucosa, were taken, since Pavy has indicated that the converting principle is situated not on the surface of the mucous membrane, but in the deeper part. The two mixtures were then placed in a bath and warmed at 48°8° C. for nearly two hours; after which they were boiled, crystals of sodium sulphate being added to aid the removal of the dissolved albumin. The individual filtrates and washings were concentrated and finally brought to a volume of 100 ¢.c¢. Of this, 25 c. ¢. were used to determine the cupric oxide-reducing power of the solution directly, while 50 ¢.¢. of each solution were mixed with sufficient ten per cent. sulphuric acid to insure a content of two per cent. and then boiled for two hours, in connection with an inverted Liebig’s condenser to prevent concentration. The acid solutions were then neutralized, concentrated somewhat and finally brought back to a volume of 50 c.c. Following are the analytical results obtained with the two solutions, the reducing power being determined by Allihn’s gravimetric method.* STOMACH. a. Before treatment with sulphuric acid. 25 c. c. gave 0°0746 gram Cu=0°0381 gram dextrose x 4=0°1524 gram dextrose. b. After treatment with sulphuric acid. 25 ¢c. c. gave 0:0728 gram Cu=0°0372 gram dextrose x 4=0°1488 gram dextrose. INTESTINE. a. Before treatment with sulphuric acid. 25 ¢. e. gave 0:0512 gram Cu==0-0265 gram dextrose x 4=0°1060 gram dextrose. b. After treatment with sulphuric acid. 25 c. ce. gave 00520 gram Cu=0-0269 gram dextrose x 4=0°1076 gram dextrose. Here, there is no evidence whatever that the glucose was affected by its two hours’ contact with the stomach and intestine of the rabbit at 48°8° C., the temperature specified by Pavy as that best adapted for the reaction. Certainly the reducing power of the glucose solu- tion is essentially the same before and after treatment with sulphuric * Zeitschrift fiir Analytische Chemie, 22. Jahrgang, p. 448. hel ‘ —-- KR. H. Chittenden—Dehydration of Glucose. 255 acid. Somewhat in accord with Pavy’s results, however, is the fact that while 200 milligrams of glucose were introduced into the stom- ach mixture and 150 milligrams with the intestines, only 152°4 milli- grams were recovered from the former and 106 milligrams from the latter, although the residues after heating with sodium sulphate, were repeatedly and thoroughly washed with hot water. Assuming that this loss of sugar in the two cases is really due to change of glucose into lower reducing bodies, the relative reducing power of the sugar before and after contact with the stomach and intestines would be 100: 76°2 and 100: 70°6 respectively. But if there had been any such change in reducing power, the treatment with sulphuric acid would certainly have indicated it. Second experiment.—A rabbit in full digestion was killed and half of the stomach and a portion of the small intestine were cleaned and finely divided. The stomach tissue was then heated at 48°8° C. for one and one-half hours, with 70 ¢. c. of water containing 0°200 gram of glucose and the portion of intestine for the same length of time, with a like amount of glucose. Treated then in the same manner as the preceding solutions, the following results were obtained : STOMACH. a. Before treatment with sulphuric acid. 25 c. c. gave 0:0901 gram Cu =0:0460 gram dextrose x 4=0°1840 gram dextrose. b. After treatment with sulphuric acid.» 25 c. c. gave 0°0882 gram Cu =0°0450 gram dextrose x 4=0°1800 gram dextrose. INTESTINE. a. Before treatment with sulphuric acid. 25 c.e. gave 0°0690 gram Cu =0°0353 gram dextrose x 4=0°1412 gram dextrose. b. After treatment with sulphuric acid. 25 ¢.c. gave 0°0726 gram Cu =0°0371 gram dextrose x 4=0'1484 gram dextrose. In this experiment, a larger amount of sugar was recovered in the case of the stomach tissue than in the preceding experiment, but in neither the stomach or intestine is there any evidence of change in the reducing power of the sugar before and after treatment with sul- phuric acid. In this conneetion it is to be remembered, that the only — ground for belief in the existence of a dehydrating ferment in the stomach is the change, noticed by Pavy, in the reducing power of the sugar under the above method of treatment. Our method of treatment with sulphuric acid, moreover, both as to the length of time the mixtures were heated and the strength of acid employed, was in accord with the method used by Pavy. In addition, the same 256 RR. H. Chittenden—Dehydration of Glucose. method of treatment was applied to a known solution of maltose with satisfactory results, viz: a rapid and complete change into dextrose as attested by the proper proportional increase in reducing power. Furthermore, we are led to infer from Pavy’s results that the action of the ferment is to be seen to the best advantage in the rabbit. Thus Pavy states,* that without having made any precise compara- tive observations, ‘I am under the impression that the stomach and the intestine of the rabbit act more energetically than the stomach and intestine of the other animals I have tried. It also appears to me that the stomach acts more energetically than the intestine, and in some instances I have noticed that the effect produced, has stood in relation to the amount of ferment material used.” The latter half of this statement would tend to indicate that the main reason for our not recovering all of the glucose is to be found either in a lack of sufficient washing of the tissue residue at the end of the experiment, or else in a slight fermentation by which a portion of the sugar might be decomposed ; for as is to be noticed in nearly all of the ex- periments recorded here, far less sugar is lost in the stomach than in the intestine, whereas if due to change in reducing power from the action of a dehydrating ferment, the greatest loss, Pavy’s statement being correct, would be observed by contact with the stomach tissue. On the contrary, our results show greatest loss in the intestine, which if due to mechanical reasons would be naturally explained, since the glairy mass of tissue, even after boiling, affords mechanical obstacles to a thorough extraction. That this is doubtless the true explana- tion, in part at least, is evidenced by the fact that a portion of the stomach or intestine, previously boiled with water to destroy its vitality, yields results after the same order as those already given, except that the amount of sugar recovered is greater, as would nat- urally be expected since the tissue being already coagulated would not enclose the sugar so completely. Thus, on warming one-half of a rabbits’ stomach, previously divided and boiled with water, with 0°200 gram of glucose for two hours, there was recovered 0°1910 gram of the glucose; while from a portion of the small intestine, likewise ~ boiled and treated with the same amount of glucose, there was recoy- ered only 0°1840 gram of the sugar. Furthermore, fermentation of the sugar would naturally occur more quickly in the intestines than in the more compact stomach tissue. Be that as it may, the reduc- ing power of neither solution was affected by boiling with dilute sul- phuric acid. * Chemical News, vol. xlix, p. 141. R. H. Chittenden—Dehydration of Glucose. 257 Third experiment.—A rabbit in a condition of hunger was killed, the stomach divided longitudinally along the curvatures, and one-half after being cleaned and finely divided, was placed in contact with 0200 gram of glucose dissolved in 70 c. c. of water, and warmed at 48°8° C. for one and one-half hours. The mixture was then heated to boiling with the addition of some crystals of sodium sulphate, the tissue and coagulated albumin filtered off and the residue washed with about 300 c.c. of hot water. The fluid was concentrated, brought to a volume of 100 ¢c.c. and then treated as in the preced- ing experiments. Following are the results obtained : STOMACH. a. Before treatment with sulphuric acid. 25 c. c. gave 0°0834 gram Cu =0°0425 gram dextrose x 4=0'1700 gram dextrose. b. After treatment with sulphuric acid. 25 c. ec. gave 0°0798 gram Cu =0°0407 gram dextrose x 4==0°1628 cram dextrose. Here, as before, there is no evidence of any change in the charac- ter of the glucose; still in spite of the comparatively large volume of wash-fluid used, the sugar was not wholly recovered. Fourth experiment.—A cat killed in full digestion was employed in this experiment. One-half of the stomach, finely divided, was plac 1 in contact with 0°200 gram of glucose in 75 c.c. of water. A portion of. the small intestine was also treated with a like amount of sugar, in the same manner. Both were warmed for three hours at 48'8° C., then treated by the same method as used in the preceding experiments. STOMACH. a. Before treatment with sulphuric acid. 25 ¢. ce, gave 0°0879 gram Cu =0°0449 gram dextrose x 4=0'1796 gram dextrose. b. After treatment with sulphuric acid. 25 c. c. gave 0°0886 gram Cu = 0°0452 gram dextrose x 4==0°1808 gram dextrose. INTESTINE. a. Before treatment with sulphuric acid. 25 ¢. c. gave 0°0668 gram Cu =0°0342 gram dextrose x 4=0'1368 gram dextrose, b. After treatment with sulphuric acid. 25 c.c. gave 0°0684 gram Cu =0°0350 gram dextrose x 4=0'1400 gram dextrose. Here again, there is no evidence whatever of any change in the reducing power of the sugar solution. Pavy has also pointed out that the stomach and intestine of the rabbit, as well as of other animals, have a transformative action on saccharose as well as on dextrose. The transformative energy how- Trans. Conn. Acap., Vou. VII. 33 Nov., 1886. 258 KR. H. Chittenden—Dehydration of Glucose. ever of the intestine, is much greater in this case than that of the stomach; which, according to Pavy, accounts for the early discovery by Bernard of the well known action of the intestine on cane sugar. Bernard supposed dextrose to be formed, but Pavy shows that maltose or a body resembling maltose in reducing properties, is-the usual product and that glucose or dextrose is formed only in the presence of considerable ferment. The ferment which produces this change, unlike the ferment which acts upon glucose, is situated on the surface of the mucous membrane and thus frequently the contents of the stomach are likewise found to possess transformative power. Fifth experiment.—A rabbit with stomach partially filled with food was killed, the stomach rinsed with water, then minutely divided and separated into two equal parts. One portion was placed in con- tact with 0°195 gram of glucose in 75 ¢. c. of water, while the other portion was mixed with a like amount of pure saccharose, also in 75 c.c. of water. A portion of the small intestine was likewise finely divided and one portion placed in contact with 0°195 gram of glucose in 75 c. c of water and the other portion with 0:200 gram of saccha- rose dissolved in 75 ¢. c. of water. All four mixtures were warmed at 48°8° C. for two hours, then heated to boiling with the addition of sodium sulphate and finally each brought to a volume of 100 c.¢, 25 c.c. of the saccharose solution, which had been in contact with the stomach tissue, gave no reduction whatever with Fehling’s solu- tion. 25 ¢.¢. of the saccharose-intestine solution, however, gave 0:0726 gram Cu, equivalent to 00371 gram dextrose. With the glucose solutions, the following results were obtained : STOMACH. a. Before treatment with sulphuric acid. 25 c,c. gave 0°0906 gram Cu =0°0462 gram dextrose x 4=0°1848 gram dextrose. b. After treatment with sulphuric acid. 25 c.c. gave 00889 gram Cu =0°0454 gram dextrose x 4==0°1816 gram dextrose. INTESTINE. a. Before treatment with sulphuric acid. 25 c.c. gave 0°0712 gram Cu =0°0364 gram dextrose x 4==0°1456 gram dextrose. b. After treatment with sulphuric acid. 25 c.c, gave 0°0689 gram Cu =0°0353 gram dextrose x 4==0°1412 gram dextrose. With glucose, the same results are to be observed here as in the preceding experiments; the only variations in reducing power, before and after treatment with sulphuric acid, being such as would come within the ordinary limits of error. In one single case, a transforma- KR. H. Chittenden— Dehydration of Glucose. 259 tion of saccharose was noticed when the sugar solution was warmed for two hours with a portion of stomach tissue from a rabbit killed in full digestion. The reduction with Fehling’s solution was quite strong. With glucose, however, many experiments have been tried in addi- ‘tion to those given above, and invariably with the same negative result. The conditions of the experiments, moreover, are in many cases identical with those of Pavy’s except in the method of deter- mining reducing power. There seems, therefore, to be no plausible explanation of the results obtained, other than that in the above experiments there was no dehydrating ferment present. Pavy states that the ferment in question, or rather ‘‘the active principle con- cerned in the transformation of glucose is susceptible of being de- stroyed by the agency of gastric digestion,” so that there is the pos- sibility of such destructive action having taken place in the stomach of the animals experimented with. It is further stated, however, that the converting principle is situated in the underlying portion of the mucous membrane, so that destruction could hardly be expected, except perhaps in the slow self-digestion occurring after death. Cer- tainly, the carefully rinsed tissue could not have retained sufficient gastric juice to affect the results. Furthermore, such decomposition would apply only to the stomach mixture and not to the intestines, unless sufficient proteolytic ferment from the pancreatic juice should adhere to the walls of the intestines to exert destructive action; but in the last experiment given, it is to be noticed that the saccharose ferment, which is presumably equally sensitive, showed vigorous action while the glucose was unaffected. It seems strange, therefore, if such a dehydrating ferment is norm- ally present in the alimentary tract, that we have not been able to obtain some tangible evidence of its presence, either in the stomach or intestines. Since the above was written, the writer has noticed that M. Ogita,* experimenting with dogs, has also been unable to confirm Pavy’s results, both in the dehydration of glucose and in the inver- sion of saccharose. *See Jahresbericht fiir Thierchemie, xv, 275. +, ee PO Mee 2 9 ee Pa es cee EIS “ . ™ ys if : . 4 ‘ ra * fel, id Y SA. 1 bev a oe QP; ee fs te ’ a. “ eee or ao >, 1 eee Z TRE EN GR eee Ree ae eoebaa ads aie tees ‘ . i ‘ ) i i‘: Lay? set; . es wh! TSR ERE daz . ; . oud 4) 4 Base are ak ; : a: = ee fll per kce Ae ed rary { PE er sete! oR ea ashy bins 2 ’ | te ‘7 Ter oS a 2 RL Ree Teoh eS eee TRE ee Paik tin Te a) oe Ut ae *) (ts , otek ee a © ° i f ; of 4 4 a ‘5 / ry | : | a a pete 9 4 : é 7 : r fe 4 s £ fy ' } . i 4 , : ag’ £ Fz te Leh : ! aD. hae aati a . . . (~ a ; ame is pote 1% arto! eT on i ate =) 7a ey ; F : ‘ j : , COIMY i . F oF “ip ses i? fons J ‘ are: . z F . ‘ , = ’ B ¥ , ri me trans. Conn.Acad. Vol. VIL. Crass III i Unique TI Unigne * Uni VI Unigue ¥, Ke) @ we © Q O Q : OQ O ; #782. «Be 43% 3732 Ba, 82 $52 Ba, ih <> 4 CP ; Q Q J @ q 852 Bd, 32° = Te48 Bk, 4 8543 Ca Ss = ca = & Bk 32 2 e » 5 % ” = B: 3 [7 5 _ VU Unigue VUL Two Forms IX Unique neces | C) 6 ©) @ O | e O ; 742 «Be = 4827652 Bh, 4H 7652 Bb, 82 7652 «BieCi(‘t‘SN 7643—B are 64% = Br 432" i XI Unique XUI Unique XIV Unique Fig. 2 be as N a x M owt = =6Ba SS Crass V . | 1 Unique II ‘Three Forms Hil ‘Two Forms . » . Geile Dey te ex es q , : Q v) \ : e AE |. Q “ re Cb, 5432! 72 Ob, , 4'92" 7632 Ce 5m 7632" Cd 82 - Se a CBG Seeex> 3 5432" 7632" 9 = & 7 ~ = eS LJ 2g m - co ~ a e 3 cf #3'2' 7632" Cg, «= 5 432" oe VIU Five Formos ‘" ( ee ee CS C$ 2 Q>CO”) 3 q 2 a 682" Little, Del. L.S.Punderson,Photo, Lith.New Haven. Trans. Conn. Acad. Vol. VIL. ] Ge S | Q 0 7542" = Ck, 413° 52 Cl, = 5432" 542 ci, 4'3°2" 1542! Ch, 5432! 7542's Cl, 432) 7542) Cm, 5432! 7542) Cm, 4'3°2" SEP, CO QO DY = + 3 4 532 5) es: a = fs 53" 2 eal (=) S a a & on = &S 2 K s 24 Q = FI ise 2 e 5 & E g « 2 s 4 & cd 54'S ct 5482 64°38" Cw, 5482" 65482 Os, 432" 7432 Cu, 4'9'2" 74320 Cv, «= 482" meow Little, Del. L.S.Punderson, Photo. Lith.New Haven. Be ee a ee - : A: ‘- ts 7 a 2. a be ee , ~* ot tie! ea ee ee ee OR ee ee ie ee ee ee eS ee SD Oe oY. Trans. Conn.Acad. Vol. VIL. PRAT ul es alee) er ere ie Pr) LD C3 74932 «Cv, «=«6«498"2" 65432 «= C¥b, «4 93*25 6453 Cf, 483328 fe () CE 5 :) 65432 Clw, 53327 74*32 Cy, 58°2* ) > ef a SUN ee . eS aX XXIX me 5 > 6 0 > ) > GQ) " 653" C#h, 4%992% 6537 653° 2) SSS 2) UU) LOI LE 23 743" 6473" 5*437 XXX Two Forms XXXI Unique (> x) ¢ ) om 6942" = O8d, 42389" 6%g82 C3 4eg828 473" CF, 489228 5943" Ctx, 48g028 (\ @) =. \ a &: eS & —_ a a J A 6582" O71, gageae 65927 om, ate 2% Cn, 54324 4p, 499828 XXXUI Three Forms p XXXIV Two Forms ° © O . a) [ 8) { A) { ex) | Bd: > eel eae - CUPP OD O § 5 Q 6592" O%m, Bae 0542" 8m, 4342? 6692" CMm, 4tg"2* ost sm mn . Toh ae. tr U.N Little, Del. L.S.Punderson, Photo, Lith.New Haven. ee | Wpans. Conn.Acad. Vol. VIL. EXXV Unique XXXVI Six Forms ae J — > (\ “6 v O 65432 C%p, 42322 65432 Cp, 43428 XXXVII To Forms XXXYVIII ThreeForms <> <> D. Ut Rak Se O Q "65432 Of 4524 5732 Oty. 4724 ~ 66432 Cty, 43422 50) CB () () 65432 C71, «5432 65432 Ct, 49392* XLIL Two Forms 2 | ; 6 a6 QO) XLIV Unique v XLVL Six Forms -) 65432 Cu 433923 65432 C¥g «4342? 6537 Ley 5432" XLVIT Unique XLYILL Four Forms 5°32 Cth 48382" C.N Little, Del. a y, <0) bE 65432 C&p, 53829 Oe, 2 \ 57492 Cth, 54324 XLII Four Forms r) 5743" Ctr 54324 5743” Ctp 5432 XLIX Unique 5482 Ctg 43828 L.S.Pundérson, Photo. Lith.New Haven. Wrans.Conn.Acad. Vel. V1 I. LATE V. LVI Unique LIX Two Forms LX Unique LXI Unique LXI Unique LXUI Unique ST) (730 (RD) (RN FP wD 643"22 643°" Da, 423" 64392 Do, 54372 = ao ee ae (PY (Se [ed ( @) (A) 7) (19) LS LS) Le Bee) Le LS) Lb a a O® oe Oi *) Ce) Be fe be i at a.) o : ea i fs PB ep B. 53428 -D% 483°2 , oe, es y, —d\ S 8 & > > fy ES (EY (AR |B) (88 [BS 8} 1°) | FOS F-) CS oy) 2 (8) (OO) (2 HS) [2 |S (BLS) ; C) Lae) Ee ER UO BRERRR ib 54832" g 8 EY De (nee i 3S 5 Oe] eye ee 54232" Dap, Amph ee D> \) Ei e 6a ast y, ) ? MON. Little, Del. L:S.Punderson,Phota. Lith.New Haven. Trans. Conn. Acad. Vol. VIL. PLATE VII > a LS ON) Mea Ca BRRB RE - eI (SS) (SF) (SD) (CB (Re 2) 8) 1S (8) CS Lg ARGGRSB 8 Fh) UH CS) [eb US) Us Be Le) 0 Ue Uo Ue 54932" D! Bia hie OD ae es PSeSeQe Ba me ) Ua UY Del L.S.Punderson, Photo. Lith,.New Haven. ° O UN, Little, D« a8 at tetris . me { 4 AR ine ta) Nae Tis fe rivets bea >a XVII.—InNFLUVENcE or Uranium SALTs ON THE AmyYLOLyTIC Action oF SALIVA AND THE ProtrEoLtytTic AcTION oF PEPSIN AND Trypstn. By R. H. Currrenpen anp M. T. Hutcn- INSON, Pu.B. Lirtie is known regarding the physiological, or even toxical action of the uranium salts. In 1825, Gmelin* reported upon the results of some experiments on the toxic action of uranic nitrate, but aside from the work done at that time, little is known regarding the action of uranium. It is our purpose, therefore, to carry out in this Lab- oratory, as opportunity offers, a series of experiments on the physio- logical and toxical action of uranium salts, and we have commenced the work by endeavoring to ascertain the influence of the above salts on the amylolytic and proteolytic action of the ferments occurring in the digestive fluids of the body. In this connection we wish to ex- press our obligations to Professor H. Carrington Bolton, of Trinity College, for his kindness in supplying us with an abundance of chemically pure uranium compounds. 1. Influence on the amylolytic action of saliva. The method employed in determining the extent of amylolytic action was much the same as that previously+ used by one of us, except that the amounts of reducing substances formed under the different conditions of the experiments, were determined volumetri- cally by Fehling’s solution, instead of by Allihn’s gravimetric method. The experiments were made in series, in which one diges- tion of each series served as a control for comparison. The volume of each digestive mixture was 100 c. c. and contained 1 gram of perfectly pure potato starch, previously boiled with a por- tion of the water, 10 ¢. c. of a diluted neutral saliva and a given per- centage of the uranium salt to be experimented with. The mixtures were then warmed at 40° C. for 30 minutes, at the end of which time, further ferment action was stopped by heating the solutions to boiling. The saliva employed in the experiments was human mixed saliva, freshly collected, filtered and made as neutral as possible with 02 per cent. hydrochloric acid, then diluted with water in the pro- * Hdinb. Med. Surg. Gaz., xxvi., 136. { Studies from this Laboratory, vol. i, 1884-5, p. 2 and 53. TRANS.. Conn. AcapD., Vou. VII. 33A Nov., 1886. 262 Chittenden and Hutchinson—Influence of A portion of 1:5. _ Hence, each digestive mixture contained 2 ¢.¢. of | undiluted saliva, The amount of reducing substances, which for the sake of conven- ience are calculated as dextrose, were, as already mentioned, deter- mined volumetrically and from the data so obtained, the percentage of starch converted was likewise calculated. Uranyl nitrate. With this salt the following results were obtained : { Total amount Relative U0.(NO3)2+6H20. reducing bodies. Starch converted. amylolytic action. 0 0-4135 gram. 37°21 per cent. 100-0 7 0:0001 per cent. — 0-4083 36°74 98°7 ; 0:0003 0°3873 34°85 93.6 00005 0°3698 33°28 89°4 0-001 0°3612 32°50 87°3 0-008 0°3131 28°17 75°5 The inhibitory action of the uranyl salt is plainly manifest in these results. A second series of experiments, with still larger per- centages of uranyl nitrate, show the retarding action still more plainly. Total amount Relative UO.(NOs3)2+6H20. reducing bodies. Starch converted. amylolytic action. 0 0-4066 gram. 36°59 per cent. 100-0 0-001 per cent. 0:4000 36°00 98°3 0-002 03880 34°92 95°4 0-008 03034 27°30 74:6 0-004 02545 22°90 62°5 0-005 0-1550 13°99 38°2 0-008 trace. The largest percentage of the salt used (0°008 per cent.), is seen to almost entirely prevent the action of the ferment, thus showing how extremely sensitive the salivary ferment is to the action of this salt. Comparing the two series of experiments, it is seen further, that a given percentage of the salt, say 0°001 per cent., is much more active in one case than in the other, indicating that the action of the salt is not constant. This is undoubtedly true to a limited extent. The action of a given percentage of the salt is constant only under like conditions. In the above series of experiments, the saliva is different in the two cases, and the real explanation of the difference in action is to be sought for in the amount of proteid matter contained in the saliva. Undoubtedly the retarding action of the uranium salt is checked, in part at least, like that of mercuric chloride,* by the * Studies from this Laboratory, 1884-85, p. 71. ' Shs. Uranium Salts on Ferment Action. 263 proteid matter of the saliva. Uranium is a well-known precipitant of albuminous matter and hence the larger the amount of albumin and globulin contained in the saliva, the weaker the retarding action of the uranium salt. Previous experiments have shown that saliva varies somewhat from day to day in its content of proteid matter, as well as in the amount of ferment, and although the former difficulty is obviated as much as possibie by diluting the saliva, still this point must be borne in mind in making comparisons of the different series of experiments. Uranyl acetate. This salt appeared somewhat more inhibitory in its action than the nitrate, due possibly to its greater acidity, The two following series show the extent of action: Total amount Relative U0.(C:H;02)2+H.O. reducing bodies. Starch converted. amylolytic action. 0 0°4049 gram. 36°44 per cent. 100°0 0-001 per cent. 02805 ‘ 20°74 56°9 0-002 0:1698 15°28 41:9 0-008 trace. 0 04032 36°28 100-0 0:00038 0:4831 38°97 107°3 0-0005 03322 29°89 82:4 0:0008 03192 28°89 79°6 0:0010 0:2882 25°93 71:4 The presence of 0:003 per cent. of the salt almost entirely stops the action of the ferment, while 0°0003 per cent. decidedly increases amylolytic action. ‘This latter influence is similar to that exerted by many other metallic salts in very small fractions of one per cent. and is doubtless to be attributed, in part at least, to the stimulating action of the acid-proteids formed, or in part, as suggested by Duggan,* to a more complete neutralization of the digestive fluid. Ammonio uranous sulphate. With this salt the following results were obtained: U(S04)2 +(NA4)o Total amount Relative SO, + H.0. reducing bodies. Starch converted. amylolytic action. 0 0°3384 gram. 30°45 per cent. 100-0 00003 0°3935 39°41 116°3 0-0005 0-3798 34:18 112:2 0-0008 0°3563 30°40 99°8 0-001 0°3550 80°28 99-4 0-002 i 0°2951 26°55 87-2 0-003 0-0805 71°24 23°7 *See Amer. Chem. Jour. vol. viii, p. 211. 264 Chittenden and Hutchinson—Influence of Here there is to be seen both stimulating and inhibitory action, both quite pronounced; and further, the salt is perfectly neutral, so that such action as is exerted must be due to the salt itself. Sodio uranic sulphate. This salt, which like the preceding was exactly neutral to test papers, shows both stimulating and retarding action, but in extent somewhat smaller than that of the uranous salt. Following are the results obtained: Total amount Relative U0280,+ Na2S0O,. reducing bodies. Starch converted amylolytic action. 0 04049 gram. 36°44 per cent. 100°0 0-0003 per cent. 0°4100 36°90 101°3 00005 0-4000 36°00 98°8 00008 04100 36°90 101°2 0-001 0-4117 37°05 101°7 0-002 025380 : 22°07 62°5 0-008 02000 18:00 49°4 0-005 trace. Potassio uranic oxychloride. UO.Cl, +2KC1 Total amount Relative +2H,.0. reducing bodies. Starch converted. amylolytic action. 0 0-4032 gram. 36°26 per cent. 100-0 0:0005 per cent. 0°3951 39°55 98-0 0:0008 04016 36°14 99°6 0-001 04088 36°74 101°3 0-002 01881 16°92 46-6 0-008 010738 9°65 26°6 0-005 trace This salt had an acid reaction and its retarding effects are seen to be somewhat more pronounced than that of the two preceding neu- tral salts. a! a - Ammonio uranic citrate. 1 (UO2)3(C65H507)2+ Total amount Relative (NH,4)3C05H;07. reducing bodies Starch converted. amylolytic action. ike 0 0:4135 gram. 37°21 per cent. 100°0 0:0003 per cent. 0-4298 38°68 103-9 00005 0-4016 36°14 97-1 00008 0-4049 36°44 97°9 0:001 0°3873 34°85 93°6 0-002 04016 36°14 97°1 0-008 038438 33°36 89°6 ———eE—eEEEy—E———s . \ Uranium Salts on Ferment Action. 26 cr (UO2)3(CgHs507)2+ Total amount Relative (NH,4)3C5H50;. reducing bodies. Starch converted. amylolytic action. 0 0:4117 gram. 37:05 per cent. 100°0 0-004 per cent. 02378 21°40 57:7 0-005 0:2144 19-29 52°0 0-006 0:2316 21°39 577 0-007 0°1845 16°60 44:8 0-008 0:1765 15°88 42°8 0-010 trace The action of this salt is mainly a restraining one, but the action is pronounced only with the larger percentages. As to the way in which these uranium salts diminish the amylolytic action of the ferment, we cannot say detinitely. What has previously* been written regarding the action of other metallic salts, under like conditions, is doubtless true here. Loss of amylolytic power is due in part, no doubt, to partial direct destruction of the ferment, as well as to change in the reaction of the fluid. Coupled with this destructive action, however, there must be in addition something in the mere presence of these salts, dependent on chemical constitution, that controls the action of the ferment. The following table shows the relative acceleration and retardation of the. various salts, compared with their respective controls expressed as 100. 2. Influence on the proteolytic action of pepsin-hydrochloric acid. The influence of uranium salts on the proteolytic action of pepsin- hydrochloric acid, was determined by ascertaining the amount of fibrin digested or dissolved in a given time, by a definite volume of a standard, artificial gastric juice, in the presence of varying amounts of the uranium salts. The gastric juice was made by dissolving 10 c.c. of a glycerin extract of pepsin in one litre of 0-2 per cent. hydro- chloric acid. The volume of each digestive mixture was 50 @.c.; composed of 25 cc. of the above mentioned artificial gastric juice and 25 c.c. of 0°2 per cent. hydrochloric acid, containing the nece. sary amount of uranium salt. The proteid material consisted ot purified fibrin, coarsely powdered and dried at 100°C. One gram o’ fibrin was used in each experiment. The digestive mixtures were warmed at 40° C. for one hour and then the undissolved residue wa collected on weighed filters and finally dyied at 100-110° C. until of constant weight. The amount of fibrin dissolved is taken as a meas- ure of the proteolytic action. * See Studies from this Laboratory, vol. i, 1884-5, pp. 70-75. TRANS. Conn. AoApD., Vou, VII. 34 Noy., 1886 Chittenden and Hutchinson—Influence of 266 8-6P 8-PP Le LG 0-6¢ LLG 9-68 1-16 9-86 6-16 1-16 6560 | Sagan | Sete eee ea 97e.14 -l0 oTUeIn O1llouLUy =e |e oulteae = ae 0 SP AEM Cat 9-9F | 8-TOL | 9-66 0-86 ie She ae a we eee a ee -£xo o1mein OI1ssejog pee IP ate ee, Bln oee 0) Soeee Slee G89 L-TOL | 8-TOT | 8:86 €-10T | ~--~° | -eyeydyns orwesn orpog SSB EE |io ae cs a eee acini eee e187 17-68 - | 8-868) enh Pegi |S" ol Se ayaa | -[ns snouvin ormouUly a [ete eee denn eal eae an 61h | FIL | 9-62 | 88 | BLOT | ~~~ [7777777 exeqeo" [Auer Qn Hees: elie oe | C6Ge [2:89 AHCGGLE +m re ea ieee > ak F698, |. O:86°=.| 2:98, =|" S** eqerzu [AUBIE) 800-0 | 400-0 | 900-0 | G00-0 | F00-0 200-0 | 800-0 | 100-0 | 8000-0 | 000-0 | 000-0 | 1000-0 |°~~“SHT¥S Jo esequeor19g ‘NOILOY OILATOTANY JO NOILVOUVLEAY GNV NOILVYATHOOY WAILVIEY ONIMOHG WIaV Uranium Salts on Ferment Action. 267 Following are the results obtained with the various salts : Uranyl nitrate. Relative UO.(NO;)2+6H.O. Undigested residue. Fibrin digested. proteolytic action. 0 0°1858 gram. 86°47 per cent. 100-0 0-025 per cent. 0°1865 86°35 99°8 0-050 0:13878 86°22 99-7 0:100 0°2397 76°03 87°9 0°500 0°5008 49°97 57°8 1-000 0:6638 33°62 38°8 Uranyl acetate. U0.2(C2H302)2 Relative +H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°1453 gram. 85°47 per cent. 100-0 0:025 per cent. 0°1581 84°19 98°5 0-050 0:°1867 81°33 95:1 0-100 0:2052 79°48 92°9 0-500 Oa" 24°98 29:2 1-000 1:0050 0 0 It is to be noticed here, that the retarding action of the acetate, as with saliva, is far greater than the nitrate, a fact which is doubtless dependent in this case on the nature of the acid united with the ura- nium. Further, it is to be noticed, that the action of uranyl sulphate falls about midway between the action of the nitrate and acetate. These facts accord with views previously* expressed, and show plainly that the extent of the retarding action of salts in general is dependent in part, of the liberation of the acid of the salt and the digestive power of the pepsin-acid formed. Experiments have shown that nitric acid of appropriate strength, united with pepsin, is about four-fifths as active as hydrochloric acid, while sulphuric acid is only a little more than one-fourth as active as hydrochloric of the same strength and that acetic acid is practically inactive. Hence, the base being the same, acetates, citrates, and other salts, the acids of which are not capable of working with pepsin will most readily retard gastric digestion. This view being correct, uranyl nitrate, sulphate and acetate should retard gastric digestion in just such relative pro- portion as our experiments show they actually do. * See Studies from this Laboratory, 1884-85, p. 94-95, 268 Chittenden and Hutchinson—Influence of Uranyl sulphate. 3 Relative U0.S0,+3H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0-1832 gram. 81°68 per cent. 100-0 0025 per cent. 0)°2545 74°55 91:3 0-050 0°2673 73°27 89°7 0-100 0°3155 68°45 83°8 0-500 06084 39°16 479 1-000 0°8225 17°75 21°7 Ammonio uranous sulphate. USO, +(Nitt,)2S04 Relative +H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°1833 gram. 81°67 per cent. 100-0 0-025 per cent. 02859 71°41 874 0-050 0°3143 68°57 83°9 0-100 0°3742 6°258 766 0-500 0-9113 8:87 10°8 , 1-000 1:0070 0 0 A comparison of the action of the two last salts, shows plainly that the ammonio uranous compound has a far greater inhibitory action than the simple uranyl] sulphate. Ammonio uranic citrate. (UWO2)s(C5H507)2 ; Relative +(NH,);C5H;0;. Undigested residue. Fibrin digested. proteolytic action. 0 0-1747 gram. 82°53 per cent. 100°0 0-025 per cent. 0°1795 82°05 99-4 0-050 0°2102 78°98 95°7 0-100 0-2180 18°20 « 94°7 0500 0°9055 9°45 11-4 - 1-000 0 0 0 Sodio uranic sulphate. UO.S0, + Na.SO4 Relative +2H.0. Undigested residue. Fibrin digested. proteolytic action. 0 0°2624 gram. 73°76 per cent. 100°0 0:025 per cent. 02666 73°34 99°4 0050 03688 63°12 85°5 0-100 04488 55°62 756 0500 08131 19-69 26°7 1-000 0-9891 1-09 age In the two last series, the ammonio uranic citrate is noticeable for not causing a gradual diminution in the proteolytic action of the ferment; but on the contrary, it gives rise to a sudden and rapid fall- ing off in proteolytic action, when a certain percentage of the salt is added. The same thing is to be noticed in the case of the uranyl —_”* Uranium Salts on Ferment Action. 269 acetate and the reason doubtless lies in the fact that the acid in these two salts is wholly incapable of forming an active compound with pepsin, and thus when a percentage of the salt is added sufficient to use up all of the hydrochloric acid of the gastric juice, digestive action comes to a full stop. Potassio uranie oxychloride. U0.Cl. + 2KCl Relative +2H.0. Undigested residue. Fibrin digested. proteolytic action. 0 0:3063 gram. 69°57 per cent. 100-0 0°025 per cent. 0:2472 75°28 108-0 0-050 0°2128 13°17 113°5 0-100 02582 74°68 107°6 0-300 02648 73°d2 105°9 0-500 0:°3578 64°22 92:5 With this salt, unlike any of the preceding, there is to be seen a direct stimulating action on the ferment. Only in the presence of 0°5 per cent. of the salt is there any retarding effect produced. This naturally suggests that possibly uranium per se, at least in small fractions of a per cent., has really a stimulating effect on ferment action, but that owing to its combination with acids, in the forma- tion of salts, its apparent effects in the case of pepsin-hydrochloric acid are those due to combination of the normal acid of the gas- tric juice and liberation of the acid of the uranium salt. In this way only, can we explain the noticeable difference in action of the oxychloride and the other uranium salts. Thus 0°5 per cent. of the former causes but slight diminution in proteolytic action, while with all the other salts, the same percentage causes on an average, a diminution in proteolytic action of at least 50 per cent. This would apply, of course, only to small percentages of uranium, for larger amounts of oxychloride would cause the formation of an indigestible uranium-albumin compound. Here, however, as in the case of all the salts, the action of any given percentage is constant only under definite conditions. Dimin- ish the amount of ferment, for example, and the amount of dissolved proteid matter consequent thereto, and then the retarding action of the same percentage of uranium salt will be correspondingly in- creased. This is well illustrated in the following series of experiments with potassio uranic oxychloride. Using the same percentages of salt as employed in the preceding series, with the same strength of acid, but with onlv half the same content of pepsin extract, and the fol- lowing results were obtained ; 270 Chittenden and Hutchinson—Influence of Relative Uranium salt. Undigested residue. Fibrin digested. proteolytic action. 0 0°1652 gram. 83°48 per cent. 100-0 0:025 per cent. 0-1982 80°18 96-0 0-050 02192 78°08 93°5 0-100. 0°2569 74°31 89-2 0°300 0°3486 65:14 780 Increasing the percentages of the oxychloride still further, either with this strength of pepsin or the preceding, and there is seen a gradual diminution in the action of the ferment. Compared, how- ever, with the action of the preceding salts, retardation is seen to be quite slow; thus even 2:0 per cent. causes a diminution of pro- teolytic action amounting to only 50 per cent. The following table of comparisons shows the relative acceleration and retardation of the various salts compared with their respective controls expressed as 100. TABLE SHOWING RELATIVE PROTEOLYTIC ACTION. Percentage of Salts_________ 0:025 | 0:05 | 0-1 0°3 0:5 1:0| 2.0 Uranyl nitrate! ie fe Se: BBB OS STO) he O78 SB Shee Uranyl acetate _--..=.. 2-.% 98°5 95-1 | 92°9| _.._ | 29.2! 0 : Uranyl sulphate __...__.... 913) 80-7] 83:8]... | 479) 217) Ammonio uranous sulphate_, 87°4 | 83:9 76°6 | = S| PHOS 0 Sodio uranic sulphate -___. | 99-4| 85°5| 75°6| ___. 26°7 | 1:5 Ammonio uranic citrate. --- | 99:4 | 95°7 | OR) se sit ue 0) Pep Potassio uranic oxychlo- ) 1 108-0 113°5 | 107°6 | 105°9 | 92°5 | - a Pets ee Oe, ses J2 96-0 | 935 892] 78:0| 81-0, 66-2| 49-4 In retarding proteolytic action, the uranium salts act in part by combining with the proteid matter to be digested, forming a uranium- albumin compound, which is indigestible. Further, in a solution at all concentrated, the uranium salt is liable to precipitate mechanic- ally a portion or all of the pepsin along with the albuminous matter. In addition to this, however, retardation is also due, as already ex- pressed, to liberation of the acid of the salt by the hydrochloric acid of the gastric juice and to the subsequent formation of a pepsin-acid only partially, or not at all, capable of digestive action, Uranium Salts on Ferment Action. 271 3. Influence on the proteolytic action of trypsin. The method employed in determining the extent of proteolytic ac: tion in this case was much the same asin the preceding. The trypsin solution was made as neutral as possible and was prepared from dried OX pancreas, previously extracted with alcohol and ether; 20 grams dry pancreas, extracted with 200 c.c., 0°1 per cent. salicylic acid and ultimately diluted to 1 litre. A little thymol was added to prevent decomposition. 50 ¢.c. of the trypsin solution were used in each ex- periment, together with 1 gram of prepared fibrin and the necessary amount of uranium salt. The first experiment was tried with uranyl nitrate, the mixtures being warmed at 40° C. for six hours. Following are the results : Relative U0.(NOs3)2+6H20. Undigested residue. Fibrin digested. proteolytic action , 0 0°2927 gram. 70°73 per cent. 100°0 0-010 per cent. 0°3328 66°72 94:3 0-025 0:3460 65:40 92:4 0-050 0°4198 58-02 | 82-0 0-100 05004 49-96 70°6 0°500 a 0 0 With this salt retarding action is seen to be gradual upto a certain point, and then suddenly all ferment action ceases. Uranyl acetate. U0.(C2H302)2° Relative + H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0-4234 gram. 57°66 per cent. 100-0 0:010 per cent. 0°4591 54:09 93°8 0-025 0:5460 45°40 18°77 0-050 0°6094 39°06 67°7 0-100 0°8173 18.27 31°6 0°500 0 0 This series of experiments was warmed at 40° C. for about five hours. The inhibitory action of the salt is seen to be more pro- nounced than that of the nitrate; indeed, there is to be seen here, the same difference in action noticed in the case of the amylolytic ferment. Uranyl sulphate. With this salt, under exactly the same conditions of time and temperature as the preceding, the following results were obtained : 272 Chittenden and Hutchinson—Influence of Relative UO.S0, + 3H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°3790 gram. 62°10 per cent. 100°0 0:010 per cent. 03868 61°37 98°8 0°025 05023 49°77 80-1 0-050 06066 39°34 63°3 0100 0-8083 19°17 30°0 0-500 -— 0 0 These results are seen to accord almost exactly with the preceding and show that both salts have an action on this ferment much more pronounced than on pepsin-hydrochloric acid. Ammonio uranous sulphate. U(SO.4) +(NHa)e Relative SO,+ H.0 Undigested residue. Fibrin digested. proteolytic action, . 0) 0:4025 gram. 59°75 per cent. 100°0 0:010 per cent. 0°4031 59°69 : 99:7 0025 0°4229 57°71 96°4 0°050 04824 51°76 86°6 0-100 05743 42°57 72:9 0500 4 0 0 Sodio uranic sulphate. U0.S04+ NasSO4 Relative +2H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0:3261 gram. 67°39 per cent. 100-0 0-010 per cent. 03414 65°86 97-4 0025 03588 64°12 94°8 0-050 0°3961 60°39 89:5 0-100 04462 55°38 82-1 0500 — 0 0 : Ammonio uranic citrate. (UO.)3(CeHs07)2 Relative +(NH,)3C¢H;07. Undigested residue. Fibrin digested. proteolytic action, 0 0:3955 gram. 60:45 per cent. 100-0 0-010 per cent. 04934 50°66 83°8 0-025 05282 4718 78-0 0050 0°6618 33°82 55°9 0-100 O-7705 © 22°95 378 0-500 0°7168 28°32 46:8 Potassio uranic oxychloride. U0.2Cl. + 2KCl Relative + 2H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°4234 gram. 56°76 per cent. 100°0 0°010 per cent. 04498 55°02 95-4 0°025 05224 47°76 82°8 0:050 9-5629 43°71 759 0°100 0°7087 29°13 50°8 0-500 —— 0 0 Uranium Salts on Ferment Action. Np With the exception of ammonio uranic citrate, the four last salts experimented with, show about the same degree of inhibitory action; the citrate, however, appears less pronounced in its action than the others. As to the manner in which the uranium salts retard the proteolytic action of the pancreatic ferment, it is probable that the main explanation is to be found in the power possessed by the former of combining with proteid matter in general; combining with and rendering indigestible the albuminous material added to the digestive mixture and perhaps precipitating, or even destroying, the ferment itself. Further, the reason why certain salts appear less active than others is perhaps to be found in the fact that in the pre- cipitation of albuminous matter by uranium salts, the uranium com- bines directly with the proteid matter, thus liberating the acid of the salt; and as trypsin is inactive in the presence of free mineral acids, and only partially active in the presence of combined acids (combined with proteids), it follows that an organic salt, such as a citrate, would naturally be less active as a retarding agent, than the nitrate or sulphate. The following table shows the relative retardation of the various salts expressed in terms of relative proteolytic action: TABLE SHOWING RELATIVE PROTEOLYTIC ACTION. Percentage of Salts__.-._._-__---- 0-01 0-025 0-05 0-1 0:5 Uranyl nitrates: =... 22-4 aes 94:3 92-4 82-0 70°6 0 Wranyl acetate... = -20..-222222 93°8 18-7 67.7 31°6 0 Wranyisculphate......5..22--22-- 98:8 80:1 63°3 30°8 0 Ammonio uranous sulphate------ 99°77. 96-4 86°6 72:9 0 Sodio uranic sulphate-.-----.------- 97°4 94:8 89°5 82:1 0 Ammonio uranic citrate_--------- 83°8 |. 78:0 559 37°8 | 46°8 Potassio uranic oxychloride------- 95-4 82:8° |, -715;9 50:8 0 It is thus seen that uranium salts, in the main, like most other metallic salts, exert a decided retarding influence on the action of the digestive ferments. Trans. Conn. Acap., Vou. VII. 35 Nov., 1886. XVIII.—Tse Retative DisrripuTtion oF ANTIMONY IN THE ORGANS AND TISSUES OF THE BODY, UNDER VARYING CONDITIONS. By R. H. Carrrenpen and Josrepnu A. Briaxg, B.A., Pa.B. OrRFILA, many years ago, proved that salts of antimony, like the salts of other metallic poisons, are absorbed and can be detected in the animal tissues and secretions, especially in the liver and kidneys; and further that the absorbed antimony is slowly discharged from these quarters through the medium of the urine. These early results were confirmed by other investigators, notably Danger and Flandin and by Panizza and Kramer, the latter of whom detected antimony, not only iu the urine, but also in the blood of a man during a course of tartar emetic.* Orfila’s work also indicated that while the elimina- tion of absorbed antimony commences very quickly, it is a compara- tively slow process; thus in one instance he stated ¢ that he found antimony in the fat, liver and bones of a dog that had taken, three months and a half before its death, 46°5 grains of tartar emetic dur- ing a period of ten days, and that similar results were obtained in a second case in which the interval was four months. Presumably, however, elimination is much more rapid than these figures would seem to indicate. Dr. Richardson, however, found antimony in abundant proportions in the liver, and in smaller proportions in the kidney and heart, twenty-one days after the last dose of antimony had been taken. As to the relative distribution of absorbed antimony, the experiments of Drs. Nevins and Richardson are the only important ones recorded. Dr. Nevins,{ experimenting on rabbits, with tartar emetic in doses of 05,1 and 2 grains four times daily, found that the weakest rabbit died after taking 12 grains, the strongest after taking 72 grains of the poison. Other rabbits were killed at varying lengths of time after taking the last dose of poison (31, 14, 4, and 3 days), and in every case antimony was found in large quantity in the liver, in smaller quantities in the spleen and stomach. Antimony was likewise found in the kidneys and urine of those animals that survived for some time, also in the lungs and in those that lived 15 days, in * See Christison on Poisons, p. 372. + Traité de Toxicologie. + See Reese, Manual of Toxicology, p. 259-260 and Woodman & Tidy, Forensic Medicine and Toxicology, p. 128-129. —— a Chittenden and Blake—Distribution of Antimony, ete. 275 the bones likewise. Dr. Nevins further states, that it was difficult to detect the poison in the muscles and in the blood, but it was found in the bones as late as the thirty-first day after discontinuing the poison. Dr. Richardson* in 1856, examined the tissues of a dog that died 1 hour and 40 minutes after a solution containing a drachm of tartar emetic had been injected into the cellular tissue. The antimony was found in the following parts, in the order given as to quantity ; blood, vomit, rectum, lungs, liver, stomach, bladder, kidneys and small in- testines. In a second experiment, a wound in a dog’s neck was dressed every morning with tartar emetic ointment, the dog dying at the end of the seventh day. In this case no antimony was found in the brain, but it was found in larger quantities in the liver and spleen than in the other organs. It is very evident, therefore, that tartar emetic, and presumably other salts of antimony likewise, will penetrate all the tissues of the body and that at the same time the antimony is constantly being eliminated by the kidneys. Further than this, the few results recorded indicate nothing definite. As Dr. Richardson well says, ‘‘the election of antimony by different parts of the body is as yet an open question; that the liver, however, would appear to be the structure in which it is most collected when the ad- ministration is slow and in small doses; and that the elimination of the poison is attempted by all the secreting surfaces.” No positive statements can therefore be made regarding the relative distribution of antimony, other than in a general way. Hence, it has been our object in the present investigation to study somewhat in detail, the relative distribution of antimiony in the dif- ferent tissues of the body under varying conditions ; both as to the form of the poison and the manner of its introduction. As with arsenic, so with antimony, the relative proportion of poison found in the different tissues after death may become of considerable medico-legal importance, provided we have sufficient confirmatory data from which to draw conclusions. Particularly is it of importance to know the way in which the form of the poison will influence its distribution. Whether as with arsenic,+ the administration of a soluble and diffusible form of the poison will lead to a noticeable accumulation in the brain or nerve tissue in general. *See Woodman & Tidy, p 129. Also Dr. W. B. Richardson, Abstract in Amer. Jour. Med Sciences, 1857, vol. 33, p. 266, and B. and F. Med. Chirurg. Rey. Oct. 1856. + See Amer. Chem. Jour., vol. v, p. 8, also Studies from the Laboratory of Phys- iological Chemistry, 8.S.S. of Yale College, 1884-85, p. 141. 