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BiociiBMicAL Researches

COLLECTED REPRINTS OF PUBLICATIONS

FROM THK

LABORATORY OF PHYSIOLOGICAL CHEMISTRY
OF COLUMBIA I'NIVERSITY

TOGETHER WITH

CONTRIBUTIONS FKnM SIMILAR I .\H( )KA Ti )RIi:S
IN OTHER INSriTLTloNS

W ILL! AM J. GILS

AND COLLABORATORS

VOLUME I

EDITED AND ISSUED BY WILLIAM J. GIES
Columbia University

July 31, 1903

GIFT

Co t\)t iflcn of

1901, 1902, 1903, 1904 AND 1905

AT

THE COLLEGE OF PHYSICIANS AND SURGEONS
THIS VOLUME,

TO WHICH SOME OF THEM HAVE MATERIALLY CONTRIBUTED,
IS INSCRIBED

IN LIVELY REMEMBRANCE OF THEIR UNFAILING
COURTESY AND MANY KINDNESSES,

AND WITH THE

REGARDS AND BEST WISHES

OF

THE AUTHOR

Digitized by tine Internet Arciiive

in 2010 witii funding from

Open Knowledge Commons

http://www.archive.org/details/biochemicalreseaOOgies

PREFACE.

This volume contains reprints of all the research papers, and
of all published abstracts of preliminary reports of investigations,
that have been issued, to date, from this laboratory since the estab-
lishment of the department in the academic year of 1898-99.
The volume also contains reprints of the several research papers
by the writer, and by the writer in collaboration with his teachers,
which have been published from other laboratories.

It has been my purpose to bring together the results of all
the investigations in which I have personally engaged. This
volume is also intended as the first of a series of biochemical
studies to be issued from this laboratory, from time to time, as
the results of our work may determine.

The investigations thus far concluded in this laboratory should
be judged in the light of the special conditions under which they
were conducted. During the first two years of the department's
history the routine work connected with its equipment, and with
the organization of laboratory instruction of large classes of med-
ical students, made it impossible for us to give more than occa-
sional attention to research. At the beginning of the second
year the writer started, in addition, two laboratory courses in ad-
vanced physiological chemistry. In the fourth year a laboratory
course in the physiological chemistiy of plants was added to
those offered in this department. During the past year the writer
has also cooperated with Professor F. S. Lee in giving an under-
graduate course in physiology, and has given laboratory instruc-
tion and assisted in directing chemical research at the New York
Botanical Garden.

The exactions of our routine work and administrative affairs
may be estimated from the figures on the next page for the total
number of students at this University who have received laboratory
instruction in physiological chemistry for not less than six hours
weekly, during a period of at least one half-year, under the writer's
constant oversight and personal direction :

5

6 Preface.

Academic \'ear. Number of Students.

1 898-1899 159

1 899-1900 177

1 900-1901 231

I9OI-1902 212

I902-1903 223

Total, 1,002

The courses lately offered in this department of the University,
and which will be given in 1903- 1904. are indicated in the follow-
ing abbreviated statements taken from the "Announcement of the
Division of Biology," issued May 23 :

1. General Physiological Chemistry. — This course is given twice during the year
and is required in the second year of all candidates for the degree of M.D. The student
is required to attend each week one lecture (i hour), one conference and recitation (i
hour), and three laboratory exercises, including frequent demonstrations (2 hours each).

Lecture. Weekly (entire class) I hour. Professor Gies.
Conference and recitation. Weekly (each section) I hour. Professor Gies.
Laboratory exercises, including frequent demonstrations. Three per week (each
section) 2 hours each. Professor Gies and Drs. Richards and Hawk.*

2. Laboratory Course in Advanced Physiological Chemistry, Including a Study
of Clinical Methods. — This course is a continuation of Course i , but gives more de-
tailed instruction in the various subjects belonging to physiological chemistrj' than the
time for Course i will allow. 6 hours. Professor Gies and Dr. Richards.

3. Laboratory Course in Special Physiological Chemistry. — This course is ar-
ranged for students who wish to make a very thorough study of the science. 12 hours.
Professor Gies.

4. Physiological Chemistry of Plants, Including a Study of Laboratory Methods. —
This course is arranged for the benefit of students of botany and of materia medica.
The course may be taken in whole or in part at the New York Botanical Garden, where
Professor Gies is Consulting Chemist. 6 hours. Professor Gies.

Courses in Physiology given with the cooperation of this department, quoting from
the same " Announcement : "

4. Laboratory Course in Special Physiology. — Given with the cooperation of the
Department of Physiological Chemistry. 3 hours. Professors Curtis, Lee and Gies,
and assistants.

6. Elementary Physiology. — Given at Columbia College with the cooperation of
the Department of Physiological Chemistry. One hour lecture, and two hours labora-
tory work. Professors Lee and Gies and Dr. Burton-Opitz.

Much of the work of investigation in this department has been
conducted by the writer with the aid of students of medicine who
have been particularly interested in physiological chemistry, but who
have had little time for special work in the subject. The character
and extent of these researches in collaboration has been deter-
mined largely by the little time remaining from that given to class

* Since the above announcement was first published Dr. Hawk resigned his
position in this department to accept that of Demonstrator of Physiological Chemistry
at the University of Pennsylvania.

Preface. 7

instruction and has depended, also, on the hmited biochemical
training and preparation of the students referred to. The writer
has given much of his time and energy to the encouragement of
the spirit of research among these men, but only such relatively
simple investigations as it was possible to conduct to advantage
during short periods, at irregular intervals and also at night, could
be undertaken with them.

My name always follows those of my associates under the
titles of the papers and reports which have been published by me,
from this laboratory, in collaboration with medical students and as-
sistants. This has occurred so regularly that it may be easily mis-
interpreted. My chief purpose in following this course, instead of
doing as present customs permit, has been to specially emphasize
the large share of credit due to those who assisted me in the prac-
tical work of analysis and experiment. Although I have encour-
aged my associates to engage in these researches with me, I have
not suggested to them, at the conclusion of our work, that they
agree to a plan of publication which might possibly magnify at
their expense my own share in the investigations. My name is
associated with another under the titles of only such of the papers
from this laboratory as resulted from researches which were strictly
cooperative and in which I myself did a large share of the actual
labor of experiment. As far as the composition of the papers is
concerned — I alone am responsible for their defects.

During the first five years of ihe department's history, Pro-
fessor R. H. Chittenden, Director of the Sheffield Scientific School,
Professor of Physiological Chemistry in the Sheffield Scientific
School and Professor of Physiology in the Yale Medical School,
was its official, non-resident Director. Professor Chittenden vis-
ited the department for several hours once a week, from October
to May, and on those occasions gave a lecture in physiological
chemistry to the class of second-year medical students. The de-
partment was organized, for the laboratory instruction of these
students, under Professor Chittenden's supervision, and with the
advice and guidance of Professor John G. Curtis of the Department
of Physiology,

The direction and stimulation of the research work in this de-
partment has devolved upon the writer from the beginning. With

8 Preface.

the few exceptions referred to below, all investigations published
from the laboratory were carried out by the writer himself or were
conducted b\' him in collaboration with others.