276 Chittenden and Blake—Distribution of 1. The quantitative estimation of antimony. There are many methods by which antimony may be detected, even when present in quite small quantities ; but there are only a few which yield accurate quantitative results, particularly in the presence of or- ganic matter. In attempting to find a method sufficiently accurate for our purpose, we first tried the method recommended by Orfila, of introducing the final antimony solution into the Marsh apparatus; using for this purpose the form of apparatus and mode of procedure, found so efficacious in the case of arsenic.* In every trial, however, there was a loss of at least 40 per cent. of the antimony; thus in the first case, with an amount of tartar emetic equivalent to 6 milligrams of metallic antimony, only 3°4 milligrams of the metal were recovered, and under exactly such conditions as with arsenic would lead to the recovery of the entire amount. And since in this trial experiment, the antimony was introduced directly into the Marsh apparatus, as tartar emetic dissolved simply in dilute sulphuric acid and without the presence of any organic matter to act as a hindrance, it follows that the loss must be due to retention of a portion of the antimony by the zine and platinum. A second trial, with the same amount of antimony salt, gave a decided deposit of metallic antimony in the heated tube, but weighing only 3°5 milligrams. In this case the ap- paratus ran for three hours, but as before, it is evident that only a portion of the antimony was converted into antimoniuretted hydro- gen, the remainder undoubtedly being retained either by the plati- num used to alloy the zinc, or by the zine itself. The presence of a small amount of platinum fused in with the zine, previous to its granu- lation, does not appear to offer any obstacles to the complete evolu- tion of arsenic as arseniuretted hydrogen. Numerous results obtained by one of ust testify to the accuracy of this statement. Bernstein had also noticed that alloying zine with platinum or silver, did not hinder the complete evolution of the arsenic, while the addition of a little platinic chloride solution to the acid fluid from which arsenic was be- ing evolved, led to the precipitation of even 50 per cent. of the - arsenic present.{ This probably is the explanation of the low results obtained by Hedden and Sadler in the estimation of arsenic by the Marsh apparatus, in the presence of platinum.§ But with antimony, * See Amer. Chem. Jour., vol. ii., p. 235. +See Amer. Chem. Jour., vol. ii, p. 235. t See Dragendorff, Gerichtlich chemische Mittheilung von Giften, p. 334, foot note. § See Amer. Chem. Jour., vol. vii, p. 342. +<.. Antimony in the organs and tissues. 277 either the platinum alloyed with the zinc is sufficient to retain fully 50 per cent. of the metal, or as is very probable, the zinc itself causes a precipitation of a portion of the antimony. That this is a point overlooked by most writers on chemical toxicology, is evident from a perusal of the literature on the subject. Whether it would be possible to obtain all of the antimony as anti- moniuretted hydrogen, in the absence of any metal other than per- fectly pure zinc, we cannot say. Certainly as a method for quantita- tive purposes, it would be too tedious a process to admit of general use, especially where such small amounts of antimony, as in our own experiments, would limit galvanic action to a minimum. That the method is capable of showing the presence of very small amounts of antimony, is unquestionable. Wormley’s experiments* are very de- cided on this point, but evidently only a portion of the antimony will be recovered. Precipitation of the antimony by hydrogen sulphide and final oxi- dation by fuming nitric acid and weighing as Sb,O,, gave fairly satis- factory results, when the weight of antimony was not less than 10 milligrams of Sb,O,.. With smaller amounts, the results were far too high, owing probably to the far greater proportional increase of sul- phur. Attempts to weigh as sulphide, after fusion of the first hydro- gen sulphide precipitate with potassium nitrate and carbonate, like- wise gave too high results when the amount of antimony was small. Further, the two latter methods are somewhat unsatisfactory, in that when the amount of antimony is very small, the nature of the final products is such, it is difficult to be certain of the purity of the mat- ter weighed. Owing to this reason partly, we next turned our attention to the electrolytic method for the separation and determination of antimony, as in this case the appearance of the metallic mirror is in itself a fair guarantee of the nature of the deposit, and its purity is easily proved: Alex. Classent has shown the accuracy of the method in general quantitative work, where moderate amounts of antimony are present (0°15—0'2 grams Sb). In his experiments, the antimony, in the form of sulphide, was dissolved in ammonium sulphide and the solution then exposed to electrolytic action. The ammonium sulphide solu- tion must be free both from polysulphides and from free ammonia. * Micro-Chemistry of Poisons, p. 229. + Quantitative Analyse durch Elektrolyse. Berichte d. deutsch. chem. Gesell., xvii, p. 2474. See also Alex. Classen and Rob, Ludwig, ibid., xviii, p. 1104. 278 Chittenden and Blake—Distribution of Classen also found that antimony was deposited quantitatively, when the sulphide was dissolved either in potassium or sodium mono- sulphide, or in potassium or sodium hydrosulphide. Polysulphides must also be absent in this case, and the potassium and sodium must be quite free from both iron and aluminum, as by long continued electrolytic action, sulphide of iron and aluminum hydroxide may be deposited upon the antimony. Classen also recommends the use of ammonium sulphydrate and a weak current; a strong current tending to cause the separation of the antimony in a pulverulent form, not closely adherent to the platinum. As it would be necessary in our work, after oxidation of the or- ganic matter, to separate the antimony as sulphide, the above method seemed particularly advantageous, and experiments were therefore tried to ascertain its value when applied to very small quantities. Weemployed a battery of four moderate sized gravity cells, giving a weak current, and as a rule, exposed the solution to the action of the current for at least 15 hours, as we found better results were ob- tained by the long continued action of a weak current, than by the quicker action of a more rapid one; particularly in such solutions as we usually had to work with, containing considerable excess of sul- phur and some organic matter. The negative pole of the battery was either a small platinum crucible or a platinum dish, while the posi- tive pole was a large piece of platinum foil welded onto a good sized platinum wire. The deposition of the antimony was much more complete, more tightly adherent to the platinum, and as a rule less mixed with sulphur under this arrangement, than when the dish was made to serve as the positive pole; due, doubtless, simply to the broader surface for the deposition of the metal. Using ammonium sulphide as a solvent for the antimony sulphide, did not give us very good results by electrolysis, the loss being cou- siderable. Much better results were obtained by using a solution of sodium monosulphide, made by saturating one-half of a 15 per cent. solution of sodium hydroxide with hydrogen sulphide, and then add- ing the remaining half of the sodium hydroxide solution. The method was tested by precipitating definite volumes of a standard antimony (tartar emetic) solution with hydrogen sulphide, dissolving the metallic sulphide in the sodium monosulphide and then exposing the solutions to electrolytic action. When the separation of the metal was complete, it was found best to wash the deposit with considerable water, without breaking the current; as sometimes, as in the presence of tartaric acid, the separated metal rapidly dissolved Antimony in the organs and tissues. 279 on discontinuing the current. The antimony was then finally washed with alcohol, any adherent sulpbur lightly brushed off and the dish dried and weighed. Following are some of the preliminary results obtained : Standard Sb Theoretical Am’t Sb Duration Solution. Am’t Sb. found. of Klectrolysis. 50 c. c. 0-0875 gram. 0:0375 gram. 16 hours. 10 0:0075 0:0077 18 10 0:0075 0:0075 24 10 0:0075 0-0078 18 10 0:0075 0:0074 8 2 0:0015 0-0012 10 0:00075 0:0008 3 These results showed the method to be perfectly satisfactory for our purposes, and we, therefore, next tried the separation of small amounts of antimony from organic matter, and its final recovery by the above method. As there was no doubt that large amounts of antimony could be satisfactorily recovered from organic matter, our experiments were confined mainly to very small quantities. In each experiment, 100 grams of either liver or beef were finely divided and to the so-pre- pared tissue, a few cubic centimetres of the standard antimony solu- tion were added and the mixture thoroughly oxidized with hydrochlo- ric acid and potassium chlorate. After removal of all free chlorine from tke filtered fluid, by careful heating, the antimony was precipi- tated by hydrogen sulphide. This precipitate, which naturally con- tained, in addition to the sulphide of antimony, considerable sulphur and some organic matter, was then treated as follows: While still moist, after being freed from all hydrochloric acid by thorough wash- ing, it was dissolved in the cold sodium monosulphide solution and then directly subjected to electrolysis. At first, we thought it nec- essary to free the precipitate from its excess of sulphur and organic matter by solution in ammonium sulphide, evaporation, fusion with potassium carbonate and nitrate, etc., obtaining it finally in the form of sulphide again, free from its former impurities. This, however, we found to be unnecessary ; in fact the loss was far greater than the gain. Provided the oxidation with potassium chlorate be a thorough one and the free chlorine entirely removed from the solution, the first hydrogen sulphide precipitate is well adapted for electrolysis directly. It was found best, however, to keep the solution on the battery until all of the sulphur and organic matter was oxidized ; that is until the reaction of the fluid had become acid. This took, 280 Chittenden and Blake—Distribution of many times, 30 to 40 hours with our slow current. A more rapid current, would, to be sure, bring about a change in reaction much more quickly, but occasionally under such conditions the antimony would be less closely adherent and loss occur. Then again as the sulphur present was gradually changed into sulphuric acid, the final, strongly acid-reacting fluid, became a good conductor and so at the last, even with our four cells, electrolytic action was quite strong. In washing the deposited antimony, the acid fluid was syphoned out and water continuously added, without breaking the current, until the original fluid was entirely removed. Following are a few of the results obtained, the antimony being added to 100 grams of tissue in each case. Standard Sb Theoretical Am’t Sb Duration Solution. Am’t Sb. found. of electrolysis. 10 c.c¢. 0:0075 gram. 0-0075 gram. 24 hours. 10 00075 0-0072 48 10 0-0075 0:0069 36 10 0:0075 0:0074 24 10 0:0075 0-0064 20 5 0-0037 00028 18 The results are certainly not all as close as those obtained in the absence of organic matter, but are perhaps as satisfactory as could be expected under the conditions of the experiment, viz: a large pro- portional amount of tissue (100 grams), a very small amount of metal and a large volume (say 500 c.c.) of fluid to precipitate from, with hydrogen sulphide. Even under the most unfavorable condi- tions, at least 75-80 per cent. of the antimony introduced into the or- ganic matter was recovered. We next turned our attention to the obtaining of some convenient and quick method for the direct determination of antimony in urine, or other like organic fluid. Preliminary experiments showed us that antimony, in the form of tartar emetic, could be separated completely from a tartaric acid solution by electrolysis. The separation takes place rapidly, but care must be taken to remove all of the tartaric acid solution by displacement with water, before breaking the cur- rent, otherwise the deposited antimony will instantly dissolve. Trial tests repeatedly gave results in close accord with theory. Excess of sodium tartrate, however, appeared to interfere somewhat with sep- aration of the antimony; while the presence of sodium chloride in the presence of an excess of tartaric acid, prevented entirely the separation of the antimony. From a sulphuric acid solution, how- ever, antimony was also deposited quantitatively, and on applying Antimony in the organs and tissues. 281 this method to antimoniacal urine, we found it possible to recover the antimony without loss. The following results testify to the accuracy of the method: In each experiment 25 ¢.c. of normal urine were employed, to which was added a number of cubic centimetres of the standard antimony solution and then 1 ¢.c. of pure dilute sulphuric acid, after which the solution was connected with the battery. Standard Sb Solution in Theoretical Amount Sb Duration of 25 c. c. urine. amount Sb, found. electrolysis. OKewe: 0:0075 gram. 0:0075 gram. 48 hours. LO” .<° 00075 ** 00074 24 =< il) 46 00075 = ** 00074 <* ORs 3) ae 0:003875 * 00088 =“ 1S) 6 Opec 0-00375 <** 0:0037 <* 1S? 3 iy ah 0-00375 <* 0:0086 << LB Ty cite Diss 0-00225 <¢ 0:0024 < HO Wess ies 00015 ** 0:0018 «* PAU Bo a ae 00015 « 0-0016 « iS eas Ry 3S 0-00075 ** 0:0008 «* Gb Es Doubtless all of these results could have been obtained equally as well in a very much shorter time, but most of the solutions were con- nected with the battery at night and allowed to run until morning, or whenever convenient. The method is evidently very accurate when applied in this manner, and we have made use of it very satis- factorily, even with 150 c.c. of urine, using in this case a platinum dish of 200 c. ¢. capacity as the negative electrode. 2. Relative distribution of absorbed antimony. As already stated, the object sought in the following experiments was to ascertain the relative distribution of antimony under varying conditions; particularly, variations in the form of the poison, as its solubility or insolubility ; in the method of introduction, as per mouth, rectum or sub-cutaneously; and lastly in the length of time during which the poison is being taken, whether in one large dose or in many small ones frequently repeated. All of these points we have endeavored to cover in the experiments about to be described. Experiment I. Hypodermic injection of a solution of tartar emetic. 0120 gram of tartar emetic, dissolved in a little water, was intro- duced under the skin (right thigh) of a cat weighing 1262 grams. TRANS. Conn. Acap., Vou. VII. 36 Nov., 1886, 282 Chittenden and Blake—Distribution of 3:10 p. m., solution injected. 3:16 ‘* vomited copiously. 3:20 <* i again, simply mucus. pinoy) << = mucus and bile. Sopa te and purged. 3:45 ‘ partially paralyzed. 3:04 ‘ vomited again. 4:30 ‘* much prostrated. BlO, 2 “dead: The various organs were then separated and the absorbed antimony determined, according to the method already indicated. Following are the results : Sh per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. eiven: feel. ues! Ui seat ety 52°0 6°35 12°21 Branly cents See ene. Se 275 0°60 2°18 Heart-and lungs]. -2 2 iee. 32°0 0°70 2°18 Kap ney Se ono io eas eee 12:0 0:15 1:25 Stomach and intestines _-_-_-__- 74:0 0:80 1:08 Mus@le from back -___.-.__-_- 138-0 1°25 0°90 339°5 9°85 These figures show the greatest absorption by the liver; the brain stands next, while the muscle tissue appears to have absorbed but a | relatively small amount of the antimony. In’this connection it must be remembered that two hours only, intervened between the intro- duction of the poison and the death of the animal, hence it is evident ° that the brain tissue must have a decided tendency to hold absorbed antimony. The antimony, however, was introduced in the form of a readily soluble salt and under conditions directly favoring rapid and wide-spread distribution. That there is certainly some decided selec- tive action, is evident from the fact that the muscle tissue, which must truly have had as good an opportunity as the brain tissue, retained per 100 grams of substance far less of the poison. Elimin- ation had evidently commenced, for the kidneys contained a decided amount of antimony. ExpERIMENT II. Hypodermic injection of a solution of tartar emetic. In this experiment, a smaller amount of tartar emetic was used than in the preceding; and further, the poison was introduced in three distinct doses, thus allowing longer time for absorption. In fact, the animal lived 22 hours after the first dose, hence the experi- a Antimony in the organs and tissues. 283 ment stands in striking contrast to No. 1, in which the animal lived but two hours; while the results, contrasted with the- preceding, show plainly the influence of time on the distribution of the poison. Following are the results of the experiment on a rabbit weighing 1295 grams. Mar. 31, at 5:20 p.m., injected under the skin of leg, 0-012 grm. tartar emetic. April 1, ‘* 8:45a.m., a6 és 0:035 ut April 1, ‘‘12:45 p.m., gs ae 0-035 ec Total, 0:082 Animal died at 3:05 p. m. Following is the distribution of the antimony: Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. HRNENOVAS S52 Soe eS oY ee 11°5 0-60 5°21 1 LATS ps a eae a alg (ee es dle 63:0 1:50 2°58 STALE awe eee a eee ie 9-0 0:20 2°22 Stomach and intestines___-_- 98-0 2-00 2°04 Heart and lungs ___.._------ 17:0 0°25 1:47 Muscleitronm back 2.2 -.- 2.222 106-0 0°70 0°66 304°5 5°25 As might naturally be expected, the results indicate a more even distribution of the poison than in the preceding experiment. Although two-thirds as much antimony was used as in experiment No. 1, the liver contains a far smaller proportional amount of the poison than in the preceding experiment, while the ‘kidneys stand first in their content of antimony. Between the brain and the liver, there is but little difference and the experiment plainly substantiates the preceding in showing the tendency of brain tissue, under these conditions, to absorb and retain antimony. In the muscle tissue the percentage of absorbed antimony is almost exactly the same as in No. 1, that is, proportional to the amount of antimony introduced. The animal had evidently lived long enough to admit of a fairly complete distribution of the poison, and elimination having been going on for some time, those parts which had originally contained the most, particularly the liver, had been drawn on to the greatest extent; so that at the time death intervened, the excretory organs, notably the kidneys, were the richest in poison. This fact further indicates that the elimination of absorbed antimony proceeds some- what rapidly. 284 Chittenden and Blake—Distribution of Experiment III. Hypodermic injection of a solution of tartar emetic. In this experiment, a cat weighing 1613 grams, had injected under the skin of its hind leg 0°150 gram of tartar emetic in one dose. There was some purging and vomiting, and the animal died in 4} hours after the administration of the poison. The object sought in this experiment, which is virtually a repetition of No. 1, with a some- what larger dose of antimony, was simply to see whether there would be found the same relative absorption of antimony by the liver and kidney as in No. 1, and if by chance there should occur a longer interval of time between the introduction of the poison and death, what then would be the relative amounts of antimony in the two organs. As stated above, the animal lived 4} hours after the administration of the poison, or 2} longer than the cat in No. 1. Following are the results of the analysis of the two organs: ‘ Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams, Li hic] eee = en Ree ee Seon 62:0 2°50 4-03 Kidneys 22-2828 220. see 14:5 0°25 1:72 These confirm to a certain extent the results of No, 1, while at the same time the smaller difference between the amount of antimony contained in the liver and kidneys, as compared with the difference found at the end of two hours (see experiment I), would seem to indicate that the liver had already absorbed its maximum amount, and that at the time of death, elimination was well under way; or in other words, that the removal of the absorbed antimony from the liver had already commenced. EXPERIMENT IV. (a.) Hypodermic injection of a solution of tartar emetic, (b.) Injection of a solution of tartar emetic per rectum. These two experiments were undertaken to ascertain whether the avenue by which the poison was introduced, would influence materi- . ally the relative absorption of the antimony. The results, however, although interesting, do not definitely answer the question. Absorp- tion by injection per rectum is so much slower than by hypodermic injection, or the effects produced are so much slower in manifest- ing themselves, that it is impossible to have the conditions exactly alike in the two cases, Either the time required to produce a given — Antimony in the organs and tissues. 285 effect, in the case of injection per rectum, will be longer than by hypodermic injection, or else the amount of poison must be corre- spondingly increased; either of which introduces an objectionable element into the experiment. (a.) Rabbit weighing 1485 grams had injected under its skin 0°80 gram of tartar emetic dissolved in a little water. Injection made at 11:35 a.m. At-3:45 p.m., 4 hours and 10 minutes after the first injection, 0°08 gram more was injected in the same manner. At 4:05 p. m. the animal died. (6.) Rabbit weighing 1512 grams had injected per rectum 0°08 gram of tartar emetic dissolved in a little water. Injection made at 11:45 a.m. At 3:50 p.m. the animal apparently not being affected at all, whereas rabbit (a) was strongly under the influence of the poison, 07160 gram more of the salt was injected per rectum as before. At 5:30 p. m. the animal was still alive, but evidently feel- ing the effect of the poison. The animal died during the night. Following are the results of the analysis of the parts from the two rabbits: Rassit (4) hypodermic injection. Sb per 100 Total weight, Weight of Sb, grams of tisssue, grams. milligrams. milligrams. UO hes ys 0 Be pet 2S 10°2 0°65 6°34 LED ea Pe Se A ee 55 0-20 3°63 bi CT te Sey eetia) ee eee a a ee 54:0 1:30 2°40 Heartand, tunes 22-72-22 - 15°5 0-30 1:93 Stomach and intestines------ 174:0 1°60 SerOr92 CULT 7ST] Ce a I Ce bd A 110°0 0°50 0:45 369°2 4°55 Rassir (b) injection per rectum. Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. Stomach and small intestines___ 172 8°89 15°30 [UES Ras Sele pa ale ned aan ene 9 0-40 4:40 Rectum and adjoining intestine. 18 0°55 3°05 LThyEie teh gh Sep i ede 3 oat nei 54 1°60 2°96 LUCIEN 9 Ree Ea 13 0°25 1:92 Miaisclomst “hs SSeS iE. esi tye te 100 1:10 gat PDO ees oI ed Se heey! J 24s 20 20 0:20 1:10 ear ARG MINES a on hy Se 17 trace 403 12°99 286 Chittenden and Blake—Distribution of Comparing first, the results obtained from rabbit (a) with those of the three preceding experiments, we see at once that the distribu- tion of the antimony is much the same as in No. 2, in which, how- ever, the animal lived nearly 22 hours after the introduction of the first dose of poison and somewhat over two hours after the last. The fatal dose, moreover, in this case was nearly the same in amount as the first dose in experiment IV a. The conditions, however, of this experiment (IV @) do not exactly accord with any of the preceding, hence close comparisons cannot well be made. The brain, as in all of the experiments with tartar emetic, contains a proportionally large amount of antimony, while the muscle contains a very small amount. The only thing in this experiment not exactly in accord with the preceding experiments, is the proportionally large amount of poison in the kidneys, as com- pared with the liver. The only apparent explanation seems to be that, the first dose being small, the liver had quickly reached its maximum absorption and elimination had been rapidly going on; so that at the end of the four hours intervening between the first and second doses of the poison, the kidneys had drawn extensively from the liver, rapidly diminishing its content of the poison. Further, after the second dose of poison, the time before death was so short that the additional absorption by the liver was not sufficient to make up the deficiency, and hence the results found. In this connection, it must be remembered that tartar emetic is very readily soluble and diffusible, and that being injected in solution directly under the skin, its absorption must necessarily be very complete and rapid. In Rabbit () the conditions are wholly different from those of the preceding experiments. In all, 0-24 gram of tartar emetic, dissolved in water was introduced into the rectum and 8-10 hours, at least, must have intervened between the administration of the first dose of the poison and death. That the stomach and small intestines should contain the largest proportional amount of antimony is perhaps not at all strange, since the antimony solution would naturally pass rapidly by osmosis through the entire alimentary tract. That this, however, is not the full explanation, is evident, when we compare the amount found in the large intestine with the former. If due simply to osmosis, the percentage amount of antimony would be about the same all through the intestines; hence we must. look to some selective action for explanation of the increased amount found in the small intestines. In all of the preceding experiments, the amount of antimony found in the stomach and intestines has been Antimony in the organs and tissues. 287 considerably greater than in the muscle tissue. Undoubtedly, the greater vascularity of the former has much to do with its greater content of the metal, but even this is not sufficient to account for all of the antimony found; for whenever blood itself has been analyzed, the amount of antimony has not been large. Unquestionably then, we must assume special absorptive action on the part of the epi- thelial cells of the stomach and small intestines. In this connection it is well to notice the work of Brinton, who proved that when tartar emetic was injected into the vein of an animal, it was very freely and rapidly eliminated by the stomach. This was also cor- roborated by Dr. Richardson who, in addition, found that a simi- lar elimination followed the inhalation of antimoniuretted hydro- gen.“ In addition, it may be that absorption of antimony from the alimentary tract goes on slowly and that hence only a por- tion was removed. This idea has considerable to support. it, when we consider the distribution of the absorbed antimony. Remembering that in this experiment, a larger amount of anti- mony was used than in any of the preceding ones, and that there was apparently ample time for absorption, one cannot help but think that the content of antimony in the remaining tissues and organs is very small. This is very evident, and must be due to one of two causes; either there has been a lack of absorption or else elimination has been going on very rapidly. The brain contains a noticeable amount of antimony, larger than found in any preceding case, while the liver and kidneys both contain a comparatively small amount. The amount of antimony in the kidneys and particularly the amount in the urine, plainly indicates that elimination was going on rapidly; but the fact that the percentage content of antimony in the liver is greater than in the kidneys, would perhaps indicate that at the time of death, absorption was not completed. Such being the case, the only inference to be drawn from the two preceding experiments, is that the introduction of tartar emetic into the rectum leads simply to a much slower absorption and distribution of the antimony than by hypodermic injection, but that there is no essential difference in the relative distribution of the poison under these two conditions. * Quoted by H. C. Wood, Therapeutics, p. 159. 288 Chittenden and Blake—Distribution of EXPERIMENT V. (a.) Tartar emetic in substance, introduced into the stomach. (.) Antimonious oxide (Sb,O,) introduced into the stomach. In this experiment there were two objects in view; one was to see the effect of tartar emetic in substance, as compared with the action of the same salt introduced into the system by hypodermic injection or per rectum; the second, to compare the absorption of an insoluble compound of antimony (Sb,O,) with that of the more soluble tartrate. In this experiment two dogs were used and the poison was fed to them at regular intervals, in small doses, for a period, in each case, of 17 days. The animals were then killed and the various parts anal- yzed.. The two experiments were exactly alike in every respect, except in the amount of poison administered. (a.) Dog weighing 7°75 kilos was fed 0°762 gram of tartar emetic during a period of 17 days, in two or three doses daily, the individ- ual doses being small enough not to induce vomiting. The first two days, the dose was 0°016 gram per day, the third 0-020 gram, the fourth 0-030 gram and so on, increasing each day until the last daily dose was 0°085 gram of the poison. The dog was then killed by chloroform, just six hours after the last dose of poison was adminis- tered. | (0.) Dog weighing 14°2 kilos was fed 2°073 grams of antimonious oxide, during a period of 17 days, in two daily doses of from 0°032 to 0°125 gram per day. The dog was then killed by chloroform, 18 hours after the last dose of antimony was given. Following are the results of the analysis of the various parts: Doe (a) with tartar emetic—(0°762 gram). Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. PaVeIe Ee ete ol ee ae 304 17°80 5°85 Salivary clamcds,s-oeeo=: 22s se 11 0°25 2°27 RGM y SU Leilene ley SET E ete 58 1:25 2°15 Bmaiina f Peps Fk poh got serene el edie 76 1:15 1°51 LOMOUO [24 eae Sees pepe da 36 0°40 144 Marseles(Ghie) eee eee ee a ee 150 1°60 1:06 PPIOEN eS een eee tek eee “29 0°15 0°80 HieGarh 22) se ere 77 0°50 0°66 Tine See eee: ee en 140 0°50 0°36 Bone (femur and tibia)----.--- 43 0:10 0:23 Bloode. center sea. Lobes 130 0°20 0°15 Westese cc co- Sena eat a eee 12 trace IPANCTOAS G. -eeee tee =- 2eces 28 trace —_— Ne) Antimony in the organs and tissues. 28 Doe (6) with antimonious oxide—(2'073 grams). Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. nivel eee same ee cee oe 452 23°70 5°24 Arena rg oe ee a RES AER EL _. 140 1°80 1°28 Muscle (fore leg) _------------ 157 1:20 0-76 [Bnaineameere hoc n8 54224 52 79 0°40 0-50 Musclei(Ghigin): a3 2-ss sta. 200 0:90 0:45 IKGIAM OY Sessa to eee Ss eo 0-10 0°12 [BIGR VARS ee sos eee ee oe 117 trace JELLO aegis Le al de te 440 trace 1667 28°10 In considering these results, we notice first that in dog (a) the dis- tribution of the poison is much the same as in the preceding experi- ments with tartar emetic, viz: the liver, kidneys and brain stand first in their content of antimony. That the liver should contain more per 100 grams than the kidneys, although the animal lived full eight hours after the last dose of poison was taken, is here to be expected, since absorption as a whole would naturally be slower than in some of the preceding experiments; and, further, in this case probably all of the antimony would be absorbed through the portal circulation. In the case of dog (6), the conditions are different from any heretofore; we have here an insoluble form of antimony con- trasted with a readily soluble and diffusible salt. Solution must necessarily be somewhat slow in this case, but the acid juices of the stomach unquestionably do dissolve and render diffusible, at least a portion of, this form of the poison. We notice first that the total amount of antimony administered, is fully three times as much as the amount of tartar emetic given, and yet the amount of antimony recovered from the different tissues and organs is but 4 milligrams more than in the case of tartar emetic. This suggests that either considerable antimony is excreted by the kidneys (more than in the case of tartar emetic) or else that consid- erable passes through the alimentary tract unabsorbed. The dog being confined in a cage of suitable construction, the 24 hours’ urine was collected on several occasions and the amount of antimony deter- mined. Thus on one day, when 0:097 gram of antimonious oxide had been administered, following after a daily dose of 0:064 gram, the 24 hours’ urine contained 13°5 milligrams of antimony (Sb). Later, at a time when the daily dose was 0°130 gram of the oxide, the 24 hours’ urine contained 22°5 milligrams of antimony. Hence it is plain that TRANS. Conn. AcapD., Vou. VII. 37 Nov., 1886. 290 Chittenden and Blake—Distribution of considerable of the antimony given was being absorbed; but bearing in mind that this latter amount was the largest excreted by the kid- neys in any one day, and further that the daily dose of antimony was being increased each day rather than diminished, it is also plainly evi- dent from the amount of absorbed antimony found, that a certain por- tion must pass through the alimentary canal unabsorbed. Further, the small amount of antimony found in the kidneys supplements this view, as does also the noticeably small amount of absorbed antimony found throughout the body, aside from the liver. One of the main objects in trying this last experiment was to see what influence the form of the poison would have on its absorption by the brain. With arsenic, it has been plainly demonstrated by one of us,* as well as by other workers in this field, that soluble and readily diffusible forms of arsenic are absorbed by the brain in appreciable quantities, while arsenious oxide for example, no matter whether taken in large or small doses, single or oft-repeated, is never found in the brain other than in mere traces. With antimony we had expected to see something of the same kind. The results, how- ever, although tending in that direction, are not quite as decisive as we should have liked. The antimony found in the brain in the anti- monious oxide case is, to be sure, considerably smaller in amount than that found in (a), although the dose of antimony given in the former was much larger than in the latter case. But it is also to be seen in the antimonious oxide case, that the amount of absorbed antimony in the brain, although very small, is still greater than the amount found in the kidneys or muséle. We attempted another experiment in the same direction with rab- bits, but as the amounts of antimony found in the brain in both ani- mals were hardly more than mere traces, the results do not give us any additional light on the matter. In spite of the fact that the experiment was a failure, so far as its main object was concerned, we venture to describe it, since it well illustrates in other respects, the greater virulence and diffusibility of tartar emetic. Two rabbits of nearly equal weight were selected, and to one potassium antimony tartrate was fed in gradually increasing doses for a period of 17 days, at the end of which time the animal died with all the symp- toms of antimoniacal poisoning. To the other rabbit, antimonious oxide was fed for the same period of time, in doses the same as given to the first rabbit; that is, doses equivalent to the antimony (Sb) contained in the tartar emetic. Each rabbit, therefore, received EE el es Se ee So 2m * See Studies from this Laboratory, vol. i, for the year 1884—85, p. 141. Antimony in the organs and tissues. 291 twice a day, the same equivalent of antimony, and at the end of the 17 days the one rabbit had taken 2°34 grams of tartar emetic, the other 1:08 grams of antimonious oxide. While each rabbit had taken the same amount of antimony, the one which had taken it in the form of potassium antimony tartrate was much more severely affected by the poison. In this case there was severe purging and finally death on the 17th day. In the case of the rabbit fed with antimonious oxide, the only apparent effect of the poison was a loss of appetite and great thirst. This animal was killed with chloro- form on the death of the first rabbit. Both forms of antimony were administered as powders, by way of the mouth, in small gelatin capsules. Following are the results of the analysis of the various parts from the two rabbits: Rapsit (a) fed with tartar emetic. Sb per 100 Total weight, Weight of Sb, grams of tissue, erams. ‘ milligrams. milligrams. DSi Te ae ek Mo 50:0 4°8 9°60 1G lla een gS BSS Ge ee ee eee perk oe 6°7 0-5 7°40 dean and. lunes) 8. 22.22.2242 18-0 0-4 2°22 Muscle from back. ....--2.4.-=- 550 0°5 0-91 iiuscle from lees... 22.2 -2 =~ 79°0 03 0:38 Pe WaIMET ris of eM i Bee he ert trace 216°4 6°5 Rassir (4) fed with antimonious oxide. Sb per 100 Total weight, Weightof Sb, grams of tissue, grams. milligrams. milligrams. ILTRYGTP SRL eaten et Pe adie ee 57:0 1:3 2:28 Muscle from back ------------ 77-0 0-7 0:90 Muscle from legs RRs Fee ae ee 100-0 0-7 0:70 mania ee eos Sate I es 2 8:0 trace earth and longs). _ 2222. -.2.2 16:0 trace [ByREnN AVES ou eee hee Ore ae 8°5 trace 266°5 ae | Looking at these results and remembering that each animal re- ceived the same amount of metallic antimony, it is evident that tartar emetic is much more completely absorbed than the oxide. With tartar emetic, however, the results are not exactly in accord with the previous ones, obtained with this salt; thus the amount of antimony absorbed by the brain is far smaller proportionally than 292 Chittenden and Blake—Distribution of Antimony,*ete. found hitherto. 'To be sure, the compound was not in the previous experiments introduced into the stomach of a rabbit in the form of powder, and it is possible that the reason for the difference in the amount of antimony found in the brain in this case and that in the brain of the dog similarly treated, lies in the fact of a slower absorption from the stomach of a herbivorous animal. XIX.—InNFivence or Antimonious OxipE on Merazorism. By R. H. Cairrenpen anv Josepu A. BLAKE. Tue physiological action of antimony has been studied mainly with potassium antimony tartrate, the form in which antimony is most commonly used therapeutically. No experiments, however, appear to have been made, even with this salt, to ascertain the influence of an- timony on the metabolism of the body. Giithgens, however, as quoted by Dr. H. C. Wood,* found in some incomplete experiments an increase in the elimination of urea after repeated non-toxic doses of antimony. It is further reported+ that antimonic acid or other preparations of the metal, when taken in half gram doses daily for about two weeks, cause a diminution in the amount of glycogen in the liver and even a total disappearance of it, and that the liver, kid- neys and heart undergo fatty degeneration. Grohe and Moslert have confirmed the latter and state that in the production of the famous fatty livers, a certain amount of the white oxide of antimony is fed to the geese daily. Aside from these facts, there appears lit- tle definite regarding the action of antimony on the physiology of nutrition. What we have, therefore, endeavored to ascertain in the present experiment is the influence of antimony on metabolism; or particu- larly, on proteid metabolism as manifested in the excretion of nitro- gen, sulphur and phosphorus. Previous experiments§ have shown that potassium antimony tartrate has a noticeable retarding action on pancreatic digestion; we have not, however, deemed it best in the present experiments to use tartar emetic, as the ready solubility and diffusibility of the compound might cause too rapid absorption and thus lead to speedy toxic action. In spite, therefore, of the fact that we have not made any experiments on the influence of antimonious oxide on digestive action, we have preferred to use the latter in the present experiments, because of its probable slower toxic action and also because it has been so extensively used as a means to induce, or to aid in the production of, fatty degeneration. * Therapeutics, Materia Medica and Toxicology, p. 156. + See Virchow’s Archiv., 1865, Band xxxiv, p. 78. + Compare H. C. Wood. Therapeutics, p. 161. § Studies from this Laboratory, 1884-85, p. 105. 294 Chittenden and Blake—Influence of Our experiments were made on a setter dog, weighing 12°6 kilos. The animal was confined in a suitable cage, so that the excretions could be collected daily without loss. The food consisted of fresh beef and crackers, together with a suitable amount of water. The beef was prepared as follows: About 40 Ibs. of fresh beef, freed from fat, tendons, ete., was finely divided by passing through a sausage machine and then dried at a low temperature until it had lost about 75 per cent. of water, and was in acondition suitable for preservation. 50 grams of this preserved meat, together with 75 grams of the sam- pled crackers, soaked in 300 ¢c.c. of water, were fed to the dog twice daily. The meat, as determined by Kjeldahl’s method, contained 12-4 per cent. of nitrogen, while the crackers contained 1°9 per cent. Hence the dog was fed daily 15°25 grams of nitrogen. On May 11th, the dog was put upon this diet and from the 17th on, the 24 hours’ urine was collected daily and analyzed. After a period of two weeks, during which daily analysis of the urine had shown a fairly constant composition, antimonious oxide was added to the diet in the quantities indicated in the table of results; the diet of course continuing the same throughout the length of the experiment. We deemed it better, as well as more accurate, to measure the in- fluence of the antimony by a daily determination of the total nitro- gen, sulphur and phosphorus of the urine, rather than to attempt a determination of urea, uric acid, phosphoric acid, etc. Nitrogen, we determined, according to the method of Kjeldahl,* modified slightly as suggested by Dr. E. H. Jenkins, of the Agricultural Experiment Station, viz: 5 c.c. of the acid urine were placed in a long pear-shaped bulb and evaporated to dryness quickly on a water bath. The resi- due was heated directly over a small flame with 10 c.c. of pure con- centrated sulphuric acid and 0:7 gram of oxide of mercury, until oxi- dation was almost complete. Then, a little finely powdered potassium permanganate was added, to render the oxidation quite complete. The solution was then diluted, an equivalent amount of potassium sulphide added to convert the mercury into sulphide, and lastly a standard solution of sodium hydroxide, after which the ammonia was driven off by boiling and collected in standard acid. Total phosphorus and sulphur were determined as follows : 50 c.c. of urine were evaporated in a capacious silver dish with 10 grams of potassium hydroxide and-10 grams of potassium nitrate and the resi- * Neue Methode zur Bestimmung des Stickstoffs in organischen Koérpern. Zeitschrift fiir analytische chemie, xxii, 366. Antimonious Oxide on Metabolism. 295 ‘due heated carefully until the organic matter was completely oxidized. The fused mass was then dissolved in water and diluted to 250 c.c. Of this, 100 ¢.c., representing 20 c.c. of the original urine, were used for the sulphur, while the second 100 c.c. were used for the phosphorous, determination. For sulphur, the 100 cc. were acidi- fied with hydrochloric acid and evaporated to dryness on a water bath in order to remove all nitrate and nitrite. The residue was then dissolved in water acidified with hydrochloric acid, and the sulphuric acid precipitated with barium chloride in the usual manner. For phosphorus, the 100 c.c. were acidified with nitric acid, evaporated to dryness, the residue dissolved in water, acidified with nitric acid and the phosphoric acid precipitated with molybdenum solution. This precipitate was then dissolved in a dilute solution of ammonia, the phosphoric acid reprecipitated as ammonio-magnesium phosphate, and the phosphorus finally weighed as magnesium pyrophosphate. Chlorine was determined volumetrically in the usual manner, with a standard solution of silver nitrate, after destruction of the organic matter by fusion with potassium nitrate, etc. The results, expressed in grams per 24 hours, are shown in the ac- companying tables. The 24 hours’ urine represents the quantity passed from 9 A. M. of one day to 9 a. M. of the next. As, however, the ani- mal was not always regular in its passage of urine, it frequently happened that the quantity on one day would be very small, while on the next it would be correspondingly increased, without any change in specific gravity, and with a daily average corresponding to the normal, as for example on May 25th and 26th. In order, therefore, to obviate the difficulty which this irregularity tends to introduce into the results, we have added to the table a daily average of each three days results; a study of which shows plainly that antimonious oxide, in the present experiment at least, does not have any noticeable influence on the excretion of any of the ele- ments determined. Numerically, there is a slight increase in the amount of nitrogen excreted during the taking of the antimony, but the increase is noticeable only in the grand average and is altogether too small to be of much significance. Further, it is to be noticed that the average for the two series does not show any corresponding in- crease in sulphur. If antimony causes an increased excretion of nitrogen, it means an increase in proteid metabolism, which should in turn give rise to an increased excretion of sulphur and phosphorus. It is to be noticed in the daily results, that the excretion of sulphur nd phosphorus runs parallel with the excretion of nitrogen; an in- ed Chittenden and Blake—Influence of io | ‘JSOT SBAA OULIN OY] JO wonj10d @ PSs PU} UO x ee 1964-0 ST G40 LhT8-0 687-61 T-8¢0T GET =. 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Oxide on Metabol LNONLOUS Ant ‘agtjodde 100d UI PUB [JOMUN YeYMoUIoS poumvses Sop oy} sv ‘pozATVUR JOU SBA OULIN oY} oUNL JO pe oy} pue ABT JO IGS OY} UdIMgoq + ! 6699-0 8049-0 TG640 ETT E4601 997 ~* “SOLOS 0} 1OJ osvicay 9908-0 9919-0 GLGKO O98 GT 8-£60L QO RON ea eS eseroae AEG at 1 g98e-0 Faee-0 099-0 =| «= SOE TT G.860T ih 2 “prow ‘g 668-0 6609-0 SELL-0 | POL GL ¢-L60T G8P *‘proe v BL8T-0 0989-0 6978-0 PlG- ET ¢-LG0T 00S ‘prow ‘g foung TL99-0 2999-0 8669-0 TTé-1T 4.8601 CUT a Alec ae esevroae ATE IT¥S-0 LEg9-0 TePL-0 FOF: TT 0-860T ccPy ‘pre “6% GGPFS-0 &P69-0 6L19-0 66S-0T 08601 VIP ‘proe "86 1619-0 8889-0 6LEL-0 889-TI ¢-6601 O&P ‘proe ‘Le | Ae "SULBIS “TBS “WURLS “URLS ‘SURI yas) enieetes “OUTLOTYO sanydjng “‘snaoydsoyg “NOS0.11N wy dg “OuUIN[O A. “MOTOR ‘ajeqd “panuiqzuoo—KNOWIINY LAOHLIAA Nov., 1886. 38 TRANS. Conn. AcaD., Vou. VII. Chittenden and Blake—Influence of 298 i) ———ernrerenr Soo “ueyBy °O*S jo qunomy 1864-0 T919-0 8964-0 GOL-8E $8601 G07 GOR Caer, advise ATE OCPE-0 LL6S-0 T8TL0 068- TT 0-860T VoV *‘proe “AL 8689-0 CLE9-0 8901-0 888- TT ¢- L601 ULP ‘prow ‘Or LOSP-0 6&89-0 922-0 189-61 0-080T C&P ‘pre 6 oun 4689-0 6199-0 90440 780-61 8-8G01 Sy (aie aoe aaa asevione Are 1989-0 O8LL-0 9F18-0 966-81 ©. 9801 09S ‘poe | *8 1097-0 8t0¢-0 1969-0 090-01 0-080T OSE ‘prow h 2 | 169-0 6719-0 T106-0 896-61 0-0801 067 ‘proe ‘9 oune “me.LO “TBI TUBA ‘stuvis 0 'O “OULOTYO rnqdjng ‘snaoydsoyg “U0SO1}IN, “9 ‘dg *OumNyO A. ‘uoyoRey ‘9yB ‘AdIXOQ SQOINOWILNY HII. Cee eS ee SS eee 299 Antimonious Oxide on Metabolism. L0¢-0 4869.0 6664-0 8Z0-6I 9-8G01 897 |“ ""SOLIOS 04} LO] oSvIOAW TITS-0 6679-0 1519-0 969-11 9-801 OV tee a gare aseroae ATE a t 9BEP-0 1689-0 L6L¢-0 86-01 0-L80T Ser ‘ploe ut 0-1 t EGPS-0 P1F9-0 P99L-0 GLL-8T 0-880T 78h ‘prow ‘91 a 29¢e.0 8819-0 089-0 LLP IT ¢.0801 F0F ‘poe ‘ey ounr | 6987-0 9969-0 8944-0 L68-61 $8601 SOEs alia ease aseroae ATE ae | TAG8-0 1868-0 06PL-0 006-1 ©-880T SLP oureyye | “FL an | CL6S-0 LOge-0 0889-0 CF9-01 0-2L601 OIF ‘ploe ‘81 OF t 988-0 STSL-0 068-0 8F9-81 0-080T P0¢ ‘prow ‘gE oune ees 0 Wes “TURIS “TBI “‘SUIBIS 0 "0. ea) OAs ‘QUILOTYO rnydyng ‘snioydsoyg “UOSOIIN "rp “dg “OUINTO A “MOL}OROY “oye jo janowy “‘panuijuod—aAdIXQ SQOINOWIINY HLIAA 300 Chittenden and Blake—Influence of Antimonious Oxide, ete. crease in the latter is always accompanied by an increase in the two former. In the grand average of the results, however, the slight in- crease in nitrogen is not accompanied by a corresponding increase in sulphur. In fact, the two series of results, indicate plainly that the antimony was without any material action. The total amount of an- timony given, 16 grains of the oxide during 13 days, was certainly sufficient in quantity to have exerted its peculiar influence if possessed of any. The antimony was certainly absorbed, and that too in con- siderable amount. Thus on the 11th of June the 24 hours’ urine con- tained 13°5 milligrams of metallic antimony; on the 17th, 22°4 milli- grams; on the 18th, 17°6 milligrams and on the 20th of June, 15:1 milligrams of metallic antimony. These quantities of absorbed anti- mony would certainly indicate the presence of sufficient antimony for some decided influence on metabolic action, if any existed. The amount of nitrogen excreted daily, is seen to be considerably below the amount of nitrogen ingested. We did not make daily examina- tions of the fecal matter, but such as were made showed plainly that the deficiency in nitrogen was contained mainly in the feces; thus on the 5th of June the 100 grams of feces excreted, contained 2°42 grams of nitrogen. At that date, the average amount of nitrogen excreted by the urine was 12°36 grams per day ; this amount, added . to the fecal nitrogen makes a total of 14°78 grams excreted, against 15°25 grams ingested; a difference to be found mainly in the hair thrown off, and in part, in the ordinary errors of analysis. We must conclude, therefore, that small repeated doses of anti- monious oxide are without influence on the excretion of nitrogen, sulphur and phosphorus, and that consequently this compound, at least when taken in non-toxic doses, has no action on proteid meta- bolism. XX.—On Some Meratiic Compounps oF ALBUMIN AND Myosin. By R. H. Currrenpen anp Henry H. Wurrenovuse, Pu.B. Ever since Lieberkiihn, in 1852, attempted to establish the molecu- lar weight of albumin by preparing and analyzing the copper com- pound resulting from the action of a soluble copper salt on a solution of egg-albumin, various investigations have been published bearing on the nature and composition of the compounds of albumin with the heavy metals. Before this time even, F. Rose, in 1833, had published an analysis of a copper compound of albumin in which he had found from 1°50 to 1:70 per cent. of cupric oxide, and Mitscherlich, in 1837, published an analysis of a similar albumin compound, in which he found from 2°8 to 3°3 per cent. of cupric oxide, while Lieberkiihn’s compound contained 4°6 percent. CuO. Compounds of albumin with other metals have also from time to time been prepared, such as zine, lead, silver and mercury, and in one or two cases provisional formule have been given. The results, however, are to be considered as quite uncertain. With platinum chloride a compound appears to have been formed* of somewhat more certain composition. Aside from the more recent experiments of Ritthausent on the vegetable -albumins (gluten-casein, legumin, etc.), egg-albumin has been the chief albuminous body experimented with, and copper the main metal. ; Recent work by one of us (C) on the albumose and globulose bodies, together with work on the products formed from casein and myosin, has led to a partial study of the metallic compounds of these bodies. As a preliminary, however, we found it necessary to study a few of the compounds of egg-albumin, and as the results thus obtained were not in accord with the more recent results of Harnack{ we have continued our work with egg-albumin and with myosin, the results of which we now present. * See Commaile, Moniteur Scientifique, 1866, and Fuchs, in Annalen der Chemie, vol. cli, p. 372. + Die Eiweisskérper der Getreidearten, ete. Journal fiir prakt. Chem., vol. xii, p. 361. ¢ Untersuchungen iiber die Kupferverbindungen des Albumins. Zeitschrift fiir phy- siologische Chemie, vol. v, p. 198. 302 Chittenden and W hitehouse— Metallic I. Ea@e-aALBuMIN. (a) Copper Compounds. In looking over the literature of the subject, it becomes evident at once that the older investigators, owing either to the nature of the compound, to adherent impurities or to faulty methods, were not able to obtain concordant results, since the copper compound of egg- albumin, as prepared and analyzed by six distinct investigators, was found to contain from 1°50 to 5°19 per cent. of CuO. In all of these cases the preparation of the copper compound was essentially the same; a solution of egg-albumin was precipitated with a solution of a copper salt, the precipitate collected, washed thoroughly with water, dried, and the copper determined by simple ignition. Naturally this method, as suggested by Harnack, might be expected to give too high results, since the copper precipitate would unquestionably re- tain considerable of the inorganic matter of the albumin. Treated in this manner, however, F. Rose,* as already stated, found the copper compound to contain from 1°50 to 1°69 per cent. of CuO. Mitscher- lich,t who held that the copper precipitate was a compound of egg- albumin with the copper salt, found in his preparations 2°8—3°3 per cent. CuO, while Bielitzki,t who demonstrated that the precipitate was an. actual compound of albumin with cupric oxide, found in his preparations 4°75—5'20 per cent. of CuO. Lassaigne, as quoted by Harnack, found 4°95 per cent. of CuO, Mulder§ 4:44 per cent., while Lieberktihn’s|| preparation contained 4°6 per cent. of CuO. Further, Ritthausen’s copper compounds of the vegetable albumins were found to contain from 11°5 to 17°0 per cent. of CuO. These results collectively, would therefore seem to indicate that when egg-albumin is precipitated by a soluble copper salt, the resulting compound does not contain a defi- nite proportion of albumin and cupric oxide, or else that there are a large number of albumin-copper compounds. More recently, how- ever, E. Harnack,§ from analysis of fifteen separate preparations, comes to the conclusion that there are two distinct compounds of albumin with copper; one containing 1°35 per cent. of Cu, the other 2°64 per cent. of Cu, indicating as Harnack suggests, a copper albu- minate in the first case of the formula C,,,H,,,N,,0,,5,Cu, in which Cu replaces two atoms of hydrogen in the albumin molecule, and in the second case, an albuminate of the formula C,,,H,,,N,,0,,5,Cu,, in * Pogeendorff’s Annalen, vol. xxviii, 1833. + Miller’s Archiv. for 1837, p. 91. { Dissertation, Dorpat, 1853. § Physiologische Chemie, 1844-51. || Poggendorff’s Annalen, vol. Ixxxvi, 1852. f Loc. cit. Compounds of Albumin and Myosin. 303 which two atoms of Cu replace four of hydrogen. The results ob- tained by this investigator would certainly seem to warrant this con- clusion, for the analytical data of the different preparations show but slight variations ; 1°34-1°37 per cent. in the one case, and 2°48—2°74 per cent. in the other. Further, Harnack worked with nearly ash-free preparations, the compounds after their first precipitation and wash- ing being dissolved in sodium carbonate and reprecipitated by care- ful addition of acid. By repeating this process several times, the ash of the preparation was almost entirely removed, while the relative proportion of copper and albumin was not affected. As to the condi- tions which determine the formation of one or the other compound, there seems to be little definite other than that in general, the com- pound with smaller content of copper was obtained when the precipi- tation took place in the presence of a slight excess of albumin, and the compound with larger content of copper when in the presence of an excess of the copper salt. In no case were the copper salt and albu- min solutions mixed in definite proportions, yet in every case one of the two compounds was formed; further, Harnack states that when an amount of copper salt exactly sufficient to form the albuminate is added to a given quantity of albumin, no precipitate results; in other words an excess of the copper salt is necessary to insure a separation of the compound. Harnack’s results, therefore, differ from those of the preceding in- vestigators in that definite compounds appear to have been formed in every case, and further, in that the compounds contain a lower per- centage of copper than found by any other investigators aside from F. Rose. This latter, it will be remembered, found 1°50-1°69 per cent. of CuO ; 1°69 per cent. being equal to 1°34 per cent. of Cu, one of the percentages found by Harnack. Harnack further states that the average of the analyses made by other investigators, aside from Rose, show about 4°4 per cent. of CuO, and assuming that the various prep- arations contained an amount of ash equivalent to about 1 per cent. (which amount Harnack found in his preparations before purification) the percentage amount of cupric oxide would be reduced to about 3:4 =2°7 per cent. Cu, or the amount found by Harnack in his highest cop- per compound. But as Rose’s preparation was made by the simple ad- dition of an aqueous solution of egg-albumin to the copper salt and the copper determined as oxide by simple ignition, it would seem neces- sary to make the same deduction of 1 per cent. also in this case, which would make Rose’s compound contain far less CuO than found 304 Chittenden and W hitehouse— Metallic by Harnack. Further, Rose* found that the serum of ox blood yielded a similar compound with cupric sulphate, which contained only 1:14 per cent. of CuO or 0°88 per cent. of Cu. Hence there would seem to be little in these earlier investigations to substantiate the results obtained by Harnack. Mérner,+ however, working with alkali-albuminate, found that on precipitating a solution of alkali-al- buminate with cupric sulphate, in the presence of an excess of alkali, he obtained a copper albuminate containing a percentage of cupric oxide corresponding closely with that found by Lieberkiibn. When, on the other hand, he precipitated a nearly neutral solution of alkali- albuminate with cupric sulphate, then the percentage of copper in the copper albuminate amounted to only one-third that found by Lieber- kiihn, or an amount about equivalent to that found by Harnack in his lowest copper compounds. Moérner further found that by precipitat- ing a calcium albuminate solution with cupric chloride, the albumin- ate combined on an average with 2°33 per cent. of CuO, or just one- half the amount required by Lieberkiihn’s formula, and considerably less than the amount contained in Harnack’s largest copper com- pounds. . Preparation of the albumin solution.—In our experiments it was thought best, as far as possible, to avoid exposing the albuminate to the action of alkalies, hence especial care was taken to prepare the egg-albumin as free from salts as possible, so that it would not be necessary to purify the albuminate by reprecipitation. The whites of a large number of eggs were finely divided by scissors and by shaking with glass, then mixed with an equal volume of water and thoroughly shaken with air, after which the solution was strained through cloth. Globulin was then precipitated by the addition of dilute acetic acid (the acid added as long as a precipitate formed), the solution finally filtered through paper, after which the filtrate was made exactly neutral with sodium carbonate and again filtered. The fluid so obtained was then dialyzed in running water for many days, a little thymol being added to prevent putrifaction. The fluid finally obtained was perfectly neutral, clear and contained but a small amount of inorganic salts. In forming the albuminate we employed both cupric acetate and cupric sulphate, using in each case the same volume of albumin solu- tion, but varying the amount of copper salt. The copper salt was generally added as long as a precipitate formed. The albumin- * Loc. cit., p. 139. + Jahresbericht fiir Thierchemie, 1877, p. 8. Compounds of Albumin and Myosin. 305 ate was washed on a pump with water, until no reaction could be obtained with potassium ferrocyanide for copper or with acetic acid and potassium ferrocyanide for albumin. The preparations were dried at 100° C., then powdered and further dried at 110° C., until of constant weight. The percentage of copper was first determined by simple ignition and weighing as cupric oxide. The oxide was then dissolved in dilute nitric acid, the copper precipitated as sulphide with hydrogen sulphide and weighed as subsulphide by ignition in hydro- gen gas with a little sulphur. Each series was made from a distinct preparation of albumin and nearly every compound made, was analyzed in duplicate. Following are the analytical results : Sertss L. With CuSO,,. No. Am’tsub.taken. Wt. CuO. Per cent. Cu. Wt. Cu.8. Per cent. Cu la 0°5621 gram. 0:0081 gram. 1:13 0:0070 gram. 0:97 b 0°5176 0:0078 1:19 0:0065 0:98 With Cu(C,H,0,),. 2a 0°5479 0:0088 1:27 ee x a b 05703 0-0091 1°26 0:0078 1:08 Seriss IT. With CuSO,, la 0:5273 gram. 0.0067 gram. 1:00 0-0053 gram. 0:79 b 0-7075 0:0083 0:93 00068 0:76 With Cu(C,H,9,).. 27 0:5697 0:0081 TeAg 0:0071 0:98 b 0:5005 0:0073 1:15 0:0061 0:95 Series III. With CuSO, la 0°6235 gram. 0°0085 gram. 1:07 age Sah b 0°6705 0:0091 1:07 0-0088 gram. 1.04 With Cu(C,H,O,).. 2a 0°8302 0:0130 1°24. 5 Ae E Tae b 0°9400 0°0151 1-27 Lane Trans. Conn. AcAD., Vou. VII. 39 Nov., 1886. 306 Chittenden and W hitehouse—Metallic Series lV. With CuSO, No. Amt. sub. taken. Wet. CuO. Per cent. Cu. Wt. Cu.S. Per cent. Cu. la 0:3621 gram. 0:0047 gram. 1°02 0:0038 gram. 0°82 b 0°3478 0:0046 1:03 00039 0°89 2a 0°4318 0-0060 1:08 00040 0-71 b 0°3288 00047 1°12 00033 0-78 3a 04083 00060 1°15 0:0047 0:90 b 04332 (0):0064 1iy 00053 0-96 4a 0°3874 00047 1:09 00042 0:97 b ():4289 00062 1:14 00050 0°90 With Cu(C,H,0,).. 5a 0°6358 ~ * 0-0090 Pela. 00073 0-91 b 0°5147 00071 1:08 0:0064 0:99 6a 0°5321 0-0075 1:12 00065 0°95 b 05125 00073 1:13 okt. ao, Ta 0-7260 00125 1:36 Lee Bhat b 06916 0:0120 1:37 0:0100 1°15 Series V. With CuSO. 1a 0:4838 gram. 0°0083 gram. 1°36 0:0074 gram. 1°21 b 0°5083 00091 1:41 ate ree With Cu(C,H,0,),. 2a 0°5044 00085 1:32 apne soe b 0°5042 00084 1°32 From the analyses of these 15 preparations it is to be seen that the percentage amount of metallic copper, determined as oxide by simple ignition, amounts on an average to 1°17 per cent. When, however, the copper is determined as subsulphide, by precipitation with hydro- gen sulphide, and thus obtained free from ash, the percentage amount of copper falls on an average to 0°94 per cent. Cu. The preparations thus contain 0°23 per cent. of ash. We were not able to obtain any copper compounds with a much smaller content of ash than this, except by the use of methods which appear to affect the composition of the compound. a Compounds of Albumin and Myosin. 307 Harnack states* that he was able to obtain the copper albuminate quite free from ash by dissolving the freshly precipitated albuminate, after it had been thoroughly washed, in sodium carbonate, filtering and reprecipitating the compound by careful addition of acid. By repeating this process several times the adhering inorganic matter was entirely removed. It seemed questionable, however, whether this treatment might not induce some alteration in the compound. The two following series of experiments were tried with the inten- tion of throwing some light upon this point. Series VI. With CuSO, | No. Am’t sub. taken. Wt. CuO. Per cent. Cu. Wt. Cu.s. Per cent. Cu. la 0°6320 gram. 0:0091 gram. 1°15 0:0080 gram. 0:99 b 0-6980 0:0099 1:13 0-0086 0:98 2a 05428 0:0077 1:12 0-0070 1-01 b 05121 - 00077 1:19 0-0061 0-95 3 0°3146 00080 2°03 0-0068 acral With Cu(C,H,O,).. 4a 0°6105 00085 are 0-0075 0-96 b 05836 0:0078 1:06 0-0072 0:99 5a 05996 00087 1°15 00081 1:06 b 05592 00082 1:16 0-0069 0:99 6 0:4139 0-0131 2°51 0-0115 2.19 In this series of experiments all of the preparations, as before, were made from the purified albumin. Nos. 1, 2, 4 and 5 were, after pre- cipitation, simply washed with water until the washings gave no reac- tion either for copper or albumin. No. 3, after being washed in a similar manner, was dissolved in very dilute sodium carbonate and re- precipitated by neutralization with dilute hydrochloric acid. No. 6 was dissolved up twice in this manner and both preparations were finally washed free from chlorine. A glance at the analyses shows plainly that this treatment has tended to increase the percentage amount of copper in the albuminate; due, doubtless, either to with- drawal of a portion of the albumin by the sodium carbonate, or else to a partial dissociation of the eee by the long continued wash- ing with water. The first reprecipitation has apparently increased the amount of copper in the compound fully 0°7 per cent., the second reprecipita- tion 0°5 per cent. more. * Loe. cit., p. 202. 308 Chittenden and W hitehouse—Metallic Srriges VII. With CuSO,. No. Am’t sub. taken. Wt. CuO. Per cent. Cu. Wt. Cus. Per cent. Cu. la 0°6326 gram. 0:0091 gram. 1:13 =e ttt b 06156 0-0086 1°10 pe ies 2a 04054 0-0081 1:60 0:0063 gram. 1°23 b 0°4995 00099 1°58 0-0074 1-19 3a 0:3870 0-0068 1:39 00049 1-02 b 03702 00066 1:40 With Cu(C,H,0,),. 4a 06175 00085 1:08 00074 0°95 b 06069 0)-0082 1:07 0-0077 1:00 5a 0°3282 00072 1°73 00055 1°34 b 0°3755 0-0088 1°75 Eee ose 6a 0°7508 00132 1°39 00104 1:10 b 0°7016 0°0121 1°38 This series was prepared in the same manner as the preceding. Nos. 1,3, 4 and 6 were simply washed with water, while No. 2 was reprecipitated once and No. 5 twice, and both ultimately washed free from all soluble matters. The results show here the same increased percentage of copper, although not so marked as in the preceding series, when the albuminate is dissolved in sodium carbonate and reprecipitatéd. Further, the percentage of ash is not, as a rule, materially changed by this process; thus in No. 2, where the albu- minate was reprecipitated once, the difference in the percentage of copper as determined by simple ignition and by precipitation as sulphide, amounts to 0°37 per cent., while in No. 3a, where the compound was not reprecipited at all, the difference is exactly the same. In precipitating the albuminate, there is formed in the fluid a small amount of either sulphuric or acetic acid. Harnack, to avoid this, states that it is better, after adding the necessary amount of cupric sulphate to the albumin solution, to exactly neutralize the mixture with sodium carbonate. If, however, the greatest care is not exercised and excess of cupric sulphate avoided, even partial neutralization of the fluid will result in the precipitation of a portion of the copper and thus show an apparent increase in the copper of the albuminate. So far, however, as our results show, the small amount of sulphuric acid libera- ted in the formation of the albuminate does not affect the character of the compound. In the following series, after each precipitation, Compounds of Albumin and Myosin. 309 the mixture was made as near neutral as possible with dilute sodium carbonate and the compounds then filtered and washed thoroughly with water. The copper in this series was determined simply by ignition. Series VIII. With CuSO,. No. Am’t sub. taken. Wt. CuO. Per cent, Cu. la 0:4273 gram. 0.0068 gram. 1:26 b 0:3867 0:0063 1:29 2a 0°3167 0:0049 1°23 b 0)°3535 0-0052 1:18 3a 0°2418 0°0080 1:00 b 0:3079 0-0089 1:00 4a 06210 0:0188 1:78 b 0:7091 0:0158 1:78 The results plainly show no appreciable difference in the composi- tion of the albuminate under this change in the conditions, unless in No. 4 where a larger amount of copper is found than usual. It is our opinion, however, that the small amount of acid liberated by the re- action is not sufficient to cause any especial change in the character * of the albuminate; neither, probably, does very dilute sodium carbon- ate in itself change the substance to such an extent that on neutrali- zation it is not precipitated in nearly its original form, or at least that the action in this case is not any greater than that produced by water alone. In fact we are much inclined to the view that the long continued action of water will gradually but surely affect the composition of the albuminate, and that doubtless the change in the composition of the compound noticed in our experiments on solution of the substance in sodium carbonate and reprecipitation is due to the combined action of the alkaline fluid and of water. Harnack states that week-long treatment of the freshly precipitated albuminate with water will gradually cause dissociation of the compound, but that it can be easily and thoroughly washed without any decomposition whatever. Our experience, however, leads us to question the correctness of this view. Ordinarily, it has taken us an entire day to completely wash the freshly precipitated albuminate, so that the wash-water should give no reaction whatever for copper or albumin. In precipi- tating the albumin solution with cupric sulphate, the albumin never appears to be completely precipitated and at the same time, as Har- nack has observed, it is necessary to add more than the proportional 310 Chittenden and Whitehouse—Metallic amount of copper salt to obtain any separation of the albuminate. As a result, the filtrate contains considerable albumin and copper, but even after several hours washing on the pump (the filtration is slow — at the best) the precipitate still gives up traces of albumin, as shown by acetic acid and potassium ferrocyanide, long after all traces of copper have disappeared. It is not impossible to wash the compound and reach a point where the wash-water contains neither copper nor albumin, but when the washing goes on slowly and the water remains more or less in contact with the albuminate for 24 hours, then fre- . quently the washings will show traces of albumin continuously, with- out our being able to reach a point where the test fails to give any reaction whatever, or to show any special change in the intensity of the reaction. EL The following series of experiments would appear to substantiate this view. The first six were washed‘for about twelve hours, when no copper reaction could be obtained in the washings and only the slightest reaction for albumin. The last six were washed for sixteen hours, and finally stood over night on wet filters with more or less water on them. At the end of this time, the washings continued to show a reaction for albumin with acetic acid and potassium ferro- - cyanide, and indeed the reaction appeared to increase rather than . diminish in intensity on further washing. The washings contained no copper. Following are the results of the analyses : Series IX.— With CuSO, No. Amt. sub. taken. Wt. CuO. Per cent. Cu. 1 0-4207 gram. 0:0052 gram. | 0-99 2 0:4676 0:0058 0:98 3 0:4881 0:0062 1:00 4 0°3162 0:0087 0°91 5 0°2882 0: 0032 0:89 6 0°1857 0:0024 1:02 7 0:2892 0:0050 1°34 8 0°4137 0:0060 ais} 9 0°4077 0:0060 1,18 With Cu(C,H,0,),- 10 06067 0°0126 1°64 11 06889 0°0111 1°27 12 0°7126 00109 1°22 Compounds of Albumin and Myosin. 311 While the difference is not very great, it is a constant difference, and it is to be remembered that the last six compounds differ in no respects whatever from the first six, except in being subjected to the longer action of water. In comparing now these different results, it is seen that we have not been able to obtain a copper albuminate with a higher content of Cu than 2°19 per cent., and this only as a result of two reprecipi- tations ; a condition, which, from our experience, tends to alter mate- rially the composition of the original precipitate. The average of the results obtained by simple precipitation, show a content of 0°94 per cent. of Cu. A study of the individual results, however, shows too great a variation to believe wholly in the existence of a single, stable copper albuminate. Either there are one or two definite com- pounds, which, being more or less unstable, are prone to change under varying conditions and thus give rise to the variations in the content of copper noticed, or else there are a number of definite compounds liable to be formed as the conditions are varied, all of which, how- ~ ever, must be more or less unstable. Glancing over the individual results, it is plain that an amount of Cu approximating to 0°96 per cent. is found altogether too frequently to be the result of chance. Doubtless this figure represents most closely the content of copper in the ordinary copper albuminate obtained by simple precipitation, while the majority of the variations from this figure are due mainly to dissociation. Taking Lieberkiihn’s formula of albumin, the following copper albuminates would be possible: (Cr2Hi12NisSOe2)3 +Cu— H2=1'29 per cent. Cu. (CrsH2N1eSOn2).+Cu—H.=0'96 “ (CroH112N1e8Oo2)5+Cu—Hy=0-77 For the first, in which Lieberkiihn’s formula for albumin is treb- led, the percentage of copper corresponds nearly to the lowest results obtained by Harnack, while in the second formula the percentage of copper accords closely with the average of our results. Whether the weight of the albumin molecule is represented more nearly by the second formula than by the first we have not sufficient data to determine, but certainly our results with the copper albuminate show a lower percentage of copper than would correspond with the first formula. , Further, it would appear that the copper albuminates are readily prone to change under slight provocation and that this point, in part, undoubtedly explains the reason for the great variation in the results obtained by so many workers. 312 Chittenden and W hitehouse— Metallic (b) Lead Compounds. Lieberktihn* states that the lead salt of albumin cannot be obtained pure; that the insoluable precipitate formed by the addition of either lead nitrate or basic lead acetate to a solution of albumin, is simply a mixture. With basic lead acetate, Lieberkiihn obtained a precipitate containing 17°86 per cent. of lead oxide, while the precipitate formed with lead nitrate contained 12°78 per cent. of lead oxide. With protein, Muldert obtained precipitates on the addition of neutral lead acetate and lead nitrate, which contained respectively 12°45 and 12.68 per cent. of lead oxide, while basic lead acetate gave a precipitate containing 30°63 per cent. of lead oxide. Berzelius{ states that neutral lead acetate precipitates both albumin and blood serum, but that the greater portion of the albumin remains dissolved in the fluid united with acetic acid. Basic lead acetate on the other hand precipitates the albumin completely. These last statements accord with our own results; with a neutral lead salt only a small precipitate was obtained, the compound being soluble apparently in both excess of the lead salt and of albumin, while with basic lead acetate the albumin seemed completely precipi- tated. Further, Berzelius|| states, on the authority of Mulder, that if a solution of potassium albuminate be made as neutral as possible with acetic acid and then precipitated with lead nitrate, the lead albu- minate so obtained contains on thorough drying 5°84 per cent. of lead oxide. Following are some of the results of our analyses. The compounds were made from thoroughly dialyzed albumin and were washed free from both lead and any excess of albumin. The preparations were dried at 110° C. until of constant weight and the lead was deter- mined first by simple ignition, with addition of a little ammonium nitrate. The lead oxide, after being weighed, was then dissolved in dilute nitric acid, the solution evaporated to a small volume, the lead precipitated with a little sulphuric acid, two volumes of alcohol added, and the lead sulphate finally filtered and washed with 95 per cent. alcohol. The sulphate was then ignited with proper precautions and from the weight obtained, the percentage of lead again calculated. * Poggendorfi's Annalen,. Band lxxxvi, p. 124. } Lehrbuch der Chemie, Berzelius, ix, p. 29. $ Lehrbuch der Chemie, ix, p. 43. || Lehrbuch, ix, p. 49. Compounds of Albumin and Myosin. 313 Series I. With neutral lead acetate. No. Amt. Sub. taken. Wt. PbO. Per cent. Pb. Wt. PbSO; Per cent. Pb. la 0:4972 gram. 0:0188 gram. 3°49 0:0209 gram. 2°85 b 0°3992 0:0150 3°48 0:0166 2°88 Serres II. With neutral lead acetate. 1a 0°5341 0:0183 3°16 0:0216 PaaS) b 0:°5881 0:0198 3°40 0:0222 2°80 With basie lead acetate. 2a 0-5832 0-0460 7°30 0-0580 6-77 b 0:5008 0:0391 ee, 0:0487 6°62 Series. III. With neutral lead acetate. la 0°8057 0:0262 3°01 00274 2°32 b 0°6522 0:0211 2°98 0:°0226 2°36 With basic lead acetate. 2a 0:'7290 0:0593 7-55 0:0615 5°74 b 0°7630 0:0631 7°66 0:0609 5°45 Series IV. With neutral lead acetate. la (0)°4121 0:0152 3°42 0:0186 2°25 b 0°4823 0-0176 3°37 0-0178 2°50 With basic lead acetate. 2a 0°7234 0:0651 8°34 b 05365 00482 8°33 Series V. With a large excess of basic lead acetate. la 0°6822 0°2119 Ps ito} hia gilli aan tae = b 0°5429 0°1714 PUSeS sue aye co Sect 2a 05836 071928 BOZDGRN. aden ese esd Eee b 0°5913 0-1960 BOsiomeear :’ Marte SL 27 ee 3a 0°5478 0:1896 Or almm ey ee ee pee b 0°5427 0°1878 POO is ite SL 222 TRANS. CoNN. ACAD., VoL. VII. 40 Nov., 1886. 314 Chittenden and W hitehouse—Metallic These results plainly indicate that more than one compound of lead is formed, especially so with basic lead acetate, the composition being dependent in this case on the amount of lead salt added. With neutral lead acetate, the variations in composition are not so marked and as it is hardly possible to prepare a lead albuminate free from salts, or to eliminate them wholly in the calculations, it is question- able how far the results should be trusted, except in a general way. The formula (C,,H,,,N,.SO,,),+Pb—H, would require 3°10 per cent. Pb, while (C,,H,,,N,SO,,),+Pb—H, would require 2°50 per cent. Pb. In the case of the albuminate formed with basic lead acetate, it is to be noticed that the compound made by the addition of a large excess of the lead salt, contains about five times as much lead as the ordinary basic lead compounds. (c) Lron Compounds. F. Rose* has made iron albuminate, both from egg-albumin and from the serum of ox-blood, by the simple addition of ferric chloride to the albumin solution. Two preparations made from egg-albumin yielded respectively 2°79 and 2°88 per cent. of ferric oxide. Rose found the albuminate, when freshly precipitated, easily soluble both in excess of ferric chloride and in excess of the albumin solution. Our preparations were made wholly from dialyzed albumin, and when so prepared and thoroughly washed the compound was found almost wholly free from adhering salts, so much so that after a few trials we deemed it unnecessary to make the determinations of iron other than by simple ignition and weighing as ferric oxide. . Following are some of our results: Series I. With Fe,Cl,. No. Amt. sub. taken. Wt. Fe.Os3. Per cent. Fe.O;. Per cent. Fe. la 0°7033 gram. 0 0094 gram. 1°33 : 0:92 b 06430 0-0086 1:33 0°93 Seriss II. 1a 04683 0-0052 111 0°76 b 0-3854 0-0042 1-08 0-75 * Poggendorff’s Annalen, xxviii, p. 140, 1833. Compounds of Albumin and Myosin. By Srriss III. No. Amt. sub. taken. Wt. Fe.Os. Per cent. Fe.O;. Per cent. Fe. la 0:4710 0:-0071 1:50 1:04 0°53829 000838 1°55 1:08 2a 07387 0:0100 1°36 0:94" 0°6355 00087 1:36 0°94 Series IV. la 04862 0:0063 1°30 0-90 b 0°5558 0:0073 1-31 0:91 2a OF oO 00070 1°36 “95 b 0:4954 0-0066 1:33 92 3a 04505 0:0068 1:39 0-97 b 0-4610 00066 1°43 0-99 4a, 0°4817 0-0071 1:47 1:01 b 04571 00065 : 1:44 0-98 5a 04819 00065 1°34 0-93 b 0°3846 0-0051 1°33 0°93 6a 04086 0:0059 1°45 1:00 b 03392 00048 1-41 0:97 Ta 03814 0:0050 1°32 0-91 b 0:4107 00058 1:29 0:90 8a 04452 0:0058 1:30 0:91 b 0-4833 0-0065 1:34 0-92 These results show a fairly close agreement with one single excep- tion, in which case the percentage of iron is nearly 0°25 below the average. The average percentage, moreover, of ferric oxide is just about one-half that found by Rose. Further, the average percentage of iron (Fe) corresponds very closely with the average percentage of Cu in the copper albuminate. Eliminating one compound with ouly 0°75 per cent. of iron, the average content is seen to be 0°95 per cent. (Cy2Hi12Nis8Oo2)4 + Fe—H;=0°86 per cent. Fe. (C72H112Ni«SO22)3 + Fe—Hs=1°14 per cent. Fe. As the iron was determined by simple ignition it would be ex- pected that the amount found would exceed the theoretical amount somewhat; hence the first formula, assuming Lieberkiihn’s formula to be correct, would be more closely in accord with our results. The results obtained indicate further, that the iron albuminate is a much more stable compound than the copper albuminate, less liable to change and less readily affected by water. 316 Chittenden and W hitehouse— Metallic (d) Zine Compounds. With zinc we made but a few experiments and those mainly to see whether the low percentage of iron found in the iron albuminate would be substantiated by a corresponding percentage of zinc in the zine albuminate. Lieberkiihn has prepared and analyzed a zine albuminate, made by the action of zine sulphate on a neutral solu- tion of alkali albuminate, and he found the compound to contain 4°66 per cent. of zine oxide. Our preparations were made by the action of a similar zine salt on a solution of purified and dialyzed albumin. Following are the results obtained with two preparations made from two distinct lots of albumin : No. Amt. sub. taken. Wt. ZnO. ‘Per cent. ZnO. Per cent. Zn. la 0:2424 gram. 0:0031 gram. 1°27 0-98 b 0:2833 0:0034 1:21 0:97 2a 02166 0-0023 1:06 0:83 b 0°2354 0:0025 1:08 0:86 The average of these two results shows a composition proportional -to that found in the case of the iron albuminate and suggests plainly that if we have to deal in these cases with a single albuminate of constant composition, the percentage amount of metal is much smaller than formerly was supposed. Further, the percentage of zinc found accords closely with the theoretical amount for a zinc albuminate formed on the type of the copper compound. (C; BGaNG “SOn2). + Zn—H.=0°99 per cent. Zn. (e) Uranium Compounds. N. Kowalewsky* has recently called attention to the use of uranic or uranyl! acetate as a reagent for albuminous matter, and. has shown that it is not only a good precipitant of albumin at ordinary temper- atures, but also that it is an extremely delicate one. Further, Kowalewsky states that the uranyl-albumin compound on ignition leaves a dark, olive green ash, composed of the green uranoso-uranic oxide, U,O,. A determination of the amount of this ash in several preparations showed 12°09 to 13-4 per cent., presumably of U,O,. * Essigsaures Uranoxyd, ein Reagens auf Albuminstoffe. Zeitschrift fir Analy- tische Chemie, 1885, p. 551. a Compounds of Albumin and Myosin. 317 Our preparations were made by adding urany! nitrate to the pre- pared albumin solution and washing the precipitated albuminate until all excess of uranium was removed. The uranium in the dried preparation was determined as uranoso-uranic oxide (U,O,) by simple ignition. The results show a fairly close agreement, but they are undoubtedly somewhat too high, owing to a small amount of adherent ash. With UO,(NO,),. No. Amt. sub. taken. Wt. U3Oz. Per cent. U;Os. Per cent. U. la 0-5980 gram. 0:0324 gram. 5°41 4-59 b 0:51938 00281 5°41 4°59 2a 08219 00428 5°20 4°41 b 0:8081 0:0418 5:17 4°38 3a 04392 00251 5°71 4°84 0:5330 0:03803 5°68 4°81 4a 0°8183 0:0427 5:22 4°43 b 0:7576 0:0394 5:20 4°41 x 06985 0:0367 5:25 4°46 6a 04269 0:0247 5°78 4:90 b 0:5496 0:03138 5:70 4°83 These results plainly do not accord at all with Kowalewsky’s. On the other hand they do agree fairly well with each other, and would seem to indicate a reasonably constant composition of the uranyl- albumin precipitate. The average of the results obtained, accords most closely with the formula (Cy2Hi12NisSOn2)s +U—H, which requires 4°73 per cent. U. (f) Mercury compounds. By the addition of an excess of mercuric chloride solution to an aqueous solution of egg-albumin, an albuminate of mercury is formed, insoluble in excess of the mercury salt. The compound can be easily filtered and admits of thorough washing with water. Rose first proved that the precipitate formed as above, is a compound of mercury with albumin, instead of a compound of the mercury salt with albumin as supposed by Bostock and Orfila. We have made a few preparations of the albuminate by adding a moderately strong aqueous solution of mercuric chloride to portions of the dialyzed albumin solution and washing the precipitates thoroughly with water. The mercury in the albuminate was deter- 318 Chittenden and W hitehouse—Metallic mined by ignition in a combustion tube with quick lime, with a pos- terior layer of calcium carbonate and sodium bicarbonate.* The mercury distilled, was collected in water and after thorough washing with alcohol to remove hydrocarbons, etc., was dried and weighed. Following are the results of our analyses of the several prepara- tions made. No. Amt. Sub. taken. Wt. Hg. Per cent. Hg. la 0-8050 gram. 0:0226 gram. 2°80 b 0°8732 0°0274 3°13 2a 0°7637 00215 2°82 b 0°7357 00201 2°73 3a 05088 00150 2°96 b 0°6261 0:0167 2°66 4a 0°9152 00300 3°28 b 08503 0:0270 317 5a 08492 0°0218 2°56 b 0°8610 00237 2°75 6a 09674 0-0284 2°98 The average content of mercury is 2°89 per cent. The theoretical amount for (C,,H,,,N,.SO,,),+ Hg—H, is 3:00 per cent. (g) Silver compounds. Silver nitrate is a well known precipitant of albumin, and Lieber- kiihn,t many years ago, assigned to silver albuminate a definite formula, calling for 6°67 per cent. of silver oxide. The preparation made by him from egg-albumin was found to contain 6°55 per cent. of silver oxide = to 6°27 per cent. of Ag. Mulder,t likewise, work- ing with alkali-albuminate, found that by neutralizing the solution as nearly as possible with acetic acid, and then precipitating with silver nitrate, the silver albuminate so prepared contained 6°14 per cent. of silver oxide. Fuchs, using ordinary egg-albumin instead of alkali-albuminate, found only half as much silver (3°28 per cent. Ag), while O. Loew,]|} working with purified egg-albumin, found still smaller percentages of silver in the albuminate made by him. Using an albumin * See Fresenius, Quantitative Chemical Analysis. + Poggendorfi’s Annalen, 1852, vol. clxii, p. 123. { See Berzelius’ Lehrbuch der Chemie, vol. ix, p. 49. § Annalen d. chem. u. Pharm., Band cli, p. 372. || Piliger’s Archiy fir Physiologie, Band xxxi, p. 393; Ueber Hiweiss und Pepton, Compounds of Albumin and Myosin. 319 solution purified simply by three days’ dialysis, Loew found that a 1 per cent. solution of silver nitrate gave no precipitate whatever in a 5 per cent. solution of albumin. On adding a little dilute sulphuric acid to the albumin solution, however, and then pouring the mixture into the silver solution a precipitate was obtained, which on thorough washing and drying was found to contain 2°17 per cent. of Ag; while a second preparation made by using a little less sulphuric acid contained 2°40 per cent. of Ag. By precipitat- ing the albumin solution directly with a 5 per cent. solution of silver nitrate, without the addition of any acid, the albuminate was found to contain in one case 4°39 per cent. Ag, in a second case 3°91 per cent. Ag. Dissolving the freshly precipitated albuminate formed in this manner, in dilute ammonia and then reprecipitating it by the addition of dilute sulphuric acid to slight acid reaction, the albumin- ate was found to contain 4°64 per cent. of Ag. Loew sees in these results a confirmation of Harnack’s views as to the copper albuminates, and an assurance that the molecular weight of albumin corresponds to Lieberkiihn’s formula three times enlarged. Using an albumin solution purified as in our previous experiments and adding to it a 10 per cent. solution of silver nitrate as loug as a precipitate was formed, four distinct series of albuminates were made* representing four distinct preparations of egg-albumin. These were all washed free from silver and also from any adhering albumin, dried at 110° C. until of constant weight and the silver determined by simple ignition. Series I. No. Amt. Sub. taken. Wt. Ag. Per cent. Ag. la 0:5900 gram. 0:0242 gram. 4:10 b 05625 00230 4:08 2a -0°5760 00230 4-11 b 0°7548 00305 4:04 3a 09005 0-0362 4-02 b 0:°7973 00325 4:07 Seriss IT, la 05859 0:0245 4:18 b 06967 0-0290 4°16 2a 0°9473 00385 4-06 b 06621 00270 4-07 3a 0°6455 0°0266 . 4°12 b 0:7000 — 00285 4:07 * The silver compounds were all made and analyzed by Mr. T. S. Bronson of this laboratory. 320 Chittenden and W hitehouse—Metallic Srriss III. la 0:5860 0:0239 4:07 b 0:6949 0:0284 4:08 2a 0:7090 0:0290 4°09 b 0:90538 0:0374 4°13 3a 0:5980 0:0247 4:13 b 0:8000 ()0328 4°10 Series IV. la 06217 00300 - 4°88 b 06810 00331 4:86 2a 05509 0:0270 4-90 b 05626 0-0278 4°86 3a 0°6955 0°0396 5°69 b 0°7515 00430 9°72 The figures show a far smaller content of silver in all of the prep- arations than found by Lieberkiihn or Mulder. In three of the series, there is seen a constancy of composition which is quite notice- able and, further, a close agreement with the second result obtained by Loew. on adding a 5 per cent. solution of silver nitrate to the albumin. In the last series, however, the percentage of silver is somewhat higher, possibly owing to incomplete dialysis of the chlorides and phosphates from the albumin solution. These figures, however, are not much higher than the highest figures obtained by Loew. A silver salt of albumin, of the composition (C,,H,,,N,,SO.,), + Ag,—H, would contain 4°28 per cent. of Ag, and while our results certainly approximate to this figure, there is variation enough to indicate an equal possibility of a mixture of two or more com- pounds. With a molecule of the size of the albumin molecule, it is possible by doubling or otherwise, to obtain a formula corresponding to almost any percentage of metal found. And inasmuch as every variation* in the method of preparing the albuminate tends to alter its composition, it seems worse than useless at present, to lay much stress on the exact constitution of the silver albuminate. A large number of albuminates are of course possible, but until we know more definitely how to separate one from another, we have no guaran- tee of the simple nature of any one. * Loew states that he has prepared a silver albuminate containing 10°7 per cent. Ag, corresponding nearly to 6 atoms of silver, and that it is possible to prepare album- inates still richer in silver. Compounds of Albumin and Myosin. 321 Examining now, all of the results obtained, we find the following average composition of the albuminates studied : Copper compound, 0°94 per cent. Cu Tron compound, 0:95" << cones Zine compound, O;ouT ss Sop Zin Lead (neutral salt) compound, BOGuacy eb Uranyl compound, 4:60 ‘* eal Silver compound, AO9N s¢* Sere Ato-+ Mercury compound, 2700) sgl a Ife Accepting Lieberkiihn’s formula of albumin as correct, then the following formulz accord most closely with the above percentages. (C72Hi12N1sSO22), + Cu— Hz requires 0°96 per cent. Cu (Cy2Hi12Ni12SO20)4+ Fe—Hs . 0°86 r Fe (Op FiiNgeSOs3) 24m — Fy int 4) O99) fei Zn (CAHN. SO. hb= ee O50 se Bh (Cy2H112NisSOo2)3 + U—He os 4-73 f U (CreHii2NisSO2e)s+Ag.—H. ‘“ 4:28 i Ag (CreHii2Nis8Oo.),+Hg—-H. “ 3:00 “* Hg We do not, however, lay much stress upon the accuracy of these formule. The results obtained in our study of these metallic com- pounds do by chance accord with them, and inasmuch as Loew and Harnack are disposed to treble Lieberkiihn’s formula for albumin, on the basis of the composition of the copper and silver albuminates, made by them respectively, we present our results as evidence that there are equally good grounds for quadrupling the above formula. We believe, however, that with the majority of these albuminates it is possible to form a large variety of compounds with the same metal, by simply modifying the conditions of precipitation. This is evidenced by Loew’s results with silver albuminate and our own with lead and copper, and since a great variety of compounds are possible, it is equally possible that in many cases we may have to do with mixtures of such compounds, which would account for the great variability in composition noticed in some of the albuminates and tor the lack of agreement in the results obtained by different workers. Coupled with this, in some cases, is the undoubted tendency of the compounds to dissociation. Il. Myosin. The myosin employed was prepared from ox flesh, by extraction with a 15 per cent. solution of ammonium chloride, after the tissue * Hxcepting one very low result. | Excepting the last series of compounds. TRANS. CoNN. ACAD., Vou. VII. 41 Nov., 1886. 