The following researches in this laboratory were carried out as
indicated below :

I. Preliminary Reports.

Under Professor R. H. Chittenden' s direetion.

ii.* The proportion of basic nitrogen yielded by elastin on
decomposition with hydrochloric acid. By R. H.
Chittenden (for Allan C. Eustis).
At the suggestion and with the cooperation of Dr. Eugene Hodenpyl.
ff. Report of a chemical examination of a knife-grinder's
lung. By Eugene Hodenpyl, assisted by Allan C.
Eustis and A. N. Richards.
Independe)itly by Dr. A. N. Ric/iards, Research Scholar of the Rocke-
feller Institute, and Mr. Charles H. Vosburgh.
ee. A modified Eck fistula, with a note on adrenalin glycaemia.

II. Papers.

hidependently by Dr. P. A. Levene.

12. On the nucleoproteid of the brain (cerebronucleopro-
teid).t
i, 15. Enibr}'ochemical studies. I. Some chemical changes
in the developing &^'g.
Under the direetion of, or done chiefly by, Dr. S. J. Meltzer.

17. On the influence of the contents of the large intestine

upon strychnine. By William Salant, Fellow of the
Rockefeller Institute.

18. A further study of the influence of the contents of the

large intestine upon strychnine. By William Salant,
Fellow of the Rockefeller Institute.
24. Studies on the influence of artificial respiration upon
strychnine spasms and respiratory movements. By
William J. Gies and S. J. Meltzer.

*The letters and numerals before this and the succeeding titles correspond with
those before the same titles on pages 25-28.

f At the conclusion of the paper, Dr. Levene acknowledges his indebtedness to
ofessor Chittenden for suggestions while the work was in progress.

Preface. 9

Under the direction of, or at the suggestion and ivitJi the counsel of,
Professor C. A. Herter.

23. Note on the glycosuria following experimental injections
of adrenalin. By C. A. Herter and A. N. Richards,
Research Scholar of the Rockefeller Institute.
25. An experimental study of the sugar content and extra-
vascular coagulation of the blood after administration
of adrenalin. By Charles H. Vosburgh and A. N.
Richards, Research Scholar of the Rockefeller In-
stitute.
The researches which were conducted in other laboratories by
the writer, under the guidance of or in collaboration with his
teachers, are Hsted in Section III of the Bibliography, page 20.
The paper by Dr. Meltzer and myself (24), also belongs to this
group in the bibliographical list, even though it was issued from
this laboratory. On the opening page of the paper issued from
this laboratory by Lesem and Gies (11), acknowledgment is made
of the fact that the research was begun by the writer, at Yale, at
Professor Chittenden's suggestion.

The general results of all the investigations may be quickly
ascertained from the index, pages 733-746, where only the data
of our own researches are classified.

The statements already made here, on a few matters of depart-
mental history relating to our researches, would be very incom-
plete without additional reference to the important parts taken in
the work of this department, from its beginning, by my colleague
Dr. A. N. Richards and by our worthy laboratory helper, Mr.
Christian Seifert. I have had help from each of them in connec-
tion with many of the details of organization, laboratory instruc-
tion and research, whenever aid was needed or desired. What-
ever success may have been attained in the work of this department
of the University has been due, in large part, to the enthusiastic,
painstaking and efficient cooperation given at all times by Dr.
Richards and Mr. Seifert. It is a pleasure to make permanent
record of the fact.

William J. Gies.

Departm:e:nt of Physiological Chemistry of Columi-ia University,
College of Physicians and Surgeons,
July 31, 1903.

CONTENTS.

FAtiE

List of Ii,lustbations lo

Bibliography ii

Divisions II

Complete list of reports and publications 13

List of reprints of papers and of abstracts of reports, in the order of

their arrangement in this voi.imf. 23

List of abstracts 25

List of papers 26

Reprints of Abstracts ok Reports 29

Names of Societies before which the reports were made 29

Abstracts 31

Abstracts of reports which have l)een followed by publications giving the

results in detail, a-r 31

Abstracts of reports of researches which have not yet been published in

greater detail, aa-mm 52

Reprints of papers 65

Titles of journals in which the papers were published 65

Papers Reprints.

A. Chemical investigations of animal tissues and tissue constituents Nos. I-15

B. Pathological and toxicological Nos. 16-28

C. M iscellaneous researches Nos. 29-35

D. Botanical studies Nos. 36-40

Index Page 733

LIST OF ILLUSTRATIONS.

No. OP
Repkint
Atwater-Blakeslee bomb calorimeter and accessory apparatus as arranged for com-
bustions 3

Vosburgh -Richards cannula 25

Erythrocytes in the blood of a patient with simple anemia. Figures i-io 27

Chart showing weekly changes in the blood of a patient with simple anemia.

Figure II 27

Erythrocytes in the blood of a patient with pernicious anemia. Figure 12 27

Patient with pancreatic cyst 28

Figure I. Shows size of cyst and its relation to adjacent parts.
Figure 2. Profile view.

Cocoanut illustrations 36

Figure I. Inflorescence.

Figure 2. Half-grown cocoanut fruit.

Figure 3. Ripe cocoanut (parts).

Figure 4. Crystals of cocoa edestin.

Figure 5. Germinating cocoanut.

" Plate 19." Sections of the germinating cocoanut and the cotyledon.

10

BIBLIOGRAPHY.

Page

I. Publications from the Laboratory of Physiological Chem-
istry, of Columbia University, at the College of Physi-
cians and Surgeons : 1900- 1903 (I-XLVIII) 13

II, Reports and papers on investigations in which all or nearly
all of the clieiiiical work was done in the Laboratory
of Physiological Chemistry, of Columbia University, at
the College of Physicians and Surgeons: 1899-1903

(XLIX-LXVI) 18

III. Reports and papers on researches carried ojat in various
laboratories by William J. Gies under the guidance
of, or in collaboration with, his teachers: 1896-1903
(LXVII-LXXXII) 20

II

BIBLIOGRAPHY.

[The titles of publications under each head are arranged in chronological order.]

I. Publications from the Laboratory of Physiological Chemistry, of Columbia

University, at the College of Physicians and Surgeons.*

1 900-1 903. I-XLVIII.

1900.

Abstracts of Preliminary Reports of Researches.

I. William J. Gies. The preparation of a mucin-like sub-
stance from bone. Proceedings of the American Physio-
logical Society, New Haven, December, 1899. American
Journal of Physiology, March; iii, p. vii.
II. A. N. Richards and William J. Gies.f A prehminary
study of the coagulable proteids of connective tissues.
Ibid., p. V.

III. William D. Cutter and William J. Gies. The gluco-

proteids of white fibrous connective tissue. Ibid., p. vi.

IV. L. D. Mead and William J. Gies. The physiological ac-

tion of tellurium compounds. Ibid. , p. xx.
V. R. H. Chittenden (for Allan C. Eustis). The proportion
of basic nitrogen yielded by elastin on decomposition
with hydrochloric acid. Ibid.,-^. xxxi.
VI. William J. Gies. Notes on the constituents of ligament
and tendon. Proceedings of the American Association
for the Advancement of Science, New York, June, 1900.
Permanent Secretary'' s Report of the Proceedings. De-
cember, p. 123.

* The department was established in 1898-1899. Research could not be effectively
started until 1899. The first reports of researches were made late in 1899, but no ab-
stracts of these reports were published before 1900. The first publications of completed
researches appeared in 1901. A list of publications of investigations carried out in
part in this laboratory, in part in other laboratories, is given on page 18.

f The names of the authors of this and the two succeeding abstracts were trans-
posed under the titles in the " Proceedings," by the Editor of the Journal. This was
done in conformity with the custom of the Journal of giving precedence to the name of
the author presenting the report. The names are here placed in the order in which the
writer preferred them. See preface, page 7.

13

14 Bibliography.

VII.* William J. Gies. New methods for the separation of
some constituents of ossein. Ibid., p. 131.

1901.

A. Abstracts of Preliminary Reports of Researches.

VIII. G. A. Fried and William J. Gies. Does muscle contain
mucin ? Proceedings of the American Physiological So-
ciety, Baltimore, December, 1900. American Journal
of Physiology, March; y, p. x.
IX. A. N. Richards and William J. Gies. Methods of pre-
paring elastin, with some facts regarding ligament
mucin. Ibid., p. xi.
X. P. B. Hawk and William J. Gies.f A further study of the
glucoproteid in bone. Ibid., p. xv.

B. Papers on our own Investkiations.
XI. L. D. Mead and William J. Gies. Physiological and
toxicological effects of tellurium compounds, with a
special study of their influence on nutrition. American
Journal of Physiology, March; v, p. 104.
XII. A. F. Chace and William J. Gies. Some facts regarding
"ureine." Medical Record, March 2; lix, p. 329.

XIII. William J. Gies. The toxicology of tellurium compounds,

with some notes on the therapeutic value of tellurates.
Philadelphia Medical Journal, March 23 ; Yii, p. 566.

XIV. William J. Gies. A note on the excretion of kynurenic

acid. Americati Journal of Physiology, April; v, p. 191.
XV. William J. Gies. An improved method of preparing and
preserving meat for use in metabolism experiments.
At7ierican Journal of Physiology, May; v, p. 235.
XVI. G. W. Vandegrift and William J. Gies. The composition
of yellow fibrous connective tissue. American Journal
of Physiology, June; v, p. 287.
XVII. P. B. Hawk and William J. Gies.f Chemical studies of
osseomucoid, w'ith determinations of the heat of combus-
tion of some connective tissue glucoproteids. American
Journal of Physiology, July; v, p. 387.

* Reported in person by the author, but, by an oversight, the abstract was omitted
by the Secretary and only the title given in the Proceedings. See page 32.

t Most of the elementary analysis and the determination of the heat of combustion,
comprising Dr. Hawk's share of the work, was done during the summer vacation of
1900, in Professor Atwater's laboratory at Wesleyan University.

Bibliography. 15

XVIII. W. D. Cutter and William J. Gies. The composition ot

tendon mucoid. American Journal of Physiology, No-
vember ; vi, p. 155.
XIX. William J. Gies. A new constituent of bone. Americmi

Medicine, November 23 ; ii, p. 820.
XX. Leo Buerger and William J. Gies. The chemical con-
stituents of tendinous tissue. American Journal of
Physiology, December; vi, p. 219.

C. Miscellaneous Publications.

XXL* A. N. Richards. Report of the Proceedings of the Ameri-
can Physiological Society, Baltimore meeting, Decem-
ber, 1900. Bosto7i Medical and Su7'gical Journal, cxliv.
First paper, January 24; p. 91. Second paper, January
31 ; p. 116.
XXII. * J. E. Kirkwood and William J. Gies. Composition of the
body (plant). MacDougaP s Practical Text-Book of
Plant Physiology, Longmans, Green & Co. Composing
Chapter ix, pp. 147-174.

XXIII. William J. Gies. Animal coloring matters. Buck' s Refer-

ence Handbook of the Medical Sciences, William Wood &
Co. Vol. iii, pp. 223-225.

1902.

A. Abstracts of Preliminary Reports of Researches.

XXIV. p. B. Hawk and William J. Gies. The composition and

chemical qualities of the albumoid in bone. Proceed-
ings of the American Physiological Society, Chicago,
December, 1901. American Journal of Physiology,
March ; vi, p. xxvii.
XXV. L. D. Mead and William J. Gies. A comparative study

of the reactions of various mucoids. Ibid., p. xxviii.
XXVI. E. R. Posner and William J. Gies. Are proteids which
are prepared by the usual methods combined with fat or
fatty acid? Ibid., p. xxix.
XXVII. I. 0. Woodruff and William J. Gies. On the toxicology
of selenium and its compounds. Ibid., p. xxix.

B. Papers on our own Investigations.
XXVIII. H. 0. Mosenthal and William J. Gies. Proteosuria.
American Medicine, March 8 ; iii, p. 387.
* Not reprinted.

1 6 BlIU.lOC.KAIMIV.

XXIX. A. N. Richards and William J. Gies. Chemical studies
ol clastin, mucoid and other protcids in elastic tissue,
•with some notes on hgament extractives. American
Journal of Physiology, April; vii, p. 93.
XXX. William J. Gies. Chemical changes in the body in which
the methyl group may be involved. Therapeutic Monthly,
April : ii. ]). 144.
XXXI. E. R. Posner and William J. Gies. Experiments to deter-
mine the possible admixture or combination of fat or
fatty acid with various proteid products. American
Journal of Physiology, J'^'lv : ^'i'? P- 33 1-
XXXII. p. B. Hawk and William J. Gies. On the composition
and chemical properties of osseoalbumoid, with a com-
parative study of the albumoid of cartilage. American
Journal of Physiology, July; vii, p. 340.
XX.XIII. P. B. Hawk and William J. Gies. On the quantitative
determination of acidalbumin in digestive mixtures.
American Journal of Physiology, September; vii, p. 460.

1903. January- July.
A. Abstr.acts of Preliminary Reports of Researches.
XXXIV. William J. Gies. Further mucoid studies. Proceedings of
the American Physiological Society, Washington, Decem-
ber, 1902. American Journal of Physiology, February;
viii, p. xiii.
XXXV. William J. Gies. A proteid reaction involving the use of

( hromate. Ibid., p. xv.
XXX\'I. William J. Gies. The influence of the H ion in peptic
proteolysis. Ihid., \>. xxxiv.
XXXVII. -^ S. J. Meltzer and William J. Gies. Studies on the in-
fluence of artificial respiration u])on strychnine spasms
and rfsi)iratory movements. Ibid., p. xlii.
XXXVIII. William J. Gies. An improved cage for metabolism ex-
periments. Proceedings of the Society for Experimental
Biology and Medicine, February. Science, March 20 ;
xvii, p. 469. American Medicine, May 2 ; v, p. 708.
XXXIX. William J. Gies. Properties of "Pence Jones' body."
find.