322 Chittenden and W hitehouse—Metallic had been thoroughly freed from salts and soluble albumin by long continued extraction with water. The myosin was separated from the ammonium chloride solution by dialysis, being obtained in this man- ner as a semi-gelatinous mass, readily soluble in salt solutions. In order to form compounds with the various metals, it was found best to use a solution of myosin in 5 per cent. ammonium chloride, the metallic compound when formed being washed with water until the washings gave no reaction for chlorine with silver nitrate. The com- pounds were then dried, first at 100° C., then at 110° C., until of constant weight. Control experiments with the metallic salt and ammonium chloride alone, invariably failed to give any precipitate whatever. No systematic attempt has apparently been made to study any of the metallic compounds of myosin; in fact, few statements are to be found regarding the existence of such compounds. Danilewsky* some time ago, showed that myosin would combine with free mineral acids, uniting with them so that with tropzolin 00 no reaction for free acid could be obtained. With strong bases, however, according to Danilewsky, myosin does not probably combine, and the state- ment is further made that a small amount of calcium oxide ordinarily exists loosely combined with myosin, which calcium by coagulation of the myosin is liberated. Further, Danilewsky found that on adding platinum chloride in excess, to a dilute hydrochloric acid solution of myosin, a myosin-platinum chloride compound was pre- cipitated, which after washing with water and alcohol and then drying at 100-105° C., contained 9°46 per cent. of platinum and 7°26 per cent. of chlorine. With copper, iron and similar salts we have not been able to obtain any precipitate in a hydrochloric acid solu- tion of myosin. By adding, however, a solution of a metallic salt of such a nature that it does not react with ammonium chloride, to an ammonium chloride solution of myosin, a precipitate is pro- duced, which as our experiments show, is ordinarily a compound of myosin with the metal or metallic oxide. This is readily seen by adding either zinc sulphate or ferric chloride to such a solution of myosin and then washing the precipitates with water, until the washings give no reaction for chlorine or for sulphuric acid. On now warming the iron precipitate with dilute nitric acid, a solu- tion will be obtained, giving a distinct iron reaction but no reaction with silver nitrate for chlorine. Similarly on warming the zine pre- * Zeitschrift fiir physiologische Chemie, v, p. 160, Compounds of Albumin and Myosin. 323 cipitate with hydrochloric acid, the solution gives no reaction for sulphuric acid with barium chloride. With cupric sulphate and cupric acetate the same is ordinarily true. It is possible, however, to prepare a myosin-copper compound in which cupric sulphate appears to unite directly with the myosin. (a.) Copper compounds. By adding either cupric sulphate or cupric acetate to a neutral ammonium chloride solution of myosin, a heavy greenish colored precipitate is obtained, which when freshly formed and after thor- ough washing with water, so that the washings are entirely free from chlorine and from copper, shows the following reactions. It is insoluble in moderately strong nitric, hydrochloric or sulphuric acid. The compound, however, is immediately broken up by the action of acids, the copper being completely removed, leaving the myosin as an insoluble residue having in the case of nitric acid a yellow color, and in the case of hydrochloric and sulphuric acids a white color. In acetic acid, the compound is more soluble, first, however, becoming semi- gelatinous. In ammonium hydroxide, the compound dissolves slowly or partially, taking on a blue color. In dilute sodium hydroxide, the compound swells up, takes on a purple color, but does not dissolve. In dilute sodium carbonate, the substance is likewise insoluble, but swells up and turns of a bluish color. Following are the results of the analyses of the various prepara- tions made. The compounds were in every case composed simply of the metallic oxide and myosin. Copper was determined, as in the case of the albumin compounds, by simple ignition and weighing as oxide. In order to ascertain how much ash was retained by the myosin compound, a few duplicate determinations of copper were made by dissolving the oxide after ignition, and precipitating the copper as sulphide and weighing as subsulphide, after ignition with a little pure sulphur in a current of hydrogen gas. Serigs I. With CuSO,. Amt. Sub. Per cent. Per cent. No. taken. Wt. CuO. Cu. Wt. Cus. Cu. la 0°5426 gram. 0:0074 gram. 1:08 0:0056 gram. .0°81 b 0°7176 0°0097 1°07 0:0073 0:80 2a 0°6359 0°0087 1:08 0:0071 0°88 b 0°5738 0:0078 1-08 0:0061 0°83 Chittenden and W hitehouse—Metallic Amt. Sub. taken. 0:5423 gram. 0-6241 0:5771 04455 0°3527 04401 0°5708 Amt. Sub. taken. 0°7425 gram. 0°7463 0:'7785 0:°7767 05488 0:°4836 0:94.77 0°8150 0-8522 08466 0°6589 06572 0:8673 0°7120 Amt. Sub. taken. 0°7213 gram. 0°6584 0°7564 0°6817 0°7820 0°62138 0°7137 0:5863 0°7732 0°7407 0°7447 0°7429 Wt. Cu.S. 0:0051 gram. 00063 Percent. CuO. Per cent. Cu. 0-79 0-81 0-73 0°72 -0°94 0-94 0°89 Per cent. Wt. CuO. Cu. 0-0072 gram. 1:05 00082 1:05 0:0109 1°50 0:0081 1°43 0:0046 1:02 0:0062 iit} 00082 td3 Series II. With CuSO,. Wt. CuO. 0:0059 gram. 0:0060 0:0057 0:0056 0:0052 0:0041 0:0085 00074 With Cu(C,H,0,),. 0-0133 0-0188 0-0124 0-0128 00156 00129 Series III. With CuSO,. Wt. CuO. 0°0171 gram. 00161 00168 00156 0:0189 00154 0-0144 0:0124 0:0164 0°0161 001838 0:0188 0-91 1°56 1°62 1°88 1:87 1-79 1°81 Per cent. CuO. Per cent. Cu. 2°37 2-44 2°22 2°29 2-41 2-48 2-01 2-11 2°12 2°18 2-45 2°53 Per cent. Cu. 0-73 0°80 0°63 0-64 0°57 0°58 0:74 0-73 0-74 0°73 1°24 1:29 1:50 1:50 1°42 1°44 1°88 1°94 Ue 1°83 1°91 1:97 1°61 1°68 1°68 1°74 1:96 2°01 ee Compounds of Albumin and Myosin. 325 With Cu(C,H,9,),. No. Amt. Sub. taken. Wt. CuO. Per cent. CuO. Per cent. Cu. Ta 0:7360 gram. 0:0161 gram. 2°18 1:73 b 0:7088 0:0154. 2°17 £73 8a 0°7789 0°0174 2°24 STEKS b 0°8111 0:0181 2°23 7p 9a 0:7376 t 0:0169 2°29 1°81 b 0°6155 0:0188 2°24 1:78 10a 0°-7815 0°0191 2°44 1:94 b 0-874 0:0213 2°43 1:93 lla 0:8048 0:0170 2-11 1°67 b 0°8308 0:0174 2:10 1°67 12a 0:6780 0:0150 2°21 Lio b 0°7695 0:0168 2:18 1°74 Comparing these results with one another, there is to be seen a very noticeable lack of agreement in composition, and further it is to be seen that. the myosin-copper compound has on an average a somewhat higher content of copper than the albumin-copper precipi- tate. The average composition of all the copper myosins shows about 1-42 per cent. of Cu, and deducting 0°25 per cent. of ash, the average content of Cu would be 1°17 per cent. Examining the individual results, it is apparent that the compounds made from the same myosin solution are approximately, at least, the same in composition and without doubt the difference in the composition of compounds made from different myosin solutions is due to variation in the concentra- tion of, and possibly also in the reaction of, the myosin-containing fluid. It would appear as if variations in the conditions of precipita- tion made a greater difference in the case of the myosin-copper com- pounds than in the compounds of copper with albumin. Several times, also, we have found that our myosin-copper precipitate con- tained sulphuric acid, even after thorough washing and when the wash-water was proved to be entirely free from any reaction with barium chloride. One such compound, after drying at 110° C., was analyzed with the following results: 0°7240 gram substance* gave 0:0343 gram BaSO, = 1:63 per cent. SOs. 0°8420 gam substance gave 0:0331 gram BaSO, = 1°73 per cent. SOs. 0°4705 gram substance} gave 00084 gram CuO = 1°78 per cent. CuO. 0°6011 gram substance gave 0:0107 gram CuO —=—- =_: 1°78 per cent. CuO. * Roasted and then ignited with pure sodium carbonate, the residue dissolved in hot water acidified with hydrochloric acid and precipitated with barium chloride. + Ignited, the residue dissolved in nitric acid, precipitated with hydrogen sulphide, etc. 326 Chittenden and Whitehouse—Metallic The molecular weight of CuO and SO, being the same, it is evident that the two are present in just the proportion to form cupric sul- phate. b. Iron compounds. By adding a solution of ferric chloride to an ammonium chloride solution of myosin, a semi-gelatinous precipitate is formed of a red- dish yellow color, and consisting of a combination of myosin and oxide of iron. The compound when thoroughly washed contains no chlorine. When freshly precipitated, it is partially soluble in dilute ammonium hydroxide, as also in sodium hydroxide, the residue becoming gummy or gelatinous and brownish yellow in color. It swells up in sodium carbonate, but is insoluble. In nitric acid the compound turns yellow, but is wholly insoluble and does not swell up. In hydrochloric and also in sulphuric acid the compound is like- wise insoluble. In acetic acid, however, it is soluble completely, forming a semi-gelatinous fluid. In this, as in other metallic com- pounds of myosin, acids simply dissolve out the metal and then exert their usual action on the myosin. The various preparations, washed free from iron and chlorine, and dried at 110° C. were analyzed with the following results: Series I. No. Amt. Sub. taken. Wt. Fe.Os. Per cent. Fe.03. Per cent. Fe. la 0°7046 gram. 0.0194 gram. 2°76 , tee a 0°7021 _0°0193 2°74 1:92 2a 0:3762 0:0128 3°40 2°38 b 0°3623 0:0121 3°33 2°33 3a 0°5837 0:0141 2°42 1:69 b 0:5618 0:0137 2°43 1-70 4a 0:5438 0:0177 3°25 2°26 b 0:6088 0:0197 3°24 2°26 5a 0:4761 0:0140 2°95 2°06 b 0:3381 0:0100 2°95 2°07 6a 0°5101 0:0178 3°40 2°37 b 0:5927 0:0194 3°28 2°29 Serizs II. 1 0°4847 0:0188 3°87 2°70 2 073200 0°0115 3°59 2°51 0°4118 0:01538 3°72 2°59 4 0.5205 0°0184 3°53 2°45 Compounds of Albumin and Myosin. 327 No. Amt. Sub. taken. Wt. Be.Os. Per cent. Fe.0;. Per cent. Fe. 5a 0-4551 gram. 0:0167 gram. 3°68 2°57 b 03668 0:01387 3°73 2°61 6a 0:3783 0:01388 3°64 2°53 b 0°3675 0:01381 3°57 2°50 Ta 04284. 0°0148 3°34 2°33 b 04041 ‘0°0186 3°37 2°36 Series III. With a large excess of ferric chloride. la 04385 00269 6°13 4-29 b 06939 00436 6°27 4°38 In none of these preparations was there any attempt made to add a definite amount of ferric chloride, but the iron salt was added until a good precipitate was obtained. Undoubtedly, the amount of iron salt added, modifies materially the composition of the compound. In series IIT it is seen that the content of iron is about double the aver- age amount contained in the other preparations. The average amount of iron (Fe) in the first two series of compounds is 2°29 per cent. ce. Zine compounds. With zine sulphate, myosin is thrown down from its ammonium chloride solution as a heavy gelatinous precipitate. Like the iron compound it is partially soluble in sodium and ammonium hydroxides, swelling up to a gelatinous mass. It is insoluble in nitric, hydro- chloric and sulphuric acids, but is partially soluble in acetic acid. In composition, it is seen to be very closely allied to the zine albu- minate. Following are the results obtained by analysis of the dried compounds : Series I. No. Amt. Sub. taken. Wt. ZnO, Per cent. ZnO. Per cent. Zn. la 0:6108 gram. 0:0049 gram. 0°81 0-64 b 06619 0:0055 0°83 0°66 2a 0°6611 0:004:7 0-71 0°57 b 0°8006 0:0059 0°73 0-59 3a 04666 | 0:0046 0:99 0:79 b 04494 0:0044 0:99 0-79 4a 0:5936 0:0048 0-80 0°64 b 06926 )-0055 0-79 0°63 328 Chittenden and W hitehouse— Metallic Series IT, No. Amt, Sub. taken. Wt. ZnO. Per cent. ZnO. Per cent. Zn. la 0°6258 gram. 0-0051 gram. 0°82 0°65 b 0:6618 0°0054 0°81 0-65 2a 0:53815 0:0064 1°21 0:97 b 0°5485 0-0065 1:18 0:94 3a 0°7925 0:0064 0°81 0°65 b 0°6913 0:0057 0°83 0°66 4a 0:4858 0:0062 1:27 1:02 b 0°5318 0:0066 1°24 0:99 da 0°6635 00056 0°85 0°68 b 0°6504 0:0050 0:76 0-61 6a 0:6263 0:0055 0°87 0°70 b 0°6639 0-0059 0°88 0°71 The average content of zinc (Zn) is 0°72 per cent. Unlike the iron and copper compounds, there is here less variation in the composition of the various preparations. d. Nickel and cobalt compounds. The extremely low percentage of zine in the zinc-myosin com- pounds, as contrasted with the iron in the iron compounds, led us to make a nickel and cobalt preparation for the sake of comparison. The results, in both cases, accord more nearly with those of the iron compound, for although containing a higher percentage of metal than the latter, it was necessary to prepare them both under just such conditions as in the iron compound led to the highest percent- age of iron, viz: a large excess of the precipitant. Following are the analytical results obtained with both substances: With Ni (NO,),. No. Amt, Sub. taken. Wt. NiO,. Per cent. NiOs. Per ceut. Ni. la 0°6165 gram. 0:0045 gram. 7:29 4:71 b 0°7016 0-0051 7°26 4°70 With CO (NO,),. fay)... ”” 8490 0-0064 9-95 6°45 b 06696 0-0065 9°70 6°28 2a 06451 00056 8°75 5°67 b 06842 0-0060 8°84 5°72 Compounds of Albumin and Myosin. 329 e. Uranium compounds. The addition of uranyl nitrate to an ammonium chloride solution of myosin produces a heavy gelatinous precipitate of a uranyl-myosin compound, which in solubility resembles the other myosin prep- arations. Washed free from excess of uranyl nitrate and from ammonium chloride and then dried at 110° C., the various prepara- tions yielded on analysis the following’ results, the uranium being determined by simple ignition and weighing as uranoso-uranic oxide: Seriszs I. No. Amt. Sub. taken. Wt. U;Os. Per cent. U;0;. Per cent. U. la 0-6373 gram. 0:0489 gram. 7-67 6°51 b 06836 00526 7:69 6°53 2a 0-7081 00592 8°36 7:09 b 0°7689 0°0655 8°51 7°22 3a 0°6418 0:0586 9-13 115 b 0:7809 0:0715 9°16 7:78 4a 06621 0°0541 817 6°93 b 0°7455 0:0608 8°15 6:91 5a 0°7525 0:0663 8°81 7°48 b (06964 00615 8:83 7:50 6a 0°7208 0°0590 8:18 6:94. 07439 0:0607 8°15 6°91 Ta 0°6648 0:0527 7°92 6°72 b 0-7101 0:0565 7:96 6°76 Series II. la 0°8736 0°0837 9:58 8:13 b 0°8570 0:0820 9°57 8:12 2a 0°5857 00598 10:12 8°59 b 0°7088 0:0715 10:09 8:56 3a 0-5715 0°0568 9°94. 8°44 b 0°6958 0:0688 9°89 8°40 4a 06929 0.0556 8:02 6°82 b 07667 « 0°0615 8:02 6°82 5a 08031 0:°0874 10°88 9°23 b 0:8272 0-0901 10°89 9°24. 6a 0-7034 0°05538 7-86 6-67 b 0°6810 00540 7-92 6°72 These results show a variation in the content of uranium, amount- ing to nearly 3 per cent. (6°51-9°24 per cent.) Further, a compari- Trans. Conn. Acap., Vou. VII. 42 Nov., 1886. 330 Chittenden and W hitehouse— Metallic son of the two series shows plainly that there is something in the nature of the second myosin solution, which tends to raise the content of uranium in the uranyl compounds; probably, the greater concen- tration or dilution of the solution. Evidently, then, the composition of the compound is, in part at least, determined by the conditions under which the uranyl] salt and the myosin solution are brought together. The average amount of uranium contained in the prepa- rations is 7°49 per cent. J. Mercury compounds. By adding a solution of mercuric chloride to an ammonium chlo- ride solution of myosin, a heavy gelatinous precipitate is formed which soon changes to a flocculent one. Freed trom the excess of mercury salt and ammonium chloride, the compound is found to be entirely free from chlorine. The substance is somewhat soluble in sodium hydroxide, swelling up first and then gradually dissolving. Dried at 110° C. and then analyzed, the following results were ob- tained. The mercury was determined as already described under mercury albuminate. Series I, No. Amt. Sub. taken. Wt. of Hg. Per cent. Hg. la 0-7609 gram. 0-0166 gram. 2718 b 1:2911 0:0268 . 2°07 2a 1°2386 00270 2°17 b 0°9525 0-0198 2-07 da 171856 0-0218 1°84 b 09261 0:01738 1°86 4a 1-3504 0°0257 1:90 b 09382 00178 1:90 Series I. 1 0°8917 0°0241 2°70 2 11196 0-0310 2°77 3a 0:95387 00271 2°84 b 088138 0:0259 2°93 4 09209 00270 2°93 5 08352 0-0241 2°89 6 08564 00217 2°53 Ta 1:0696 00844 3°22 b 0°8550 0:0264 3°09 Compounds of Albumin and Myosin. 331 The average content of mercury (Hg) is 2°43 percent. The results of each series show a fairly close agreement, but the two series do not compare with each other at all. Thus, the average amount of mer- cury in the compounds of the first series is 1°99 per cent., while in the second series the average amount rises to 2°87 per cent. This is another good illustration of the influence of the strength of the solu- tion on the composition of the precipitate, and as in this case the presence of any ash could not interfere with the ultimate result, since the mercury was separated by distillation, it follows that the appar- ently higher content of mercury in the second series must be due to combination of the myosin with a larger amount of the metal. Fur- ther, it has been claimed* that in the case of the silver albuminate, it is possible under certain circumstances for the albuminate, when formed in a concentrated solution, to inclose a variable amount of albumin mechanically, and thus the apparent percentage of silver in the albuminate be reduced. If such was true of the myosin-mercury compounds, a far greater variation would be expected in the per- centage of mercury in the different preparations of the same series. The following table of comparisons shows the average content of metal in the albuminates formed from the two kinds ot proteid matter. Kgg-albumin. Myosin. Copper compound, 0:94 per cent. Cu 1:17 per cent. Cu Tron s 0°95 Fe 2:29 Fe Zine se 0-91 Zn 0°72 Zn Uranyl ee 460 U 7-49 oh Mercury ‘ 2°89 Hg 2°43 Hg Lead 2°56 Pb Lee pe! Silver gs 4:09 Ag eee wae Nickel ae 4°70 Ni Cobalt ss 6:03 Co Apparently, the two forms of albuminous matter, the albumin and globulin, do not form corresponding compounds with the metallic salts experimented with. * See Loew, Pfliiger’s Archiv fiir Physiologie, Band xxxi, p. 393. XXI.—Ecc-ALBumiIn anp A.tsumosEs. By R. H. CuirrenpEeNn AND Prrcy R. Bouron, Pu.B. Ever since the albumose bodies were first separated from the pro- ducts of fibrin digestion* with pepsin-hydrochloric acid, it has been our intention, already expressed, to subject the various individual albuminst to the action of purified pepsin under like conditions, and thus ultimately to acquire a comparative knowledge of the albumose bodies obtainable from these different sources. Already the albumose bodies from fibrin and the globuloses{ have been subjected to a care- ful study and we present here the result of a study of the albumoses from egg-albuniin. In doing this, we have to report at the same time, the results of a study of the composition of egg-albumin itself. For we have made it a rule, in the series of experiments shortly to be described, to analyze a sample of each lot ofalbumin prepared for digestion, In this manner we have obtained data for a direct comparison of composition between the original sample of albumin and the products formed by its digestion. This we have deemed of considerable importance, for the data so obtained may throw considerable light on the nature of the changes involved in the formation of the albumoses; particu- larly, as to whether they are hydrolytic in their nature. Four distinct samples of albumin were prepared, three of which were prepared in large quantities and served as material for the subse- quent digestions. Albumin A. This was a preparation of coagulated egg-albumin, prepared espec- ially with the view of obtaining a product wholly free from globulin. The method employed was essentially that recommended by Ham- marsten.§ The whites of 120 eggs were freed from the yolks, then *W. Kiihne and R. H. Chittenden, Ueber Albumosen, Zeitschrift fiir Biologie, Band 26.6), 5 1S + W. Kihne and R. H. Chittenden, Ueber die nachsten Spaltungsproducte der Hiweisskérper, Zeitschrift fir Biologie, Band xix, p. 159. { W. Kiihne and R. H. Chittenden, Globulin und Globulosen, Zeitschrift fir Biologie, Band xxii, p. 409. § See K. V. Starke, Beitrage zur Kenntniss des Serum- und Kialbumins, Jahres- bericht fiir Thierchemie, 1881, p. 18. ——— ee ee Chittenden and Bolton—Kgg-Albumin and Albumoses. 338 finely divided by shaking with glass, the fluid mixed with an equal volume of water or more, then shaken vigorously with air and finally filtered through cloth. The solution so obtained, was then saturated with crystals of magnesium sulphate at 20° C., for the com- plete removal of the globulin. The mixture was filtered through paper and the clear filtrate saturated with sodium sulphate. The precipitated albumin was then filtered and washed with a saturated solution of sodium sulphate, after which it was dissolved in water and dialyzed in running water until the magnesium and sodium sulphates were entirely removed. The fluid was then again filtered and the albumin finally coagulated by being poured into eight litres of boil- ing water, slightly acidified with acetic acid. The great bulk of the coagulum so obtained was at once placed in four litres of 0-4 per cent. hydrochloric acid, while a small sample for analysis was washed with 95 per cent. alcohol, finally with absolute alcohol and then dried, first at 100° C., and finally at 106° C., in vacuo, until of con- stant weight. The following table shows the results of the analysis of the product. The various determinations were made as described in the previous articles on these subjects, the sulphur being deter- mined by fusion with potassium hydroxide and potassium nitrate in a silver crucible, according to the method designated by Hammar- sten* as la. Albumin B. This albumin was prepared from the whites of 126 eggs by a some- what different method. The albumin solution, after dilution with water, was made very distinctly acid with acetic acid, and the heavy precipitate of globulin, after it had well settled, removed by filtration. The acid fluid was then made exactly neutral with sodium carbonate and again filtered ; it was then thymolized and dialyzed in running water for eight days. A little globulin, not precipitated by the acetic acid, was found in the bottom of the dialyzers when the salts had diffused out. This was filtered off and the perfectly clear fiuid evap- orated at 35-45° C., to perfect dryness. A sample of this preparation was ground fine, dried at 106° C. im vacuo and analyzed. It is perhaps questionable, whether all of the globulin is removed by this method. The precipitate with acetic acid was quite heavy, and as H. Dillnert has recently shown that the amount of globulin in egg-albumin, as * See Zeitschrift fiir physiolog. Chemie, Band ix, p. 289. + Ueber die Globuline im Hihnereiweiss, Jahresbericht fiir Thierchemie, 1885, Dp: 1. Chittenden and Bolton—Egg-Albumin and Albumoses. 334 00-00F 60-86 roe a aes Py oe aie oi 3 | 777" 18h BG | 899-0 | TO-4 |. OS8T-0 | 0868-0 | I ira te = es ai | - : = ; eaatl| "mRIs movies SOE eal eee ‘ : qe | 5 S i Unica) 9) Bet ai 3 et dd y | ‘wes WBS wes S iy so te feat | NS a arse 4) Tae) H O*H gouRysqng | FN ‘O NINOATY AO SISATVNY TRANS. Conn. AcAp., Vou, VIL. 00-00F s Gk EC Soh cee a aes card es +h) mM 60-6 60-6 60-6 BTL ‘is 2 a ee ee g 89-91 ws ae, L9-CT - — 69-ST ee: Te SN = T8-9 atte Aes are att 9L-9 98:9 H = 741 ork Piva ae pe 6L-TS 69-1 O “9.0B10A V = ‘aounysqns aatf-ysp ayy fo Uo1isodwmoo abn, Uad1agq 5 oe = 2 : = Ss ieee ee whe ee SS eee Oe peste | Seto Peat 9 2 Be el ine eee S 9F-0 100-0 | CL60-1 > Sia Foon ee ees So a gremtren eties fola| eee Se SAe | Noe Re a i CP:0 8900-0 | ~~ “tel L066: T as . Picket cise tt? So AE kd Eater |e eee pete So sae 60-6 6860-0 Se leak ae 7999-0 SS in BOA Bele 2 aks 10-6 6LET-0 Pert eae Bite reads" cin ed ROS) oor att GO0F6-0 § See? ea ie Soa epee 09-s St Sree Ae eee S | Se-Sgaar eeobe| BOT aOR ee Se ea ee IS eee eee ----- gel anes a SAE ee SPA) TSS ro” Se GOS RAE, | Geb mal Be Ee gee SE een Oe oe re Ee Oe ate ag pegces 9 Rhee ap tS | Sp St a) RS] ge ah| aoe seal ies 2 | seme ep ep eee Gs ana an ea espe SE as “Sime aaa ae Se AES QE SS Fal SS Sef Se | SRO ee 2 SS eo Oe Oeste SO Ree ev oeea) ont an “¢ wa | leat | ~~ . 3 Ne % “URLS : | “cows |. 8 “coud Oe oe ‘meg | | ‘meus “mBIs aes JOS) % “ONA + HOW Pant TRS 2 )-panoy 2S) OMNAEATS| CL ee a into eae “panoy ‘posn | ‘ON myanpep| § | WA UOIRNE | io g| OUP OF] UBV | yey | N |———— ——!| 0 209 H O*H | eourysqng' 19}JB S ioe *OSeg %o9rq | “punojy N | ‘ASOWATTIVOLOUG ‘q dO SISATVNY Chittenden and Bolton—Egg-Albumin and Albumoses. 351 Digestion of albumin B. 340 grams of the dry albumin, non-coagulated, were soaked in 2 litres of 0-4 per cent. hydrochloric acid for 24 hours, then warmed up to 45° C. and 1 litre of the purified pepsin-hydrochloric acid added, also warmed at the same temperature. The mixture was kept at 45° C. for 16 hours, then neutralized and filtered. The filtrate, con- taining the albumose bodies formed from the non-coagulated albu- min, was then treated as already described under Albumin A. B. Protoalbumose. The protoalbumose isolated from this digestion, was purified in much the same manner as A protoalbumose, and did not differ from it in its reactions, except that with water it did not dissolve to quite so clear a solution; in fact its solution in water resembled more closely the aqueous solutions of protoalbumose from fibrin. During its final purification, it was dialyzed in running water until no chlorine reaction could be obtained with silver nitrate. In spite of this fact, however, the preparation contained a large percentage of ash, con- sisting mainly of calcium sulphate, ferric oxide and a little calcium phosphate. The accompanying table shows the composition of the substance after drying at 106° C., im vacuo, until of constant weight. In the purification of this protoalbumese, the substance was repre- cipitated three times by saturating the aqueous solution of the pre- cipitate with sodium chloride. By this treatment, as already stated, there is considerable loss, inasmuch as the precipitation of proto- albumose with salt in this manner is never complete, considerable remaining each time in the salt-saturated fluid. By adding a very little acetic acid, however, the protoalbumose is completely precipi- tated from the salt-saturated solution. The filtrates therefore, from the second and third precipitations of protoalbumose with salt alone were united, and the albumose remaining in them precipitated by the addition of a little acetic acid, saturated with sodium chloride. Our object was to see whether the protoalbumose which had at one time been precipitated by salt alone. and then had finally become soluble in the salt-saturated fluid, differed at all in composition or in reaction from the protoalbumose still insoluble in the salt solution. The albumose separated in this manner was purified by being dis- solved in water, the solution made exactly neutral with sodium car- bonate and dialyzed for several days. The fluid was then con- Chittenden and Bolton—Egg-Albumin and Albumoses. 352 00.005 8646 86.1 76-1 68-9 16-0¢ ‘OSRIOAW 86-1 68-9 T6-0¢ Oi aS ‘aounjsqns aadf-ysp ayy fo Uuorrsodmos abnyuaolag 16-1 CUGM0" wu ceeracclaeer = ae ee ee le ee ae] ag aloe Shee eee gg cee eae ae Gc:0m | 60U-Oae ae | = SA | ene ee, ER eg SSE Bese ae eae oh eee | 18-3 (SFT0-0 sa Sige | | Sateen ee a sees eee a eet et pe eae Savalas ce G0) | 6b e-0%, tare eee peer ea ee ee Sao fe cig Sorelaeers Se S| ae Saat a“ i ele “= las ao per Ieeceou | ‘ % ured ; meds p S “WU Oi aes ; | “ISV JO g SOMIGE MOM Lice he Aas leans guiseougll ergo ee ge ee ae Sanonpep UJI woIsny Ee oR oy} WOE} “YSV | 1. F e.g Benes 1oyyJe loye 'ogeg | SV J9S) toseg ENE ‘punoy N | 09 “(Uedls “punoy O°H F80S-0 CPh6P-0 Ch6T-0) FITE-0 cO8¢-0 meds ‘pesn aouRysqug ‘HOOD HO Aq ‘aomnjos [QVN wou pozesidioerg “ASOWNATVOLOUd ‘Gq dO SISATVNW Chittenden and Bolton—Egg-Albumin and Albumoses. 353 centrated to a syrup and the albumose precipitated by alcohol. This precipitate was again dissolved in water, the solution made exactly neutral and again dialyzed. After suitable concentration, the albumose was again precipitated by alcohol, washed with alcohol and ether and finally dried at 106° C. in vacuo. The results of the analysis are seen in the accompanying table. The ash in this preparation is seen to be much smaller than in the protoalbumose precipitated by salt alone. In other respects the two analyses are closely comparable, particularly the carbon and sulphur. The reactions were in almost every case the same as with the pre- ceding preparation, excepting perhaps a somewhat greater solu- bility. B. Deuteroalbumose. This body was separated and purified in exactly the same manner as in the preceding digestion. The analysis of the product is shown in the accompanying table. The ash contained some calcium sul- phate and a little ferric oxide. The reactions of the body were the same as those of A deuteroalbumose. From this digestion, more or less heteroalbumose was separated but no analysis was made of the product, as the amount was rather small for the necessary purification. Digestion of Albumin C. In the digestion of this sample of coagulated albumin, a much more vigorous pepsin-hydrochloric acid was employed than in the preceding digestions. The freshly coagulated albumin was placed in 3 litres of 0°4 per cent. hydrochloric acid and brought to a tem- perature of 45° C., then 400 c.c. of a pepsin solution, made from a pure glycerin extract of pepsin, were added and the mixture kept at 45° C. for 24 hours. The fluid was then neutralized, filtered and the clear filtrate saturated with sodium chloride. The albumose bodies were then separated and purified according to the methods already described. The several bodies showed the same reactions as observed in the preceding preparations. Protoalbumose and deuteroalbumose were analyzed. ‘The results are shown in the accompanying tables. The ash of the deutero- albumose contained no sulphate, but was composed almost entirely of ferric oxide. TRANS. Conn. ACAD., Vou. VII. 45 Nov., 1886. Chittenden and Bolton—Egqg-Albumin und Albumoses. 354 . 00.00T 80-46 Sear ea ir ee ‘ora aera O 60-6 60-6 60-6 Ries. iia coast) aA S$ hike GT a ee: 08-ST CL: CT ree 5 N 76-9 Ser ae Bie Beis 66-9 96-9 H 61-19 aan ree ae = 9T-TS T6-1¢ 0) ‘QBBIOAY ‘aounjsqns aatf-yso ay, fo uorrsodwos abnzuelag Se ae ee ea ES ee ce ae Pea: dee ae | i, ee G06 ~—soTL6 0SC0-0 fmm = Agee aia, 5 ad | eae oy'liory ae rs are | ey ie Ne a is. oe 2 ite aioe cane 8L¢§-0 ITA 60-6 | G16 | UD) Ba eg Bega cae ian olga cs 2 OS a a “a Fite | |"mon is a | ee aha a ea 32 666-0 TA re. ‘oat |e ee 60:0 GENO liga ee clte eadi ap ees ae cee etl ge gt ee 6 |e aE Pers-0 = QA fe aie! 2h ne ee eth ee tee 92s S8000— Sasa ) eteue pote ete Rae ena as ie ae et | Fore-0 =A Smeal sees | ign re aang es: ee ee ET GROG | teaGiie | Ase, Ce eee as oe See ee OSS ON Ad Se Beaty 6 | =) oe Same ihn Be ee [aes ons agen! TOOL, s| Geetey keeGont imines lie omeese Se sl a | Crer-0 =—«dTIT | | | 1 Se At ie aie dal| | dee ee ey eae gi fleet (ees EA hp “"™7 | =" "| 18-08 | SShP-0 | 68-9 | OSFT-O | Tl¥e-0 II eee Lease ae last | Rasen Sar ell wap. -eeaes Ue Sent ee spe | SS SSseS Mer eOU mG Ono rOmeae Preeti eT CRE eines ae Pa? a "i | . pt 900818 |, Bias m WRIS y jeamsselg | “YL One F "mes % “mes “TUB Ysy JoS| »% ONX+HO® fang jo y/ TV OU} 4 | -punoy| | % | spumos ‘punoy | ‘pasn ‘on Ssuyoupep | S Wy WOIsNny lqsV jo g TOA | UsVv UsV N @) TOYO) H | OfH /PoTLBISQNyG roqyFe S| | Jaye FOgeq | | tosed | ‘punoy N | : “HSOWNATVOURLOANC “GC dO SISATVNY Chittenden and Bolton—Egg-Albumin and Albumoses, 355 Dysalbumose. This form of albumose was found in all three digestions, but the amount was much smaller than noticed in the fibrin digestions. The substance was obtained as an insoluble residue, after extracting the first sodium chloride precipitate successively with 10 and 5 per cent. salt solutions and with water. It was then dissolved in 02 per cent. hydrochloric acid, and after filtration precipitated by neutralization. After precipitation in this manner, a portion of the substance was found soluble in sodium chloride and on dialysis of the solution, sep- arated in much the same manner as heteroalbumose. The portion still insoluble in salt solution was then washed thoroughly with water and lastly with alcohol and ether. Not enough of the albu- mose was separated from any one digestion for analysis, but by unit- ing the products from all three, sufficient was obtained for the following analytical data: I. 0°2537 gram substance gave 01500 gram H,O=6°57 per cent. H and 04550 gram CO,=48'92 per cent. C. II. 0°3700 gram substance gave 46°3 c.c. N at 13°8° C. and 761°3™™ pressure= 14°96 per cent. N. II. 0°1354 gram substance gave 0:0069 gram ash=5'09 per cent. The ash-free substance therefore contained 51°52 per cent. C, 6°92 per cent. H, 15°79 per cent. N. The ash was composed wholly of ferric oxide. The peculiar behavior of dysalbumose after solution in either dilute acids or sodium carbonate and neutralization, shows plainly that the substance is simply heteroalbumose rendered insoluble by action of the sodium chloride. Dysalbumose, wholly insoluble in sodium chloride, is readily dissolved by sodium carbonate of 1 per cent., and on neutralization of the alkaline fluid is in great part pre- cipitated. The substance however, is now soluble in sodium chlor- ide and has evidently been reconverted into heteroalbumose. It is very apparent, however, from our results that heteroalbumose from egg-albumin is not so readily converted into dysalbumose by the action of sodium chloride, as heteroalbumose from fibrin. In all three of our experiments, the amount found was very small. Further, it would seem as if heteroalbumose from albumin was somewhat more resistant to the action of alcohol and ether than heteroalbumose from fibrin. Still the former did become quite rapidly insoluble in sodium chloride after standing under alcohol d Albumoses. Chittenden and Bolton—Kgg-Albumin an 356 % "YUsV Jos) suoupep | LOTT SE | 18-6 | | | OOTTL-0 “TUR. “ONM+ HOM TIM WOISN) lage 'OSeg 00-001 86.86 mee BY ae tiie RS 00-6 00-6 ao ces a ST-9L % 8L-9L Cas Tae OT +h esta =< c0-4 7) TT 1G ware dae SP Tg 6&-TS OSRIOA VW ‘aounysqns aatf-yso ay, fo uoyrsodwuos abpzUasag 96-6 QOLO 0% ee ra Say cee ae Es Oe re ar | 7" | GO-TS | BLBL-0 | 66-9 6896-0 | C9GP-0 I an OES § 7 ‘ | | | i ( yx ‘wea % | 79) ee ae % pifteees ve | cy % qHeig. . =|. 2% "TUBIS | pe ; wy | ey | 8 | a gosty | = p | ‘paner*o0.| H -| PUNO} O'H | oouesgng | | | Toye 'OSed punoz | | - 8358 Chittenden and Bolton—Egg-Albumin and Albumoses. for a short time. The reactions of dysalbumose, aside from its behavior towards sodium chloride, were found to be much the same as those of heteroalbumose. Relation of the albumoses to albumin. In composition, the albumoses from albumin are seen to differ from each other somewhat more than the albumoses from fibrin ; collec- tively, however, there is less difference in composition between the albumose bodies and the albumin from which they are formed, than noticed in the case of the albumose bodies from fibrin.* In the latter, however, there is no guarantee that the fibrin employed in the experiments had the actual composition assigned to pure blood- fibrin. The fibrin-albumoses collectively contained about 50°6 per cent. of carbon and 17:1 per cent. of nitrogen, while Hammarsten found fibrin itself to contain 52°6 per cent. of carbon and 16°9 per cent. of nitrogen. In our experiments, on the other hand, we have for comparison the composition of the albumin actually used in the experiments, and in the accompanying table the differences in com- position of the various products are plainly to be seen. Examining these in detail, we see that all of the products show a somewhat smaller content of carbon than albumin itself. With nitrogen, however, there is a very close agreement throughout, and with sulphur likewise. In the case of the fibrin-albumoses it was considered that the diminished percentage of carbon indicated plainly that the albumoses were hydration products, and that they were formed from fibrin by simple hydrolytic action. The results obtained with the globuloses did not appear to confirm this view, but in this case it must be remembered that the digestion of globulin by gastric juice may be quite a different process from albumin diges- tion. With albumin, however, the results, although less pronounced, also indicate hydrolytic action and that the products formed are hydration products. The following table shows the extent of these differences, and also shows the close agreement in composition between proto- and deutero- albumose and the so-called soluble and insoluble hemialbumose from egg-albumin, isolated and analyzed by Kiihne and Chittenden at the commencement of their study of these bodies.* * See Zeitschrift fiir Biologie, Band xix, p. 174. 309 7 Albumoses. yt ANA Chittenden and Bolton—Egg-Album ‘ayeqidioead proe oyooy + -skq -01099F] 66.0 60°S 98°T 8-T ogre | 80-1 A ie 19-8¢ | 28-88 | 80-78 | ee &8-T Bee 69-1 80-2 Z0-2 08-1 og-cr | eho | cect | sect | Lect | 8h-ST 86-9 66-9 06-9 @0-2 76-9 | 16-9 eg-e¢ | ecte | 90-2¢ | 09-19 | @LIe | 20-8¢ ies oraiv| v¥ 9 q V “TLV psi oben Vag aaa ‘9sOuINg|eOIOyNI ‘areyidiooid aprazopyo wnpog ». 18-3 19-81 86-F3 68-82 86-1 10-6 F6-GT S1-9L 68-9 €0-L 16-0 ¥6-0¢ ta *«d “OSOUING][BOJOIg 360 Chittenden and Bolton—Egg-Albumin and Albumoses. Fibrin products. Egg-albumin products. | | Kepees A lee ee ie kee oo ae eee eben 69 a We cc ie = Sao See) Se oOo |] we | BBY SE) E |Se8 608) 8) So |B) As) £ |ahs ere| a8 | Be | Me aes |e ae a|7 S| 3 “ci S Csr s2 ae 50°77 50°65 | 52°68 50°89 | 51°04 | 51-07 = -51°62 | 52°33 Eileeshee ee See 6-78 | 6:83 | 6°83 | 6°81 | 6°89! 698 | 6°97) 6:98 ING ae nes ae -| 17-14 | 17-17 | 16-91 | 15°98 | 15-79 | 16-00 | 15-82 | 15°85 wane: esce Sy: 1:08) | 099%) [2:107) Wes) Sse |b aESRAY SB aaa 0 sees | 24°23 | 24°38 | 22°48 | ._-- ---- | 24°00 23°63 | 23-02 Deuteroalbumose is seen to contain 0°7 per cent. less carbon than egg-albumin, while protoalbumose contains fully 1:25 per cent. less. The nitrogen in the two compounds is in accord with the content of carbon and the composition of the two products certainly suggests hydrolytic action. The lower content of carbon in the albumose bodies has been ex- plained by some writers on the ground that the precipitants used, principally acetic acid, would tend to form an acid compound, and that even when dried and ready for analysis, the compound would still contain some acid; which fact would be sufficient to account for the low content of carbon found. It is to be noticed, however, in these results that the deuteroalbumose, not only on an average but in each individual case, contains more carbon than protoalbumose, which was precipitated by sodium chloride alone without the addi- tion of any acetic acid whatever. Further, comparing the two preparations of protoalbumose B, one of which was precipitated by salt alone and the other by acetic acid, it is seen that the percentage of carbon is the same in both. Unquestionably proto- and deuteroalbumose both do combine with acids, but after neutral- ization and long continued dialysis the albumose body certainly exists in a free state, uncombined with either acid or alkali. The greater portion of this work was completed before we had any knowledge of Dr. Neumeister’s work on the complete separation of protoalbumose from deuteroalbumose.|| It was, therefore, too late for * See Kiihne and Chittenden, Ueber die nachstén Spaltungsproducte der Hiweiss- kérper. Zeitschrift fiir Biologie, Band xix, p. 174. + Average of all the products analyzed. Zeitschrift fiir Biologie, Band xx, p. 40. {¢ According to Hammarsten. § Average of all products. || Zur Kenntniss der Albumosen. Zeitschrift fiir Biologie, Band xxiii, p. 381. Chittenden and Bolton—Egg-Albumin and Albumoses. 361 us to take advantage of his method of separation. Our preparations of deuteroalbumose unquestionably were not wholly free from proto- albumose, but that they did not contain much of this body is evi- denced by the small precipitate obtained with cupric sulphate ; for, as Dr. Neumeister has recently shown, deuteroalbumose entirely free from protoalbumose gives no precipitate whatever with cupric sul- phate. TRANS. CONN. ACAD., Vou. VII. 46 Nov., 1886. X XITI.—CasEIn anp 1Ts Primary CLEAVAGE Propucts. By R. H. Carrrenpen anp H. M. Parnter, B.A., Pu.B. FoLiow1ne out the general plan of procedure indicated some time ago with other albuminous bedies,* we have endeavored to prepare and study the primary products formed in the digestion of pure casein with pepsin-hydrochloric acid. Assuming that casein in its conver- sion into peptone by artificial gastric juice, passes through certain intermediate stages, in which bodies akin to the albumose bodies are formed, we have applied the methods of separation used so suc- cessfully in the past and have been able to isolate a class of bodies bearing the same relationship to casein that the albumoses do to albumin. For this class of bodies we propose the name of caseoses. In studying these substances and particularly their composition, we deemed it essential to be certain of the purity and composition of the casein to be digested; particularly in view of the recent con- troversy between Hammarstent and A. Danilewsky as to the single nature of this albuminous body. Assuming, as claimed by Dan- ilewsky,{ that casein is a mixture of two albuminous bodies and that the uumerous analyses recently made by Hammarsten of various preparations of casein are incorrect, particularly in the percentage of sulphur, made it incumbent on us to obtain some data on these points confirmatory of one or the other view, before advancing to a study of the products formed by the digestion of casein. Of the various methods used for the preparation of pure casein, that depending on repeated precipitation by acids and re-solution in alkalies has been the most in vogue; for although theoretical objections might be advanced as to the possibility of change in the nature of the substance under the influence of acids and alkalies, the . results obtained have in some respects, at least, been very satisfac- tory. Alex. Schmidt, in conjunction with Kapeller,§ showed plainly that dialysis of milk and then precipitation of the casein by acetic acid, while it gave fairly good results, was not sufficient in itself to * See the preceding articles on globuloses and albumoses. + See Zeitschrift fiir physiologische Chemie. Band vii, p. 227. Zur Frage, ob das casein ein einheitlicher stoff sei. Also same volume, p. 427. + See Zeitschrift fiir physiologische chemie, Band vii, p. 433. § Beitrag zur Kenntniss der Milch, Jahresbericht fiir Thierchemie, 1874, p. 154. Casein and its Primary Cleavage Products. 363 wholly remove the inorganic salts, and thus recourse was had to repeated precipitation by acids, after solution in dilute alkalies. By this method, Hammarsten* came to the conclusion that milk con- tains but two albuminous bodies, viz: casein and lacto-albumin, and the same investigator has repeatedly made use of this method for the preparation of pure casein. Lundbergt has plainly shown the notice- able resistance of casein to the action of acids, and Hammarsten has indicated the possibility of using acetic acid, if necessary, in place of the stronger hydrochloric acid. Millon and Commaille, however, have claimed that in the precipitation of casein with either acid, the’ precipitate does not consist of free casein, but is a compound of casein with the acid used. This erroneous view, Hammarsten shows depends simply on the great difficulty of washing the precipitated casein completely, and he suggests that it is perhaps impossible to prepare large quantities of casein absolutely pure. For the preparation of the substance, however, Hammarsten recommends acetic acid in preference to hydrochloric acid and final drying of the compound at a temperature of FL0°°C. Danilewsky and Radenhausen,} however, prefer to use hydrochloric acid in the preparation of pure casein, and for this purpose they use skimmed milk, diluted with 4-5 volumes of water, to which the dilute acid is added little by little, until a good precipitate is obtained. After filtration and repeated washing with distilled water, the casein is rubbed fine, then dissolved in water to which a little ammonia has been added, the fluid filtered and the clear filtrate again precipi- tated by the addition of a little dilute hydrochloric acid. Casein, so prepared, after being washed with distilled water, reacts acid to test papers and shows the usual reactions of this body; but if when freshly precipitated, the substance is boiled with perfectly neutral 50 per cent. alcohol and filtered hot, according to Danilewsky and Radenhausen, the casein is separated into two bodies, one of which is partially soluble in hot alcohol and separates out on cooling, while the other is insoluble. The soluble portion is termed caseoprotalbin, the insoluble portion caseoalbumin. The former, it is stated, gives * Zur Kenntniss des Caseins und der Wirkung des Labfermentes, Jahresbericht fir Thierchemie, 1877, p. 158. + Kleinere Beitrage zur Kenntniss des Caseins, Jahresbericht fiir Thierchemie, 1876, p. 11. ¢ Untersuchungen tber die Kiweisstoffe der Milch, Jahresbericht fiir Thierchemie, 1880, p. 186, 364 Chittenden and Painter— Casein and its no sulphur reaction when boiled with 2 per cent. sodium hydroxide, but contains 1°13 per cent. of sulphur, while caseoalbumin is stated to contain 1:23 per cent. of sulphur. Hence, according to these investi- gators, casein is a mixture of two bodies, one of which is rich in sul- phur, while the other contains a somewhat smaller amount. The objection which these investigators make to the use of acetic acid in the preparation of casein, is that from a sodium acetate solution, casein itself is not precipitated, or only in part, but that the precipi- tate consists mainly of the protalbin body. Further, Danilewsky and Radenhausen claim that caseoalbumin dissolved in 1 per cent. sodium hydroxide and allowed to stand for 24 hours at the tempera- ture of the room, is changed almost completely into caseoprotalbin, with loss of sulphur and calcium phosphate. In a similar manner, protalbin dissolved in lime water, with addition of alcohol and phos- phoric acid, can be changed into caseoalbumin. Hammarsten,* however, takes exception to these views and points out that the peculiar behavior of Danilewsky’s casein towards boiling 50 per cent. alcohol, depends in part upon its content of calcium phos- phate, the presence of which impurity depends upon the use of hydro- chloric acid in the precipitation of the casein, which acid does not favor the removal of the salt as well as acetic acid. Using acetic acid as the precipitant and then employing a casein three times so precipitated, and which analysis showed to be almost entirely free from calcium phosphate, Hammarsten, by treatment with boiling alcohol, was unable to. obtain anything more than a trace of sub- stance corresponding to caseoalbumin. Further, casein is unques- tionably changed by boiling with alcohol, as Hammarsten clearly shows; in fact it is well known that heating an albuminous body in water is liable to change its nature, at least its solubility, and there is no reason why treatment with 50 per cent. alcohol should not lead to a like result. Again, Hammarsten points out clearly another inconsistency in the reasoning of Danilewsky and Radenhausen in connection with the so-called conversion of caseoprotalbin into caseo- albumin. The former body is stated to be poorer in sulphur than the latter, and yet we are told that the protalbin body can be con- verted into caseoalbumin by simple solution in lime water and addi- tion of phosphoric acid, with or without alcohol. Yet how it is possible by this method of treatment to convert a body with a small * Zur Frage, ob das Casein ein einheitlicher Stoff sei. Zeitschrift fir physiologische chemie, Band vii, p. 227. a | Primary Cleavage Products. 