*The report was not abstracted. The paper was published in the succeeding
number of the Journal, i-ee first footnote on page 17.

Bibliography. 17

XL. A. N. Richards. A modified Eck fistula, with a note on
adrenalin gl}xaemia. American Medicine, May 2 ; v,
p. 709. Science, May 8 ; xvii, p. 742.
XLI. W. A. Taltavall and William J. Gies. The influence of
chinic acid on the elimination of uric acid. Proceed-
ings of the American Physiological Society, JVashington,
May, 1903. American Journal of Physiology , July; ix,
p. xvi.
XLII. William J. Gies. Peptic proteolysis in acid solutions of
equal conductivity. Ibid., p. xvii.

B. Papers on our own Investigations.
XLIII. Gordon Lindsay and William J. Gies. Some notes on
Pollacci's new method of detecting albumin in the
urine. American Medicine, January 31 ; v, p.

175-

XLIV. William J. Gies. Chemical studies of the pitcher plant,
Sarracenia purpurea. Journal of the New York Bo-
■ tanical Garden, February; iv, p. 37.
XLV.* William J. Gies and S.J. Meltzer. Studies on the influ-
ence of artificial respiration upon strychnine spasms and
respiratory movements. American Joiirnal of Physiology ,
March ; ix, p. i.

XLVI. Charles H. Vosburgh and A. N. Richards. An experi-
mental study of the sugar content and extravascular
coagulation of the blood after administration of
adrenalin. Ame7'ican Journal of Physiology, W-dsoh; ix,
P- 35-

C. Miscellaneous Publications.

XLVII.f William J. Gies. On the normal occurrence of arsenic in
organisms. Letter to the President of the Medico-Legal
6'(9«V/}', New York, February 17. Medico-Legal Journal
March ; xx, p. 541.
XLVIII. f William J. Gies. Proceedings of the Society for Experi-
mental Biology and Medicine. SecretaiJ s Rep07-ts.
Science, March 20 ; xvii, p. 468 : May 8 ; xvii, p. 741.
Also Americaji Medicine, May 2 ; v, p. 707.

*This paper is also included with those listed in Section III. See page 22.
f Not reprinted.

1 8 BlBLIOGKAI'UV.

II. Reports and papers on investigations in which all or nearly all of ihe chem-
ical work was done in the Laboratory of Physiological Chemistry,
of Columbia University, at the College of Physicians
and Surgeons. 1899-1903. XLIX-LXVI.

1899.
A. AnsiRACT OK A Premminarv Rp:i'ort of Research.

XLIX. Eugene Hodenpyl, assisted by Allan C. Eustis and A. N.
Richards. Report of a chemical examination of a
knife-grinder's Kmg. Proceedings of the New York
Pathological Society, November 8. Medical Record,
December 23; Ivi, p. 942.

?). Papers ox ovk own Investigations.

L. P. A. Levene. On the nucleoproteid of the brain (cere-
bronucleoproteid). Archives of Neurology and Psycho-
pathology, ii, p. I.
LI. P. A. Levene. Embryochemical Studies. I. Some chem-
ical changes in the developing egg. Archives of Neu-
rology and Psychopatholoi^y, ii, p. 557.

1900.
Abstracts of 1'reliminary Reports of Researches.

LIL* P. A. Levene. Some chemical changes in the developing
egg. Proceedings of the American Physiological Society,
New Haven, December, 1899. American Journal of
Physiology, March ; iii, p. xii.

LIII. J. E. Kirkwood and William J. Gies. Some chemical
notes on the composition of the cocoanut. Pro-
ceedings of the New York Academy of Sciences, May.
Science, June 15 ; xi, p. 951. Also, Annals of the
Neiv York Academy of Sciences, 1900-1901, xiii, p.
489.

LIV. J. E. Kirkwood and William J. Gies. The composition of
the endosperm and milk oS. the cocoanut. Proceedings of
the American Association for the Advancement of Science,
New York, June. Science, October 19 ; xii, p. 585.
Also Permanent Secretary' s Report of the Proceedings,
December, p. 275.

* This report was made shortly after the publication of the preceding paper.

Bibliography. " 19

1901.

A. Abstract of a Preliminary Report of Research.

LV. J. E. Kirkwood and William J. Gies. Changes in the
composition of the cocoanut during germination. Pro-
ceedings of the American Physiological Society, Balti-
more, December, 1900. American Joiiriial of Physiol-
ogy, March ; v, p. xiv.

B. Paper on our own Investigations.

LVI. Rolfe Floyd and William J. Gies. x\n extreme case of
simple anaemia. Medical Record, April 27 ; lix, p. 650.

1902.

Papers on our own Investigations.

LVII. C. A. Herter and A. N. Richards. Note on the glycosuria
following experimental injections of adrenalin. Medi-
cal News, February i ; Ixxx, p. 201.

LVIII. J. E. Kirkwood and William J. Gies. Chemical studies
of the cocoanut, with some notes on the changes during
germination. Bulleti?i of the Torrey Botanical Club,
June 20; xxix, p. 321.
LIX. Francis W. Murray and William J. Gies. A case of pan-
creatic fistula of three years' duration, with a chemical
study of the fluid eliminated. American Medicine, July
26; iv, p. 133.
LX. William Salant. On the influence of the contents of the
large intestine upon strychnine. Americait Medicine,
August 23; iv, p. 293.
LXI. William J. Gies. On the nutritive value and some of
the economic uses of the cocoanut. Journal of the
New York Botanical Garden, September ; iii, p.
169.

LXII.* W. W. Lesem and William J. Gies. Notes on the " pro-
tagon " of the brain. American Joitrnal of Physiology.
December; viii, p. 183.

* This research was begun by the writer in the Sheffield Biological Laboratory at
the suggestion of Professor Chittenden.

20 Bibliography.

1903. January- July.

A. Titles of Prki.imixakv Ri.i'orts ok Researches.

LXIIL* D. T. MacDougal for William J. Gies. " Alkaverdin,"
a hitherto unknown pigment found in leaves of Sarra-
cenia purpurea. Proceedings of the Botanical Society of
America, December, 1902. Secretary s Report. Sci-
ence, February 27 ; xvii, p. 338.

LXIV.* D. T. MacDougal for William J. Gies. The digestive ac-
tion ensuing in the \nicherso( Sarracenia purpurea. Ibid.
LXV.-= D. T. MacDougal for B. C. Gruenberg and William J.
Gies. Chemical studies of various kinds of logwood.
Ildd., p. 339.

B. Paper on our owx Investigations.
LXVI. William Salant. A further study of the influence of the
contents of the large intestine upon strychnine. Amer-
ican Medicine, June 27 ; v, p. 1027.

III. Reports and papers on researches carried out in various laboratories by

William J. Gies under the guidance of, or in collaboration with,

his teachers. 1896-1903. LXVII-LXXXII.

A. Abstracts oe Preliminary Reports of Researches.

1899.
LXVII. William J. Gies. On stimulation and excitability of the
ancemic brain. Report of the meeting of the British As-
sociation for the Advancement of Science, Dover, Sep-
tember, p. 897.

1900.

LXVIII. Leon Asher and William J. Gies.j- The influence of pro-
toplasmic poisons on the formation of lymph (with a
note on lymph formation after death). Proceedings
of the American PJiysiological Society, New Haven, De-
cember, 1899. American Journal of Physiology, M.axch.;
iii, p. xix.
LXIX. William J. Gies. The influence of protoplasmic poisons
on the formation of lymph. Second report. Proceed-
ings of the New York Academy of Sciences, January.
Science, February 16 ; xi, p. 269. Also, Annals of the
New York Academy of Sciences, 1900-1901, xiii, p. 434.

* Report not abstracted. See paper No. 40 (LXIII-LXIV).

f The statement in the second footnote on page 13 applies here also.

Bibliography.

21

1903. January- July.

LXX. * R. H. True and William J. Gies. The physiological ac-
tion of heavy metals in mixed solutions. Proceedings
of the Botanical Society of America, December, 1902.
Secretary' s Report. Science, February 27; xvii, p. 339.
LXXI. Jacques Loeb and William J. Gies. Further studies of the
toxic and antitoxic effects of ions. Proceedings of the
American Physiolo^i^ical Society, Washingto?i, December,
1902. Ainericati Journal of Physiologv, February ; viii,
p. xiv.
(XXXVir.) t S. J. Meltzer and William J. Gies. Studies on the in-
fluence of artificial respiration upon strychnine spasms
and respiratory movements. Ibid., p. xlii.

B. Papers on our own Investigations.

1896.
LXXII. R. H. Chittenden and William J. Gies. The mucin of
white fibrous connective tissue. Journal of Experimental
Medicine, \, p. 186.

1898.
LXXIII. R. H. Chittenden and William J. Gies. The influence of
borax and boric acid upon nutrition, with special refer-
ence to proteid metabolism. American Journal of Physi-
ology, January; i, p. i.

1900.
LXXIV. Leon Asher and William J. Gies. Untersuchungen iiber
die Eigenschaften und die Entstehung der Lymphe.
IV. Ueber den Einfluss von Protoplasma-Giften auf die
Lymphbildung. N . Einiges iiber Lymphbildung nach
dem Tode. Zeitschrift filr Biologic, November; xl, p.
180.

1901.

LXXV. William J. Gies. Do spermatozoa contain enzyme having
the power of causing development of mature ova?
American Journal of Physiology, October; vi, p. 53.

* The report was not abstracted. The paper was published shortly afterward.
(LXXIX.)

fThe report was not abstracted. The paper was published in the succeeding
number of the Journal. See footnote on page 16.

2 2 Bibliography.

LXXVI.* William J. Gies. On the nature of the process of fertili-
zation. ATedical Ne^cs, November i6; Ixxix, p. 767.

1902.

LXXVII. Jacques Loeb and William J. Gies. Weitere Untersuch-
ungen iiber die entgiftenden lonenwirkungen und die
Rolle der Werthigkeit der Kationen bei diesen Vorgiin-
gen. Archiv fiir die gesammte Physiologic, December ;
xciii, p. 246.

1903. January-July.

(XLV.) t William J. Gies and S. J. Meltzer. Studies on the influ-
ence of artificial respiration upon strychnine spasms and
respiratory movements. American Journal of Physiol-
ogy, March ; ix, p. i.
LXXVIII. William J. Gies. On the irritability of the brain during
anaemia. American Journal of Physiology, y\.z.y ; ix, p.

131-
LXXIX. Rodney H. True and William J. Gies. C;n the physio-
logical action of some of the heavy metals in mixed so-
lutions. Bulletin of the Torrey Botanical Club, July:
XXX, p. 390.

C. Miscellaneous Public.'XTIoxs.

1895-
LXXX.+ William J. Gies. Preparation of a new beverage from
milk. Dietetic and Hygienic Gazette, S.-^x\\; xi, p. 212.

1896.

LXXXI.i" William J. Gies. On the normal occurrence of iodine in
the animal body. Dietetic and Hygienic Gazette. First
paper, March; xii, p. 158. Second paper, June; xii,

P- 352-

1898.

LXXXII. William J. Gies. On the decomposition and synthesis
of ])roteids in living plants. Yale Scientific Monthly,
February ; iv, p. 204.

* In conducting the researches referred to in this and the preceding paper, the
author enjoyed the use of the investigator's room, at Wood's Holl, reserved for the De-
partment of Physiology of Columbia University.

I See footnote on page 17.

% A translation. Not reprinted.

List of Reprints

OF

PAPERS AND OF ABSTRACTS OF
REPORTS,

IN

The Order of their Arrangement in this Volume.

Page.

List of abstracts 25

List of papers 26

23

LIST OF THE PUBLISHED ABSTRACTS OF REPORTS

AND PAPERS INCLUDED IN THIS VOLUME.

ARRANGED IN THE ORDER OF

THEIR PRESENTATION.

[Refer to the Bibliography, pages 13 to 22 inclusive, for names of authors and
journals, for dates, etc. The Roman numerals, wherever they occur below, correspond
with those for the same articles in the Bibliography. ]

ABSTRACTS OF PRELIMINARY REPORTS OF RESEARCHES.

A. Reports which have been followed by publications giving the re-
sults IN detail. [The letters preceding the titles correspond
with those at the he.\ds of the reprinted abstracts. The
nu.mbers in parenthesis at the ends of the titles indi-
cate the corresponding detailed puelica-
tidns listed below.]

Page of

the
Volume.

Chemical Investigations of Animal Tissues and Tissue Constituents.

a — I. The preparation of a mucin-like substance from bone (2, 3) 31

b — VII. New methods for the separation of some constituents of ossein

(2, 3-6) 32

c — X. A further study of the glucoproteid in bone (3) t^},

d — VL Notes on the constituents of ligament and tendon (4, 5, 7, 8).... 34
e — II. A prelimjnar}' study of the coagulable proteids of connective tis-
sues (4) 35

f — IX. Methods of preparing elastin, with some facts regarding ligament

mucin (4) 36

g — III. The glucoproteids of white fibrous connective tissue (5) 37

h - XXIV. The composition and chemical qualities of the albumoid ' in

bone (6) 39

i — LII. Some chemical changes in the developing egg (15) 40

Pathological and Toxicological.

j — IV. The physiological action of tellurium compounds (20, 21 ) 40

k — LXVIII. The influence of protoplasmic poisons on the formation of lymph

(with a note on lymph formation after death) (19) 42

1 — LXIX. The same — a second report (19) , 43

m — LXVII. On stimulation and excitability of the anaemic brain (26) 44

Miscellaneous Researches.

n — LXXI. Further studies of the toxic and antitoxic effects of ions (29) 47

o — XXVI. Are proteids which are prepared by the usual methods combined

with fat or fatty acid (35) 48

25

26 Titles of Reprints.

I'age ol

the
Volume.
Butiiniial Sliidies.

p — LI II. Some chemical notes on the composition of the cocoanut (36, 38) 48
q — LIV. 'riie composition of the endosperm and milk of the cocoanut