365 content of sulphur into a body richer in sulphur, is hard to see. Much more plausible is it, as suggested by Hammarsten, that in these two bodies we have to deal with the same substance, in the one case united with calcium phosphate, and in the other uncom- bined with this salt; or in other words that the so-called protalbin body in the absence of calcium phosphate is soluble in boiling 50 per cent. alcohol, while in the presence of that salt it is insoluble. This view being correct, and Hammarsten’s observations would tend to show that it is, it is obvious that the caseoprotalbin of Dan- ilewsky is simply a portion of the casein, which, owing to lack of a sufficient amount of calcium phosphate, passes into solution on being boiled with dilute alcohol; while caseoalbumin, on the other hand, is likewise a portion of the casein, insoluble on account of.the presence of calcium phosphate; changed, however, more or less by action of the boiling alcohol. Further, the reason why casein precipitated several times by acetic acid does not contain as much calcium phosphate as when precipitated by hydrochloric acid, and thus reacts differently with alcohol, depends on the far greater insol- ubility of freshly precipitated casein in excess of acetic acid than in hydrochloric acid. In the precipitation of casein with hydrochloric acid only the slightest excess of acid can be added, on account of the ready solubility of the precipitate in this dilute acid. With acetic acid, however, a moderate excess can be added without solution of the precipitate, and thus in the latter case, a larger proportion of mineral salts are removed at each re-precipitation. Danilewsky and Radenhausen have further called attention to the fact that casein precipitated with hydrochloric acid yields a larger amount of alkaline sulphide than when precipitated by acetic acid. This statement, Hammarsten has several times been able to verify, but the latter investigator seeks an explanation for this fact in the occasional: presence of a second albuminous body, richer in sulphur, presumably serum-globulin, precipitable like casein by acids. Serum- globulin too, is readily soluble in excess of acid, even more so than casein, and hence by the acetic acid method of precipitation, which allows a far greater excess of acid, the casein would be much less liable to contamination by this hypothetical globulin than by the hydrochloric acid method. In this connection it may be well to notice that Musso and Menozzi* claim the presence in milk of a peculiar albuminous body containing 53°74 per cent. C, 15°52 per * Studien iiber das Eiweiss der Milch. Jahresbericht fiir Thierchemie, 1878, p. 139. 366 Chittenden and Painter— Casein and its cent. N and 1°55 per cent. S, for which they claim a_ position midway between serum-albumin and casein. It can be partially pre- cipitated from fresh milk at ordinary temperatures by the addition of acetic acid. Further, Lebelien* has proved the presence in milk of a globulin-like body, lacto-globulin, which can be precipitated by satu- rating the fluid remaining after removal of the casein with sodium chloride, with magnesium sulphate. The substance appears to be identical with paraglobulin, and thus this fact, just discovered, would seem to confirm Hammarsten’s theory as to the cause of the greater content of sulphur sometimes noticed in casein precipitated by hydro- chloric acid. Accepting then, Hammarsten’s views as correct, it is obvious that casein precipitated by acetic acid, if not a single body, must be com- posed of two bodies, more or less alike and both precipitable by dilute acids. In attempting to settle this point definitely, Hammars- ten has sought by analysis of a large number of preparations made under different conditions, to obtain data as to the exact composition of casein variously prepared. Naturally in this connection, consider- able attention was paid to the content of sulphur, since, Danilewsky and Radenhausen’s views being correct, variations in the content of sulphur would naturally be expected. If casein is a mixture of equal parts of caseoprotalbin and caseoalbumin with 1°13 per cent. and 1°23 per cent. of sulphur, respectively, then casein itself would nat- urally contain 1°18 per cent. of sulphur; an amount somewhat higher than has been found heretofore. Recently, Dainlewsky+ has modified his views somewhat, and now considers casein, as before, to-be a mixture of two distinct bodies, but of nucleoalbumin with nucleoprotalbin instead of caseoalbumin and caseoprotalbin. As the reactions of these two bodies are appar- ently much the same as those given as characteristic of the caseo- bodies, this change of view appears to be mainly a change of name. Danilewsky still claims the correctness of the high content of sul- phur in casein and assumes that the variation in the results obtained by different workers is due simply to difference in the methods of determination, and that unquestionably pure casein contains over 1°0 per cent. of sulphur. The content of sulphur in casein as determined 30-40 years ago by * Beitrag zur Kenntniss der Eiweisskorper der Kuhmilch, Jahresbericht fir Thier- chemie, 1885, p. 184. +See Zeitschrift fiir physiologische chemie, Band vii, p. 433. Primary Cleavage Products. 367 Lehmann, Riihling, Volckel and others,* varies from 0°85 per cent. to 1:10 per cent. Ritthausen,t from several analyses of the copper compound of casein, found a content of sulphur equivalent to 0°80— 1:12 per cent. in the free casein. Schwarzenbach,f{ by a study of the platinum cyanide compound of casein, ascribed to casein itself a con- tent of 0°19-1°10 per cent. of sulphur. Hammarsten, however, found by analysis of eight distinct preparations of casein, some of which had been reprecipitated even ten times with acetic acid, a content of sulphur ranging from 0°619 per cent. to'0°775 per cent.§ Later, Hammarsten|| analyzed four other preparations of casein and each by six distinct methods. Omitting two or three results, which were altogether too low on account of inaccuracies in the method, Ham- marsten found in these different preparations of casein, as a result of twenty-nine distinct determinations by five different methods, 0-798 per cent. as maximum, 0°726 per cent. as minimum, or 0°758 per cent. as the average, content of sulphur. Taking, however, the results obtained by what Hammarsten considers as the more correct methods the average content of sulphur is raised to 0°77—0°78 per cent. Inno case did Hammarsten obtain results in any way con- firmatory of Danilewsky’s views. Hammarsten further made a large number of phosphorus determinations, and these as well as the results obtained for carbon and nitrogen showed too little variation to war- rant the idea of a mixture of two bodies of unlike composition. While therefore, Hammarsten’s results would seem to point conclu- sively to the unit-like nature of casein, we have, however, made quite a number of different preparations of the substance, both from fresh milk and from skimmed milk, with the idea of obtaining confirmatory data, with which to make direct comparisons between the composi- tion of,casein and its primary cleavage products. Preparation and composition of Casein. The casein was precipitated in some cases by acetic and in others } p by hydrochloric acid. In both cases the acid used was very dilute, * See Gmelin-Krauts’ Handbuch der Organische Chemie, Band iv, Abtheilung, iii, 1870, p. 2254. + H. Ritthausen und R. Pott, Untersuchungen tiber Verbindungen der Kiweisskorper mit Kupferoxyd. Journal fiir prakt. Chemie, 1873, Band vii, p. 361. t{ Annalen der Chem. u. Pharmacie, Band exxxiii, p. 185. § Zeitschrift fir physiologische Chemie, Pand vii, p. 259. || Ueber den Gehalt des caseins an Schwefel und iiber die Bestimmung des Schwefels in Proteinsubstanzen, Zeitschrift fiir physiologische chemie, Band ix, p. 273. 368 Chittenden and Painter— Casein and its the hydrochloric acid being 0-2 per cent. In dissolving the casein for re-precipitation, a very dilute solution of ammonium hydrox- ide was employed; in fact so dilute as to consist of hardly more than water with a trace of ammonia. We used ammonia, in preference to sodium or potassium hydroxide, as this alkali would seem less liable to induce any alteration in the content of sulphur. Further, in dis- solving the casein in ammonia, the solution at no time became more than very faintly, if at all alkaline; usually being hardly more than neutral to test papers. The general method of procedure was to dilute fresh cow’s milk with about four volumes of water (skimmed milk diluted considerably less) and then to precipitate the casein with either hydrochloric or acetic acids, adding the precipitant cautiously, until complete precipi- tation was obtained. The precipitate was then washed as completely as possible with large quantities of water, both by decantation, tritura- tion with water in a mortar and on a cloth filter. The casein was then dissolved in the ammonia water, filtered through paper and reprecipitated, each time being thoroughly washed with water. In the portion used for analysis, the final precipitate was further washed with alcohol and ether and lastly soaked in a mixture of alcohol and ether for the more complete removal of any fat. The preparations were then dried in the air and lastly on a water bath at a gentle heat. When dry, they were powdered and extracted with boiling ether in a fat extractor for several hours, to insure complete freedom from fat. Ultimately, the products for analysis were dried at 105° C. in vacuo until of constant weight. In all, seven preparations of casein were made for analysis, as follows: No. I. From fresh milk, precipitated twice with hydrochloric acid. ‘© TI. From fresh milk, precipitated twice with acetic acid. «* JII. From skimmed milk, precipitated three times with acetic acid. «« TV. A portion of No. III, precipitated a fourth time with acetic acid. « V. From skimmed milk, precipitated three times with hydrochloric acid. « VI. A portion of No. V, precipitated a fourth time with hydrochloric acid. «* VII. From skimmed milk, precipitated four times with hydrochloric acid. The methods of analysis were essentially the same as those em- ployed by Kiihne and Chittenden in the analysis of the various albu- mose bodies. Carbon and hydrogen were determined by combus- Primary Cleavage,Products. 369 tion with oxygen in an open tube, the gases passing over a long layer of granular oxide of copper at a bright red heat, a layer of lead chro- mate at a dull red heat and a roll of freshly reduced metallic copper. Nitrogen was determined as nitrogen gas by combustion with oxide of copper, the gases passing over a long anterior layer of heated oxide, a short layer of metallic copper and a final layer of oxide of copper. The tube was exhausted with a Sprengel pump before and after the combustion and the nitrogen was collected in a Schiffs’ azo- . tometer, provided with a jacket tube for rapid cooling of the gas toa constant temperature. In the determination of sulphur and phospho- rus, the substance was fused with a mixture of potassium hydroxide and potassium nitrate (10 grams of the former and 1°5 grams of the latter) in a silver crucible, according to the method designated by Hammarsten * as 1a. In order to economize time, a single fusion was made to serve for both a sulphur and phosphorus determination ; in other words, a sufficient amount of casein (usually 1:2 grams) was fused with potassium hydroxide and nitrate, the fused mass dis- solved in water, the solution made up to a known volume and then one-half, representing one-half of the original substance, was used for the sulphur, the other half for the phosphorus determination. Both the alkali and the nitrate were free from sulphur and phosphorus; at least to such an extent that in a blank experiment, the resultant solutions gave no precipitate whatever, with either barium chloride or with molybdic solution. The oxidations were made at as low a temperature as possible, except towards the end when the temperature was raised, and occasionally a little more nitrate added, to facilitate complete oxidation. As the percentage of sulphur was quite an important point, we took particular pains to have the final acid fluid entirely free from nitrate and nitrite, as well as from any excess of hydrochloric acid, so as to avoid as much as possible any solvent action on the barium sulphate. For the determination of sulphur, therefore, the alkaline solution of the fused mass was acidified distinctly with hy- drochloric acid and the acid solution evaporated to perfect dryness on the water-bath. In this way the objectionable nitrate and nitrite were removed. The residue was then moistened with hydrochloric acid, taken up in water, and the solution allowed to stand until any chloride of silver present, had settled out. The fluid was then fil- tered and precipitated as usual with barium chloride. For phos- * Zeitschrift fir physiologische Chemie, Band ix, p. 289. TRANS. Conn. ACAD., Vou. VII. 47 Nov., 1886. Chittenden and Painter— Casein and its 370 00-001 90-86 sho ae a © ey Pc 98-0 98-0 ye os i Ps the or. &8-0 * 68-0 ie Pa aS, cs. 66-91 ze aa 6-81 — 6B-GT age ena OF -d aoe ree pie es 60-2 ney) 66-9 ane ee oe cee 16-€G &6-6¢ ‘aSBIOAV OmMznho ‘aounjsqns aatf-ysp ay, fo Uorpisodmon abnquarwag 6-0) /-OFO0-0sh IS race fy. ieee nae rene SS eege aoe iein'| Rp rate eee eee eee ae 960) | E7000 |= -* = le 9 = eur s rap ais mee TSA ee lor le a aera age sel Ones eee ---- | ---= | og.9 6810-0 wae aS Bee fede ee er ca a ate | 7 | mmm | O8F-0 | TIT Soe ese | om oS re ee DST Me Bers GP-S SSA. OL -2o HeOLe-0:| SOeerieeed (Gore C4 Th Ba | ad ek tite agilltae vg ae: SEO oat TS) OLB | SE6L-0 |°S0-L | G098-0 | FOLF-0 | I | ‘TURIS | "WRIs “COU : | ; ; : Oiesalae : F TRIS 2 : et z “ONY + HOM y “ON + HOW % eae each ‘ih me y 5 ae 2 Rae eae ON ‘USV poe d YM woisny 8 YIM dong eg 5} ee H F ) gour)s t UsV "00 Joe +O* ges Jayze FOSRg “‘punoy NY -qug ‘T ‘ON NIASVD JO SISATVNW 371 Primary Cleavage Products. 00.00E 9E-66 &8-0 94-0 O8-GT TO-4 61-69 ‘QOB10A V 68-0 88-0 82-0 ‘Q0UDISQNS datf-YsD bL-0 98-CT GL ST ay, fo uoyrsodwuos abnjualad 90° UGS OoHwAmao 9-0 | 0800-0 69-0 ¢800-0 ‘med | ‘punoy | “” USV d 2 ‘qsV “wes ‘SON + HOW Wy dorsny 1oye +O%q* oq meds % | §*ONN+HOH Ss YIM UoIsny layye 'Ogeg LUST 99-1 bb N G-9G2, 6-8EL “WO o1nssodd GLY 6-67 96-6¢ T8-6¢ 0696-0 LETL-0 60-2 66-9 OGTE-0 0086-0 VokS-0 9gc¢-0 80PL-0 986¢-0 S0FL-0 986¢-0 GFSE-0 &C9E-0 8&6P-0 C898-0 *punoy N Ores ‘TUBIs ‘punojy 500 fy se mRIS *punojy O°H “TRS ‘posu ours “qug ‘TIL ‘ON NIQSVOD AO SISATVNY Chittenden and Painter— Casein and its 372 GFT ¥é-T 1900-0 £900-0 “med *punoj qsSv 88-0 00-00L 78.16 68:0 68-0 66-91 60-4 08-8 *OSBIOA VY “TRS “ON + HOW WIA WOIsNy Jayye O° q°3Wl 68-0 68-0 aie Ee OPO 96ST ‘aounysqns aatf-yso ayy fo worprsoduoo abnuaolag ames 18-&¢ OMAnmwO 0880-0 ie ae “TRIS “ONN+HOM | % WA uorsny N 19}jyB "OStg TLL | OTS | TES PLGL | 0-16 | 87 “UU 10) 6 : amsseig| “7 | °° *panoy NT LG-6G 8-6 Orr CESL-0 ECPL-0 mURIS “punoj °00 | ‘IIT (ON NIQSVOD HO SISATVNY ~~”: | 0667-0 | ITIA “""" | SS8F-0 | ITA ~~" | 997S-0 | IA 5 I ODPS-0"|, Ac “~~ | 9898-0 | AT “""" | 99¢E-0 | TIT CLEC-0 | G08E-0 | IT TSP6-0 | S98E-0 | T “WBS Taehle poy |B ox O°8 "| -qng 373 Primary Cleavage Products. , 00.00T 66-16 os £8+0 68-0 ASST 90-4 68-8 “OSBIOA V 18-0 c8-0 61-0 86-T LUST ‘aounysqns aatf-yso ay) fo uoyrsodwos abnyucdlag 6600-0 ¥&00-0 18-0 98:0 “mes |*ONX+HOM d AIM WoIsny Toye 40% qs 8-0 82-0 le wes “ON + HOM YIM OISNy loye 'ogeg 48-ST 99-ST wees 9-V9L 9-79L T-g GS-87 % N “TOU ‘Ors einsselg | “L ‘punoy N ‘AI ‘ON NIGSVD JO SISATVNYV 0GL8-0 TLE9-0 IT-k 67-ES 66-9 60-4 TmR3 “punoy °00 fd se ‘ON . Chittenden and Painter— Casein and its 374 00-00F 86-66 78-0 GL-0 GL-T TT +k | £Or-EG ‘OSBIDA YW 08-ST OL: ST 80-2 FI-8¢ CE-8S ‘aounjsqns aaif-ysp ay? fo Uuorrso0dwuos abnUadlag 9700-0 CP00-0 -s-- 68-0 vL-0 % ‘USV “TRS “panos YsV pb d TURLS “ONM+ HOM YA WoIsny I9ye +O*q*av “BLS “ONM+HON | YIM UOIsny 1aqje "OSeg L9-ST LG-GT p N “Wud OINSS8oI q F-61 P-8T ‘0. AL “*punoy NV PTS 8-8P 0969-0 VE6T-T GLST-T 60-2 60-2 OS6T-0 S188-0 9TES-0 6LES.0 OSE8-0 Of&8-0 8688-0 6996-0 F808-0 6819-0 OL6S-0 URIS *‘punoj} 509 b H mRIs *punoj O°H “mes *pesn 90uR\s “Qug XI TITA IIA TA A Al ‘ON ‘A ‘ON NIASVD JO SISA IVNY 375 Primary Cleavage Products. 00-00F [6-16 480 48-0 80.91 : GO-k GGEG ‘Q0BIOAW | SF00-0 | ~~~” 9700-0 £80) 18-0 66-0 18-0 OL-9T 40-91 ‘aounjsqns aaif-ysn ayy fo uowprsodwmos abnyUwaalag TUR. . *‘punofy p ysy | @ "mes “ONM + HOM Uji uoOIsny Jaye 4Q%g*aK OMAnuno “mURIS “ONMN+HOM WIM wosny Joqye 'OSeg 96-ST 66-ST N tustuns 0. einsseig | ‘, “punojy NU ‘TA ‘ON NIQSVD JO SISATIVNY OFOL-0 602-0 66-9 | P666-0 ¥6-9 | LO&s-0 | 8687-0 | 9EPF-0 0669-0 6169-0 069-0 6169-0 OF9E-0 06Sé-0 &V9E-0 PLLE-0 TUBIS *‘punos "00 eden fo} *punoy % H | oH “URIS ‘posn eourys “qug Chittenden and Painter— Casein and its 376 00-001 SNES Mec Mies Pe te Biak dea Pee 8 eae Oe ee O 88-0 peer eres pee sat ee eee ie oh Reh a a BG ha POs SG | anges RM Pace page se pu g CN) ail eat a se 10-91 MOu eae eee ak ae ee N GD Or ECE Pe Re eta ay CEPT SS IY Regen Te PT-L LO-L H UGS seal a aha’ SAT 0 Races, Oat aie 7M aee a oS 6F-8g 8G-2¢ 9 “OSVIOA YW ‘aoupnjsqns aauf-yso ayy fo uowrsodwoos abnzuao1ag | Gestcoun ier. se sacra We Gus eee ne pear | Seen cel ue weal Secs ui ones SIIF-0 ILA L0:119900-0|-" Tie eet" Pie Wgiaace aes SSP Ses Ci ie al ee eens Gall a eee: 620F-0 |ILA Span 27S Geile SOT esOie lost la aeane os pee er ented eos ec ried Mic By ers B819-0 |IA Baresi || Matec 98-0 68F0-0 Be thst ae oe Soap ee eee ee ie eae Cas 6829-0 |A eC aealeey, (eee ees: Po BOT |G-00h: <6) (greg. ae Cart haere 0868-0 |AT = Pearse ore a Spain jel eee Sas Peele Ute desc lees ollsss < S Bec haga ecLy-0 [TIL eae lead aac ens De MERE ce Reece “--" | --7- | gee] o2gt-0 | 80-2 | 68¢¢-0 | 2907-0 [II as a oa hd ai Sond fetes c sages pees ““-- | =--- | gg.g¢ | 0926-0 | 86-9 | T9Te-0 | Te0g-0 {I ‘ 3 “Wels “weg we Do cD mURIS “a1eIs weiss % |ounor| 2 (ONH+HON) % |“*ONN+HOM| % ‘|omsserg] “1 Boch et eee ease eee ‘ysy| PUNO! a | qua aon | g WA woIsny N 0 rac pes ed ao n 3 vy ree “OF f3N qoye 'OSeg ‘punoy N 08 pS eae ‘ILA ‘ON NIDSVO AO SISAIVNY Primary Cleavage Products. 377 phorus, the alkaline fluid was acidified with nitric acid, evaporated to dryness, the residue dissolved in a little water acidified with nitric acid, filtered, and the phosphoric acid precipitated in the usual man- ner with ammonium molybdate. After standing 24 hours at 40° C. this precipitate was filtered off, dissolved in ammonium hydroxide and the phosphoric acid re-precipitated as ammonio magnesium phos- phate and ultimately weighed as magnesium pyrophosphate. The accompanying tables show the results of the analyses of the different samples of casein. Comparing now the average composition of these different prepa- rations of casein, it is to be seen that they all show a very close agreement throughout. Thus the percentage of phosphorus in the seven preparations varies only from 0°84 to 0°89, sulphur from 0°75 to 0°89, nitrogen from 15°75 to 16°08, hydrogen from 7:01 to 7:11 and carbon from 53°19 to 53°53 ; or leaving out one preparation which for some reason showed a high content of carbon, from 53°19 to 53°39 per cent. The results therefore show a constancy in composition fully as marked as observed by Hammarsten and thus tend to confirm the latter in the view that casein is a single body of definite com- position. : Comparing our results collectively, with those obtained by Ham- marsten (see table showing average composition), we find a fairly close agreement throughout, although minor differences are tobe observed. First, all of our preparations show a content of carbon somewhat higher than found by Hammarsten. The latter investiga- tor found the carbon in bis preparations to vary from 52°78 to 53-09 per cent., while in all of our preparations, the content of carbon cal- culated to the ash-free substance is above 53 per cent. The possi- bility of our preparations still containing some fat was rendered improbable by the thorough treatment with ether which they had received, and further by the fact that the nitrogen in our preparations was also somewhat higher than found by Hammarsten. One of the preparations, however, with a high content of carbon, was extracted again for several hours with boiling ether, but on analysis the con- tent of carbon was found unchanged. ‘The content of phosphorus agrees exactly with Hammarsten’s results, while the sulphur is, on an average, 0°] per cent. higher. There is nothing in the content of sul- phur, therefore, to even suggest confirmation of Danilewsky’s views. The amount of ash in our preparations was somewhat larger than found by Hammarsten and further, there is no especial connection to be seen between the content of ash and the precipitant used; the Trans. Conn. AcApD.. Vou. VII. 48 Nov., 1886. Chittenden and Painter— Casein and its 378 "ga ‘d ‘engi ‘olmaqosory INJ JYOUoQsolyee 99g ‘S}[NSer 9aIY} JO O5BIOAB ‘UJosBo JO punodutoo z0ddoo oy} jo stsAyvue mos uesnegyyiy Aq pozepnopep + ‘g9z ‘d ‘TA pueg ‘otwoyo oyosteojorsdAyd any 4JLIyOs}10Z 90g + 4-108 “ney OF Sup io00V | JO ecRloAy 86-0 T&T 8L-66 60-66 C9-16 c8-0 18-0 88-0 GLO 68-0 88-0 ¢9-CT 16-1 96-GT C0-4 L0-4 OTL 96-6¢ 0&-&¢ §G-€G ep Inu-ywon iit Beret! aoerie pee le TEA ONT TOH WE poqeqidio -o1d sow, p ‘TA ‘ON TOH yy poyeydro -o1d sauly ¢ “AON “HOOO-“HO poyeqidro yin -a1d sow) Pp “AT “ON ‘HOOO“"HO YIM pozeqidio -oid sauly ¢ TIL ON ‘SNIASVO DHL JO NOILISOdNOD ANVUAAY AHL ONIMOHS WAV], 6-0 ‘HOOO-"HO TIM poyey -1dioe1d a0TA\} TI ON TOH YIM poqey -1droaid 9014 IeoN : Primary Cleavage Products. 379 amount in the acetic acid precipitate being fully as large as in the casein precipitated by hydrochloric acid. Compared with Ritthausen’s results (see table showing average composition), obtained by analysis of the copper compound of casein, the percentage of carbon comes very much too high. It is question- able, however, how close a comparison should be drawn between indirect results obtained by analysis of a metallic compound of casein and those obtained by analysis of casein itself. In conclusion then, we must affirm that our results accord closely with those of Hammarsten’s, while the two together make it very improbable that in casein we have to do with a substance composed of two bodies of unlike composition. Digestion of Casein and Formation of Caseoses. In the digestion of casein with pepsin-hydrochloric acid, the casein was prepared by precipitation and reprecipitation with acetic acid, and rendered as pure as possible by thorough washing with water. While still moist it was placed in 0°4 per cent. hydrochloric acid, as preliminary to its treatment with pepsin. Pure pepsin solution, free from peptone and albumose, was prepared from the mucous mem- brane of pig’s stomachs by the method already described.* Digestion A. 1300 grams of moist casein in 4 litres of 0-4 per cent. hydrochloric acid were brought to a temperature of 45° C. and 600 cc. of pure pepsin solution added. The mixture was kept at a temperature of 45° C. throughout the digestion. The casein began almost immedi- ately to swell up and in less than an hour the entire mixture was con- verted into a semi-solid, jelly-like mass. Thereupon, one litre more of 04 per cent. hydrochloric acid was added, together with a little more pepsin solution. At the end of three hours, the mixture was quite fluid, but contained considerable gelatinous matter in suspension. Neutralization of a filtered portion, produced no précipitate whatever. The addition of crystals of sodium chloride gave a heavy white pre- cipitate and the filtrate from this precipitate gave a further precipi- tate on the addition of acetic acid. At the end of four hours, the entire mixture was made neutral with sodium hydroxide and then filtered through paper. The undigested residue, when dry, amounted to about 30 or 40 grams. This residue of so-called casein dyspeptone, * See the preceding article on egg-albumin and albumoses. 380 Chittenden and Painter— Casein and its which appeared in every digestion in greater or less quantity, was apparently wholly insolubie in fresh portions of gastric juice and was similar in its reactions to the like-body previously described by Lubavin.* | When fresh, it appeared as a more or less jelly-like mass, much like starch paste. It was readily soluble in dilute alkalies and precipitated by neutralization, but insoluble in excess of acid. It was precipitated from its solution in dilute sodium hydroxide by addition of salt in substance. This body we did not attempt to study further, but hope to do so later. The filtrate from the undigested residue, when cold, was not per- fectly clear but became so on the application of a gentle heat. On boiling the solution, a very slight precipitate was formed. The addi- tion of acids, hydrochloric, nitric or acetic, either concentrated or dilute, caused a heavy white precipitate, not wholly soluble in excess of acid, even of concentrated hydrochloric. The precipitate was likewise more or less permanent when warmed or even boiled with the acid. In the filtrate from the precipitate produced by acetic acid, the addition of potassium ferrocyanide gave no precipitate. The precipitate produced by acids was readily soluble in dilute alkalies. On addition of nitric acid, of any strength, and the appli- cation of heat even to boiling, the mixture turned first rose color then reddish, and as the boiling was continued the color deepened and finally became brownish red. The change from rose color to brown also takes place in the cold. With concentrated nitric acid the color is nearer the yellow of the xanthoprotein reaction, but still shows plainly the brown or reddish tinge. For separation of the individual caseoses, the entire solution with- out being concentrated, was saturated with sodium chloride, by which an exceedingly heavy precipitate, more or less curdy, was obtained, which was finally filtered off and washed with saturated salt solution. The washing was made more thorough by grinding the mass with the salt solution in a mortar. This precipitate, by analogy, would naturally be composed mainly of a body correspond- ing to protoalbumose with possibly something corresponding to hete- roalbumose. The precipitate was washed thoroughly with saturated salt solution, dissolved in water, filtered, and again precipitated by saturation of the fluid with sodium chloride. All of the substance, however, was not reprecipitated; quite a little remained in the salt- * Ueber die Kiinstliche Pepsin-Verdauung des caseins, ete., Hoppe-Seyler’s Med. Chemische Untersuchungen, p. 467. Primary Cleavage Products. 381 saturated fluid and was thrown down as a white curdy precipitate by the addition of a little acetic acid, evidently some protocaseose not precipitated by salt alone. The main precipitate of protocaseose, ete., twice precipitated by salt, was treated with 3 litres of 10 per cent. salt solution, the residue with 3 litres of 5 per cent. salt solution and the residue still remaining, with 3 litres of water. In this man- ner, all of the proto and heterocaseose was dissolved, leaving a small residue wholly insoluble in dilute salt solutions and in water; presumably dyscaseose. The latter, however, was in exceedingly small quantity. It was dissolved in 0:2 per cent. hydrochloric acid and reprecipitated by neutralization of the solution with sodium . carbonate. A. Protocaseose. The 5 and 10 per cent. salt solutions of protocaseose together with the aqueous solution, were united and the mixture saturated with sodium chloride. Here, as before, all of the protocaseose was not precipitated ; a portion remained in the filtrate and was precipitated only on the addition of a little acetic acid. The main portion of the protocaseose precipitated for the third time with salt in substance, was dissolved in water, the solution filtered and divided into two parts. One part was thymolized and dialyzed in running water until all chlorine was removed from the solution (Protocaseose A 1). The other part was again saturated with salt, the precipitate washed with saturated salt solution, then dissolved in water and like the former dialyzed until all chlorine was removed from the solution (Protocaseose A 2). In protocaseose 2, there was more evidence of the presence of a body resembling heteroalbumose than in No. 1. Thus in No. 2, quite a little gummy substance separated from the solution on dialysis, but the amount even here was not large. When the dialysis was finished, both solutions were perfectly neutral to test papers and in both cases the protocaseose was separated from the clear fluid by evaporation and precipitation with alcohol. For analysis, both products were washed thoroughly with alcohol and ether and finally dried at 105° C. in vacuo. Their composition is shown in the accompanying tables. As already stated, every time protocaseose was dissolved in water and reprecipitated by saturating the solution with sodium chloride, a certain amount of the substance remained in solution, precipitable only on addition of a little acetic acid. Protocaseose precipi- tated from the salt-saturated solution in this manner by acetic acid, 382 00-001 99-86 cae aces Psy Ping fee O 96-0 96-0 36. aes Pe =; S$ Gh6GT ys) 8L-ST 89-T so os N GT h Sei’ me ae 5 Ses CT-L H 09-69 oa. pee ae eed LG- 6G FPS @) "Q.0R10A VY ‘gounysqns aauf-ysp ay, fo wor1rsoduos abnuaolag | GarOF i eTeO: Ohi) ia) carl) eee g 2 GAN eA ay | Poccae atc rae ye ek ts een aes _ &B0-0 IA “gee ae ie La 98-0 0880-0 FOAM Ik wpe sedans alta ae allt Sasha calle a een eS Ne 0909-0 A iy ee, | Se Ga a ae ak aad TSBs) oe GO sie POL Ge io Se aie a ae Re ee og 0L8E-0. AT 5b | PO = le pager Ue): sere TeeeE || sesOOti PeOe |. BR ah So ee) oe Get ee eo ae 008-0 Ill sod eee ee ade 7 ee ee Sy) So eee a eg, Se eer 0809-0 ete ae ales 096E-0 II Soi ae ee ii ~-oi | betes eae ga al x, |, Pe OG OF 6128-0 01-9 1286-0. CGLF-0 I ; "uIBAs ses Oo | 3:0 ‘ baie % |*OoNu+HON| % | SSS) ab > “waes8 Vg “seas Bef ‘ON USV Sy Ss YA DoIsny Niall > sew ¥ ®) "panoy *OO | H | PUNY O°H | gouvysqng 2 ‘punoy N 1918 Foseg ‘LV aSoasvOOLOUg JO SISKIVNY 383 Primary Cleavage Products. “YSy JOS Suyonpep 19}JBS O $ N H e) 88-0¢ | S9SL-0 GP-0S | 2689-0 69-9 08-9 00-001 61-66 goes ore Ss =e net Be 86-0 86-0 ae Tad 5 See ear 18-91 _— 68-ST 98-CT ae ite z Tok Te Raa. ah OL-2 LoL G8-EG oat Tes ey 68-S¢ L8-6G ‘9.0R10A V ‘gounysqns aauf-ysv ay) fo worpisodwmos abn,Ud0laq 6G: T GOO. Seamer es ok ae Se See arias et tia (Me a | Were ee Se | 9-0 POCO te cee ltege ah ee Ratio ae SES cy | aan opal ers me oe ie Re SN oe 2 hn Se ot GRO OREO Oil ce aes es oe Ege $l ea a tee ee Fite. je Tc seen eet (a) aa aa a “Fa 1 B8-FL ie PON- | 2-062) 6 Vhs ko a Pa Ale. hee oe ae ae geen ee Lace 2s 2: SBaP | E08), 1-06 | 8h | WeIs : “TURAS “co 1D) e we % | *ONU+HOH | ang jog) Ee | 2 ae % |emeserg] “1 | | 2 Ss WM TONE | pay yo | CUM) “USV | ysy | N 4) Joye 'OSPE “OS ‘punoj N ‘8 VY ASOUSVOOLOUd AO SISATVYNY “meR1s *punoy °00 Wc eae cc6¢-0 | IA See ok €c6F-0 | QA pier, ate 6667-0 | A ae c9cs-0 | AT Lae 9696-0 | IIT 9966-0 | Fe6E-0 | IT F8ce-0 | 6eLE-0 | I “mes “MOBI “punoy ‘pesn | ‘oN O°H | eoueysqng 384 Chittenden and Painter— Casein and its was found to be quite different in its nature from protoalbumose or protoglobulose. The two latter undoubtedly combine with acetic acid, when precipitated from a salt-saturated fluid; the eompound, however, is readily and completely soluble in water. With proto- caseose on the other hand, acetic acid produces a precipitate, not only insoluble in the salt-saturated fluid, but also more or less insolu- ble in water containing a little acid. It is easily soluble in dilute alkali and alkali carbonate, and is not precipitated by neutralization with hydrochloric or acetic acid. Addition of acid, however, beyond neutralization immediately causes precipitation of the caseose. Protocaseose once precipitated with salt and which on the second precipitation, failed to separate from the salt-saturated fluid, was precipitated by a little acetic acid, washed somewhat with water, dissolved in very dilute sodium carbonate solution, neutralized and then dialyzed. in running water for nearly a week. The solution was then perfectly neutral to test papers and was likewise perfectly clear. It was evaporated to a syrup and the caseose precipitated by alcohol. After being washed with alcohol and ether, it was dried at 105° C. in vacuo until of constant weight, and then analyzed with the following results: Protocaseose A 3. I. 0°3703 gram substance gave 0:2160 gram H.O =6°:48 per cent. H and 0°6500 gram CO, =47°86 per cent. C. II. 0°4200 gram gave 50°9 c. c. N at 21:2° C. and 765:0™™ pressure = 14°21 per cent. N. Ili. 0°7053 gram fused with KOH and KNO; gave 0:0418 gram BaSO, = 0°81 per cent. S. IV. 0:4076 gram gave 0:0386 gram ash = 9°47 per cent. The ash-free substance therefore contained 52°594 C. 717% H: 15°70% N. 0:90% 8S. Like all of the caseose bodies, this contained a large percentage of ash in spite of its long continued dialysis. The ash was mainly cal- cium phosphate with some oxide of iron, obtained in part doubtless from the salt used in precipitation. It is not difficult to see how protocaseose, precipitated by salt alone, should take up and retain semi-mechanically considerable inorganic matter, But in the pres- ent case, where the great mass of the albuminous substance has been precipitated by the salt added, it seems somewhat surprising that the caseose should separate from the clear fluid in the presence of Primary Cleavage Products. 385 considerable acetic acid with such a large percentage of adherent mineral matter, unless the latter is chemically combined with the albuminous substance. Oft-repeated and long continued dialysis appears to have but little influence in diminishing the amount of this impurity. Our experience has taught us that where the caseoses have once been brought in contact with lime salts, reprecipitation and other methods of purification avail but little. But very little heterocaseose was found in this digestion, not as much as was found in some of the others, later on. Still, during the first dialysis of the protocaseose, some little heterocaseose separated from the fluid, as the last traces of salt dialyzed out. A. Deuterocaseose. Deuterocaseose was separated from the filtrate from the first sodium chloride precipitate of protocaseose, by acetic acid. Asastudy of the reactions of protocaseose had shown plainly that this body is never completely precipitated by salt alone, a little acetic acid was added to the salt-saturated filtrate, and the precipitate, presumably a mix- ture of proto and deuterocaseose, thrown away. The remaining deu- terocaseose was then precipitated by adding about 200 c.c. of a salt- saturated acetic acid (30 per cent. acetic acid) to the fluid. The total volume of the mixture was nearly 16 litres. In this manner an abund- ance of a finely divided precipitate was obtained, which at first, seemed insoluble in water and in dilute sodium chloride. It was readily soluble in water containing a trace of alkali and was not precipitated by neutralization. On being washed, however, with a saturated salt solution for some time, the washings were found to have dissolved considerable of the substance, which could be precipitated from the solution by strong acetic acid. Further, after being washed with salt solution, the substance remaining appeared quite noticeably solu- ble in water. Evidently then, this body on being washed more or less free from acid, becomes soluble in water to a certain extent, its aqueous solution then giving a strong reaction with acetic acid and potassium ferrocyanide. The great bulk of the precipitate was therefore washed with saturated salt solution until the washings were nearly free from acid; then, having become partially soluble in water, it was placed in about 2 litres of water, the solution ultimately saturated with sodium chloride and the caseose again precipitated by addition of about 20 ¢.c. of acetic acid. This second precipi- tate was washed somewhat with salt solution and finally with a large volume of water. At first, the washings gave no reaction with Trans. Conn. Acap., VoL. VII. 49 Nov., 1886. ee Chittenden and Painter— Casein and its 386 00-00T E096 ie es me eae hte then O G40 cl-0 PL-0 de “SS aa ee es Ek GL tne ee 8L-ST 89-CT se. ee 2 86-9 Rabe i, “aes ee 00-4 96-9 HH 65-19 rig ee — tag t 6G-T¢ 99-I¢ DO ‘O5RIOA V ‘aoungsqns aatf{-yso ay} fo Woriso0dwuos abnzUWadLagq 96-0T 8980-0 | - oy a cies ete ae ae ace ae geht See ay eae 6878-0 ITA we--- | ----- 99-0 6620-0 a eae er ie eee ae ae ee 0609-0 TA Soe. |} Seek 19-0 F60-0 ke a a St? = age ad oy Bee FG09-0 A : rw on 3 °c aks ete LEP | = G-99L VBb = eVGP a ee = eel et OSCE-0 AI ee |S een 5 i ek LO-FT | 6-992 O-GE SSP ee sr ae ee |lmet 9ELE-0 Tir et ee aes en = ee |" | F697) L8L9-0 | £69 | 2968-0 G00F-0 II Te ee oe or 7 o 4 fen eels "|" "" 1 88-97 | 0889-0 | 8-9 | 1982-0 LOOP-0 I “WBLS ‘ ; ae ee 1) 6 ao - “URIS “mUBIs “URLS 4 ‘punojy z 9 are {Se nee ig oanseoud L panoyz : panoy ‘pesn ‘ON YsV roe rogeg ‘puno} N OY) O°H doUBISGUG ‘VY GSOHSVOOUNLAACG AO SISATYNY —_——- Primary Cleavage Products. 387 acetic acid and potassium ferrocyanide, but later on, these reagents gave a heavy precipitate, showing plainly that the substance was dissolving. The entire precipitate was thereupon dissolved in very dilute sodium carbonate, the solution made exactly neutral with hydrochloric acid and then dialyzed. After remaining in the dial- yzers for nearly a week the fluid was removed, filtered from some heterocaseose which had separated, evaporated to a syrup on the water-bath and precipitated with alcohol. This precipitate was re-dissolved in water; the solution made exactly neutral to test papers and again dialyzed. From this solution, the caseose was finally precipitated by alcohol, after suitable concentration of the fiunid, washed with alcohol and ether and dried at 105° C. in vacuo. The final solution, prior to precipitation by alcohol, was perfectly neutral and quite clear, showing no evidence of the presence of any heterocaseose. " The composition of the substance is shown in the accompanying table. Digestion B. In this digestion, 750 grams of freshly prepared casein were mixed with 4 litres of 0-4 per cent. hydrochloric acid, the mixture warmed at 45° C., and then 800 c. c. of pure pepsin solution added. The mix- ture was warmed at the above temperature for one hour and a half, then neutralized and filtered, and the clear filtrate saturated with sodium chloride. This precipitate, as in the preceding digestion, was washed thoroughly with saturated salt solution, then succes- sively extracted with 10 and 5 per cent. salt solution and finally with water, leaving a small residue of dyscaseose soluble only in 0-2 per cent. hydrochloric acid. The united filtrates, containing proto and heterocaseose, were again precipitated with salt and then treated as described under protocaseose B. B. Protocaseose. The protocaseose formed in this digestion and twice precipitated by salt, was dissolved again in water, filtered through paper and then dialyzed until no chlorine reaction could be obtained with silver nitrate. Quite a little heterocaseosé separated from the solu- tion during dialysis, which was removed by filtration. The fluid was then concentrated, the substance precipitated by alcohol, again dissolved in water and dialyzed. This time, as no heterocaseose separated from the fluid, the solution was concentrated, precipitated ats in and . Chittenden and Painter— Case 388 00-001 87-86 SEY =. aa ody ates O 06-0 06-0 oi a on Se See i) 99-ST ae 69-T T9:CT E,. 7 N 90+k os Sue ee F0-2 60:2 Eh 16-29 a. eee ee 88-69 ¥6-6G @) ‘QSBIOAW - ‘aounjsqns aatf-yso ayz fo wo1risoduos abnzuaotag s : ve LL-0 6G- 1 DCU SU pee | 9 sees Fs red ell BR see eee ay ae: iI ak be oe ae a {1 ae rea | re o69-0 | IA A ae eg eee ne ¢L-0 | 0060-0 MC TPa GS ape Sopra cn | oan Soy ae a Sa espe pS hee eee 6698-0 | 2A ier Fees Ree eee pe a ek eek aa NP we se 00 Lo BiiGl- Ole = ett Lee ‘ie | ey SS oe a Slo” eVeE 6696-0 | A “a oa, Be Sta oa mo | ica ie Neh a So iat Se OSE Ta BOR clieGeGe, arian) hiro) ee Bae || a ee PS0F-0 | AT oh ee Ea ite ser Be eae 8 oa Se ae ae noe Bec* (MO sas ee leases eR ah EES le lee wR Repeat pes ae Te ee ee Tea Sm beck Sa cee (Rte oa “="" | """" | 68-87 | GP9L-0 | 19-9 | POSS-O | 89Cr-0 | II | | Fae “28 PT BETS es bes eth Tar ke Keer GL GOs OR GOCO- Ol) Mane eilee amice= |e NP Verscc en cil P : : wut kde cays Md mgs 900848 ee TmUBIs elnsselg | ‘“L cep “mes “URIS “URIS UsV JOS} % | “ONM+HOH |. y|USV out) 2 my. ts 7 eas Joop Suyonpep| qt WOISNy qng jo % wo. 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Ce oT ae Rie ® ae O 06-0 68-0 16-0 Aare a) =. ar a Ss - 61T-9L Bos Se TG-91 LI-9T ee 3 N TO-4 See Ce ies = ote 00-2 60-k H 81-9 eg = as ie ais 7 9F-GS TP-6G 8) ‘aBBIOAV & ‘aounysqns aauf-yso ay2 fo uoyrsodumos abnyuaovaq S S = S = eS A (ole ees re “See eae ee aoe ates | Sh sce atest SBE alee Boe Q 18-6 | 6880-0 ; 0F6E-0 ITA <0) = Pa | ae ee 660-0 saad | eed Nae oo ee ee te ae aE ee ose 0109-0 IA r=) Se ee &8-0 1920-0 Se ge ee ae Pe tee al ace ee ee e090 | A S Se ln ee = ee ee = a dae ey a= ae Eee 69-FT | §-G9L | P06 | G-29 OLOS-0 AI & 5 Pe BAe a po tp tee Cotes be SOME |= oeon= | O-OT |0r0gS| eo |) Ses ese te TecPr-0 Til = ESM ee ea le Pe ee tc ec Sen he ea ei 0&19-0 1&9 1006-0 06SE-0 II Se nr ges inca cn in ae mae Gee ie ern Oe Posie spectre hes 74 2727 8088-0 &&-9 1686-0 F80E-0 I “mes “TOUT “Oye z : , aes % |\*onnt+Hon | % |oemsseig| ‘1 20 % “mB % ‘mes he 3 USsV Zs J S YUM dorsny N 0 “punoy *OD H *punoj O° H sone N ey. r9yye 1OSe ‘punoy N 5 ‘*6 G ASOASVNOLOYUT AO SISATVNY 390 Chittenden and Painter— Casein and its with alcohol and the substance finally dried at 105° C. in vacuo (Protocaseose B 1). Its composition is shown in the accompanying table. Such portion of the protocaseose as was not precipitated the second time by salt alone, was precipitated by a little acetic acid. At first, the precipitated substance was insoluble in water (no reac- tion with acetic acid and potassium ferrocyanide), but after being washed with salt solution until the acid reaction had nearly disap- . peared, it then dissolved quite appreciably in water, as evidenced by the reaction with acetic acid and potassium ferrocyanide. The bulk of the precipitate, after being washed, was dissolved in a little very dilute sodium carbonate, the solution made exactly neutral with hydrochloric acid and then dialyzed until all chlorine was removed from the solution. The caseose, after concentration of the solution, was precipitated by alcohol, washed with alcohol and ether and finally dried at 105° C. im vacuo. The composition of the substance (Protocaseose B 2) is shown in the accompanying table. B. Deuterocaseose. Deuterocaseose was separated from the filtrate from the first sodium chloride precipitate, in the same manner as in the preceding digestion. Like A. deuterocaseose, this precipitate was at first in- soluble in salt solution and in water, but after being washed for some time with saturated salt solution, it was found to be gradually dissolved, as shown by the rapid disappearance of the precipitate and the pronounced reaction with acetic acid and potassium ferrocy- anide in the wash-fluid. - Evidently the acid is easily removed from the compound by simple washing and when that has been effected, the substance becomes soluble, or else the acid compound is more insoluble in water containing a little free acid, than in water or salt solution alone. Hydrochloric acid and acetic acid seem to act alike. The substance is readily soluble in dilute sodium car- bonate and is not precipitated by neutralization, but is quickly thrown down by a slight excess of hydrochloric or acetic acid. This compound was not analyzed, but was used in studying the reactions to be described later. Digestion C. In both of the preceding digestions, the products formed resulted from the action of an exceedingly vigorous pepsin mixture. In the Primary Cleavage Products. 391 present case, the pepsin solution employed was much weaker and the pepsin-casein mixture was warmed at 45° C. for several days instead of hours. 750 grams of casein were employed and 4 litres of 0:4 per cent. hydrochloric acid, to which was added a reasonable amount of pure pepsin solution. About an hour after the addition of the latter the mass began to gelatinize, and at the end of 18 hours the whole mix- ture was a perfectly stiff jelly. Thereupon, 2 litres more of 0:4 per cent. acid were added together with a little pepsin. The mixture was then kept at 40-45° C. for three days longer. Quite a large residue of undigested matter remained, semi-gelatinous and soluble only in alkalies. The mixture was then neutralized and filtered. The filtrate contained considerable caseose, as evidenced by the heavy precipitate obtained on saturating a portion with sodium chloride. In the preceding digestions, the caseoses were separated directly from the dilute ‘solutions, without previous concentration, thereby avoiding any possible change due to the action of heat. In the pre- sent case, however, the perfectly neutral solution was evaporated to a small volume and then all of the caseoses were directly precipitated by saturating the fluid with ammonium sulphate. The precipitate produced was exceedingly gummy, but was washed as thoroughly as possible by trituration with a saturated solution of ammonium sulphate. The caseoses were then dissolved in water, the solution filtered from the small residue of insoluble matter and saturated with sodium chloride. The protocaseose so separated was freed from heterocaseose, etc. by repeated precipitation and dialysis. Carbon and hydrogen were determined in a portion of the dried substance (Protocaseose C 1) with the following results: I. 05107 gram substance gave 0°3101 gram H.O=6:74 per cent. H and 0:9386 gram CO.