(36, Z^) ■•••••••• 49

r — LV. Changes in the composition of the cocoanut during germination

(36) 51

B. Reports of Rese.arches which h.we not yet keen Pi bi.ished in
Greater Detail.
Chemical Investigations of Animal Tissues and Tissue Constituents.

aa — VIII. Does muscle contain mucin ? 52

bb — XXV. A comparative study of the reactions of various mucoids 53

cc — XXXIV. Further mucoid studies 54

Pathological and Toxicological.

dd- XXXIX. Properties of " Bence Jones' body " 55

ee — XL. A inodihed Eck fistula, with a note on adrenalin glyciemia 55

ff — XLIX. Report of a chemical examination of a knife grinder's lung 56

gg — XXVII. On the toxicology of selenium and its compounds 58

hh — XLI. The influence of chime acid on the elimination of uric acid 59

Miscellaneotts Researi hes.

ii — V. The proportion of basic nitrogen yielded by elastin on decompo-
sition with hydrochloric acid 59

jj — XXXV. A proteid reaction involving the use of chroniate 60

kk — XXXVI. The influence of the H ion in peptic proteolysis 61

11 — XXXVIII. An improved cage for metabolism experiments 62

mm — XLll. Peptic proteolysis in acid solutions of equal conductivity 62

PAPERS.

[The letters in parenthesis at the ends of the titles indicate the corresponding pre-
liminarj' reports listed above. The " number of the reprint "' corresponds with the large
numeral at the top of the opening page of the reprint.]

Chemical Investig.a.tions of Animal Tissl'es and Tissue Constituents.

Number Pages of Page of

of the the the

Reprint. Reprint.* Volume. t

1. An improved method of preparing and preserving meat for use

in metabolism experiments (XV.) 235-239 69

2. A new constituent of bone (XIX.). (a, b) 1-5 75

3. Chemical studies of osseomucoid, with determinations of the

heat of combustion of some connective tissue glucoproleids.

(XVII.). (a, b, c) 387-425 81

* In most cases these numerals are those of the pages of the original articles and of
the reprints obtained when the papers were first published. Only a few of the papers
have been specially reprinted (Xos. 12-17 and 38-40).

fThe page numerals of the volume Ark printed on reprints Xos. 12-17 and 38-40.
See preceding fo tnute.

Titles of Reprints. 27

Number Pages of Page of

of the the the

Beprint. Reprint. Volume.

4. Chemical studies of elastin, mucoid and other proteids in elastic

tissue, with some notes on ligament extractives (XXIX.).

(d, e, f) 93-134 121

5. The composition of tendon mucoid (XVIII.). (d, g) 155-172 163

6. On the composition and chemical properties of osseoalbumoid,

with a comparative study of the albumoid of cartilage.

(XXXIL). (b, h) 340-358 181

7. The composition of yellow fibrous connective tissue (XVI.).

(d) 287-297 201

8. The chemical constituents of tendmous tissue (XX. ). (d) 219-231 213

9. Do spermatozoa contain enzyme having the power of causing

development of mature ova? (LXXV.) 53~76 227

10. On the nature of the process of fertilization (LXKVI.) i-ii 251

11. Notes on the " protagon " of the brain (LXII.) 183-196 263

12. On thenucleoproteid of the brain (cerebronucleoproteid) (L.).. 277-285 277

13. The mucin of white fibrous connective tissue (LXX IT.) 287-301 287

14. Animal coloring matters (XXIII.) 303-313 303

15. Embryochemical studies. I. Some chemical changes in the

developing egg (LI.), (i) 315-321 3I5

Pathological and Toxicological.

16. The influence of borax and boric acid upon nutrition, with

special reference to proteid metabolism (LXXIIL) 325-352 325

17. On the influence of the contents of the large intestine upon

strychnine (LX.) 353-355 353

18. A further study of the influence of the contents of the large

intestine upon strychnine (LXVI) I-3 357

19. Untersuchungen iiber die Eigenschaften und die Entstehung

der Lymphe. IV. Ueber den Einfluss von Protoplasma-
Giften auf die Lymphbildung. V. Einiges iiber Lymphbil-
dungnach demTode (LXXIV.). (k, 1) 180-216 361

20. The toxicology of tellurium compounds, with some notes on

the therapeutic value of tellurates (XII I.), (j) 1-20 399

21. Physiological and toxicological effects of tellurium compounds,

witha special studyoftheirinfluence on nutrition (XL), (j) 104-149 419

22. Chemical changes in the body in which the methyl group may

beinvolved (XXX.) 1-3 467

23. Note on the glycosuria following experimental injections of

adrenalin (LVII.) 1-7 47i

24. Studies on the influence of artificial respiration upon strychnine

spasms and respiratory movements (XLV. ) 1-25 479

25. An experimental study of the sugar content and extravascular

coagulation of the blood after administration of adrenalin

(XLVI.) 35-51 505

26. On the irritability of the brain during ansemia (LXXVIIL).

(m) 131-137 523

28 TiTLKs OF Reprints.

Number Pages of Page of

of the the the

Reprint Reprint. Volume.

27. An extreme case of simple anaimia (LV I. ),, 1-16 531

28. A case of pancreatic fistula of three years' duration, with a

chemic study of the fluid eliminated (LIX.) i-l? 547

MiSCELLANEOrS RESEARCHES.

29. Weitere Untersuchungen iiber die entgiftenden lonenwirkun-

gen und die RoUe der Werthigkeit der Kationen bei diesen

Vorgangen (LXXVII.). (n) 246-268 565

30. A note on the excretion of kynurenic acid (XIV.) 191-195 589

31. Some facts regarding " ureine " (XII.) 1-12 595

32. Some notes on Pollacci's new method of detecting albumin in

the urine (XLIII.) 1-3 607

3;^. Proteosuria (XXVIII. ) 1-4 611

34. On the quantitative determination of acidalbumin in digestive

mixtures (XXXIII.) 460-491 615

35. Experiments to determine the possible admixture or combina-

tion of fat or fatty acid with various proteid products

(XXXI.). (o) 331-339 647

Botanical Stldies.

36. Chemical studies of the cocoanut with some notes on the

changes during germination (LVIIL). (p, q, r) 321-361 659

37. On the physiological action of some of the heavy metals in

mixed solutions (LX.XIX.) 390-402 701

38. On the nutritive value and some of the economic uses of the

cocoanut (LX I.), (p, q) 7I5-7I7 7I5

39. On the decomposition and synthesis of proteids in living plants

(LXXXII.) '. 719-727 719

40. Chemical studies of the pitcher plant, Sun-tuenia purpurea

(XLIV.) 729-731 729

The following miscellaneous publications have not been reprinted, as was indicated
in the Bibliography, pp. 15-22 : XXI, XXII, XXXVII, XLVII, XLVIII, LXIII,
LXIV, LXV, LXX, LXXX, LXXXI.

ABSTRACTS

OF Preliminary Reports of Researches, Published in Vari-
ous Journals (see Bibliography) and Made Before
THE Following Societies :

British Association for the Advancement of Science, 1899 — m.*

New York Pathological Society, 1899 — ff.

American Physiological Society, 1 899-1903 — a, c, e, f, g, h,
i, j, k, n, o, r, aa, bb, cc, gg, hh, ii, jj, kk, mm.

American Association for the Advancerhent of Science, 1900
— b, d, q.

New York Academy of Sciences, 1900 — 1, p.

Society for Experimental Biology and Medicine, 1903 — dd,
ee, 11.

* The letters following the names of the societies correspond with those before the
titles listed on pages 25 and 26.

29

A. ABSTRACTS OF REPORTS OF RESEARCHES
WHICH HAVE BEEN FOLLOWED BY PUB-
LICATIONS GIVING THE RESULTS
IN DETAIL, pages 31-51 ; a-r.

Reprinted from the American Journal of Physiology, I goo, iii ; Proceedings of
the American Physiological Society, p. vii.

ar^ THE PREPARATION OF A MUCIN-LIKE SUBSTANCE

FROM BONE.

By William J. Gies.

Young, in 1892, working under Halliburton's direction, was
unable to separate mucin from bone. This negative result has
gained general acceptance in spite of the fact that the method em-
ployed by Young could hardly have been expected to yield any
other. The bone powder and shavings, in quantities ranging from
2.5 to II grams, were extracted with 100 to 500 c.c. of lime or
baryta water, and, after several days, the filtered solution was
treated with acetic acid. Failure to obtain precipitates under these
conditions led to the conclusion that bone does not contain mucin.
Calling attention to the main defect in this procedure, it is sufficient
to suggest that the inorganic substances in bone must necessarily
impose a mechanical obstacle to the action of the dilute alkali,,
and that their removal ought to be the first step in any attempt to
get at whatever glucoproteid might be contained in the tissue.

I have prepared a mucin- like substance from the rib and femur
of the ox by the following method : The perfectly clean bones
were kept in 0.2 per cent, to 0.5 per cent. HCl. As the inorganic
matter dissolved out, the bones were shaved and the shavings ac-
cumulated in 0.2 per cent. HCl. These were finally run through
a meat chopper, then washed free from acid, and extracted in half-
saturated lime water. The filtered extract gave a heavy precipi-
tate with 0.2 per cent. HCl in excess. 1,700 grams of femur
shavings yielded 7 grams of this material : 875 grams of rib shav-
i^''gs gave 3.5 grams. This substance appears to be mucin, though

*The letters preceding the titles correspond with those in the list on pages 25-26^

31

32 Abstracts of Reports.

it may be chotidromucoid or a relative of each. It dissolves
easily in o. i per cent. Xa.,CO.,. It is acid to litmus. It gives the
proteid color reactions, yields a reducing substance, and contains
ethereal sulphuric acid. The nitrogen and sulphur content of the
unpurified substance approximates that of tendon mucin and chon-
dromucoid. The filtrate from the mucin precipitate contains a
substance which has many of the qualities of chondroitin sulphuric
acid. A careful investigation of the composition and character of
the mucin-like substance, and the body supposed to be chondroitin
sulphuric acid, is now being made. The general method em-
plo}'ed for their detection and separation promises, also, to yield
material well suited for the studies we shall make of bone gelatin
and the various organic bone constituents.

It is evident from these results that ordinary compact bone,
just like the other forms of connective tissue, does contain mucin
substance, and, further, that in the process of ossification the con-
nective tissue matrix is not completely removed.

Reported in person by the author before the American Association for the Ad-
vancement of Science at the June meeting in 1900, but, by an oversight, the abstract
was omitted by the Secretary from the Proceedings of the Society and only the title
there given ; on p. 131.* 'Jhe abstract below is the one presented to the Secretary for
publication.

b. NEW METHODS FOR THE SEPARATION OF SOME
CONSTITUENTS OF OSSEIN.

BV WU.LIAM J. GlES.

The author improved the method of preparing ossein by plac-
ing the compact bone in 0.2 to 0.5 per cent. HCl for three or
four hours, and then scraping off with a scalpel the thin, softened
layer of tissue in somewhat elastic, translucent shavings. The
dilute acid has little or no effect on the bulk of the organic con-
stituents and the latter may be separated easily from the shavings,
especially after the latter have been put through a meat chopper.
The method was demonstrated.

The new constituents of bone, prepared by the author from the
ossein obtained in this way, are a chondromucoid-like substance

*This abstract and that on page 34 are the only ones in which additions have
been made to the original form of publication.

Abstracts of Reports.

33

and another glucoproteid having some of the quahties of para-
mucin. The filtrate from these precipitates also contains a sub-
stance apparently identical with chondroitin-sulphuric acid. Chem-
ical analysis of these bodies is now in progress.

Ossein prepared in this manner may be kept in artificial pan-
creatic juice at 40°C. for several days without perceptible decrease
in quantity. This method suffices, therefore, to remove gluco-
proteid and nucleoproteid and elastin in the preparation of bone
collagen. The gelatin obtained from the latter is especially pure
as a result of this preliminary treatment. An elastin-like albumi-
noid remains after the finely minced ossein has been extracted with
dilute alkali for several days and has been boiled, also, in water
for a week or more. Both the gelatin and the elastin prepared by
these methods are about to be carefully studied.

The author concludes that this general method of preparing
ossein will be favorable, also, to studies of the fat, the bone pig-
ment and such nucleoproteid as may be assumed to exist in osse-
ous tissue.

Reprinted from the American Journal of Physiology, 1901, v ; Proceedings of the
American Physiological Society, p. xv.

c. A FURTHER STUDY OF THE GLUCOPROTEID IN BONE.
By p. B. Hawk axd William J. Gies.

Five different preparations from the femur of the ox have been
analyzed since the figures for the first two (from rib and femur of
the ox) were reported to this society a year ago. The elemen-
tary composition of the seven varies between the extremes here
given in percentage figures :

C. H. N. S. Ash.

45. 75-48. oS 6.66-7.29 II. 97-14. 15 1.36-2. 21 0.33-2.72

The ash-free substance does not contain phosphorus. The
amount of sulphur that could be split off in the form of ethereal
sulphate varied from 0.49 to i.io per cent.

The following figures show the average percentage composition
of the preparation of osseomucoid which we have good reason
to think is the purest, and also of chondromucoid, as determined
by Morner :

34 Abstracts of Reports.

C. H. N. S. O. S (as eth sulph )

Osseomucoid 46.41 6.76 12. oS 2.04 32.71 1.08

Chondromucoid 47-30 6.42 12.58 2.42 31.28 1.72

Compared with the glucoprotcid of cartilage, osseomucoid
appears to contain more hydrogen and oxygen and correspond-
ingly less of the other elements. In its reactions it is practically
the same.

This abstract was abbreviated unsatisfactorily in the Proceedings of the American
Association for the Advancement of Science, 1900, p. 123, and is here given in the
form in which it was presented to the Secretary for publication.

d. NOTES ON THE CONSTITUENTS OF LIGAMENT AND

TENDON.*

Bv William J. Giks.