=50°11 per cent C. II. 0°4088 gram gave 0:0194 gram ash = 4°80 per cent. The ash-free substance would, therefore, contain 52°64 per cent. of carbon and 7:08 per cent. of hydrogen. In the filtrate from the first salt precipitate, the protocaseose re- maining was precipitated by the addition of a little salt-saturated acetic acid. The precipitate after being washed was dissolved in a little dilute sodium carbonate, the solution neutralized and dialyzed. As no heterocaseose separated from the solution, it was concen- trated and then precipitated by alcohol. After purification by re- solution, dialysis, etc., it was dried at 105° C. in vacuo and analyzed with the results shown in the accompanying table (Protocaseose C 2), Chittenden and Painter— Casein and its 392 00-001 84.46 a a 3 thes i Ma O 64-0 61-0 eo “Pee = E $ $8.1 ee 66-ST Lb ST a ad N ; GT ok RSs — : GEL 8th H GLTG RV a i gg't¢ 18-T¢ @) ‘ODB10A VW ‘aoupjsqns aatf-ysp ay? fo Uuowrsodwmos abnzuawag pesets. | GOGO: On| =e |e" ee met ce Si ah: ee Sietloee e pa ae eae PIEP-0 TIA SP-6 | 9860-0) 2°) Fae wees || eg ee mae ar ae al em les a ree tes ela 8907-0 IA en at GL-0 CT0-0 Sees Sh yale Oe sl en | ea ce Sk eee es 8009-0 A ese || = sae eee | Nae ete Paes OPE) 6-90) 7 | L0G) SCE = |e Se $a Wea vie ae Oscé-0 | AT ---- | ------ esc Scat OG Pip | ROG Lsy CeGhs |. eles | ew lee 2 eee a a ae ae S686-0 It ae dee aa elas, sare Sc ae | mies ae “""" | $L-9F 8C6L-0 cP-9 8976-0 CECP-0 Il 1 del MR ieee ae ap | ae Se eee ae % eel ee DB OF 8c0L-0 T¢-9 8696-0 O80F-0 I “ “mes Ee ae | as oe : B| ounogs|~% 2 | PONE +HOM |B a “mead % ‘umes mae ac UsV . Ss YM aoIsny N 0. | spumoy *O9* | “4s "puno; OFF: | Sr asane ae royye "OSea “punoy NN ee ‘6 QO WSOUSVOOLOYUdG AO SISATVNY Primary Cleavage Products. 393 Deuterocaseose was then separated from the filtrate from the acetic acid precipitate, by saturation of the fluid with ammonium sulphate, as recommended by Neumeister* for deuteroalbumose. It was then purified by dialysis, ete., and its reactions carefully studied. So far as we could see, it differed from deuterocaseose A and B in two respects only, but these points showed so marked a difference it was plainly evident that the deuterocaseose separated by ammo- nium sulphate was quite different from deuterocaseose A, separated by acetic acid. Thus deutero C was not precipitated at all in an aqueous solution by acetic acid, nor by acetic acid and potassium ferrocyanide, neither was its aqueous solution precipitated by cupric sulphate. The significance of these points of difference will be dis- cussed later on. After studying the reactions, there was not enough substance remaining for analysis. Digestion D. In this digestion, 2 kilos of freshly prepared casein were used, together with 6 litres of 0:4 per cent. hydrochloric acid and an appro- priate quantity of strong pepsin solution. The mixture was warmed at 45° C. for five hours, then neutralized and filtered from the semi- gelatinous residue. D. Protocaseose. The neutral fluid was concentrated to about 14 litres and then filtered from the slight flocculent precipitate which had formed. Saturation of the fluid with sodium chloride gave an exceedingly heavy precipitate, somewhat more gummy than usual. The entire fluid, however, was only partially saturated with salt, with a view to see whether the precipitate produced in this manner would agree wholly with the precipitate produced on complete saturation. Thus a fractional precipitation was made, in which the first fraction represents that portion of the caseose precipitated by about two- thirds saturation of the fluid with salt. This precipitate was there- fore filtered off, washed as usual, dissolved in water and re-precipi- tated with salt. As dyscaseose was generally found only in traces, the precipitate, after being washed, was dissolved at once in water and dialyzed until all chlorine was removed from the solution. On opening the dialyzing tubes, quite a large quantity of heterocaseose was found adherent to the sides of the paper., The clear solution of * See Zeitschrift fiir Biologie, Band xxiii, p. 381. TRANS. Conn. AcaD., VoL. VII. 50 Nov., 1886. Chittenden and Painter— Casein and its 394 89-0 69-0 00-001 00:66 0 8-0 GO-9T ADek §6-8G ‘OGRIOA VW 88-0 cy 60-7S ‘gounjsqns aauf-ysv ayy fo worprsodwuos abpzUaolag 16-h ¥8-G oHAnmo LPLO-0 6710-0 ‘ysB jo d Sut -jonpep | » d Ioye J mUeIS *20TLB YS, “ONS + HOM j-4n8 Jo YIM WOISNT Ioyye 4*O* q* 3 % “yse jod “UIBIs “yse % ey Moly |S V LO* dN 8910-0 ten} ‘punoj qsv 6910-0. | -- | [8-0 “URLS “ONM+ HOW YjIA WoIsny Jaye OORT Awe ‘O10 sorg| © ‘pauo) N GPLL-0 98-9 08-TS 0686-0 “mes ‘panoj *00 F009-0. F109-0 6F0F-0. T3er-0. ELFT-0 4009-0 F109-0 GL9S-0 “""" 198GP-0 8082-0 290F-0. 8968-0 112-0 | ‘ON ‘. @ ASOMSVOOLOUd AO SISATIVNY Primary Cleavage Products. cP-9 68-9 usV ITTA ‘ON 00-00F IT 8S a ak Gat ag Spee mits ae ar O 0.1 OFT 66-0 :s tis (hee ara! S) 98-91 Bic be ee ¥8-CT 68-GT eee — N OL +k < Ros ao aoe OTL OT-2 H 78-69 es 2 =. ee 88-69 61-86 ©) ‘O0BI1OA VY ‘aoungsqns aauf-ysv ay) fo Uuorprsodwmon abpyuadsag OSG020s lke ea iitie West eta MI i = en ro eae le cael (Ig ae § a a Pie gee Sana 98TF-0 1960-0 iil ne a ele ae |i, et ae * re a t eareat| | See ach Ala SE OGCF-0 —s vO-1 68P0-0 aS rah Ge. ey (a Ne | REST |e | Soe AR 0¢89-0 “See (66:0 FOO | eeep] 8189-0 | 89-9 | 80T8-0 TT¢&-0 Ne hele ere seme fal ae a “~~ | "5" | SP°6P | «2094-0 | 99-9 | TTSe-0 96TF-0 : “Wes Bostny Fone oe ; : , ee % | “onut+Hom | % |omssrg} “p | °° | 4 ae % ney ah Sg: YMA WOIsny N sao) & H & eourysqn qsy Toye *ORue Mitts 00 O°H qug ‘°8 @ ASOUSVOOLOUT AO SISATVNY 396 Chittenden and Painter— Casein and its protocaseose was concentrated, precipitated with alcohol, the precipi- tate dissolved in water, again precipitated with salt in substance, the precipitate dialyzed, this time without showing any heterocaseose, and finally precipitated with alcohol, washed with alcohol and ether and dried at 105° C. in vacuo (Protocaseose D 1). The product was analyzed with the results shown in the preceding table. On adding more sodium chloride to the first filtrate from the above precipitate, thereby completely saturating the solution, a second precipitate of protocaseose was obtained, which was purified in the same manner as the preceding preparation. The only difference noticed while purifying the substance was that, on dialysis, nothing corresponding to heterocaseose separated from the fluid. After final washing with alcohol and ether, the substance was analyzed with the results shown in the accompanying table (Protocaseose D 2). The difference in the results will be discussed later on. The original salt-saturated filtrate was precipitated with a little acetic acid, and the protocaseose so precipitated dissolved in dilute sodium carbonate. The solution was then neutralized and dialyzed in running water until all chlorine was removed. The substance was then separated by precipitation with alcohol, and ultimately purified as described previously (Protocaseose D 3). The addition of a little more acetic acid to the acetic acid and salt-saturated filtrate from the above, gave a still further precipitate of caseose ; presumably proto- caseose with perhaps a trace of deuterocaseose, which was filtered off, washed with saturated salt solution and then freed from acid and purified in the same manner as the preceding preparation (Proto- caseose D 4). The two last products, after being dried at 105° C. in vacuo, were analyzed with the results shown in the following tables. Comparing these two tables, it is seen that the difference in per- centage composition of the two ash-free substances is not very great, and taking into consideration the large percentage of ash, it is proba- ble that the two latter precipitates have approximately the same composition. D. Deuterocaseose. The filtrate, from which certainly all protocaseose had been re- moved by acetic acid, and in fact nearly everything precipitable by acid from the salt-saturated fluid, was treated with ammonium sulphate in substance. A gummy precipitate resulted, which natu- rally enclosed considerable salt, and which for purification was dis- 397 00-001 79-86 eat ee a ikod a0 cep aS O 90-1 F0-T 80:1 eee eer Ba Aen S oL-9T VaR, Te 8T-9T 10-91 iam OS N 81h tafe Les Kaioal Pa 9T-2 TT-d H 90-69 a he ps is ak 5 OT-6S 10-6 ) “OSVIOAV 3s ‘aounpsqns aaif-yso ayy fo worpsodumos abnzuavad S = S na 96-0 6160-0 can “Se ap ae ae ore egy Pee co Die pas cate S 00-1 LEE0-0 ae | eee Bre oh haee e Sulit Sel ee ae ann ee es ea S Sats ae. Soot 9660.05 ame eres ee oy ee 5% ose aad Ss sa Lio need |e LOGE | SsS9k" |-2-8E | rSh | s ice Pa as. > : eee haat Deals = 1.68:FE| O'990 3) PBs) 2h crs 80-0 |69-0 Wee ee > Eee ge S Ss a orem alicee nw Se ee 4600-0 Tres *ONN+ HOM YIN TOIsHy qaqye ‘O° g*sWq |g0uR4S -qns jo | % ‘yse | Jod “TURIs “yse ay} WOT 40% q°3W ysVy “mes “punoy US. OFAnwo 6-991,0-81 2-67 T-992) 9-81 /8-68 “mes “ON + HOW YIM WOIsNy % 8 Joye 'OSPd \ Few Staten ‘eins -So1g ‘punoy N Do anf ‘O°O 1699-0) L06-0 “meds “punoy °00 fd se 0809-0 7809-0 BCOF-0 @s0F-0 €80F-0 (0609-0 ‘809-0 0988-0 BITF-0) 006-0 FF9E-0 0808-0, 666F-0 “mURIsS od “Tae 3 | ~~ | :pesn “punojy O*H 9oue}s -qng ON ‘gq wsOmSVOONmaLOAAdG AO SISATVNYV 400 Chittenden and Painter— Casein and its solved in water and dialyzed until the greater portion of the salt was removed. The solution was then concentrated and the substance reprecipitated by saturating the neutrai solution with ammonium sulphate. This precipitate, after solution in water, was then dialyzed until all of the ammonium sulphate was removed, after which the solution was concentrated and precipitated with alcohol. When dry, the substance gave by analysis the results shown in the accom- panying table. This body differs from all of the preceding preparations, in that it is not precipitated from an aqueous solution by acetic acid. Neither is it precipitated at all by the addition of salt in substance; but the addition of a little acetic acid to the salt-saturated fluid gives a heavy precipitate which, however, does not represent all of the deutero- caseose, since the filtrate gives an additional precipitate with ammonium sulphate. Apparently about one-half of the substance is precipitated by acetic acid. Further, the acetic acid precipitate in this case differs from the protocaseose precipitate with acid, in that it is readily and completely soluble in water. This body, therefore, which certainly must represent pure deuterocaseose, shows a close resemblance to the pure deuteroalbumose separated by Neumeister. Like the latter, it does not give any precipitate whatever with cupric sulphate nor with ferric chloride and only the faintest turbidity with acetic acid and potassium ferrocyanide. D. Heterocaseose. In each digestion, evidence was obtained at various points in the process of separation, noticeably on dialysis of the first protoalbumose precipitate, of the presence of a body insoluble in water but soluble in dilute sodium chloride solution. The quantity of the sub- stance, however, was in most cases exceedingly small, so much so that nothing more than a few reactions could be tried with it. In the present digestion, however, the amount was somewhat larger, and sufficed for a partial analysis. The substance was obtained as a more or less gummy residue, on dialysis of protoalbumose 1. It was purified by solution in 10 per cent. sodium chloride and separation by dialysis. Like heteroalbumose, the whole of the substance was not now soluble in salt solution, for a portion had apparently been converted into a body resembling dysalbumose, insoluble in salt solution but soluble in 0°2 per cent. hydrochloric acid. Primary Cleavage Products. 401 Analysis of the dried substance gave the following results : I. 0-4011 gram substance gave 0°2463 gram H.O=6°82 per cent. H and 0°7434 gram CO.=50-54 per cent. C. _ II. 0°4287 gram gave 53°6 c.c. N at 18°6° C. and 760°1 mm. pressure =14-70 per cent. N. Ill. 0:4118 gram gave 0:0256 gram ash =6:21 per cent. The ash-free substance would therefore contain - 53°88 per cent. C, 7°27 per cent. H, 15:67 per cent. N, Reactions of the caseoses. Under this head little need be said. The reactions characteristic of the albumose bodies in general will apply here. Certain differ- ences, however, have already appeared in our description of the processes incident to separation of the caseoses.. Protocaseose, unlike protoalbumose, is precipitated from an aqueous solution by acetic acid. The precipitation, however, is not complete; satura- tion of the acid filtrate with sodium chloride, invariably gives an additional precipitate which is the heavier, of the two. Further, long-continued washing of the acid precipitate with water or salt solution appears to partially remove the acid from the caseose body. Protocaseose is likewise precipitated from an aqueous solution by hydrochloric, nitric and sulphuric acids; the precipitate, however, is far less soluble in excess of sulphuric or nitric acid than in excess of the other two acids. In very dilute acids, protocaseose is soluble and is partially precipitated by addition of stronger acid of the same kind; ¢hus the substance is readily soluble in 0°4 per cent. hydrochloric acid, from which solution it is precipitated by the addi- tion of a little concentrated acid, this precipitate dissolving on the addition of more acid. Evidently then, protocaseose as fast as formed by the action of pepsin-hydrochloric acid, would dissolve in the acid gastric juice and not be mixed with the jelly-like insoluble residue. Boiled with dilute or strong acid, protocaseose is appar- ently not changed; at least no precipitate is obtained on neutraliza- tion of the acid fluid. The acetic acid solution of protocaseose gives avery heavy precipitate with potassium ferrocyanide. In an aque- ous solution of the substance, cupric sulphate gives a heavy curd-like precipitate, while ferric chloride gives a similar precipitate read- ily soluble in excess of the precipitant. Like protoalbumose, proto- caseose is precipitated by saturation of its aqueous solution with sodium chloride, but never completely ; there always remains in the Trans. Conn. AcaD., VoL. VII. 51 MARCH, 1887. 402 Chittenden and Painter— Casein and its filtrate, a portion of the substance precipitable only on addition of acetic acid. . Of the several preparations of deuterocaseose, those precipitated by ammonium sulphate are evidently the only ones perfectly pure. Further, it is evident that this substance can be obtained pure only by complete removal of all protocaseose from the solution, which implies precipitation of a large portion of the deuterocaseose also, and then precipitation of the small amount of dentero remaining, by saturation of the fluid with ammonium sulphate. D deutero, pre- pared in this manner, shows several very marked points of differ- ence from protocaseose. In the first place, it is not precipitated in an aqueous solution by acetic acid. Further, the addition of potas- sium ferrocyanide to a solution acidified with acetic acid gives no precipitate whatever. Cupric sulphate and ferric chloride both fail to produce any precipitate in an aqueous solution. Pure dentero- caseose, as already mentioned, is not precipitated by saturation of its aqueous solution with sodium chloride; addition of acetic acid, however, to the salt-saturated solution gives a heavy precipitate, which represents perhaps half of the deutero, the remainder of which is precipitated only by saturation of the fluid with ammonium sulphate. It is thus evident that in the precipitation of protocaseose from a salt-saturated solution by acetic acid, more or less deuteroca- seose will be likewise precipitated, the amount depending probably on the concentration of the solution and other minor circumstances. Hence, A deutero is unquestionably contaminated with some proto- caseose, and on the other hand protocaseose D 3 and 4, and perhaps protocaseose C 2, without doubt contain some deuterocaseose. That A deutero contains some protocaseose, is evident from the fact that it gives a precipitate with cupric sulphate, and further its aqueous solution is rendered decidedly turbid by acetic acid. Moreover, the protocaseose precipitated by acetic acid, and which may contain some deutero, appears to differ in one or two respects from either proto or deuterocaseose. Thus, an aqueous solution of the purified substance is precipitated like pure protocaseose by acetic acid, but the precipitate is only partially soluble in excess of the acid and even that requires a large excess. : With nitric acid, pure deuterocaseose gives no precipitate, but on warming the solution the xanthoprotein reaction comes out strongly. Composition of the caseoses and thetr relation to casein. In studying the composition of the various caseoses we have been hampered by the large percentage of ash invariably present in all of Primary Cleavage Products. 403 TABLE SHOWING RELATIVE COMPOSITION OF THE CASEOSES. Protocaseose. C EE N Ss O MeN precipitate sel oe ke. 28. U2. 52°50 | 7:15 | 15°73 | 0°96 | 23°66 Or a Cy hai hy Sie een ee fori oe 53°85 | 7°21 | 15°84 | 0°98 | 22°12 A 3 Acetic acid precipitate.._.--.----.--- | 52°59 | 7°17 | 15:70 | 0-90 | 23°64 haha precipitates 4 s20.e. -22i4i. 02022 52°91 | 7:06 | 15-65 | 0°90 | 23-48 B 2 Acetic acid precipitate___......-.---- 52°48; 7:01 | 16°19; 0:90 | 23-47 Gaia Nae@laprecipitate.:+. 52.524. -2- >. -2- eG OSs ps tace li aes als C 2 Acetic acid precipitate. ---..-.---.---- (51-73 | 7-15 | 15-85 | 0-79 24°48 ete ae! Precipitates oe. oe .ss—2-+. 2s —- 53°93 | 7:17 | 16:05 | 0°85 | 22-00 1D)ee)e aS GORY NI pe Sh esa ees 2 52°84 | 7:10 | 15°86 | 1:04 | 23-16 D 8 Acetic acid precipitate___.--..--..---| 52°05 | 7:13 | 16°12 | 1:06 | 23°64 D4 « Riga ers 20) Ue dct Ey 52-88 | 7-07 16-13) 0-98 | 22-94 Deuterocaseose. A. Acetic acid precipitate. ......__....- 51:59 | 6°98 | 15°73 | 0-75 | 25-03 D. (NH,),S0, Feta reat hie he I Sh ee 51°79 | 7:05 | 16:00} 1°17 | 23:99 Heterocaseose. 1D al Sa gee Rao 53°88 | 7:27 | 15°67 | -- ae Casein. pverace OL Nos: L=VIE ie 2s ie 2 53°30 | 7:07 | 15°91 | 0°82 | 22-03 404 Chittenden and Painter— Casein and its the preparations. We have already commented on the difficulty, in fact, impossibility, of removing certain inorganic salts after they have once been brought in contact with a caseose body. Repeated precipitation appears to affect the percentage of ash but little. The reason for the large percentage of ash lies in the precipitation of the caseoses from such large volumes of fluid. We thought it unwise at first, to expose the bodies to the long continued evaporation neces- sary for precipitation with a small amount of salt. To avoid the possible danger of change, therefore, the large volumes of fluid resulting from the several digestions were saturated directly with salt, and as this involved the use of large quantities, calcium salts and some iron as impurities in the sodium chloride, were unavoid- ably introduced. These, the caseoses seemed at once to catch hold of and retain, in spite of oft-repeated purification. In the digestion D, in which the fluid was concentrated somewhat before precipita tion, the percentage of ash is seen to be somewhat smaller than in preparations from the other digestions. In comparing the composition of the individual protocaseoses (see the accompanying table) it. is seen that two of the bodies show a content of carbon somewhat higher than casein itself, while the average of all the others, with one exception, shows a content of carbon a little lower than casein. Leaving out the acetic acid pre- cipitate C 2, the average of the remaining ten preparations of proto- caseose shows the following composition for this substance: Cie: H N Ss 0 erOLOCASGOSO a5. = 7- mae eee 52°89 4-10 15:94. 0°95 23°12 Wasein= en. ee De a ee 53°30 7-07 15°91 0°82 22°03 Plainly, the average of our results would indicate that protocaseose does not differ essentially in composition, from the casein from which it is formed. A slightly smaller content of carbon is the only notice- able difference. To be sure the individual results show noticeable variation in the percentage of carbon, but bearing in mind the large amount of ash present in the preparations, it is evident that the aver- age result is of more value than the results obtained in any one case. As to the lower content of carbon in so-called protocaseose C 2, it is probable that this body is composed mainly of deuterocaseose. The two caseoses being precipitated together in this digestion by ammo- nium sulphate and then separated afterwards from a fairly concen- trated solution by saturation with salt and addition of acetic acid, renders it probable that the protocaseose was more completely precip- Primary Cleavage Products. 405 itated than usual by salt alone; and further, it is probable that on addition of acetic acid to the concentrated and salt-saturated fluid, a much larger proportion of deuterocaseose was precip- itated. In confirmation of this view it was noticed that the amount of deuterocaseose obtained by the later precipitation with ammonium sulphate was quite small; far smaller proportionally than obtained in D. That the body contained some protocaseose, was evident from its reaction with cupric sulphate and with acetic acid. Pure deuterocaseose evidently contains a smaller content of car- bon than protocaseose. It is equally evident that it is a body further removed from casein than protocaseose. Its general reac- tions show a closer relationship to peptone than to casein or the proto-body. Heterocaseose, on the other hand, judging from analysis of a single preparation, contains fully as much if not more carbon than casein itself. Nearly all of the caseoses show a somewhat higher percentage of sulphur than casein, but probably the increase (0:1 per cent.) is due mainly to a trace of sulphate in the ash, not accounted for. Owing to the large amount of phosphate in the ash of the different prepara- tions, phosphorus was sought for only twice. In both of these, how- ever (protocaseose D 1 and deuterocaseose D), the phosphorus in the ash was the exact equivalent of the total phosphorus found after fusion with potassium hydroxide and nitrate. This might indicate that in the cleavage of casein with pepsin-hydrochloric acid, the phosphorus of the casein is removed in the form of a phosphorized body, leaving the thus non-phosphorized matter to break down into the caseoses. With this thought in mind, we propose to study later the nature and composition of the insoluble, semi-gelatinous body separated in the first stage of digestion. We also hope to extend our work by a study of Weyl’s commercial ‘ casein-peptone,” pre- liminary examination of which -has shown us the presence in large quantities of caseoses. In this way and by a somewhat different method of isolating the individual caseoses, we hope to verify our present work and at the same time obtain products comparatively free from ash, with which to establish beyond question the composi- tion of the caseoses. We are also occupied in a study of pure casein- peptone, purified according to the method made use of by Kiihne and Chittenden in the study of fibrin-peptone. XXTI1.—Isrivence or Some OrGanic anp INoRGANIC SuB- sTANCES ON Gas Merasonism. By R. H. CutrrenpEN AND G. W. Cummins, Pu.B. Wuite much time has been spent during the past few years in studying the influence of various substances on proteid metabolism, far less attention has been paid to the effects of these substances on the consumption of oxygen and the elimination of carbonie acid. Naturally in studying the influence of any substance on the nutrition. of the body, we need to know not only its action on the excretion of nitrogen but also its influence on the production of carbonic acid. In this way only can we arrive at a true understanding of the influence of the substance on total metabolism, and obtain the necessary data from which to draw conclusions as to its influence on the consump- tion of either nitrogenous or non-nitrogenous matter. The difficulties, however, in the way of carrying on consecutive determinations of the relative amount of carbonic acid eliminated by the lungs are consid- erable, and in the absence of the necessary respiration apparatus, the difficulties are greatly increased. We have, however, endeavored to carry on some experiments in this direction, and although lacking the ordinary apparatus we have still been able with the means at our disposal to obtain some interesting results, a portion of which are simply confirmatory of previous work, while others are wholly new. The apparatus employed in measuring the amount of carbonic acid eliminated is shown in the accompanying illustration (see Plate). The chamber in which the animal was placed during the experiment, was a bell jar of 32 litres capacity, with ground edge fitting closely upon a smooth glass plate. This when coated with grease made a perfectly tight joint, but in order to avoid any possibility of error, the jar and plate were placed in a shallow pan of galv anized iron, and water poured in to the depth of 2-8 inches, thus insuring a perfectly air-tight joint. In the top of the bell jar was an opening, closed with a doubly perfor- ated rubber stopper, through which passed two tubes; one bringing air into the chamber, the other carrying it to the absorption apparatus. The inlet tube (to the left of the figure) was prolonged so as to admit the air nearly at the bottom of the jar, while the outlet tube came just through the stopper, thus insuring a perfect circulation of air, Air was drawn through the chamber by means of three aspirators, Chittenden and Cummins— Gas Metabolism. 407 two of which had a capacity of 15 litres and one of 74 litres. The three aspirators working together would therefore draw through the chamber 374 litres of air at every filling, and the flow was so regulated that 30 minutes were required to draw that amount of air through the apparatus. The flow of water from the aspirators was quite regu- lar, since the inlet tubes went to the bottom and the air had to bubble up through the water, as the latter ran out, on the principle of Mariotte’s bottle. The rate of flow was regulated by carefully changing the difference in height between the inlet tube (for air) of the aspirator and the outlet tube (for water). This of course, at the outset, was a tedious operation, but when once perfected and the apparatus permanently set up, the three aspirators ran exactly to- gether, with a maximum variation of 15 seconds for the half-hour, which variation, however, was seldom observed. In addition, each aspirator was marked off into eight divisions, the last one of which was equal to only one-half of the others. In the two large aspirators these divisions indicated exactly the same volume, while in the small aspirator the divisions represented half the capacity of the former ; but the flow of water in the latter was regulated to consume the same amount of time as in the former. Hence four minutes were required for the water to flow by each of the first seven divisions, and two minutes for the last, making a total of thirty minutes for the entire volume of water to flow from each aspirator. The tube drawing the respired air from the chamber in which the animal was enclosed, was divided a short distance from the chamber, as seen in the figure, and two-fifths of the mixed air was drawn successively through three absorption tubes filled with a standard solution of barium hydroxide for absorption of the carbonic acid. The absorption tubes were about two-thirds of a metre long and the lower tube (a) contained 100 ¢. c. of a standard baryta solution, the middle tube (0) also 100 ¢. ¢. of the solution, and the upper tube (c) 50¢.¢. The amount of carbonic acid absorbed was, at the end of the experiment, determined by titration with a standard solution of oxalic acid, using phenol-thalein as an indicator. Two titrations were made, one of the contents of tube @ and one of the contents of the two tubes 6 and e«. By using the three tubes, absorption of the carbonic acid was quite complete. In order to aid absorption, the air was broken into small bubbles by being forced through a small tube dip- ping beneath the barium hydroxide. Frequent blank experiments showed that all of the connections were perfectly tight, and further, all of the tubes being in the same position, that the flow of water 408 Ohittenden and Cummins—Influence of Some Organic from each aspirator was perfectly uniform, and that the aspirators could be relied upon to draw the given volume of air through the apparatus in the time designated without any appreciable variation. In addition, the two-fifths drawn through the absorption tubes for determination of the carbonic acid was always exactly two-fifths of the aspirated air; since the aspirators, as already remarked, worked with perfect uniformity. Any tendency to variation, either in the time, or in the action of the individual aspirators, was noticed at the very outset of the experiment, as the water reached the level of the different marks on the aspirators, and could be at once checked or controlled by moving slightly the water outlet tube so as to either increase or diminish the difference in height between the latter and the inlet tube for air. Theoretically, variations in the temperature of the water in the aspirators might affect somewhat the volume of air analyzed, but a constant determination of the temperature of the water showed such slight variations that they did not seem to justify us in making any corrections for possible change in the amount of air aspirated. Naturally, all of the supports for the three absorption tubes were permanently placed, so that there could be no change of position; the tubes themselves placed in the same position in the holders; the volumes of baryta solution invariably the same, so as not to increase or decrease the pressure to be overcome; and lastly the aspirator tubes and stoppers fastened so as not to admit of any change. With these precautions, the results obtained, both as to the volume aspirated and the time consumed, were quite satisfactory. ; As already mentioned, the total capacity of the three aspirators was 874 litres or 54 litres more than the capacity of the bell jar. This amount of air drawn through the chamber in 30 minutes, was more than enough to supply the largest rabbit experimented on, with the necessary amount of oxygen. But there must have been a slight accumulation of carbonic acid in the air of the chamber; this, how- ever, was a constant factor throughout the experiments. Further, the results obtained, expressed in milligrams of CO,, do not represent the total amount of carbonic acid eliminated by the rabbit during the thirty minutes of the experiment, but simply the amount of CO, contained in the 374 litres of air aspirated during that time. Such a result, however, ought certainly to show just as plainly any influence on the elimination of carbonic acid, as a determination of absolute quantity and thus be equally valuable as an indication of influence or lack of influence on the gas metabolism of the body. Further, the results thus obtained ought to express equally as well, the comparative action of the various substances experimented with. / and Inorganic Substances on Gas Metabolism. 409 In every experiment, the time at which the animal was introduced into the bell jar was exactly noted and then two minutes were allowed before starting the aspirators, to make all of the connections properly. The rabbit was therefore under the bell jar, in each determination of carbonic acid, for exactly thirty-two minutes. The aspirators were started simultaneously and their progress carefully watched, in order to check any slight irregularity that might show itself. The animals experimented with were wholly rabbits, and prelimi- nary trials showed us plainly that it was very necessary to have them in a condition of hunger during the experiment, in order to avoid the irregularities incident to change in digestion. Further, we soon found that this was best accomplished by depriving the animal of food for three days, after which the experiment was commenced and allowed, as a rule, to extend through three con- secutive days, the animal being deprived of food during the entire period. On the first of the three days, eight determinations of carbonic acid were made and the results obtained were used as a control, with which to compare the results obtained on the two fol- lowing days, when the animal was being dosed with the substance experimented with. This, as a rule, we found to be the most satis- factory method of procedure, since small differences could not be relied upon as expressing anything of importance; for the varying restlessness of the confined animal, involving more or less muscular activity, would many times lead to variations in the amount of car- bonic acid excreted, as may be noticed in the control experiments on those days when the animals were not dosed. Hence, the average of several consecutive results must necessarily express more correctly the average elimination of carbonic acid than any single result. Further, we deemed it better to allow the experiments to extend, as a rule, over several days and thus study the action of small, repeated doses of the various substances rather than to observe the effects of a single large dose, where violent action might naturally be expected. The following table of results illustrates the way in which our experiments have been conducted, and at the same time. shows the extent of variation, in the amount of carbonic acid, to be expected under normal circumstances from day to day. In this experiment, the rabbit had been deprived of food for three days, and the results show the amount of carbonic acid in the 37:5 litres of aspirated air for four distinct periods, during the fourth and fifth days. As already stated, the total amount of baryta solution employed in the three absorption tubes was 250 c. ¢., of which 100 c. c. were used in the first TRANS. ConN. ACAD., Vou. VII. 52 . MARCH, 1887, 410 Chittenden and Cummins—Influence of some Organic or lower tube (a), while the remainder was used in the two other tubes b and c. Several solutions of oxalic acid were employed, the average strength of which was such that 1 c. c. equaled about 20 milligrams of carbonic acid. ; ' - : | A a Oxalic acid to) Sree eek 3 a. 3 neutralize ba-| © = é 5 S = a 2 3 ryta solution.| sg [So °| 9 5 4 3 . a : wo. “ f= ia o Time. Ss () BS N48) gc re — 5 a, H a Ow oo a on S| . S++ Ag re] oO : 25 os ee) o Cie =e ~ x a Oo mao a = O ota) . ev rs ey Sh Nl ye Sh) eh ah ass O:= mn) ee = | &O ie) 3 is; S ‘ eS = gs = a= oe fo) g Oo g | elo cS) 2 ; S ar call eet o) a Ss) o | March 18. 9:48 to 10:18 10°4 | 25:0 | 35:4 | 46:1 9°47 180-2 | 450°5 | 38°8 ) 38°1 Q =< =i oe J > io) N ° 11:51 to 12:21 10-6 | 25:2 | 35-8 | 45°1 | 2:59 to 3:29 10°5 | 25°83 5:03 to 5:38 10°4 | 24:9 | 35°3 | 45-1 < =) [e.2) co me lo 2) vo we (=) > On ou - (ss) @ ie) March 19. 8:59 to 9:29 10-20 Sool le Sb:3 | Aba 9°8 182°0 455°1 38°3 10:53 to 11:23 | 10-9 | 25:3 | 36-2 | 2:58 to 3:23 | 10-2 | 24-2 | 344) 4541] 10-7 | 198°7 | 4968 | 38-7 4:51 to 5:21 | 10°6 | 24-9 | 35-5. Average, 10°5 | 25:0 | 35:5 | Action of uranyl! nitrate. As stated in a preceding article,* the physiological action of ura- nium salts has been little studied. Experiments are now in progress to show the influence of uranium on proteid metabolism, and our present results show the influence of this substance on the excre- tion of carbonic acid. The rabbit first experimented with was de- prived of food for three days, and on the fourth day the experi- ment was commenced, extending through three entire days, during which time the animal was without food. The accompanying tables show the results obtained. The body temperature was ascertained by inserting a self-registering thermometer into the rectum. * Chittenden and Hutchinson, this volume. and Inorganic Substances on Gas Metabolism. 411 A study of the first results shows plainly a decided action on the part of the uranium salt. The influence of the salt, however, mani- fests itself somewhat slowly, and it is not until the third day that its action becomes very pronounced, when the increased excretion of car- bonic acid becomes very noticeable, accompanied with a slight rise in temperature. The first action of the uranium appears to cause a diminution in body temperature and in the amount of carbonic acid eliminated. The total amount of uranium salt given was quite large (1:175 grams in divided doses), and although no especial toxic symp- toms showed themselves, the animal died on the day following the conclusion of the experiment. A second series of experiments was tried, using smaller amounts of uranium nitrate and extending through four days, the results of which are also shown in the accompanying tables. The rabbit was ~ deprived of food for four days prior to commencing the experiment. The amount of uranium nitrate given was considerably smaller than the quantity employed in the first series of experiments, and the animal did not suffer any permanent iH effects from its use. The following table shows the average daily result, expressed in milli- grams of CO, contained in the 37°5 litres of aspirated air, together with the average body temperature. May 3. 38°9° C. 574°3 milligrams CO, ger 4. 39-0 540°8 : e¢ Ca 39°9 581°2 He «6 coal; 38°5 716°3 ss ue The uranium nitrate was introduced by hypodermic injection in the following quantities : May 3. 0 mez: 5:18 p. m. ’ 0:080 gram of the salt. ia De 8:40 a. m. 0-090 * fg Ata 10:20 a. m. 0:100' “* -¢ oe Os 12:40 p. m. 07150‘ or cee 0: 3:25 p. m. (eiles (eres es SS say 5:15 p. m. 0200.4" se BG. 0 0-770 In this second series of experiments it is to be noticed that the first two days are given up wholly to determining the normal excretion of carbonic acid, and the results show fully how close an agreement may be expected under normal circumstances. Taking the results 412 Chittenden and Cummins—Influence of some Organic First SERIES OF EXPERIMENTS WITH URANIUM. Normal period, without uranium nitrate. Oxalie acid to Male ids ; Ae ae neutralize ba-| = ei baile Ps & ob = si 4 (5) a (2) . 7 Date. ryta solution. Se el S 5 = 3 a5 IBN 8) gd = i ee ARE April 19. . [eet kK le2n| ge Ee 33 = 3 — ike: S22 ® rah q 1S fo od |S0d|/ ea |jSaa| 5:2 "= bp area be 23 (496|8 7.4 "| £2 | 68 | seen 8° |s Eve IS A S) 5 = A. M. 9:47 to 10:17 6:5 | 24:8 | 31:3 | 463 | 15-0 278°9 697°3 | 38:7 10:45 to 11:15 7-8 | 25°38 | 33°6 | 46:3 | 12-7 285°3 588°3 | 38°7 11:46 to 12:16 | 7-9 | 25°8| 33-7 | 46:3] 19:6 | 288-4 | 5836 | 386 Ms 2:04 to 2:34 | 8:4 | 26-3 | 34:7 | 46-3] 11°6 | 214°9 | 537°3 | 384 2:57 to 3:27 | 8-4 | 26-1 | 34:5 | 46-3| 11°8 | 218-6 | 5466 | 38-7 3:58 to 4:28 | 8-2 | 26-1 | 84:3] 46:3) 12°0 | 221-4 | 553°5 | 38°6 4:52 to 5:22 | 8-5 | 26-2] 34-7 | 463] 11°6 | 2149 | 537°3 | 38-7 Average, 79 | 25:9 | 38°8 | 46-3 | 12°5 | 281:0° | 577-7 | 38-9 April 20. With uranium nitrate. A. M. 9:04 to 9:34 | 7-8 | 25°9 | 83:2 | 46:3] 181 | 242-7 | 606°8 | 38-4 9:57 to 10:27 | 8-3 | 268 | 35:1 | 46:3 | 11:2 | 207-5 | 5x8°8 | 384 10:59 to 11:29 | 9-8 | 266 | 36:4 | 46:3 99 | 188-4 | 458°6 | 38:2 11:53 to 12:23 | 8-7 | 263 | 35°0 | 46:3 | 11°3 | 2093 | 523-5 | 381 159 t0.229 | 88 | 263) s51/ 463) 11-2 | 2075 | 5188 | 88-4 2:51 to 3:21 | 88 | 265) 353 463 | 11:0 | 2048 | srz-0 | 388 3:46 to 4:16 | 8-4 | 26-1 | 845 | 46-3, 11:8 | 2186 | 446°6 | 39-1 4:41 to 5:11 | 6-8 | 25:2 | 32:0 46:3 14:3 | 264-9 | 662-4 | 38-9 Average, 8-4 | 262 346 | 463] 11-7 | 217-3 | 535°9 | 38-5 and Inorganic Substances on Gas Metabolism. 413 With uranium nitrate—continued. |Oxalie acid to AC ey ; rote ; neutralize ba-| 3 = saapeiaits veh a oO E Date. ryta solution. | & 5 z Sits 3 oe S 25 |sa 4) gg | 2 oe April 21. Sonn as a Coens Se oe ah Sle aera | Ete | RBS Ses oO oO o fant 2 Ss S os ag a ° A. M. 9:02 to 9:32 5:0 | 22:4 | 27-4) 46:3 18:9 350-1 875°5° | 39°2 9:58 to 10:28 6:70 | 24:9 | 30°99 | 46:3 15°4 285°1 712°8 | 38°9 10:55 to 11:25 6:0 | 24:7 | 380°7 | 46°3 15°6 289-0 722°6 | 39:0 11-47 to 12:17 | 5-7 | 24-8 | 30:5 | 46:3] 15°8 | 292-7 | 731-9 | 39-4 P. M. 2:00 to 2:30 | 7:3 | 25-7 | 33:0 | 46°38 13°3 246°4 6161 | 3971 | 2:55 to 3:25 «60 | 25:0 | 31:0 | 46°3 15:3 283°4 708°7 | 39-1 3:47 to 4:17 | 8-4 | 26-2 | 346 | 463] 11-7 | 216-7 | sqr-5 | 39-2 4:42 to 5:12 9°3 | 26°6 | 35:9 | 46°3 10°4 192°6 481°7 | 391 Average, 6°7 | 25:0 | 31-7 | 46:3 14°6 269°5 673°9 | 39-1 The following figures give the average daily result in body tem- perature and in the amount of carbonic acid contained in 37°5 litres of aspirated air: April 19, 38°9° C. ae 38°5 “< co) 4248 39-4 <« The uranium nitrate was the following quantities: April 19, 5°40 p. ce 20, 8°55 a. 20, 10°35 a. 20, 12°40 p. 20, 1:35 p. 20, 5:30 p. 21, 8°55 a. 21, 2°45 p. 577°7 milligrams CO, 535°9 673°9 ce ce ce ce introduced by hypodermic injection in m m. m The animal died on the 22d. 0:050 gram of the salt. 0-100 0°100 0-150 0°150 0°300 0-200 0°125 1:175 ee ee 414 Chittenden and Cummins—Influence of some Organic SECOND SERIES OF EXPERIMENTS WITH URANIUM. Normal period, without uranium. A. M. 9:08 to 9:38 10:08 to 10:38 11:03 to 11:33 11:57 to 12:27 P. M. 2:07 to 2:37 2:59 to 3:29 3:50 to 4:20 4:44 to 5:14 Average, May 4. A. M. 8:50 to 9:20 9:49 to 10:19 10:44 to 11:14 11:37 to 12:07 P. M. 1:56 to 2:26 2:48 to 3:18 3:41 to 4:11 4:32 to 5:02 Average, ‘Oxalic acid to cc Total oxalic acid used. eo et 30°1 32°8 34°7 32°5 30°2 d0°2 30°4 Oxalie acid equiva- lent to 250 c. ¢. Ba(OH), c. c¢. | = | Difference. . ¢. ox- oo 44°8 44°8 44°8 44:8 | 44:8 44-8 44-8 44:8 prey | alice acid, OSes Normal period—continued. | neutralize ba- | ryta solution. | page tes Set al oe ke 9-2 | 24:3 9:5 | 25:0 87 | 24-4 8°6 | 24:2 9-7 | 25°0 8:3 | 24:2 8-7 | 24:5 8-7 | 24:5 8:9 | 245. 9°2 | 24:7 9:5 | 24:4 8°9 | 24°6 9:5 | 24:8 91 25-0 9°6 | 25°2 9°4 | 25:0 9°3 | 24:8 30°9 33°9 33'5 34°3 84-4 34:8 34-4 34-1 34°1 44°8 44:8 44°8 | 44°8 10°9 10°9 11°3 10°5 10°7 10:0 10°4 10°7 | CO, in a, b and e. mg. 220°3 220°3 228-4 212°2 216'3 202°1 210°2 216°3 10°7 216°3 E So ) es 5711 | 38°9 520°5 | 38°8 591°3 | 38-9 606°5 | 38°7 510°4 | 388 621°6 | 38°9 586-3 | 38-9 586°3 | 39°4 - 374°3 | 389° 550°9 | 38°9 550°9 | 38°9 571°r | 38:9 530°6 | 38°9 540°8 | 391 505°6 | 39°2 525°6 | 39-1 540°8 | 39°0 5g0°8 | 39-0 A115 and Inorganic Substances on Gas Metabolism. With uranium nitrate. Oxalic acid to (ol Mee 6 3 2. ‘ & neutralize ba-| © feo | a s 2 = TAGE: ryta solution.| = 5 21s rales) = a 3S Jd loa sl og 2 1S) g, ay 5. | Soh] gs ‘ eens a May eB IaS 6g aS gs a hs We cs ie od |\Sa6\ aa |Sas| oa ae a aie = ee ES | RSs | emia ga oa oF | o* So Neyo! SL n|@ A =) ) 6a ALM. Ce Fae ee 8:48 to 9:18 8:7 | 24:6 | 33:3 | 44:8 11°5 232°4 | 581°2 | 39-1 9:41 to 10:11 8:5 | 24:5 | 38°0 | 44:8 11°8 238°5 | 596°3 | 39°3 10:39 to 1119 | 9-0 | 24:8 | 33-8] 44:8] 11:0 | 2223 | s55‘9 | 39°6 11:34 to 12:04 8:7 | 24:6 | 33°3 | 44°8 Talay 232°4 | 581°2 | 39°9 LES | 1:57 to 2:27 8:5 | 24:7 | 33:2 | 44:8 11°6 234°4 | 586°2 | 40:0 2:49 to 3:19 | 9:0 | 24:8) 33:8) 44:8] 11:0 | 2223 | se5-9 | 40-0 3:45 to 4:15 8:4 | 24:6 | 33:0 | 44:8 11°8 238°5 | 596°3 | 40°4 4:40 to 5:10 8:6 | 24:4] 33:0 | 44:8 |- 11:8 238°5 | 5963 | 40° Average, 8-7.| 246 | 383 | 448 | 11:5 | 282-4 | g8r-2 | 39-9 May 6. With uranium nitrate—continued. A. M. 8:52 to 9:22 | 7-0 | 2-4 | 29:1 | 44:8.| 15-7 | 317:3 | 793°4 | 40-0 9:52 to 10:22 | 56 | 233 | 28-9] 448 | 15-9 321-4 | 803°5 | 39°6 10:48 to 11:18 6°9 | 24:0 | 30°9 | 44:8 13°9 280°9 | 702°5 | 38-2 11:48 to 12:13 6°9 | 24:0 | 30°9 | 44:8 13-9 ‘280-9 702°5 | 379 P. M. | 2:11 to 2:41 75 | 24:6 | 32:1 | 44:8 12°7 | 256°% | 642°8 | 37-4 3:02 to 3:32 75 | 24:4 | 81:9 | 44:8 12°9 261°7 | 654°4 | 38-1 Average, 6-9 | 23°7 | 30°6 | 44:8 14-2 286°5 | '716°3 | 38°5 416 Chittenden and Cummins—Influence of some Organic EXPERIMENT WITH CUPRIC SULPHATE. Normal period, without copper. A. M. 9:05 to 9:35 10:00 to 10:30 10:57 to 11:27 11:49 to 12:19 P. M. 2:07 to 2:37 3:00 to 3:30 3:02 to 4:22 4:42 to 5:12 Average, May 11. A. M. 9:04 to 9:84 10:03 to 10:33 10:58 to 11:28 11:54 to 12:24 P.M. 1:56 to 2:26 2:49 to 3:19 3:48 to 4:13 4:39 to 5:09 Average, Oxalic acid to! ls ee i : ane neutralize ba-| < set Aca! Pies bs a op jryta solution. | 2g |SS°] = 3 5 | | 26 [ea al oc 2 rans | S j BS 2 & $5 = a caw] Beles Oe Pes eNO alo feta s 3 gS |$95\ 82 ess| gs a) | ees Bee oe tee a S S 11:3 | 27-7 | 39-0] 47-7 | 8-7 | 175-8 | 430°7 12°3 | 28:0 | 40:3 | 47-7 | 7-4 149°5 | 3740 11°8 | 27°8' | 39°6 | 47-7 8-1 163°7 | 409°4 12°4 | 28-0 40-4) 47-7 73 147°5 | 3689 | | 12:2 | 27:9 | 4071 | 47:7 7:6 153°6 | 384°1 | 11-9 | 28-0} 39:9 | 47-7| 7-8 1576 | 394°2 | 12-4 | 28-0 | 40-4] 47-7} 7:3 | 1475 | 368-9 12:5 | 27:9 | 40°4 ) 47-7 7-3 146°5 | 36674 12127295 40-08 aie TT 155°2 388°2 With cupric sulphate. 130 | 28:3 | 41:3] 47-7 | 6-4 129°3 | 323°4 12-0 | 28-0 | 40-0 | 47-7) 7-7 | 155°6 | 389:1 12-7 | 28:0 | 40-7 | 47-7] 7-0 | 141°5 | 353°8 125 | 27-8 | 403 | 47-7) 74 | 1485 | 3715 11:8 | 27-9 | 39-7 | 47-7) 8-0 | 161-7 | 40473 13-0 | 28-2 | 41:2 | 47-7] 65 | 181:4 | 328°5 13-2 | 28-2 | 41-4] 47-7] 683 | 127°3 | 318-4 13-7 | 28:3} 42:0 | 47-7] 5:7 115-2 | 288-1 12-7 | 28-1 | 40-8 | 47-7] 69 | 1888 | 347-2 Body temperature. 379 87:2 37:3 38-2 38°3 36°7 (807 36°6 30°8 37°0 and Inorganic Substances on Gas Metabolism. With cupric sulphate—Continued. 417 Oxalie acid to) Lisi ; ‘Bh neutralize ba-| a (es is @ eb Date. ryta solution. | 3 5 Bie Silents 2 SI May 12 FET ee 5 oe se 3 = . Y oo Y y Seles é Zc 3 2 ol ae ipo ie od |\2ad| ae ISA a| os “a eis Sd Stef Syelee | SIGs TS hs = Oo = Ss oO < IS Q = = Z A.M. 9:11 to 9:41 13°3 | 28°3 | 41°6 | 47:7 671 123°3 | 308°3 10:06 to 10:36 13°6 | 28°3 | 41:9 | 47-7 5°8 Lees) 2035 11:60 to 11:30 14:7 | 28:3 | 48:0 | 47:7 4:7 95:0 | 237°5 11:52 to 12:22 13°6 | 28:4 | 42°0 | 47-7 5:7 115°2 | 288'1 P. M. 2:15 to 2:45 13°7 | 28:2 | 41:9 | 47-7 5:8 117°2 | 293°1 3:09 to 3:39 | 13°7 | 28°3 | 42°0 ; 47-7 a7 114:°2 | 285°5 3:59 to 4:29 14:4 | 28:3 | 42°7 | 47-7) 5:0 LODO) | 252.9 4.53 to 5:23 14:0 | 28:4 | 42:4 | 47-7 5:3 107-1 | 267°9 Average, / 13:9 | 283 | 42°2| 47-7] 5:3 | 111°3 | 2783 ody temperature. aC: |B qo CL x} . a (Se) oO co 30° 30°8 30°9 36°71 36°2 36°2 30°8 Following are the average daily results, expressed in milligrams of CO, contained in 37°5 litres of aspirated air, together with the average body temperature: © May 10. 37°9° C. 388°2 milligrams CO, prot. 37-0 847-1 eee «12, 35:8 278-3 gah Mee The cupric sulphate was introduced by hypodermic injection, following amounts: May 10. 5°34 p. m 0:025 gram CuSO, CON oa lale S:0t ae a, 0:005 = ot Saale 9:55 a. m. OZ020 meee OC see tt 12:29 p. m 0-050)“ at Jor han 9:08 a. m OrO25y) <5 ce 0-130 Rabbit died on the 13th. rm Trans. Conn. ACAD. VoL. VII. 53 Marcu, in the 1887. 418 Chittenden and Cummins—Influence of some Organic of these two days for comparison, it is seen that the action of the uranium is somewhat slow, but that it produces on the first day (May 5) a noticeable rise in temperature, without any appreciable change in the elimination of carbonic acid. The full effect of the uranium, however, shows itself on the day following the last dose of the salt, and we then see the same noticeable increase in the elim- ination of carbonic acid noticed in the first series of experiments. We have to conclude, then, that uranium nitrate, when taken in sufficient quantity, tends to raise materially the body temperature and to increase very noticeably the excretion of carbonic acid. Action of cupric sulphate. Falck, as quoted by Dr. H. C, Wood,* has found that cupric sul- phate acts upon dogs, pigeons, rabbits and similar animals as an irritant, neurotic poison; producing great depression of temperature, with progressive general paresis ending in death, apparently from failure of respiration. Our experiments on rabbits show a marked influence of the salt in depressing body temperature and a still greater influence in diminishing the production of carbonic acid. The results of one experiment are shown in the preceding table. Although but 130 milligrams of the copper salt were used altogether, the animal finally died on the day following the conclusion of the experiment. Action of arsenious oxide. C. Schmidt and Stiirzwaget have shown by experiments on cats, that arsenious acid tends to produce a noticeable diminution in the excretion of both nitrogen and carbonic acid. Voit, however, has pointed out that in these experiments, the diminished excretion de- pends simply on the loss of a large portion of the food by vomiting, and Bolckt has shown that small doses of arsenious oxide given to hungry dogs, is wholly without influence on the decomposition of proteid matter. With large, toxic doses of arsenic, Gihtgens§ and Kossel|| have shown that a very noticeable increase in the elimina- tion of nitrogen takes place. These facts constitute about the sum * Therapeutics, Materia Medica and Toxicology, p. 46. + Moleschott’s Untersuchungen, vi, p. 283. + Zeitschrift fir Biologie, vii, p. 430. § Centralblatt f. Med. Wissen., 1875, p. 529. || Archiy. f. exper. Path. u. Pharm, v, p. 128. and Inorganic Substances on Gas Metabolism. 419 total of our knowledge regarding the action of arsenic on tissue changes. Our experiments were made with rabbits in a condition of hunger, deprived of food for three days prior to the experiment, and the results appear to show that arsenious acid, in the case of rabbits, has a tendency to diminish the excretion of carbonic acid, presumably through its action on the metabolic activity of the tissue cells. The amount of arsenic given was quite small and the animal seemed wholly unaffected by the poison. Action of potassium antimony tartrate. Voit* states that antimony in large doses affects proteid metabo- lism in the same manner as arsenic, and since Saikowsky has shown that both arsenic-and antimony tend to produce a fatty degeneration of the various organs, in which presumably the fat is formed from the decomposition of proteid matter, the non-nitrogenous moicty of the albumin molecule being stored up as fat instead of being burned to carbonic acid, it seems natural .to expect that these two substances when taken in large quantity at least, should like phosphorus diminish both the consumption of oxygen and the elimination of carbonic acid. With rabbits our results with antimony certainly lead to this con- clusion. Even small doses of tartar emetic quickly !ead to a dimin- ished excretion of carbonic acid and also to a noticeable fall in tem- perature. In the first series of experiments, the results of which are shown in the accompanying tables, the excretion of carbonic acid fell from 363°6 milligrams per 37°5 litres of aspirated air to 203°8 milli- grams and with a fall in temperature of from 39° C. to 346° C. The total amount of tartar emetic given was 82 milligrams. In the second series of experiments, where as before, the rabbit had been deprived of food for three days prior to the experiment, still . smaller quantities of antimony were given with even more pronounced results, both in the diminution of carbonic acid and in the depression of temperature. Thus while in the normal period the excretion of carbonic acid amounted to 396 milligrams per 37°5 litres of aspirated air and with a normal temperature of 38°6° C., tartar emetic (0°055 gram) given in divided doses reduced the carbonic acid to 106°5 mil- ligrams per 37:5 litres of aspirated air and the temperature to 270° C. Ackermannt has already called attention to the great decrease in animal heat produced by antimony, notably in the case of rabbits: * Hermann’s Handbuch der Physiologie, Band vi, p. 184. + See H. C. Wood, Therapeutics, etc., p. 158. 420 EXPERIMENT WITH ARSENIOUS OXIDE. Normal period, without arsenic. Chittenden and Cummins—Influence of some Organic Date. June 7. A. M. 10:04 to 10:34 11:08 to 11:38 12:06 to 12:36 2:27 2:52 to 3:22 3:00 to 4:20 4:44 to 5:14 Average, June 8. A.M. 9:00 to 9:30 9:52 to 10:22 10:45 to 11:15 11:37 to 12:07 P. M. 2:14 to 2:44 3:08 to 3:38 3:58 to 4:28 4:48 to 5:18 Average, 'Oxalic acid to c. Cc. Total oxalic acid used. Oo ow © zt w 30°4 33°9 34°3 | 82-4 34:0 30°6 c. C. Jent to 250 ec. ce. Oxalic acid equiva- Ba(OH)s. : 45:5 45:5 45°3 | alice acid. S | Difference. c. ¢. Ox- S 12°8 10°1 11°6 11:2 13.1 10°5 11°6 With arsenious oxide. neutralize ba- ryta solution. ce ae 7:8 | 24:5 FB | 25°2 | 92 | 26-2 8°3 | 25°6 8:6 | 20°7 6°9 | 20°5 9-1 | 24:9 8-2 | 25-4 9:0 | 25°8 / 10°3 | 26-4 10°1 |- 2673 9:8 | 26-4 » Ot | 26°0 10°4 | 26°5 10:0 | 26°4 9-4 | 26°3 98 | 26:3. 34:8 06°7 36:4 | 36:2 | 30°1 36°9 36°4 30°7 36°0 45°5 45°5 45°5 45:5 45°5 45°5 45°5 45°5 45°5 10°7 8°8 9-1 9°3 10°4 | CO, in a, b and ec. | CO, in 37°5 L. aspi- a ° b= | rated air. mg. on roy aS a Ne) 510°4 *586°3 566°0 662°0 528° 586°6 495°3 480°4 Body temperature. 38°7 38°9 39°2 38°9 ——— VS and Inorganic Substances on Gas Metabolism. 421 With arsenious oxide—continued. Sail | 4 4 Oxalic acid to Z BS 5 : 3 a S 3 neutralize ba-| = seq tec ad ° = ‘ | 5 ryta solution.| & Seis I WA = Date. ay) ¢ x Si oo ow = Olse ae, 1 . eee oO Ye) te 2 : Se) See oc iF ace =a June 9. Bi eee ren eS od aatae 2 q 2 5) os 5 oO ® Jd = 3S fos] = oe. (=S) A= 80 ao mn s| + O lege o= ad atte) em bo ov oct O| om o OD faa eae} ag a © Sao SUSE ape gel ine oe lacey Oey (eit abies 5 = i Oo = oO O aa) A. M, 9:00 to 9:30 9:4 | 26-1 | 35°5 | 45°5 10°0 202'1 505'4 39°0 9:52 to 10:22 | 10-4 | 26-5 | 36-9 | 45°5 10:41 to 11:1 | 9-1 | 26-1 | 35-2 | 45°5 | 10-3 | 208-2 | s20-5 | 39-1 11:36 to 12:06 | 9°6 | 263 | 35-9) 455 | 9-6 | 194-0 | 485-2 | 39-0 ie 2) oO e a © @ - Ww N ei oo le 2) l=} P.M: 2:15 to 2:45- | 9°6 | 26°5 | 36-1 | 45:5 | 9-4 | 190°0 | 475¢x | 39°2 3:08 to 3:38 | 9:6 | 26°5 | 361 | 45:5 | 9:4 | 190-0 | 475" | 38-9 3:57 to 4:27 | 10-3 | 26:5 | 868 | 45-5] 87 | 175°8 | 430°7 | 39:2 4:47 to 5:17 | 10-0 Average, 9°7 | 26:4 | 3671 53 9-4 189°3 473°5 | 39°71 b ' Average daily excretion of carbonic acid expressed in milligrams of CO, per 37°5 litres of aspirated air, and average temperature is as follows : June 7 38°9° C. 586°6 milligrams CO, “é 8 38°9 480°4 ee 66 meg 39-1 AT3*5 = S The arsenious oxide was introduced by way of the mouth in small gelatin. capsules, in the following doses : ou oS June 7 5:25 p. m. 0:005 gram As.O; ts 8:47 a. m. O05), , ** * 2/5 12:12 p. m. 0:005 rf ath 8 5:25 p. m. 0-005 = ** e a. 8:50 a. m. 0-005“ x abet, 12:12 p. m. OFO10). 5:45 : 0°085 422 Chittenden and Cunvmins—Influence of some Organic FIRST SERIES OF EXPERIMENTS WITH ANTIMONY. Normal period, without tartar emetic. Oxalie acid to) oo al aes . ee ; neutralize ba-) © se x: iz if pa a z Date. ryta solution. | & 5; [et oe a=, a J S ib id 15-28 egte lrregeeali es 2 1 70 ee March 31. 9 |) jog | Se (225) go | an 22 [eze| ge eae) 28 | SE | oa [ie 25) ‘ > Ss a) s] a ° ee jess se 15 a S 8 3 A. M. | 9:03 to 9:33 13°9 | 30°9 | 44:8 | 52°6 78 144:5 | 361°3 | 38°6 10:01 to 10:31 13°8-| 81:0 | 44:8 | 52°6 78 144.5 | 361°3 | 38°8 10:56 to 11:26 15°0 | 31:0 | 46:0 | 52°6 6°6 122°2 | 305°7 | 38°9 11:52 to 12:22 13°7 | 80°8 | 44:5 | 52°6 8:1 150°0 | 375°2 | 39°2 P. M. 1:55 to 2:25 | 125 30-0 | 42-5 | 52-6 | 10-1 | 187-0 | 467-7 | 39-4 2:47 to 3:17 15:0 | 31:1 | 46-1 | 52°6 6°5 120°4 | 301°r | 39-2 | 3:41 to 4:11 14:4 | 30°4 | 44°8 | 52°6 78 144°5 | 361°3 | 39°2 4:36 to 5:06 13°7 | 30°8 | 44:5 | 52°6 8:1 150°0 | 375°2 | 39°3 Average, | 14:0! 30-7! 44-7! 52°6| 7.85 | 145-4 | 363°6 | 39-0 April 1. With tartar emetic. A. M. | | 8:57 to 9:27 14:1 | 30°8 44-9 52°6 Tiff 141°7 | 3544 | 381 9:54 to 10:24 14:5 | 31:0 | 45°35 | 52°6 aA 131°5 | 3289 | 36°9 10:50 to 11:20 15-4 | 31-0 | 46°4 | 52-6 6:2 114°8 | 287°2 | 36°4 11:44 to 12:24 15°4 | 31:0 | 46°4 | 52°6 6:2 114'8 | 287°2 | 30°7 P. M. 2:01 to 2:31 | 17:0 | 31°2 | 48°2 | 52°6 4:4 81°55 2038 | 346 Average, 15°3 | 31:0 | 46:3 | 52°6 6°3 1169 | 292°3 | 36°3 The antimony was given in the form of tartar emetic and was introduced by hypodermic injection as follows: March 31. 5:20 p. m. 0-012 gram tartar emetic. April 1. 8:45 a. m. 0-035 fs fe oe 1. 12:48 p. m. 0:035 i s 0-082 Rabbit died at 3:30 p. m., April 1. a and Inorganic Substances on Gas Metabolism. 423 SECOND SERIES OF EXPERIMENTS WITH ANTIMONY. Normal period, without tartar emetic. Oxalie acid to os apes i : ‘a é neutralize ba-| = a S 5 = S 5 Tate: rytasolution. | Sg |S °| 9 = 4 s =m6é |Oa a iS = 2 es a, 2 Mid st PE a es | Eee a eee ae, oy Se bee See iar «| BE preal hve inne 9:14 to 9:44 | 14:4 | 30°99 | 45°3 | 526 73 135°2 | 3381 | 38:4 10:09 to 10:39 | 13:0 | 30°3 | 43°3 | 52°6 9°3 172°3 | 430°8 | 38°5 11:04 to 11:34 | 18:1 | 30°5 | 43°6 | 52°6 9:0 166°7 | 416°9 | 38-4 11:57 to 12:27 14°5 | 30°8 | 45°3 | 52°6 73 185°2 | 338°: | 88-6 P.M. 2:05 to 2:35 | 13°8 | 30°7 | 44:5 | 52°6 8:1 150°0 | 375°2 | 38°9 2:58 to 3:28 | 13:1 | 30°5 | 43°6 | 52°6 9:0 166'7 | 416-9 | 38:8 3:52 to 4:22 | 12°7 , 30:5 | 48°2 | 52°6 9-4 174-1 | 435°4 | 38°9 4:45 to 5:15 12°9 | 30°7 | 486 | 52°6 9-0 166°7 | 416°9 | 38:7 Average, 13-4 | 306 ' 44:0 526 8°5 158°4 | 396°0 | 38°6 April 6 With tartar emetic. A. M. 8:59 to 9:29 | 14:5 | 30°38 | 45°3 | 52-6 73 135°2 | 3381 | 37:4 9:57 to 10:27 | 15°2 | 31:1 | 46°3 | 52°6 6°3 LG) SZ2O5°Sr | og 10:52 to 11:22 | 14:5 | 30°6 | 45:1 | 52°6 75 188°9 | 347°4 | 36°6 11:46 to 12:16 | 14°5 | 31:0 | 45°5 | 52°6 Wel 131°5 | 328°9 | 37:3 yf Be aE, 1:58 to 2:28 | 15°3 | 30°9 | 46:2 | 52-6 6°35 117°6 | 294°1r | 36:2 2:50 to 3:20 | 14:5 | 30°9 | 45:4 | 52°6 7:2 133°4 | 333°5 | 36:2 3:42 to 4:12 | 15:4 | 31:2 | 46°6 | 52°6 5:95 110°2 | 275°6 | 35:4 4:38 to 5:08 §=15°7 | 31:2 | 46:9 | 52°6 57 115°6 | 289°0 | 35°7 Average, | 14:9| 31:0 | 45°9 | 52°6/ 6-68 | 124:9 | 312°2 | 36:7 424 Chittenden and Cummins—Influence of some Organic With tartar emetic—continued. | Oxalie acid to hee K z oat Y neutralize ba-| ao s | oo Date. ryta solution. | @ 5 |SS°| 9 8 ee ox Ire ay OS 2 | 1g gt April 7. Be Sof og oe me 3 ore od le < G| gs eae ates Ses | Snes | ee fae cael ee eee = tbl) cae tO ue es eel He ESI here os Ss ss SoBe eae ae a 8 S - ALM. 9:00 to 9:30 17:6 | 31:2 | 48°8 | 52°6 3°8 70°4 | 176°0 9:59 to 10:29 18:5 | 31:3 | 49°8 | 52°6 2°8 51°8 | 129°7 10:58 to 11:28 18°9 | 31°4 | 50°3 | 52:6 2:3 42°6 | 106°5 Average, | 183| 31°3| 496| 526| 2-97 | 54:9 | 13774 Body temperature. Average daily excretion of carbonic acid expressed in milligrams of CO, per 37°5 litres of aspirated air, together with average temper- ature is as follows : April 5. 38°6° C. 396-0 milligrams CO, 6. Maint 312°0 7 Bs if 28°3 137-4 3 Dy The following amounts of antimony were injected: April 5. 5:30 p. m. 0-015 gram tartar emetic. 6. 8:45 a. m. 0-015 eH ‘s 6. 12:39 p. m. 0:015 ss ES 6. 5:24 p. m. 0-010 Hs a 0-055 Rabbit died at 12 m., April fi and Inorganic Substances on Gas Metabolism. 425 Action of morphine sulphate. Boeck and Bauer* have already made a careful study of the action of morphine on the elimination of carbonic acid and the absorption of oxygen. By experiments on a cat and ona dog they found that the action of morphine on metabolism was mainly an indirect one, affecting especially the consumption of non-nitrogenous matter. Further, that its action hinged mainly on its power of affecting mus- cular activity ; thus in the case of a cat the first action of morphine was to increase the elimination of carbonic acid and the consumption of oxygen, due to the increased muscular activity induced by the poison, while in the case of a dog, where narcosis was half induced, there was a diminution in the amount of carbonic acid eliminated amounting in one case to 27 per cent. This diminished excretion was due almost wholly to the quieting action of the morphine and was followed by an after period in which there was increased produc- tion of carbonic acid, due to the increased activity of the muscle tissue. ; In these experiments the dose of morphine was 0°05 gram, in the form of chloride, and was introduced by subcutaneous injection. The injection of the poison was followed soon after by convulsions, ete., indicating vigorous toxic action. In our first series of experiments we endeavored to have the toxic action less pronounced, and for this reason the morphine was introduced by way of the mouth in repeated doses, the experiment extending through three days and into the fourth. The rabbit was deprived of food through the entire period and had also been kept without food for three days prior to the experiment. The data are to be found in the accompanying tables. The results do not show any very marked action, either on the excretion of carbonic acid or on the body temperature. At no time was there any noticeable indication of increased muscular activity, the rabbit remaining fairly quiet in the chamber and showing no symptoms of tetanic convulsions. On the other hand there was no very profound narcotism. A study of the individual results, how- ever, shows that directly after each dose of morphine, the excretion of carbonic acid fell quite noticeably for one or two periods. Such action as was produced, therefore, in this experiment, is to be consid- ered simply as incidental to the semi-somnolent condition of the animal. In a second shorter series of experiments with a rabbit, one single * Zeitschrift fiir Biologie, Band x, p. 339. Trans. Conn. AcaD., Vou. VII. 54 Marou, 1887. 426 Chittenden and Cummins—Influence of some Organic FIRST SERIES OF EXPERIMENTS WITH MORPHINE. Normal period, without morphine. Oxalie acid to Site Howe baled is ; neutralize ba-| Vera ‘ [rte a eh 5 A (5) S20 ° | os g 2 Data: ryta solution. | © 5 |S °| © es = = s eS) [INN Mis nae Ad ge 1 sled eee March 24. Rae fey ee OS Meta | Sls Pes Xl os ON Bore Pte fe. tea. oS tors 3) Ge, pa Bel Ga) Bl Re auc: 37 |e = el es = SF Zo 5 5 H Oo = | oO : 'S) -Q A.M, 8:54 to 9:24 | 11.0] 255 | 36:5 | 45-1 | 86 | 1598 | 399°5 | 37-9 9:53 to 10:23 1a OP 20388 Bir | 45d 74 137°5 | 344°0 | 38:0 | 10:50 to 11:20 164 Bore | Sidr 450 78 | 144:9 | 362°5 | 37°9 11:46 to 12:16 Se easey (alu (ay il 8:3 154°2 | 385°7 | 38:0 P. M. 2:14 to 2:44 10°6 | 26:1 | 86°7 | 45-1 8:4 156°1 | 390°3 | 38°2 3:08 to 3:38 | 10:2! 261) 363/| 451/ 88 | 163-5 | 4088 | 38:3 4:02 to 4:32 10°3 | 26°71 | 36:4 | 45-1 8:7 161°6 | 404°2 | 38:2 4:55 to 5:25 11:4 | 26°71 | 37:5 | 45-1 76 141:2 |; 353'2 7) Sas Average, | 11-0! 25-9! 369| 451! 82 | 152-4! 381-0 | 38-0 March 25. \ With morphine sulphate. A. M. 8:51 to 9:21 | 11-7 | 26:5 | 88-2] 45:1) 69 | 1283 | 320°8 | 38-2 9:53 to 10:23 | 11:8 | 265 | 683] 45-1) 68 | 126-4 | 316-2 | 37°8 10:47 to 11:17 | 10:9 | 26-2 | 37-1] 45:1 | $0 | 148-7 | 37z-8 | 38-0 11:44 to 12:14 | 11-1.) 26-4] 375 | 451] 76 | 1412 | 3532 | 38-0 P.M. 2:01 to 2:31 | 1171 |. 26:2 | 37-3 | 45:1 7.8 144:0 | 360°1 | 38°7 3:08 to 3:38 | 11-7 | 262] 37-9 | 45-1) 72 | 1838 | 33477 | 38-7 4:04 to 4:34 | 11:0 | 25-7; 36-7] 45:1) 84 | 156-1 | 390°3 | 886 4:57 to 5:27 10°0 | 25°2 | 35:2 | 45-1 9°9 184'9 | 462°3 | 38°8 Average, ALD 26-1 | ~80522) Ant 79 145°4 | 363°7 | 38.4 and Inorganic Substances on Gas Metabolism. 427 With morphine sulphate—continued. ‘Oxalie acid to neutralize ba-| 3S Date. ryta solution. = 3 SS) March 26. a 2s om oS |o uw OS 3 2 2 AER Aas A. M. ‘ 9:24 to 9:54. 17-6 | 30:8 | 53-4 10:19 to 10:49 16°4 | 35°6 | 52:0 11:14 to 11:44 16°5 | 35:7 | 52:2 12:09 to 12:39 16°5 380°7 | 53°2 P. M. 1:58 to 2:28 17-4 | 35°38 | 58:2 2:55 to 3:25 10) 4 302% | 522% 3:49 to 4:19 LGA Shion OG 4:48 to 5:18 16:0 | 35:3: | 51°3 Average, 16°7 | 35°6 | 52:3 March 27. A. M. 8:33 to 9:03 16°2 | 35°6 | 51:8 9:30 to 10:00 gE eine || owls c. C.. lent to 250 c. ¢@. Oxalic acid equiya- Ba(OH)s. 60°5 | 60°5 : bs 3 men | & : zz Bet = ° q ‘| s * fan} _ o . im -° 2 ox my NE ee lee he | ee re r= a So es Ss) io) © A oO j@) ica) 71 | 131-5 | 3289 | 38°8 85 | 157-4 | 393-7 | 388 8-7 1611 | 403°0 | 38°9 9:3 172°3 | 430°8 | 38-9 Following is the average daily excretion of carbonic acid, expressed in milligrams per 37°5 litres of aspirated air, together with the aver- age body temperature : March 24. SG ain, oc 26. a PE (8 38°0° C. 38°4 38°8 38°9 381:0 milligrams CO, 363°7 ee 66 378°4 ee 66 416°9 sé 66 The morphine was introduced into the stomach in solution in the following amounts: March 24. 6:00 p. m. Sone: 8:35 a. m. ee Say 12:35 p. m. oo iy. 5:50 p. m. CEN play 9:15 a. m. LU i 3 12:50 p. m. 0-075 0-075 0-075 0-100 0-100 0-100 gram morphine sulphate. ee ee 428 Chittenden and Cummins—Influence of some Organic Jarge dose of morphine sulphate was given, and given by hypo- dermic injection. In this case, as before, there was no increased muscular activity, but there was a sudden and rapid fall both in tem- perature and in the amount of carbonic acid excreted, the latter of which was stili quite pronounced on the following day, although the temperature has gone back to normal. This diminution in carbonic acid was accompanied by a profound narcotism, the animal lying almost motionless and with a respiration ranging from 8 to 20 per minute. The results, which are to a certain extent corroborative of Boeck and Bauer’s, are shown in the accompanying table: SECOND SERIES OF EXPERIMENTS WITH MORPHINE. Normal period without morphine sulphate. ' A ' i i ao © : 2, ; Oxalic acid to if gos > 3 & si S neutralize ba- = 3) $e é = Sj 2 ryta solution.| «5 |5S S A | z Time aS |SN S| rg 2 ee | eB ; Es ie) orn > aS 3g Pat ace s+ an) qj 3 os] oD 5 = 2 3 oud ro) faS) oO it.. as aa fas} aea(cd) 1 —< HO “= op eats Sas i Co Oo = bo oo |oSo| $s Sl H's we af So Gis 12-8 5171S ia = oO iS) e) iB S ~ o) a) o io) ea March 12. A.M. ; 9:12 to 9:42 15.4 | 34:1 | 49°5 159°251 9°75 180°6 465°0 | 39-1 10:00 A. M., injected subcutaneously 0:100 gram morphine sulphate. A. M. | 10:23 to 10:53 | 16-9) 34:5 | 51-4 59:25 785 | 145-4 | 363°5 | 366 11:29 to 11:59 | 17:3 | 34°5 | 51°8 59:25, 7-45 | 1880 | 3425 | 34-4 12:32 to 1:02 | 16:3 | 34:1 | 50:4 | 59-25) 8-85 | 1640 | qro‘o | 333 Pow 9:48 to 3:18 | 17-0 | 34:2) 51-2 |59-:25| 8:05 | 149°2 | 373°0 | 34:4 3:49 to 4:19 | 17°8 | 34:6 | 52:4 |59-25| 685 | 126-9 | 317°3 | 85°0 4:47 to 5:7 =| 17-1 | 34:3 | 51-4 | 59-251 7°85 | 145-4 | 3635 | 363 March 13. | ; 10:08 to 10:38 16°9 | 34°3 51°2 | 59°25 8:05 149°2 373°0 39°2 Action of quinine sulphate. The importance of quinine as a therapeutic agent and particularly its value as a febrifuge has led to a thorough study of its physiolog- — a eS and Inorganic Substances on Gas Metabolism. 499 ical action. In this connection, the action of quinine on proteid metabolism has been very thoroughly investigated, but as to its exact influence on the decomposition of non-nitrogenous matter, as shown by its effects on the elimination of carbonic acid, there is less una- nimity of opinion. This is naturally a point of considerable import- ance for if, as is generally supposed, the alkaloid has the power of diminishing body temperature, it would presumably be due to its in- fluence on the combustion of non-nitrogenous matter in the body. Ranke, Kerner, von Boeck and others have plainly shown the power of quinine to diminish proteid metabolism, but Strassburg, by an elaborate series of experiments* found that the alkaloid had no very decided effect upon the elimination of carbonic acid, either in healthy or fevered rabbits. Boeck and Bauer,+ however, from experiments on cats, claim that quinine in the first stage of its action diminishes somewhat the pro- duction of carbonic acid, owing to its inhibitory action on the tissue cells; but when large doses of quinine are given, so that con- vulsions appear, then there is an increased production of carbonic acid, owing to the greater decomposition of non-nitrogenous matter incident to increased muscular activity. With small doses of the alkaloid, it is to be presumed that the slight diminution in carbonic acid noticed by Boeck and Bauer comes simply from diminished proteid metabolism. In our experiments, rabbits only were used and these in a con- dition of hunger, having been deprived of food for three days prior to the experiment. In the first series of experiments, the total amount of quinine given was quite large, so that at last the animal finally died from its effects. No decided action on the production of carbonic acid was noticed until just before the animal’s death, when both the body temperature and the amount of carbonic acid fell quite noticeably. On the second day of the experiment, when the quinine was first being given, the body temperature, as taken per rectum, fell quite gradually until it finally reached a point 1°5 °C. below the average of the normal period. The results of the experi- ment are to be seen in the accompanying tables. The quinine given was in the form of hard, gelatin-coated pills and possibly was not as rapidly absorbed as might otherwise have been. At no time was the rabbit in convulsions. * Quoted from Dr. H. C. Wood, Therapeutics, p. 75. + Zeitschrift fir Biologie, Band x, p. 350. ———_ 430 Chittenden and Cummins—Influence of some Organic First SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. = | Oxalic acid to gS © # | Be g neutralize ba-| BZ |e9s| ¢ ae of | 3 Dates | ryta solution.| ¢0 |S$Qo| ° & a 3 25 |oX az fas 2 5i FS April 12 me Reon ties ite = | 8 z ou (ee ay Pe et oS Sie a's | oe os |$o5/82 eee] 8S | ce | sf | go ES lessee g- |S A e) co) ea) A.M. haw | 9:11 to 9:41 95 | 24:2 | 38-7 | 41°5 78 144°5 361°5 | 38:7 10:05 to 10:35 10°8 | 24:4 | 85:2 | 41°5 6:3 116°7 291°8 | 38-7 10:58 to 11:28 10°8 | 24:2 | 35:0 | 41°5 6:5 120°4 30r°r | 38°7 11:54 to 12:24 10°7 | 24:2 | 34°9 | 41°5 66 =| 122°2 305°7 | 38°3 Ps M. 1:58 to 2:28 10°0 | 24:0 | 34:0 | 41°5 75 138-9 347°4 | 38°6 2:51 to 3:21 10°0 | 24:2 | 34:2 | 41°5 73 135°2 3381 | 38-4 3:49 to 4:19 10°5 | 24:3 | 34:8 | 41:5 6-7 =| 124-1 310°4 | 38°7 4:41 to 5:11 11:0 | 24:3 | 35°3 | 41°5 62 | 114°8 287°2 | 38°8 Average, 10-4 | 242 | 346] 41:5 | 68 | 127-1 | 317°8 | 38-6 April 13. With quinine sulphate. A. M. 9:14 to 9:44 | 9:3] 23-9] 38-2) 41:5 | 83 | 158-7 | 384°5 | 38-6 } 10:10 to 10:40 | 11:3 | 23:9 | 35°2 | 415) 63 | 116-7 | 291-8 | 386 | 6-1 | 113-0 | 2826 | 382 : 11:05 to 11:35 | 11:0 | 24:4 | 35-4 | 41°5 11:58 to 12:28 | 10-3} 242] 84:5) 41:5 | 7-0 | 129-7 | 314°3 | 38-2 1:55 to 2:25 | 9-4{ 240| 88-4] 415) 84 | 1500 375°2 | 88-1 2:46 to 8:16 | 101 | 24:2] 34:3] 41:5 | 7-2 | 188-4 | 333°3 | 37-6 8:40 to 4:10 | 93| 23:8] 3381] 415) 84 | 4:32 to 5:02 | 10:3 | 24:1 | 344 | 41:5) 7-1 143-5 | 282°6 | 38:0 a f=) @ = w > ss uo oo aS pe Average, 10'1 | 24:1 | 84:2] 41:5 | 78 138°6 | 325°6 | 38-1 and Inorganic Substances on Gas Metabolism. 431 With quinine sulphate—continued. PR oa 5 ‘Oxalie acid to! 1a 5. . ; Be D neutralize ba-| Be E 9S 3 7 2 ae Se a 2 Date. ryta solution; «3 |32 | 4 = S x April 14. q. [a g os Seis j Me See eee |e | Hashes 25 ees owe 4 =° Ss) a a wen | | | | 8:54 to 9:24 | 100 243) 843) 415) 7:2 | 198-4 | 333°5 | 38°6 9:50 to 10:20 | 10-2 | 241 | 343 | 41:5) 7-2 | 188-4 | 3335 | 38:2 10:43 to 11:18 | 9:8 | 240) 388 )| 415) 77 | 1496 | 356-7 | 38:3 | | 11:38 to 12:08 | 10°9 | 24°3 | 35°2 | 41°5 | 6°3 116°7 291°8 | 37°6 reer” i | | P. M. | | | | | 1:57 to 2:27 11°3 | 24:5 | 35°83 | 41°5 | 57 105°6 264°0 | 34°7 Average, | 10-4 | 24-2 | 34:6| 41.5-; 6-9 | 1263 | 315-9 | 37-5 The following amounts of quinine were given by way of the mouth: April 12 5:20 p. m 0:130 gram quinine sulphate. eo 1S 9:10 a. m 0:260 << ss Be copeorliss 1:00 p. m. Ors 00 Rees ec ie als 5:15 p. m Or020h < a es om 8:45 a. m. 0-520 < sf os oe ie 12:00 m. OF SOM wee oy a 2°600 Rabbit died at 3 p. m. April 14. Following are the average daily results in temperature and in the amount of carbonic acid excreted per 37°5 litres of aspirated air. April 12 38°6° C. 317°8 milligrams CO,. ee 13 38-1 ee 325°6 66 66 66 14 37°) “6 315-9 «6 66 432 Chittenden and Cummins—Influence of some Organic SECOND SERIES OF EXPERIMENTS WITH QUININE. Date. May 17. par er 8:40 to 9:10 9:33 to 10:08 10:29 to 10:59 11:24 to 11:54 P. M. 1:55 to 2:25 2:48 to 3:18 3:43 to 4:13 4:35 to 5:05 Average, May 18. A. M. 9:05 to 9:35 10:00 to 11:30 10:51 to 11:21 11:46 to 12:16 P. M. 2:14 to 2:44 3:07 to 3:37 4:01 to 4:31 4:58 to 5:28 Average, Normal period, without quinine su Oxalic acid to | ao. é neutralize ba-) ees Sol ue ryta solution. Ee 38 ‘ a e. 382) se So 169 ae. ee ae ae g : 25 : 3 2 Boe ae 53 | 22°2 | 27-5 | 41°8 14'3 6:1 | 22°8 | 28:9 | 41:8 12°9 6:2 | 22°6 | 28°8 | 41°8 13°0 4°9 | 21:9 | 265 | 41°8 15:0 5:0 | 221 | a7-1 | 418) 14-7 | 4°5 | 22:0 | 26°5 | 41:8 15°3 | 6-7 | 23-1 | 29-8 | 41-8) 120 6°7 | 23:0 | 29:7 | 41°8 12:1 67 | 224 | 284 | 418) 139-7 With quinine sulphate. 6-2 22°5 | 28°7 | 41°8 13:1 75 | 23:0 | 30:5 | 41°8 11:3 56 | 2271 | 27-7 | 41:8 14-1 6:0 | 22:5 | 28°5 | 41°8 13:3 6°5 | 22°8 | 29:3 | 41°8 12°5 6:5 | 22°9 | 29:4 | 41°8 12:4 7:0 | 28:1 | 38071 | 41°8 rs 6:0 | 22:1 | 28-1 41°8 13°7 64) 226 | 290 418) 128 lphate. Nlepe ) 2. | Sale 5 | eee seh | Se | ae -) S) a 289°0 | 722°7 | 388°8 260°7 | 651°9 | 388 262°7 657°0 | 38°8 3032 | 7581 | 39-1 29771 742°9 | 39°0 309°2 | 773°2 | 38:9 242°5 | 606°5 | 38°8 244-5 | 611°5 | 38°9 2763 | 690s | 38-6 264'8 | 662°0 38°6 2284 571°r | 38-4 285°0 | 712°6 | 38:3 268°8 | 672°2 | 38°8 252°6 | 631°7 | 38°9 249°6 | 624'r | 390 236'5 | 59r°3 | 39-0 276°9 | 692°4 | 38-4 257°8 | 64qr7 | 38-7 and Inorganic Substances on Gas Metabolism. 433 With quinine sulphate—continued. | | | : O) UI Q [o) — Xe Oxalie acid to so. Z S Ss g ; ro} Z2a2 iS} 2 ap q neutralize ba-| = Bos : = ie 5s Date. ryta solution.| 3 5 2S Y ia ie Sj & | om ste | Air al les Ses | gs ; oS j= | as a = May 19. | See oe 1 een ee = el 8 ; : r= 65 oS |opO Des eet ee Jd ® ge sey Stn = || ce eet TS Fo lath) co) BO Sea ors Ss Sees Oia, mira | ag af Se Se Oreille ong iene = oe) o) On s0 js ao = (e) [S| Ss) 2 a ~_ > | —= ——— A. M. 1 9:02 to 9:32 | 7:3 28-0] 30°3| 41°8| 11:5 | 282-4 | 582-2 | 38-9 9:57 to 10:27 | 7-6 23-2 | 30°8| 41°8| 11:0 | 222°3 | 555°9 | 38-4 10:50 to 11:20 | 7-2! 23-1 | 303] 41°8| 11:5 | 282-4 | 581-2 | 38-7 11:43 to 12:13 6°9 | 23:0 | 29:9 | 41°8 11°9 | 240-5 6014 | 38°6 Poy Me 2:03 to 2:33 75 | 238°2 | 80-7 | 41:8 ALL | 223°3 5584 | 38:9 2:54 to 3:24 | 82 | 236 3:47 to 4:17 | 74 | 233) 30-7] 41:8] 11-1 | 2243 | s6r-0 | 38:8 30-2 | 41-8} 11:6 | 2345 | 586-3 | 38:8 31:8 | 41:8 | 10-0 | 2021 | 505-4 | 38-5 4:40 to 5:10 G1 | 23-1 Average, | 74] 23:2 | 806) 41°83 11:2 | 2264 | 566-4 | 38:7 The quinine was given by way of the mouth in gelatin capsules, in the following quantities : May 17 5:30 p. m. 0°250 gram quinine sulphate. OS alls) 8:50 a. m. 0:250 ‘ ‘ « be 18 12:30 p. m. 0-250 “* as “i ps 18 5:30 p. m. 0250; 75 sd ee edg 8:50 a. m. 0°325 Es eh ts 1:55 p. m. 0°250 * we fy 1:575 Following are the average daily excretions of carbonic acid ex- pressed in milligrams of CO, per 37-5 litres of aspirated air, together with the average body temperature : May 17 38°6° C. 690°5 milligrams CO. ee 18 38°7 ee 644:°7 ee ee tee to) 38°7 “ 566-4 Us A TRANS. CONN. ACAD., Vout. VII. 55 Marcu, 1887. 434 Chittenden and Cummins—Influence of some Organic THIRD SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. Oxalic acid to ls Soa & S) i ep x neutralize ba-| -= SS sl. ce} cobra igen! = 4 FE 2: 5 a Sere) 4 = S| }) Time. ryta solution.| © 5 |Fo ° a — | & 2). a 4 AO x 10 =t [<) ray oe eS es = aa a March 8. Hola e Bt lene ce a 75. algae ; 235 ons oO | ip 3 ey a aage oS oO fe aS) = 2 8 = @.S [once | tsp | S| = ors rae ts bo as i\2eels "in aso") oan iS ee A Ss Ss) aa) A. M. 9:50 to 10:20 15:5 | 33-9 | 49°4 | 59°25 9°85 182°5 456°3 | 39°2 10:58 A. M. injected subcutaneously 0°083 gram quinine sulphate. 11:15 to 11:45 | 16°9 | 34:5 | 51:4 | 59°25) 7-85 145°4 | 363°5 | 38-4 12:25 to 12:55 | 15:3 | 33-9 | 49-2 | 59°25 10-05 186°2 | 465°5 | 39-9 P. M. 3:00 to 3:30 15°6 | 34:0 49°6 | 59°25 | 9°65 178°8 | 447°0 | 39°8 4:06 to 4:36 5 | 15°8 | 83-7 | 49-5 59°25 9°75 | 180-7 | 451°8 | 39:9 Average, _ | 15°9 | 84:0 | 49°9 | 59:25 | 9°35 | 1728 | 432'0 | 39°5 FouRTH SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. Ener: F 4 + Oxalic acid to Ss | Ss Bain © neutralize ba- ee ce alt ae s bt =| & Time. ryta solution.| @95 | Fs S a A = acim egies i 2 1D 53 = March 10, a [8 off) os e Pa EY | 2 A ° ee) 2s so [>] : 1) Sirs Nis gs SO 5) sl se ao aes So . mn = =a OD = 94 a a a) “= 8p = me oo ovo ine] a a oy Os a =I a & > O°. | |O) Si ai b4 Qs S O# 5° 2 o zg a) = 2 Q ep) | ie) jee) A. M. 9:11 to 9:41 15:0 | 33°8 | 48°8 | 59°25 | 10:45 193°6 | 484'0 | 38°8 10:15 A. M. injected subcutaneously 0:15 gram quinine sulphate. 10:33 to 11:03 | 16°5 | 34°6 | 51:1 | 59:25) 8-15 151°0 | 377°5 | 38°4 11:39 to 12:09 | 15:4 | 33:9 | 49°3 | 59°25] 9°95 184-4 | 46r'0 | 38°9 12:38 to 108 | 15°3 | 84:1 | 49-4 | 59-25] 9:85 | 1825 | 456-3 | 38-4 Pp. M. R 2:54 to 3:24 16:0 | 84°3 | 50°3 | 59°25 |) 8-95 1658 414'5 | 391 4:07 to 4:37 15°9 | 34:1 | 50-0 | 59°25 | 9°25 171-4 Average, 15°8 | 34:2 | 50°0 | 59°25} 9°25 171-0. | 42776.) Sem and Inorganic Substances on Gas Metabolism. 435 EXPERIMENT WITH CINCHONIDINE. Normal period, without cinchonidine sulphate. |Oxalic acid to Bo | : ; | eee S neutralize ba- | = a ze a cS) a Beste a 2 Date. ryta solution.) @© 5 | Fo | os Sg = S 8 [rs Eid ae) = ap 2 June 24. | | a.) | Som te ef oe h B italia sc Aven es Se a Sey eee ge )22°) e2 ea) 2s | se | st |e. eenlie SOS. eit tes ij) )) ee A. M. | 9:00 to 9:30 =| 106 | 28:8 | 39:'4 | 50°2 | 10°8 218°3) 545°8 | 36-4 9:56 to 10-26 | 11-2 291 40-3 50-2) 9:9 | 2001 | 500°3 | 37-4 | 10:47 to 11:17 | 12:0 | 29-0] 41:0 | 50:2) 9-2 185°9 | 465°0 | 38-1 11:38 to 12:08 | 106 28-7 | 39:3 | 50-2 | 10-9 220°3 | 550°9 | 36:3 2:03 to 2:38 | 103 285 | 888) 50-2 11-4 | 280-4 | s76xr | 36-9 2:51 to 3:21 | 9-4 | 28-2| 876 | 502) 12:6 | 2549 | 6368 | 37-1 $4210 4:12 | 9-7 | 28°5 | 38-2 | 50°2 |. 12:0 | 242°5 | 606-5 |: 37-0 4:30 to 5:00 | «9-7 | 285 | B82 502 120 | 2425 6065 | 36-4 Average, | 10:5 | 28-6 | 30-1 | 50-21 11-41 | 2043 | s6z0 | 37-0. June 25. With cinchonidine sulphate. A. M. 9:03 to 9:33. =: 10°8 | 286 39-4 | 502) 10-8 | 2183 | 5458 38-6 9:58 to 10:28 11-4 | 28-9 40:3 | 50:2 9:9 | 200-1 | 500-3 386 10:53 to 11:23 11:1 | 28-7 | 398 | 502 10-4 | 210-2 5286 386 1148 to 12:18 | 11-6 | 28-7 40:3 | 50-2 9:9 | 2001 | 500°3 | 38-7 P. M. | 2:04 to 2:34 =| 11:1} 28°8 | 39:9 | 50:2! 10°83 208°2 | 520°5 | 38-9 2:59 to 8:29 | 11°5 | 29:0 | 40:°5| 50:2) 9:7 | 196-0 | 490-2 | 39-0 3:56 to 4:26 , 11°6 | 29:0 | 40°6 | 50:2) 96 194°0 | 485'2 | 39-0 4:50 to 5:20 12-1 | 29-1") 41:2 )--50-°3. |) 920 181°9 | 454°8 | 89-1 Average, 11-4 | 28°8 | 40-2 | 50:2 | 10:0 201°1 | 502°8 | 38:8 Following are the average daily amounts of carbonic acid excreted, expressed in milligrams per 37:5 litres of aspirated air, together with the average body temperature. _ June 24, Bet OB. 561:0 milligrams CO,:. tee Oh, 38°8 502'8 " ie Following are the doses of cinchonidine : June 24, Blo Bay M; 1:000 gram cinchonidine sulphate. CaO 10:35 A. M., Oe2 5007 5° nt oh Fe 205 12:35 P. M., 0:°325—S* ye a 1575 Rabbit diced at 5:40 vp. M., June 25, in convulsions, 436 Chittenden and Cummins—Influence of some Organie In the second series of experiments, smaller doses of quinine were employed, so that no special toxic action was observed. In this case there seemed to be a gradual falling off in the amount of carbonic acid produced; such a decrease as might be assumed would naturally result from diminished proteid metabolism. The results are shown in the accompanying tables. The body temperature, as determined per rectum did not show any change whatever under the influence of this quantity of quinine (total dose 1:575 gram of quinine sulphate). There does not appear to be any proof that moderate doses of quinine lower the body temperature of healthy animals or even man, and Kerner found in his experiments that a full dose of quinine given to a healthy man would prevent the usual rise of temperature resulting from vigorous exercise, but did not affect the temperature under ordinary circum- stances. Liebermeister* has also reported that the alkaloid has no constant depressing action on the bodily heat in health. Two other short series of experiments were tried with quinine, also with rabbits in a condition of hunger. In both of these cases the quinine was introduced by sub-cutaneous injection in the form of sulphate. In both cases there was a slight fall in temperature, accompanied with a noticeable decrease in the amount of carbonic acid eliminated, directly after injection of the quinine. This effect, however, was only temporary, for the temperature quickly rose to the normal, and even somewhat above the normal point, while the carbonic acid in the third series came quite back to the normal and in the fourth series remained only a little way below. It would appear, therefore, from our experiments, that in a healthy, hungry rabbit moderate doses of quinine sulphate exercise at the most only a very slight depressing influence on body temperature, and have but a minimum effect on the production of carbonic acid. Action of cinchonidine sulphate. Previous experiments + on man have shown that cinchonidine has the power of lessening materially the elimination of nitrogen, pre- sumably through its inhibitory action on proteid metabolism. Cinchonidine is supposed to have much the same physiological action as quinine and cinchonine, only weaker. Our present experi- # Deutsch. Archiv fiir Klinische Medicin, Band iii. + See Chittenden and Whitehouse, Studies from the Laboratory of Physiological Chemistry, vol. i, p. 164. a ee and Inorganic Substances on Gas Metabolism. 437 ments with the alkaloid, using quite large amounts of the sulphate, show a somewhat different action from quinine. Using a large rab- bit, without food for three days, and giving it by way of the mouth, in gelatin capsules, large doses of the alkaloid, there was a very noticeable and constant rise in temperature up to the very time of death, accompanied by a slight but gradual diminution in the amount of carbonic acid given off. In all, 1°575 grams of cinchonidine sul- phate were given; an amount exactly equal to the quinine sulphate given in the second series of experiments with quinine. With cin- chonidine, however, the rabbit was much prostrated, showed symp- toms of tetanic convulsions, and finally died in a vigorous tetanic spasm at the end of the second day. The results are shown in the preceding table. Action of Antipyrine. Antipyrine or dimethyloxychinicine has of late been much experi- mented with. Among the many statements which we have seen recently concerning its action are the following, which are of interest in this connection, Arduin* found that 3 grams given to a rabbit produced cataleptic stiffness, diminished reflexes, etc., followed by violent convulsions. There was also a very marked fall of bodily temperature. Anseroff,+ by experiments on animals, found that the alkaloid caused an increase of blood pressure and a decrease of inter- nal temperature, as shown by a thermometer in the rectum, but a considerable rise in the external temperature, sometimes as much as 12°C. Pavlinoff{ has reported that antipyrine produces a very con- siderable quickening of the respiration, while Dr. Walter, of St. Petersburg,§ is reported as having found that the alkaloid while reducing febrile temperature, also reduces nitrogenous tissue changes ; and further, that the assimilation of proteids is materially favored by the drug. F. Miiller|| has also found that in fever antipyrine dimin- ishes the excretion of nitrogen. Coppola, however, states that in the case of a dog, 0°3-0°4 gram of antipyrine was wholly without action on its excretion of nitrogen. Coppola has further found that the * Abstract in Therapeutic Gazette, 3d series, vol. i, p. 677. + Abstract in Therapeutic Gazette, 3d series, vol. ii, p. 315. ¢ Abstract in Therapeutic Gazette, 3d series, vol. ii, p. 339. § Abstract in Therapeutic Gazette, 3d series, vol. li, p. 53, || Jahresbericht fiir Thierchemie, xiv, 242. 4 Jahresbericht fiir Thierchemie, xv, 97. 438 Chittenden and Cummins—Influence of some Organic FIRST SERIES OF EXPERIMENTS WITH ANTIPYRINE. Normal period, without antipyrine. Oxalie acid to Pee i f ey : neutralize ba-| ZB |-B eS Pe z @ eo | & Date. ryta solution. | = 3 eae aes 3 iS a S Oi ee . o May 24. 5 5 z capO ea tee ba eso] fs on 23 Bec Se |SB5| 22 (g24) = | ot | SE | ge £ a =I ) A a) iS) ia) A.M. 8:54 to 9:24 8°6 | 26°4 | 35:0 | 46:1 11°1 224°3 56r1°0 | 38-4 9:52 to 10:22 8°8 | 26°5 | 35°3 | 46-1 | 10°8 218°3 | 545°8 | 38-4 10:47 to 11:17 95 | 26-4 | 35:9 | 46-1 10°2 2051 513°0 | 38°4 P. M. 1:55 to 2:25 9-1 | 26°3 | 35°4 | 46-1 | 10°7 216°3 | 540°8 | 38°3 2:47 to 3:17 9°5 | 26°4 | 35°9 | 46-1 | 10°2 205°1 | 513°0 | 38°6 3:40 to 4:10 9-1 |. 265: | °35°6: | 46-1 | 103 212°2 | 530°6 | 38:4 4:35 t0 5:05 | 88 | 26°3 | 85-1 | 46-1 | 11:0 | 222-3 | sss5°9 | 38-4 Average, 9-0 | 26-4 | 35-4 | 461 | 10:7 | 2148 | 535-7 | 38-4 May 25. With antipyrine. A.M, 8:56 to 9:26 9°6 | 26°4 | 36°0 | 46°1 | 101 2041 | 5104 | 38:2 9:53 to 10:23 9°6 | 26:4 | 36-0 | 46-1 10-1 204:1 510°4 | 381 10:49 to 11:19 77h | 25°6 | 83:1 | 46:1 13°0 262-7 657°0 | 38:0 11:47 to 12:17 8-4 | 25°9 | 34°3 | 46-1 | 11°8 238°5 | 596°3 | 38-1 P. M. 2:15 to 2:45 7-9.) 26:0 | 33°9 | 46-1 | 12:2 246°6 | 616°6 | 38-1 3:15 to 3:45 8'°7 | 25°8 | 34:5 | 46:1 | 11°6 234'5 | 586°3 | 38:3 4:15 to 4:45 8-4 | 26°0 | 84:4 | 46-1 | 11:7 236°5 | 591°3 | 38:0 5:09 to 5:39 74 | 25°6 | 33:0 | 46-1 | 18-1 264'8 | 662°0 | 881 Average, 8-5 | 25°9 | 34-4 | 46-1 | 11°7 | 2365 | 5or°3 | 38-1 May 26. A. M. 9:02 to 9:32 | 11°3 | 26°8 | 38:1 | 461 8:0 161°7 | 404°3 | 36°2 9:59 to 10:29 9-4 | 26:0 | 35:4 | 46-1 | 10°7 | 216°3 | 540°8 | 35°3 eee : : : 4 and Inorganic Substances on Gas Metabolism. 439 alkaloid not only has a noticeable antipyretic action in conditions of fever, but also reduces the temperature in healthy organisms. The extent of reduction, however, is not great, ranging only from 0:1 to 0°6 of a degree. Further, the diminution in temperature is to be ascribed, according to’Coppola, not to diminished metabolic activity, but to increased giving up of heat, due to dilatation of the blood- vessels by the antipyrine. Jacubowitsch * also claims for antipyrine an inhibitory action on the excretion of uric acid. So far as our knowledge extends, however, no experiments have been tried as to the influence of this therapeutic agent on the production of carbonic acid. Our experiments have been confined wholly to rabbits, and those in a condition of hunger. In the first series of experiments, during the antipyrine period, the alkaloid was given in large and oft-repeated doses, in the form of powder, in gelatin capsules, at follows: May 25, 8:33 A. M., 0:2 gram antipyrine. fe 20; 9:35 Ss Or2p= 88 re S25; 10:30“ Ooze FS Soe! op pNP yaaa O20 a5 a HG ha. 12:27 P. M., (I OG oe ey Pati neta O65 = da 3:54“ O65 os aos 4:51 OrGrs % a anaes 5:46“ OsG7 es Seu: 8:45 A.M, O26) 9°" Gap: O38: «6! Te0S 45 ee 5:4 The accompanying tables show the results obtained. At the end of the last period of the second day (May 26) respiration was very rapid, about 208 per minute, and during that period the amount of carbonic acid excreted was larger than in any other. In spite of the large dose taken, however, the alkaloid appears to have had no special action on the production of carbonic acid, and further, the temperature was only very slightly lowered until on the last day, just prior to the animal’s death. On the last day of the experiment (May 26) the rabbit appeared much prostrated, and at 10.35 a. M. was seized with a convulsion, followed soon after by three others, dying at 11.30 a, M. In the second series of experiments somewhat smaller amounts of antipyrine were given, but here, as in the first series, there was no * Jahresbericht fiir Thierchemie, xv, p. 444. 440 Chittenden and Cummins—Influence of some Organic SECOND SERIES OF EXPERIMENTS WITH ANTIPYRINE. Normal period, without antipyrine. | | ' . - | K ‘ ‘Oxalic. acid to) Ee at Mie . rf 3 Ineutralize ba-| co | °°%| he @ t | § r Intien: | 2 |S Sees Ss ; | ss Date. | yta solution | 5 [Se EI s | | feet Bega baa ete 2 2 ye May 31. | Be eo ee = oo | tees Oe Ne ety eg ON es Ey ay seas | -@-? 2od| BB Sas) Ose 7 EH) Tiss bo 2s |£85/ 55 |RaA| Se oF SH | Be ee }a |0 g co) =) FQ A. M. | 9:02 to 9:82 8°5 1 26 9:57 to 10:27 | 8-1 | 25: 10:59 to 11:29 | 9-1 | 25:5 | 34:6 | 443 | 9-7 |, 196-0 | 490-2 | 889 ~~ | 95 | 191:0 | 477°6 | 38-9 3 | 838 | 44.3 | 10:5 | 211-2 | g28-z | 389 1/332 443 | 114 | 9243 | sér0 | 38-9 © oD no oH on 9 = fo oo 11:51 to 12:21 P. M. | 1:44 to 2:14 98 | 25°7 | 35°5 | 44:3 | 88 176°8 442°2 | 38°7 3:30 to 4:00 | 9:3 | 25:5 | 848 443 | 95 | 1920 | 480-r | 38-9 4:21 to 4:51 | 9-7 | 25-7 | 85-4 | 44:3 | 8-9 | 179-9 | 449°8 | 88:8 ' Average, 9-1 | 25°5 | 84°6 | 44°3 9°7 195°9 | 489°9 | 38°8 June 1. With antipyrine. A.M. 8:53 to 9:23 | 9:9 | 25:5 “54 | 448 | 9 | 179-9 449°8 | 38-7 9:50 to 10:20 | 9-4 | 25°5 | 34:9 | 44:3 | 9-4 | 190°0 | 475*x | 38°9 10:43 to 11:13 | 10-1 | 25-7 | 85-8 | 44:3 | 85 | 170-8 | 427°0 | 38-9 11:40 to 12:10 | 9-7 | 25:7 | 85-4 | 44:3 | 89 | 179-9 | 449°8 | 38-7 P. M. 2:15 to 2:45 9:6 |. 25:6) || 3552) |) 443 9-1 183-9 459°9 | 39°2 3:12 to 3:42 | 9-9 | 25°8 | 35-7 | 44:3 | 86 -| 1738 | 434°6 | 88-4 4:11 to.4:41 | 9-7 | 25-6 | 353 | 44:3 | 9-0 | 181-9 | q5q-8 | 383 5:05 to 5:35 8°7 | 25°3 | 34:0 | 44:3 | 10°3 208°2 | 520°5 | 38-4 Average, 9:6 | 25°6 | 85:2 | 44:3 | 9.08 | 188-5 | 458-9 | 88-7 and Inorganic Substances on Gas Metabolism. 441 With antipyrine—continued. “4 ‘ ° é ' ro) = A \Oxalic acid to a E 3 Ba go neutralize ba-| 3S ¢ : a ae E Date. ryta solution.| @©35 |Zo 2 =| 4 x Saye EE a et oe 2 ane 2 June 2 2808 Be fe Seer & a oo 8 Seine 2S La Le a ao eee re} ) La = oS = oe Ey zo > 85 \|28,5| 85 |g2n| ga Be) Se ee Fe s |O A eS) S) sa) A. M. 8:56 to 9:26 | 10:0 | 25°5 | 35:5 | 44:3 8°8 1778 | 444°7 | 38-7 9:53 to 10:23 9-8 | 25° 30°4 | 44:3 8-9 179°9 | 449°8 | 38°6 6 10:47 to 11:17 8°8 | 25°3 | 34:1 | 44:3 10°2 206-1 515°5 | 37:8 11:43 to 12:13 | 8-6 | 25-1 | 38-7 | 443 | 106 | 214-2 | 535-7 | 87-4 P. 2:02 to 2:32 86 | 25:1 | 33-7 | 44:3 10°6 213°2 | 533°2 | 37-1 2:55 to 3:25 8°7 | 25:1 | 33°8 | 44:3 10°5 212°2 530°6 | 373 3:54 to 4:24 8-8 | 25°3 | 34:1 | 44:3 10:2 206°1 515°5 | 37-6 il 4:47 to 5:17 8:5 | 25: 33°6 | 44:3 10°7 216°3 | 540°8 | 37-5 _ Average, 8-9 | 25:31 34-2 | 44-3 | 10-06 | 20382 | so8-2 | 37-7 Following are the average daily excretions of carbonic acid, ex- pressed in milligrams per 37°5 litres of aspirated air, together with average daily body temperature: May 31 38°8° C. 489-9 milligrams CO. June 1 38:7 4589 “ ‘6 re 37°7 508°2 a es Antipyrine was given in the following quantities by mouth, in gelatin capsules: May 31 4.57 p.m. 0-2 gram antipyrine. June 1 8:40 a. m. 0:2 of roe a Grilles 5s 0-2 Ss Ce | 10:25“ 0:2 eS 5 yt: alikeA0) 0G 0-2 ee ae 12:17 p. m 0:2 gs Co") “al 2:52 0-2 ag 6c 1 3:49 ce 0:2 ee OG) ea 4:48 § (0-2 Be sa 5:40 << 0-2 ek See 8:45 a.m 0:5 ue LOND 9:34 < 0-5 ge a 1. LOS 0-5 oe SE mi 11:24 <** 0-5 Ot 5o 4:45 p.m. 0°2 a6 4:2 TRANS. Conn. ACAD., Vou. VII. 56 Marcu, 1887. 449 Chittenden and Cummins— Gas Metabolism. discernible action on the excretion of carbonic acid. Further, the alkaloid did not noticeably lower the body temperature until toward the close of the second day, when the quantity given had reached an amount nearly sufficient to produce a fatal result. We have then to conclude that antipyrine, at least in therapeutic doses, has no special influence on the production or elimination of carbonic acid by the rabbit. XXIV.—New EnGrAND Spipers or THE Faminy CINIFLONID A. By J. H. Emerton. Tue spinning organs of the Ciniflonide differ from those of all other spiders. They have in front of the usual spinnerets an ad- ditional spinning organ, the cribellwm, with spinning tubes like the other spinnerets, but much finer, and they have on the metatarsus of each hind leg a row of stiff hairs, the calamistrwm, by which the thread is combed from the cribellum in a loose curly band. This band of loose thread forms part of every cobweb made by these spiders (Pl. x, fig. 1y.) and is easily distinguished in new webs by its width and white color and in old webs by the amount of dust which it collects. The feet have three claws and some species have a few curved and toothed spines under the claws, like Hpeitride and Theridide. The trachee are large and open in a wide slit in front of the cribellum. The colors are generally dull brown and grey. A double row of oblique light markings on the back of the abdomen, which shows most distinctly in Amaurobius, is in a modified from the usual mark- ing of the abdomen throughout the family, often varying greatly in shape in the same species. These spiders were first placed together in one family by Black wall, who in 1839 noticed their peculiar webs and spinning organs. Before that time they had been scattered among various families according to their size, form, and habits. They have been treated in the same way by Thorell in his book on the genera of European ‘spiders, and by Menge in the spiders of Prussia. Simon divides the French species into two families, Dictynidw and Uloboride. Bert- kau in his latest revision of the family, in 1882, carries the division into families still further and unites them all into a sub-order, Cribellata. I have followed Blackwall in considering the group as one family, and use his name Ciniflonide. The sub-family Uloborine of Thorell J. Blackwall. On the number and structure of the mammul employed by spiders in the process of spinuing. Trans. Linn. Soc. London, vol. xviii, 1839. P. Bertkau. Cribellum and Calamistrum. Archiv fiir Naturgeschichte, 1882. JULY, 1888. 444 J. H. Emerton—New England Spiders of Tuse in the same sense, transferring it from the Epeiride to this family. Several of the spiders described by Hentz under the name of Theridion are probably Dictyna. Of these, 7. sublatuwm, morologum, and foliaceum belong to this genus without much doubt, though I cannot identify them with any species here described. Blackwall mentions among spiders from Canada, Ergatis (Dictyna) diligens, var. annulipes, Ann. and Mag. of Nat. Hist., 1871. B. G. Wilder describes the webs and habits of Hyptiotes cavatus under the name H. Americanus, in Popular Science Monthly, 1875. E. Keyserling has described in Transactions of the Zool. Botan. Gesellschaft of Vienna, 1881 to 1884, the following species: Dictyna sedentaria, Baltimore, Coll. of L. Koch. D. volupis, Museum, Cam- bridge, Mass. D. volucripes, Museum, Cambridge, Mass. DD. foli- ata, Colorado, Vienna Museum, J vittata, Washington, D. C., War- saw Museum. D. arundinaceoides, Cation City, Colorado, Coll. of G. Marx. Dictyna Sundevall. The genus Dictyna is composed of small spiders that live in loose webs of various shapes on fences and on plants, especially on the ends of stalks and among the flowers of Solidago, Spirea, and other slender plants with clusters of small flowers. The head is generally high, but not so wide as in Amaurobius- The sternum is very wide and convex and the labium large, often nearly as long as the mandibles. The trache are large and the opening generally distinct. The difference between the sexes in most species is very great. The male palpi are very large and the palpal organs conspicuous. The tibial joint of the male palpi has on the outer side a process with two short spines. The mandibles of the males are bowed outward (Plate 1x, fig. 2d) and are much longer than those of the female. They are bent forward at the tips, and at the base of each mandible is a short tooth projecting forward. Plate 1x, fig. 20. The colors are usually dull yellow and brown covered with white or gray hairs. The cephalothorax is usually lighter in front and dark at the side, and the abdomen has a double row of light mark- ings in the middle on a dark ground, but these markings are ex- tremely variable even in the same species. PI. 1x. The webs of Dictyna usually radiate irregularly from a hole or hiding place where the spider hangs. Some species, living on walls, the Family Ciniflonide. 445 make a round patch of web with the hole near the center, but usually the shape of the web depends on that of the plant on which it is made. The principal threads of the web, if they are parallel or radiating slightly, are often crossed by a number of parallel short threads, like a segment of a web of Zpeira (PI. x1, fig. 3) and the curled band is carried back and forth on these as in the figure of the web of Amaurobius. Pl. x, lg. Dictyna muraria, new sp. Pu, IX, FIGURES 1 TO 1g. Length about 3"". The cephalothorax is dark brown, a little lighter on the top of the head with a few gray hairs in longitudinal rows. The abdomen resembles that of D. volucripes Keys, but the middle dark markings are wider in front and more broken behind. In the middle of the front half is a wide dark patch, extending about to the middle of the abdomen. Behind this are two rows of dark spots connected by transverse lines, more or less complete, forming an Epeira-like marking. PI. 1x, figs. 1 to le. The markings of this species and of volucripes vary greatly, so that they often cannot be distinguished by them. The metatarsus of the hind legs is nearly straight, not so much curved as in volw- cripes. : The males are darker, but usually have the same markings. Their abdomen is smaller than that of the females, but the cephalothorax is fully as large. The male palpi resemble those of volucripes Keys. The tibia is similar in shape, but is proportionally longer, and the two-spined process shorter than in volucripes. Pl. 1x, Lf, 14g. This spider is found all over New England. It is the most com- mon species on fences, but is found also on plants, and, in winter, under leaves. It sometimes tries to fly, oftener in the spring than in the fall, which is the usual flying time of most spiders. I have speci- mens from Mt. Washington, N. H.; Portland, Me.; Salem, Mass.; Albany, N. Y.; New Haven, Conn.; Wood’s Holl, Mass.; and several places around Boston. A female in the Museum of Zodlogy, Cambridge, Mass., named by Keyserling, D. arwndinaceocides Keys., is perhaps this species. It has the abdomen very much distended, so that the epigynum shows much plainer than usual. The spider first described by Keyserling as D. arundinaceoides came from Colorado, and I have not seen it and do not feel sure enough of its identity to adopt the name for this species. 446 J. H. Emerton—New England Spiders of Dictyna volucripes Keyserling, Zodl. Botan. Gesellschaft, Vieuna, 1882. Pr. IX, FIGURES 2 TO 2f AND Pu. XI, FIGURE 3. Female, 3:5" long or longer. The male is nearly as large, but the cephalothorax is larger and the abdomen smaller. The cephalothorax is dark reddish brown, and partly covered with white or gray hairs, most of them arranged in several lines from the dorsal groove to the eyes. The abdomen has an irregular dark figure in the middle, narrow in iront and widening backward. On each side of this is a light gray area, which becomes yellow in alcohol, and below these the sides and under surface of the abdomen are dark brown with some light markings. The abdomen is covered with gray hairs which modify the color. The legs are brown, usually lighter than the thorax, and covered with gray hairs. The male is a little darker than the female. The male palpi are short, and large at the end. The patella is as wide as long. The tibia is a little longer than the patella and widened on the outer side at the distal end, so as to be as wide there as long. ‘The two-spined process is as long as the tibia is wide, and is on the upper side of the tibia. The tarsus and palpal organ are large. PI. 1x, figs. 2e, 2f. The two spined process varies in form. It is usually curved forward, but in some specimens is nearly straight. This species lives most commonly in thick and irregular webs on the ends of plants. The dried tops of Spircwa and Solidago are favorite places for it. It also lives occasionally on fences. All over New England. Dictyna longispina, new gp. Pu. IX, FIGURE 4. This species resembles volucripes, but is a little smaller. The markings of the abdomen are similar, but the cephalothorax and legs are lighter and redder. The plainest difference of this species from the others is in the shape of the tibia of the male palpus. This is very long, as long as the femur, and stouter. The two-spined process is as long as the tibia, and extends backward nearly parallel to it. Pl. 1x, fig. 4. The palpal organ extends farther backward than usual. The end of the tube and the accompanying process extending in a spiral nearly to the base of the tibia. A young female, similarly marked and colored, accompanies the male and is probably the same species. Meriden, Conn., one male and one female. the Family Ciniflonide. 447 Dictyna bostoniensis, new sp. Pi. TX, FIGURES 3 TO 3d. This is a rather large species, measuring 4"™™ or more in length, but the cephalothorax is small and the abdomen much larger than in most species. The legs are whitish. The cephalothorax of the female is the same color in the middle, but darker and streaked with radiating brown lines on the sides. The abdomen is white with gray or black markings. PI. rx, figs. 34, 3c, 3d. In the mid- dle of the front half of the abdomen is an irregular dark stripe extending over the first and second segments. Behind this are two rows of irregular spots, about one-third the width of the abdomen apart. The sides are marked by a few dark spots in broken ob- lique lines. The sternum and under side of the abdomen are white, with a few irregular dark spots. The spider resembles a piece of bird dung. The cephalothorax of the male is larger and darker colored. The male palpi are short and slender. The tibia is short and as wide at the distal end as it is long. The two-spined process is short and on the outer side. The tarsus and palpal organ are small. Pi. 1x, fig. 3a. In 1873, this spider lived in great numbers on the iron fence around the public garden in Boston, making webs in corners, with an open tube in which the spider stood. Single specimens were found in Beverly and Brookline. In 1886, it was rare on the pub- lic garden, but common on the fences of the Back Bay park on Beacon street. I have not found it in other parts of New England. Dictyna minuta, new sp. Pu. IX, FIGURES 5, 5a. About 2™" long. The markings are like those of D. muraria, but the colors are lighter and redder than in that species, and the only two specimens are much smaller. The legs are very light brownish yellow, darker toward the base. The sternum and labium are reddish brown, and both are large and wide in proportion to the size of the spider. The tibia of the male palpus is about twice as long as the pa- tella, and nearly straight. The two-spined process is short and turned slightly forward. The spines are large and black. The point of the palpal organ is long and slender and twisted loosely. In both specimens it reaches backward half the length of the tibia. Pl. rx, figs. 5, 5a. Two specimens only, from Hamden, Conn., and Providence, R. I. 448 J. H. Emerton—New England Spiders of Dictyna rubra, new sp. Pu. IX, FIGURE 7. Female 2°5™" long. The color is more red than in the other species. The cephalothorax and legs are light orange-brown. The abdomen is darker reddish brown, with several yellow cross lines on the hinder half, and in some individuals a yellow patch on the front half. The sternum, maxille, and mandibles are light, like the legs. The male palpi are moderately large. The tibia is a little longer than wide, and as thick at the base as at the tip. The two-spined process is on the upper side of the tibia, close to the base. It is as long as the tibia is thick. The tarsus is small. PI. 1x, fig. 7. The abdomen is more pointed behind than in most species. | It lives on plants, but I do not know its web. Common in eastern Massachusetts and around New Haven, Conn. Dictyna cruciata, new sp. Pu. IX, FIGURES 6, 6a. This is the lightest colored species. The cephalothorax of the female is yellowish white in the middle and light brown at the sides. In the male the whole cephalothorax is dull yellow. The abdomen is white in the middle, the color spreading down the middle of each side, forming in many individuals a cross-shaped marking (PI. 1x, fig. 6). The sides are light brown. The legs are yellowish white. The male palpi are large and the palpal organs wide. ‘The tibia is short, not much longer than wide, and the two spines are short and on a low process of the tibia. Pl. rx, fig. 6a. Eastern Mass. ; New Haven, Conn. Dictyna volupis Keyserling, Zoé]. Botan. Gesell., Vienna, 1882. PL, IX, FIGURES 8 TO 8c. This is one of our most common spiders throughout the summer. It lives under leaves and between the twigs of trees and shrubs of all kinds, making small and thin webs. The female is about 3™" long. The legs and front part of the cephalothorax are yellowish white. The sides of the cephalothorax are brown. The abdomen has usually an irregular light yellow marking in the middle and is light brown or reddish at the sides. Pl. rx, fig. 8. Some individuals are without the yellowish marking on the back and have the abdomen brownish all over, covered with whitish hairs. The reddish markings all become redder in alcohol. The sternum and under side of the abdomen are light yellow. 1 the Kamily Ciniflonide, 449 The colors of the male are very different. The whole cephalo- thorax is orange-brown, not much darker at the sides. The abdomen is reddish brown, darker than in the female, with only a little yellow in the middie, and sometimes none. The legs are darker yellow than in the female. The males and females are about the same size, but differ in form as much as they do in color. The front of the head is low in both sexes, and rises backward to its highest point midway between the eyes and the dorsal groove. In the males the mandibles are so long that the distance from the top of the head to the end of the mandibles is as great as the length of the cepbalothorax. The male mandibles are concave in front and bowed widely apart in the mid- dle. Even the females have the mandibles a little concave in front. The male palpi are long and large. The tibia is twice as long as wide; thickened at the end, and curved downward. The two-spined process is short and a little in front of the base of the tibia. PI. Ix, fig. 8¢. The palpal organ is unusually large, and the long tube can be seen passing around it under the edges of a large thin appendage. PI. 1x, fig. 8a, 80. The webs are spread under leaves or between twigs. I have twice seen the pairing of this species. In one case the female stood across a forked twig and the male reached up from below, his head being under hers and his mandibles paraliel to her sternum. In the other the male and female stood head to head in the web, the cephalothorax of the female being tipped up in front, and resting upon the head and mandibles of the male. Common all over New England. In winter they are often found under leaves, half grown, and soon get to their growth when warm weather begins. Several small, flat ezg-cocoons are fastened under a leaf and there may be several broods in each season. Dictyna frondea, new sp. Pu, [X, FIGURES 9, 9a. This species is a little smaller than volupis and is similarly col- ored in the female, except that it is usually a little darker and less red. The cephalothorax is light brown, a little lighter in the middle of the head, but not so much so as volupis.. The abdomen is brown at the sides and yellow in the middle. The yellow stripe is narrower and straighter than in volwpis, and often forms a regular herringbone figure. PI. rx, fig. 9. The under side of the abdomen Trans. Conn. Acap., Vou. VII. 57 JULY, 1888, 450 J. H. Emerton—New England Spiders of is nearly as dark as the upper side, and the sternum has the same color while volupis is usually light colored beneath. ‘Fhe males differ less from the females than in volupis. They are colored a little darker than the females. The light stripe on the ab- domen is narrower and in some individuals wanting. The male palpi are long and slender. The tibia is more than twice as long as wide. The two spined process ts very small and close to the base of the tibia. The tarsus is smaller than in volupis, and the palpal organ very much smaller and more simple, PI. 1x, fig. 9a. On grass and low bushes all over New England. This species (or 2. volupis) is probably Hertz’s Theridion foliaceum. Amaurobius ©. Koch. The genus Amaurobius contains our largest spiders of this family. In general appearance they resemble the stouter species of the genus Tegenaria, but do not have the long upper spinnerets of that genus. The head is large and high, and wide in front. The eyes are in two rows, only slightly curved, and are all small and of nearly the same size. The maxille are long, and at the tip curve inward a little over the labium. The labium is longer than wide and a little narrowed at the tip. The mandibles are very large and strong. The whole body is thickly covered with fine, short hair, giving it a soft velvet-like appearance. The spines on the legs are small and concealed by the hair. The calamistrum consists of two rows of hairs, those of the outer row being much curved and close together, and those of the inner row three or four times as far apart. Pl. x, fig. 1f The cribellum is long and narrow and divided in the middle, PI. x, fig. le. The colors of all our species are much alike. The cephalothorax and legs are dark brown, except in freshly moulted or young speci- mens, and the abdomen is dark gray with a double row of oblique light markings. The webs are large and loose, often filling a cavity in a rotton log or under stones. There appears to be little regularity in the shape of the web or arrangement of the threads. The whole web is made of smooth silk and the band of curled threads is afterwards attached to parts of it as in Pl. x, fig. 1g. The sexes are about equal in size. The male palpi are large. Their tibial joints are short and wide and furnished with large pro- cesses of various shapes. the Kamily Ciniflonide, 451 Amaurobius sylvestris, new sp. Pu. X, FIGURES 1 TO lg. This is the common Amaurobius all over New England. The female is 10"" long with the cephalothorax 5™™ long, The head is nearly as wide as the thorax. It is low in front and rises to its high- est point half way to the dorsal groove. The cephalothorax is dark brown, darkest on the front of the head. The legs are dark brown, usually lighter than the thorax. In the young the colors are all much paler. The abdomen is oval, widest behind. It is dark greenish gray with a double row of oblique yellow or white markings on the hinder half, and two curved markings of the same color on the front. These markings run together, forming a figure which varies greatly in form and size in different individuals, Pl. x, fig. 1. The males differ but little from the females. The male palpi are large. The tibia is short and wide and has three long processes, the inner of which is slender and pointed and nearly twice as long as the tibia (Pl. x, figs. 1a, 10.) but not.so much curved as in figures of the European A. claustrarius. The epigynum is small, the middle lobe is small and the side lobes meet behind so as to completely surround it (PI. x, fig. 1¢) which is very different from the epigynum of A. claustrarius as figured by Koch. This species lives under stones, under leaves, and in the hollows of rotton trees and stumps. Fig. ly is part of a web, showing the arrangement of the curled threads. All over New England. In the White Mountains up to the high- est trees. Three specimens of this species in the museum of Comp. Zodlogy, Cambridge, are named by Keyserling A. claustrarius, which this species closely resembles. I have only young claustrarius for com- parison, but judging by descriptions and figures, especially those of L. Koch in Abh. Nat. Gesellsch. of Nuremburg, 1868, I do not be- lieve them the same species. Amaurobius ferox (Walck.) Koch., Ciniflo ferox Blk. PL. X, FIGURES 3 TO 3c. This is our largest. species. It is found only about houses and cel- lars, and is probably imported, as it is a common spider in Europe. The female is 12™™ long. Cephalothorax 6™" long, 4°" wide. The head is 3™™ wide and highest half way between the eyes and the dorsal groove, 452 J. H. Hmerton—New England Spiders of The cephalothorax is yellowish brown, darkest in front and nearly black around the eyes. The legs are the same color as the thorax, darkest toward the tips. The abdomen is dark gray with light yel- lowish marks on the back. On the front half of the abdomen are a middle and two lateral stripes and behind these four or five pairs of oblique markings. The eyes are all small and about equal in size. The front row is about half the width of the head and the eyes equidistant. The upper row is longer and the lateral eyes considerably farther from the middle ones than these are from each other. The mandibles are large and strong. The calamistrum is a double row of spines, half the length of the hind metatarsus. The male differs but little from the female, except that the ab- domen is a little smaller and the front legs longer. The male palpi are very large. The tibia is as short as wide. It is bent inward, and has a large spine on the outer and another on the upper side, each nearly as long asthe tibia. On the inner side is a third smaller spine. PI. x, figs. 3¢, 30, 3c. The tarsus and palpal organ are large and round. Fig. 3a. The epigynum is large and dark colored. The middle lobe is large and enclosed by the others only at the sides. Pl. x, fig. 3. Boston, Salem, Beverly, Mass.; Providence, R. I; Albany, N. Y.; New Haven, Conn., in cellars and houses. . Amaurobius tibialis, new sp. Pu. X, FIGURES 3 TO 3c. Female 8™™ long. The cephalothorax is light brownish yellow, not darker in front. _ The legs are of the same color and not much darker at the tips. The light markings on the abdomen are united into a middle band with oblique branches at the sides on the hinder half. The middle lobe of the epigynum is entirely concealed, the lateral lobes divided by a groove in the middle. PI. x, fig. 2. The middle process on the tibia of the aie palpus is short, but the other processes are much larger than in the other species. The outer one is about as long as the tibia is wide, and has a large hook on the inner side. The inner process is long and slender, curving over the back of the tarsus and extending nearly to the end of it. Piix, figs..24,.20; This species is found on Mt. Washington, N IL., up to the highest trees. the Family Ciniflonide. 453 Titanceca Thorell. Titanceca americana, new sp. Pu. X, FIGURES 4 TO 4d. This spider resembles 7: guadriquttata of Europe, but is usually without markings on the abdomen. The female is 5 or 6™™ long, re- sembling in size and shape the common Steatoda borealis, from which, however, it is readily distinguished by its black color. The cephalothorax is dull orange-color, blackish around the edges and toward the front. The rest of the body is deep black and cov- ered with long hair, except in some individuals a few light gray spots in pairs on the abdomen. The sternum is as wide between the second legs as it is long. The labium is as wide as long, a little narrowed and rounded at the tip. The maxille are nearly straight on the inner edges not curved in- ward at the tips, as in Amauwrobius. The head is not so wide as in Amaurobius. The eyes have nearly the same arrangement, but are proportionally larger. PI. x, fig. 1. The spinnerets are short. The cribellum is divided in the middle as in Amaurobius, and the calamistrum is like that genus. The claws of the feet are large and strong, proportionally larger than those of Amaurobius. Like most of the genus, this lives under stones in the driest and hottest places. Under the loose stones under the trap hills around New Haven and Meriden, Conn. it is common. Ihave a few from Mt. Monadnock, N. H., but have not found it elsewhere in New England. Titanceca brunnea, new sp. Pu. X, FIGURES 5 TO 5c. This species is about as large as 7. americana, but is a little more slender and less hairy. The cephalothorax is light or dark brown, like dead oak leaves among which it lives. The joints of the legs are darker toward the distal ends. The abdomen is similarly colored, but becomes redder than the rest of the body in alcohol. Across the back are four or five lines of light yellowish spots, and there are larger irreguiar spots along the sides, as in many species of Dictyna. PI. x, figs. 5, 5a. Under the abdomen between the spinnerets and epigynum are two large light spots. Fig. da. : Besides the color, the only plain difference between this and the black species is in the palpi of the female, which in this species have 454 J. H. Emerton—New England Spiders of the last two joints a little stouter than in 7. americana. In both species the palpi are very spiny at the end, though the spines are concealed by hairs. I have found this species three times under leaves in woods near New Haven, Conn. Uloborine Thorell. These spiders have been classed by most authors among the E/peiride on account of their resemblance to Tetragnatha, and espe- cially on account of their round, or at least radiate, webs. The arrangement of the eyes, the mouth parts, and the trachez are all different from the Epeiride. The spinning organs include the eri- bellum and calamistrum, like the other Ciniflonide, and the cross- threads of the webs are partly made of curly threads spun by the calamistrum and not covered with a liquid in drops like the webs of Epeiride. cs The adhesive thread of these spiders is not made separately and attached to old threads as it is by Amaurobius and Dictyna, but both threads are spun at the same time. PI. x1, figs. 27, 27 show both sides of a piece of the cross-threads of the web of Hyptiotes. Uloborus Latreille. Véeleda, Blackwall. Phillyra, Hentz. Uloborus plumipes Lucas = Phillyra ripaira, Hentz. Pu. XI, FIGURES 1 TO lf. The female is about 5™™" long. Cephalothorax 1:5"™. The cephalo- thorax is flat in front, and extends forward in the middle beyond the mandibles. Behind it is wide and swelled up on each side, where the abdomen hangs over it. Pl. x1, fig. 16. The abdomen is narrow and slightly notched in front and extends over the cephalothorax a quarter of its length. The abdomen is widest and highest ‘a third of its length from the front, and at this point has a pair of humps. The colors are very variable. A dark, plainly marked female has the femur and patella of the front legs dark brown or nearly black, and the tibia dark brown, except a white ring at the base; at the end of the tibia is a brush of coarse, dark brown hairs. The tarsus and metatarsus are white. In lighter individuals the color of the femur or tibia may be broken by a white ring near the middle. The other legs have femur, tibia and metatarsus dark brown, divided near the middle by a white ring. Patella and tarsus brown, lighter at the ends. The cephalothorax is dark brown with a narrow, indis- the Kamily Ciniflonide. 455 tinct light line in the middle. In lighter individuals this stripe is wider. The dorsal markings of the abdomen are more variable and less definite. On the front of the abdomen are two light spots, behind which are two very dark ones, sometimes united into one. Behind these are two white spots half as far apart as the humps and a little in front of them. The humps are generally dark on the inner side and light on the outer. Farther backward are two or three pairs of light spots, surrounded by a darker brown area, darkest in the mid- dle and toward the spinnerets. The sternum is brown, and the under side of the abdomen is dark in the middle and light at the sides. In lighter individuals most of these markings can be seen, the darker ones being light brown or yellow, and the lighter ones yellow or dirty white. In some no markings can be defined. The first pair of legs is twice as long as the second, and much longer than the fourth pair. The terminal joint of the palpus is more than twice as long as the one before it. The palpal claw is large, with two or three teeth. flor, fio 10. The eyes are in two rows. The upper are largest and appear still larger on account of being surrounded by dark rings. PI. x1, figs. la, 1b. They are on the top of the head, the lateral pair farthest back. The front row of eyes is close to the edge of the head, just over the mandibles. The mandibles are small and rounded forward at the base. The maxillz are as wide as long, with the front ends nearly square. The Jabium is triangular. (See figure of same parts in Hyptiotes, Pl. x1, fig. 2h.) The male is much smaller than the female. The cephalothorax is more pointed in front and lower behind. The abdomen is not much larger than the cephalothorax and not so plainly humped as in the female. The legs are but little shorter than those of the female and the markings and colors are the same. The first tibia does not have a bunch of hairs at the end like the female. The palpal organ is nearly spherical, all the parts being wound closely together. PI. xi, figs. ld, le. The femur of the male palpus has, at the base, a short process on the outer side. Pl. x1, fig. 17 The webs are round and nearly horizontal, the cross-threads usually less regular than in webs of Epeira. The webs are commonly made between loose stones, but sometimes in low bushes. The cocoons are 456 J. H. Emerton—New England Spiders of half an inch long and quarter as wide, narrowed at both ends, and with numerous short points by which they are attached to the web around them. I have found them with the female under stones. The cocoons are light brown, and each female appears to make sey- eral of them. The cocoons are made in July. This spider is found all over New England, but is not common anywhere. I have taken them from several places around Boston, Mass., in New Haven, Conn., and in Simsbury, Conn. I have specimens of both sexes from the southern part of France, given me by Mr. E. Simon. It is found in Italy and Spain. The common Uloborus of the north of Europe (U. walckencerius) is a very different species. Hyptiotes Wlk. = Mithras, Koch. Hyptiotes cavatus. Pu. XI, FIGURES 2 TO 2h. This peculiar spider is without much doubt the one described and figured by Hentz under the name of Cylopodia cavata, although he saw but six eyes and four spinnerets, and otherwise described it in- correctly. Its habits have been well described by B. G. Wilder in the Popu- lar Science Monthly, 1875, where he calls it Hyptiotes americanus. This spider resembles a shortened Uloborus. The adult female is ‘about 4™" long, and is colored like the end of one of the dead pine branches among which it usually lives. The cephalothorax is as broad as long, highest in the middle just behind the eyes, and hollowed behind under the front of the abdo- men. The abdomen is oval, a little flattened in front. On the back are four pairs of low humps, the second largest, on each of which are a few stiff hairs. The arrangement of the eyes resembles that in Uloborus, but the eyes are farther apart and farther back on the thorax. PI. x1, figs. 2, 2a. Sar The legs are short and thickest in the middle, tapering distinctly — from the patella to the claws. The hind metatarsus bearing the calamistrum is curved inward on the outer side. The claws have three or four teeth and under the claws are a few curved spines, some of which are toothed as in Apeira. Pl. x1, fig. 2e,4,g. The palpal claw has four or five teeth. The mandibles are very small and slightly arched forward near the base. the Family Ciniflonidee. 457 The maxille and libium are like those of Uloborus. (Eloxt, fit 2h3) The spinnerets are long. The cribellum is small and not divided in the middle. The male is about half as large as the female. The abdomen is much smaller and the humps lower. The palpal organ is very large. The tube is long and slender and extends one and a half times around the organ, supported by the edge of a thin appendage. At the tip it lies against two small flexible processes and over them is a large dark horn. The whole apparatus is so large as to cover the patella as well as the tarsus. PI. x1, figs. 2¢, 2d. The epigynum is simple externally, but the inner tubes correspond in length to those of the palpal organs. The colors of both sexes are various shades of brown, covered with white or gray hairs. The markings on the cephalothorax and legs are usually indistinct. The eyes are surrounded by black rings. The humps on the abdomen are usually darker than the rest of the back. Dark markings follow the dorsal vessel and two or three pairs of its branches. Other individuals have the front, and some the whole back of the abdomen very dark brown. The web consists of four rays crossed by a dozen or more threads. The point where the rays meet is attached to a thread which extends to the spider’s roost, usually the end of a twig. Here it holds by the hind feet and draws the thread tight with the fore feet. When an insect strikes the web the spider lets go with the hind feet, the elasticity of the web draws the thread out with a snap, and slides the spider along it toward the web. When it reaches the center it feels the rays to find where the insect is, runs out on the nearest one, covers the prey with silk, and carries it out of the web. The making of this web is fully described by Wilder. Having finished the rays, the spider begins with the cross threads farthest from the center, walking along the upper ray until it is near enough the next to step across, then it crosses and walks outward again on the second ray. The new cross thread is elastic enough to shorten to the proper length when she reaches the point to attach it. When the cross thread is finished to the fourth ray, the spider walks back to the center and out on the upper ray to the point for beginning another. This spider is common all over New England and the Middle States. I have seen cocoons near their webs, like that described by Wilder, but have never traced it to them or any other spider. 458 J. H. Emerton—New England Spiders. EXPLANATION OF PLATES, PLATE IX. Dictyna. Figs. 1, la, 1b, lc, 1d, le. Dorsal markings of different individuals of Dictyna muraria. Figs. 1f, lg, Palpi of male D. muraria. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 2, D. volucripes, sternum and mouth parts: 2a, side of female; 2b, side of male; 2c, front of head and mandible of female; 2d, front of head and man- dible of male; 2e, 2/, palpal organ and male tibia of different individuals. . 3, 3a, palpus of male D. bostoniensis; 3b, 3c, 3d, dorsal markings of D. bostoniensis. Tibia of male palpus of D. longispina. 5a, Male palpus of D. minuta. Palpus of male D. rubra. Dorsal markiugs of D. volupis; 8a, 8b, male palpus of D. volupis; 8c, tibia of male palpus seen from below. 4, 5, . 6, Common dorsal marking of D. cruciata ; 6a, male palpus of D. cruciata. 7, 8, . 9, Dorsal markings of abdomen of D. frondea; 9a, palpus of male of D. frondea. Prats X. Amaurobius. 1, Amaurobius sylvestris, x 4; la, tibia and patella of right palpus of male; 10, tarsus and palpal organ; 1c, epigynum; ld, foot; le, cribellum; If, calamistrum; lg, part of web showing the arrangement of the curled threads. 2, Amaurobius tibialis, epigynum: 2a, upper side of right palpus of male; 26, outer side of the same. 3, Amaurobius ferox, epigynum ; 3a, palpal organ of male; 30, tibia of male palpus, upper side; 3c, the same, outer side. 4, Head of Titaneca americana; 4a, 4b, palpus of male; 4c, palpus of female; 4d, cribellum. 5, Dorsal markings of Titaneca brunnea; 5a, under side of abdomen of the same; 5b, sternum, maxillee and labium; 5c, palpus of female. PLATE XI. 1, Uloborus plumipes, side of female; la, top of cephalothorax and eyes; 1b, side of cephalothorax and mouth parts; Ic, male; 1d, le, palpus of male. 2, Hyptiotes cavatus, x 8, female; 2a, the same, male; 2, calamistrum; 2c, palpus of male; 2d, palpal organ from below; the place of the large termi- nal process is shown by a dotted line; 2e, 7, g, first and second feet; 2h, labium and maxille; 27, 2j, thread of web of Hyptiotus cavatus, showing opposite sides; 2k, Diagram of web. 3, Web of Dictyna volucripes. EN bee. Absorption of Arsenic by the Brain, R. H. Chittenden and Herbert E. Smith, 149-152. Acetate, Lead, 64, 86, 11], 313. Uranyl, 262, 267.. Acid, Arsenic, 65, 90, 112. Boracie, 97. Free, 58. Pepsin-hydrochlorie, 84-107. peptone, 54. Phosphotungstic, 244. proteids, 51. solutions, 143. Albumin, 332, 333, 336, 341, 351, 353, 358. Albumin, Metallic Compounds of, 301- aol, Albumoses, 244, 332-361. Alkaline solutions, 143. Alkaloid salts, 106, 120. Allen, 8. E., Influence of Various Inor- ganic and Alkaloid Salts on the Pro- teolytic Action of Pepsin-hydrochloric Acid, 84-107. Amaurobius, 443, 444, 450, 453, 454. ferox, 451. claustrarius, 451. sylvestris, 451. tibialis, 452. Ammonio uranic citrate, 264, 268, 272. uranous sulphate, 263, 268, 272. Ammonium arsenate, 65, 112. bromide, 153-165. chloride, 101. oxalate, 98. Amphopeptone, 221, 223, 224, 228, 229. Amylolytic action, Influence of Bile on, 134-148. of Diastase of Malt, as modified by various conditions; studied quantita- tively. R. H. Chittenden and Geo. W. Cummins, 44-59. of Saliva, Influence of Salts on, 261-273. of Saliva, Influence of Temperature on, 125-133. of Saliva, Influence of Therapeutic and Toxic Agents on, 60-83. Antimonious oxide, 288. Influence of, on Metabolism, 293— 300, Antimony, Distribution of, in the organs and tissues, 274-292. tartrate, Potassium, 66, 113, 419. Antipeptone, 228, 230, 221, 232, 234, 235, 23%, 239. Antipyrine, 437. Arsenate, Ammonium, 65, 112. Arsenic, Absorption of, by the Brain, 149-152. acid, 65, 90, 112. Arsenious oxide, 64, 89, 111, 418. Atropine sulphate, 74, 121. Barium chloride, 114. Bile, Bile Salts and Bile Acids, Influence of, 134-148. Blake, Joseph A., Influence of Antim- onious Oxide on Metabolism, 293-300. Relative Distribution of Antimony in the organs and tissues of the body, under varying conditions, 275-292. Boracie acid, 97. Borax, 97. Bolton, Perey R., Egg- albumin and Al- bumoses, 332-361. Bromide, Mercuric, 62, 88, 110. Potassium, 71, 104, 119. Influence of, on Metabolism, 153- 165. Brucine sulphate, 75, 121. Carbohydrates, 186. Carbonate, Sodium, 45, 47, 49. Casein and its Primary Cleavage Pro- ducts. R. H. Chittenden and H. M. Painter, 362-405. Caseoses, 379, 401. Chittenden, R. H., Absorption of Arse- nic by the Brain, 149-152. Amylolytic action of Diastase of Malt, as modified by various condi- tions; studied quantitatively, 44-59. Casein and its Primary Cleavage Products, 362-405, Dehydration of Glucose in the Stomach and Intestine, 252-259. Egg-albumin and Albumoses, 332- 361. Globulin and Globulose Bodies, 206- 460 Chittenden, R. H., Influence of Anti- monious Oxide on Metabolism, 293- 300. of Bile, Bile Salts and Bile Acids on Amylolytic and Proteolytic Action, 134-148. of Certain Therapeutic and Toxic Agents on the amylolytic action of Saliva, 60-83. of Cinchonidine Sulphate on Meta- bolism, 166-178. of Potassium and Ammonium Bro- mides on Metabolism, 153-165. of Some Organic and Inorganic Sub- stances on Gas Metabolism, 407-442. of Temperature on the Relative Amylolytic Action of Saliva and the Diastase of Malt, 125-133. of Uranium Salts on the Amylolytic Action of Saliva and the Proteolytic Action on Pepsin and Trypsin, 261— 273. of Various Inorganic and Alkaloid Salts on the Proteolytic Action of Pepsin-hydrochloric Acid, 84-107. of Various Therapeutic and Toxic _ Substances on the Proteolytic Action of the Pancreatic Ferment, 108-124. Relatiye Distribution of Antimony in the organs and tissues of the body, under varying conditions, 275-292. On Some Metallic Compounds of Albumin and Myosin, 301-331. Peptones, 220-251. Post-mortem Formation of Sugar in the Liver, in the presence of Peptones, 179-206. Chlorate, Potassium, 70, 96, 117. Chloride, Ammonium, 101. _ Barium, 114. Ferric, 68, 92, 113. Manganous, 91, 114. Mercurie, 62, 79, 86, 109. Potassium, 101, 118. Sodium, 71, 100, 118. Stannous, 67, 89, 111. Cinchonidine sulphate, 73, 122, 436. Influence of, on Metabolism, 166- 178. INDEX. Culbert, W. L., Influence of Potassium and Ammonium Bromides on Meta- bolism, 153-165. Cummins, Geo. W., Amylolytic action of Diastase of Malt, as modified by vari- ous conditions; studied quantitatively, 44-59. Influence of Bile, Bile Salts and Bile Acids on Amylolytic and Proteo- lytic Action, 134-148. Influence of Some Organic and In- organic Substances on Gas Metabo- lism, 407-442. Influence of Various Therapeutic and Toxic Substances on the Proteo- lytic Action of the Pancreatic Fer- ment, 108—124.: Cupric sulphate, 63, 80, 85, 111, 418. Cyanide, Mercuric, 62, 88, 110. Potassium, 69, 95, 115. Cyllopodia cavata, 456. DeForest, K. L., On the Law of Error in Target-Shooting, 1-8. Dehydration of Glucose in the Stomach - and Intestines. R. H. Chittenden, 252-259. Deuteroalbumose, 345, 353. ‘ Deuterocaseose, 385, 390, 396. Deuteroglobulose, 213. Diastase of Malt, Amylolytic action, 44- 59, 125-133. Dichromate, Potassium, 95, 115. Dictyna, 444, 453, 454. arundnaceoides, 444, 445. bostoniensis, 447. -cruciata, 448. foliata, 444. frondea, 449. longispina, 446. minuta, 447. muraria, 445, 447. rubra, 448. sedentaria, 444. vittata, 444. volucripes, 444, 445, 446. volupis, 444, 448, 449, 450. Dictynidee, 443. Dysalbumose, 355, Cinchonine sulphate, 73, 122. Ciniflonidee, 443-458. Citrate, Uranic, 264, 268, 372. Cobalt compounds, 328. Compounds, Cobalt, 328. Hee-albumin, 302. Hgg-albumin and Albumoses, R. H. Chittenden and Percy R. Bolton, 332- 361. Copper, 302, 323. Tron, 314, 326. Lead, 312. Mercury, 317, 330. Nickel, 328. Silver, 318. Uranium, 316, 329. Emerton, J. H., New England Spiders of the Family Ciniflonidee, 443-458. Kpeira, 445, 455, 456. Kpeiridee, 443, 444, 445, 454, Ergatis (Dictyna) diligens, var. annuli- pes, 444. Extensions of certain Theorems of Clif- Zine, 316, 327. Copper Compounds, 302, 323. Cribellata, 443. ford and of Cayley in the Geometry of n Dimensions. LEliakim Hastings Moore, Jr., 9-26. a INDEX. Ferric chloride, 68, 92, 113. Ferricyanide, Potassium, 70, 116. Ferrocyanide, Potassium, 69, 95, 116. Ferrous sulphate, 68, 92, 113. Fibrin, 102, 226. Gases, Influence of, 77, 119. Gas Metabolism, Influence of some Sub- stances on, 406-442. Gastric juice, 223. Gland peptone, 235, 237, 239. Globulin, 208, 210. Globulin and Globulose Bodies. W. Kiihne and R. H. Chittenden, 206- 219. Globulose Bodies, 212. Glucose, Dehydration of, 252-259. Glycogen, 184, 185. Heteroalbumose, 347. Keterocaseose, 400. . Heteroglobulose, 215. Hutchinson, M. T., Influence of Ura- nium Salts on the Amylolytic Action of Saliva, the Proteolytic Action on Pepsin and Trypsin, 261-273. Hyptiotes, 454, 455, 456. Americanus, 444. cavatus, 444, 456. Influence of Antimonious Oxide on Met- abolism. R. H. Chittenden and Jo- seph A. Blake, 293-300. Bile, Bile Salts and Bile Acids on Amylolytic and Proteolytic Action. R. H. Chittenden and Geo. W. Cum- mins, 134-148. Certain Therapeutic and Toxic Agents on the Amylolytic Action of Saliva. R. H. Chittenden and H. M. Painter, 60-83. Cinchonidine Sulphate on Metabol- ism. R. H. Chittenden and Henry H. Whitehouse, 166-178. Potassium and Ammonium Bro- mides on Metabolism. R. H. Chit- tenden and W. L. Culbert, 153-165. Some Organic and Inorganic Sub- stances on Gas Metabolism. R. H. Chittenden and G. W. Cummins, 407-442, Temperature on the Relative Amy- lolytic Action of Saliva and the Dias- tase of Malt. R. H. Chittenden and W. #. Martin, 125-133. Uranium Salts on the Amylolytic Action of Saliva and the Proteolytic Action on Pepsin and Trypsin. R. H. Chittenden and M. T. Hutchinson, 261-273. Various Inorganic and Alkaloid Salts on the Proteolytic Action of Pepsin-hydrochloric Acid. R. H. Chittenden and 8. EK. Allen, 84-107. 461 Influence of Various Therapeutic and Toxic Substances on the Proteolytic Action of the Pancreatic Ferment. R. H. Chittenden and Geo. W. Cum- mins, 108-124. Iodide, Mercurie, 62, 88, 110. Potassium, 71, 104, 119. Tron compounds, 314, 326. 223 wav. Juice, gastric, pancreatic, 230. Knots, with a Census for Order Ten. C. N. Little, 27-43. Kitihne, W., Globulin Bodies, 206-219. Peptones, 220-251. and Globulose Lambert, Alexander, Post-mortem For- mation of Sugar in the Liver, in the presence of Peptones, 179-206. Law of Error in Target-Shooting. E. L. DeForest, 1-8. Lead acetate, 64, 86, 111, 313. compounds, 312. Little, C. N., On Knots, with a Census for Order Ten, 27-43. Malt, Diastase of, 44-57, 125-133. Magnesium sulphate, 69, 94, 114. Manganous chloride, 91, 114. Martin, W. K., Influence of Tempera- ture on the Relative Amylolytic Ac- tion of Saliva and the Diastase of Malt, 125-123. Mercurie bromide, 62, 88, 110. chloride, 62, 79, 86, 109. cyanide, 62, 88, 110. iodide, 62, 88, 110. Mercury compounds, 317, 330. Metabolism, Influence of Bromides on, 153-165. Influence of Antimonious Oxide on, 293-300. Influence of Cinchonidine Sulphate on, 166-178. Metallic Compounds of Albumin and — Myosin. R.H. Chittenden and Henry H. Whitehouse, 301-331. Metallic Salts, 78. Mithras, 456. Moore, Jr., Eliakim Hastings, Extension of certain Theorems of Clifford and of Cayley in the Geometry of » Dimen- sions, 9-26. Morphine sulphate, 72, 120, 425. Myosin, Metallic compounds of, 301-331. Narcotine sulphate, 121. Neutral peptone, 48. solutions, 143. New England Spiders of the Family Ciniflonidee, 443-458, 462 INDEX. Nickel compounds, 328. Nitrate, Potassium, 70, 96, 117. Uranyl, 262, 267, 410. Organic and Inorganic Substances, in- fluence of, on Gas Metabolism, 407- 449. Oxalate, Ammonium, 98. Oxide, Antimonious, 288, 293-300. Arsenious, 64, 89, 111, 418. Oxychloride, uranic, 264, 269, 272. Painter, H. M., Casein and its Primary Cleavage Products, 362-405. Influence of certain Therapeutic and Toxic Agents on the Amylolytic Action of Saliva, 60-83. Pancreatic Ferment, Proteolytic Action of, 108-124. juice, 230. Pepsine, 140, 224, 261-273. Pepsin-hydrochloric acid, Influence on, 265. Proteolytic Action of, 84-107. Peptone, Acid, 54. Gland, 235, 237, 239. Neutral, 48. Peptones, 179-206. W. Kiuhne and R. H. Chittenden, 220-251. Permanganate, Potassium, 68, 94, 115. Phillyra, 454. ripaira, 454, Phosphotungstie acid, 244. Post-mortem Formation of Sugar in the Liver, in the presence of Peptones. R. H. Chittenden and Alexander Lam- bert, 179-206. Potassio uranic oxychloride, 264, 269, 272. Potassium antimony tartrate, 66, 113, 419. bromide, 71, 104, 119, 153-165. chlorate, 70, 96, 117. chloride, 101, 118. cyanide, 69, 95, 115. dichromate, 95, 115. ferricyanide, 70, 116. ferrocyanide, 69, 95, 116. iodide, 71, 104, 119. nitrate, 70, 96, 117. permanganate, 68, 94, 115. Proteids, Acid, 51. Proteolytic action, Influence of Bile on, 134-148. of Pancreatic Ferment, Influence of various Substances on, 108-124. of Pepsin, Influence of Salts on, 261-273. of Pepsin-hydrochlorie Acid, Influ- ence of various Salts on, 84-107. Protoalbumose, 342, 351. Protocaseose, 381, 384, 387, 393. Protoglobulose, 212. Quinine sulphate, 72, 122, 428. Relative Distribution of Antimony in the organs and tissues of the body under varying conditions. R. H. Chitten- den and Joseph A, Blake, 275-292. Saliva, Amylolytic Action of, 60-83, 125-133, 261-273. Salts, Alkaloid, 106, 120. Influence of various, 84-107. Nature of the action of, 78. Uranium, Influence of, on Ferment Action, 261-273. Silver compounds, 318. Smith, Herbert E_, Absorption of Arsenic by the Brain, 149--152. Sodio uranic sulphate, 264, 268, 272. Sodium carbonate, 45, 47, 49. chloride, 71, 100, 118. sulphate, 117. tetraborate, 71, 97, 116. Solutions, Acid, 143. Alkaline 143. Nentral, 143. Stannous chloride, 67, 89, 111. Steatoda borealis, 453. Strychnine sulphate, 75, 121. Sugar in the Liver, Post-mortem For- mation of, 179-206. Sulphate, Atropine, 74, 121. Brucine, 75, 121. Cinchonidine, 73, 122, 166-178, 436. Cinchonine, 73, 122. Cupric, 63, 80, 85, 111, 418. Ferrous, 68, 92, 113. Magnesium, 69, 94, 114. Morphine, 72, 120, 425. Narcotine, 121. Quinine, 72, 122, 428. Sodium, 117. Strychnine, 75, 121. Uranic, 264, 268, 272. Uranous, 263, 268, 272. Urany], 268, 271. Zine, 67, 80, 91, 114. Target-shooting, Law of Error in, HE. L. DeForest, 1-8. Tartar emetic, 281, 282, 284, 288. Tartrate, Potassium antimony, 66, 113, 419. Tegenaria, 450. Tetraborate, Sodium, 71, 97, 116. Tetragnatha, 454. Theorems of Clifford and Cayley, 9-26. Therapeutic and Toxic Agents, Influence of, on Saliva, 60-83. Substances, Influence of, on Pan- creatic Ferment, 108-124. Therididze, 443. Theridion, 444. foliaceum, 444, 450. morologum, 444. : : INDEX. Theridion, sublatum, 444. Titanceca, 453. americana, 453, 454. brunnea, 453. quadriguttata, 453. Trypsin, 119, 120, 143, 147. Influence of Salts on, 261-273. Uloboridze, 443. Uloborinz, 443, 454. Uloborus, 454, 456, 457. plumipes, 454. walckeneerii, 456. Uranic citrate, Ammonio, 264, 268, 272. oxychloride, Potassio, 264, 269, 272. sulphate, Sodio, 264, 268, 272. Uranium compounds, 316, 329. Salts, Influence of, on Ferment Ac- tion, 261-273. 463 Uranous sulphate, Ammonio, 263, 268, 272. Uranyl acetate, 263, 267. nitrate, 262, 267, 410. sulphate, 268, 271. Veleda, 454. Whitehouse, Henry H., Influence of Cinchonidine Sulphate on Metabolism, 166-178. On Some Metallic Compounds of Albumin and Myosin, 301-331. Zine compounds, 316, 327. sulphate, 67, 80, 91, 114. et wiki et irda Plate VIII. Trans. Conn. Acad., Vol. VII. ‘SNINWOD GNV TT NOILVYIdSayY A 5 LLL) ‘ a % “one Mt am | l a | ee im mn — ay tii: . 1 y 7 ) r . ; i ¥ ‘ - ' . ~ * 5 “ c / i . \ j t . : . . , - - * ' ; e 2 : : : ~ ‘ ‘ ih 5 > i 7 . » ‘ . : . i 4 a . ‘ + - . Aj . A a . ; ‘ : : ‘ 6 > > + ‘ P: “a > - £ 2 - - s = f ta ry - ° ; . - ‘ x « . * . ¥ * - ' be ; E 1 ¢ * 7 . : * \ * p ¥, 4 p . ; Toe ; 5 re = 4 , . Shin Trans. Conn. Acad .VoL.VIL PLATE IX to Ide” wy ve Va f 7 If fo dip ey Ls ‘\ , i yy, ad 4] | be Wl | Mi) I . 0% * het w i on aS 4 \ if } i Ha) (WI ars THEmerton fromnature. LS.Punderson &Son,Photo-lith New Haven DICTYNA. es . COS Tre ¥ ¥ es 4 a ; A ? Trans.Conn Acad Vol. VI. underson &Son,Photo-lith New Haven. aS) JHEmerton from nature. ce me TAN clits Gy Ee haloes TIT AR COROA Trans.Gonn.Acad.Vol. VIL. i. cw” pe Man, “ TE m i fl ANN My) ti WYP. nN} 2) 3 y KES WET Ll Ae 1 h;' Y i | q 14 = Witenes ih Au K a SVE fs Sy) JHEmerton fromnature. LS.Punderson &Son,Photo Iaith New Haven. WhO ROR LTS —HyPTrOTRS: LG ay ‘y La at x) ake" ah mt