Qualitative. — Ligament consists mostly of elastin. The author
and his assistants find that it contains considerable glucoproteid ;
also, coagulable proteid in appreciable quantity. These facts have
not been duly considered, heretofore, in the preparation of elastin,
and may account for variations reported in the composition of
elastin as also in the character of its decomposition products.

There appears to be more than one glucoproteid in tendon.
Various products separated by differential methods show nitrogen
content varying from 11.5 to 14.7 per cent.; sulphur content, from
1.3 to 2.8 per cent. These facts seem to explain the discrepancies
in former analyses of tendon mucin. A large proportion of the
sulphur of the molecule is in the form of ethereal sulphate and it
is quite probable that mucin and chondromucoid are more inti-
mately related than had been supposed.

Both ligament and tendon contain crystalline nitrogenous ex-
tractives. Thus far creatin has been separated from each.

These points are being worked out in detail with the help of
Messrs. A. N. Richards and William D. Cutter.

Quantitative. — The author presented the following figures for
averages of many analyses of ligament and tendon, the first to be
reported for these tissues. They show relative general composi-
tion.

* This abstract and that on page 32 are the only ones to which additions have

been made.

Abstracts of Reports. 35

Fresh Ligamentum Nu

chae (Ox).

Fresh Tendo AchilHs (Ox)

Per cent.

Per cent.

Water,

57-57

62.87

Solids,

42.43

37-13

Inorganic matter,

0.47

0.47

Organic matter,

41.96

36.66

Collagen,

7-23

31-58

Elastin,

31-67

1.63

Coagulable proteid.

0.62

0.22

SO3 in the ash,

5.64

6.65

The high content of SO.^ in the ash is noteworthy. In all
probability much of it arises on incineration from the ethereal sul-
phate in the glucoproteids of each tissue. The results for elastin
and collagen are particularly instructive.

The data in this connection have been obtained in work in
which Messrs. Leo Buerger and G. W. Vandegrift are cooperating.

Reprinted from the American Journal of Physiology, igco, iii ; Proceedings of
the American Physiological Society, p. v.

e. A PRELIMINARY STUDY OF THE COAGULABLE
PROTEIDS OF CONNECTIVE TISSUES.

By a. N. Richards and William J. Gies.-^

This investigation was prompted by the belief that there is, per-
haps, more metabolic activity in the connective tissues than their
"passive mechanical functions" suggest, and, therefore, that an
increase of our knowledge of their chemical units will be of some
value. Ligament, tendon and hyaline cartilage are the represen-
tative forms of connective tissue we have studied thus far in a pre-
liminary way. Aqueous and magnesium sulphate extracts of the
thoroughly clean tissues were made, examination with the spectro-
scope showing the absence of haemoglobin. Cartilage has thus
far given entirely negative results. Tendon seems to contain two
coagulable proteids ; one separates at 54°— 57° C, the other at 73°.

Ligament contains much more coagulable proteid than the other
forms. Quantitative determinations with the ligamentum nuch^
of the ox show that that particular form of ligament contains
0.65 per cent, of coagulable proteid in the fresh moist tissue and
1.98 per cent, in the dry. Proteid is precipitated regularly in the

* See second footnote, page 13.

36 Abstracts of Reports.

various extracts at 42^-50°, 54°-58°, 66°-70°, 74-76^ and 83°-
85° C. W'c do not ha\'e sufficient faith in the heat coagulation
method to conclude from these results alone that there are as many
coagulablc proteids in ligament as these temperatures may indi-
cate. We think these results are suggestive rather than conclu-
sive, and expect, by fractional precipitation methods and chemical
analysis, to determine definitely the number present. Upon ex-
traction with half saturated lime-water, ligament yields mucin-like
material, which later investigation ma\- show is closely related to
the glucoproteids in tendon.

These results with the ligament suggest that, in the prepara-
tion of elastin, due regard must be paid to the fact that the tissue
contains a fairly large proportion of soluble and coagulable pro-
teid. Possibly some of the variations in the figures reported for
the composition of elastin, and in the nature of its decomposition
products, ma}- be due to proteid which had not been removed in
its preparation.

Along with this research a study of connective tissue extrac-
tives is being made. Ligament has been found to contain an un-
expectedly large quantity of creatin, and the concentrated extract
yields a fairly heavy, brownish precipitate with silver nitrate
in the presence of ammonia. Future results in this connection,
also, may bear directly on the question of metabolism in the
connective tissues.

Reprinted from the .American Journal of Physiology, 1901, v; Proceedings of the
American Physiological Society, p. xi.

f. METHODS OF PREPARING ELASTIN, WITH SOME FACTS
REGARDING LIGAMENT xMUCIN.

Bv A. X. Richard;^ .vnd \Viij,i.\m J. Gies.

In continuation of the studies reported at the previous session
of the Physiological Society, we find that the ligamentum nuchae
of the ox contains an appreciable quantity of mucin, having all
the qualities of the glucoproteids separable from white fibrous
connective tissue. The nitrogen of five different preparations varied
from 12.90 per cent, to 13.86 per cent.; the sulphur from 1.32 per
cent, to 2.05 per cent.

Abstracts of Reports.

37

In order to insure removal of mucin and coagulable proteids
from ligament in the preparation of elastin, we have extracted the
finely divided tissue for several days in large excess of cold half
saturated lime-water. This preliminary process makes extraction
of the tissue with hot alkali unnecessary, and thereafter, when the
usual method is continued, neither albumin nor globulin is present
to be coagulated and there is no mucin to be decomposed.

By this improved method we have made three different prep-
arations of elastin from the ligamentum nuchae of the ox. Each
contains less sulphur than elastin obtained by the old method, the
quantity varying from 0.13 to 0.17 per cent, (not deducting S of
the ash, amounting to O. ii per cent, of the purified substance).
We have observed in two preliminary experiments that all the sul-
phur in the elastin prepared by our own method is firmly united
in the elastin molecule and is not broken away on boiling with i
per cent. KOH. This result is not obtained with elastin prepared
by the older method, in which extraction with alkali is avoided.

Using Schultze's method, the distribution of nitrogen in the
elastin prepared by the improved process as contrasted with that
of the old was found to be as follows :

Ammonia.

Bases.

Amido Acids.

Total Percentage.

A. Old method f I)

(2)

B. Improved method.

2.26

2.34

1-73

2.98
2.26
3.08

95-44

98.42
95-23

100.68
103.02
100.05

Our results in this connection seem to indicate that elastin does
yield organic bases, as Kossel and Kutscher have contended in
opposition to Bergh and Hedin.

Reprinted from the American Journal of Physiology, 1900, iii ; Proceedings of
the American Physiological Society, p. vi.

g. THE GLUCOPROTEIDS OF WHITE FIBROUS CONNEC-
TIVE TISSUE. ■

By William D. Cutter and William J. Gies. *

Thus far two series of continuous fractional extractions of ox
tendon have been made with half saturated lime-water and the

* See second footnote No. 2, page 13.

38 Abstracts of Reports.

mucins precipitated from each of the extracts analyzed. The
semi-cartilaginous character of the sheath in which the divisions
of the main shaft of the Achilles tendon move suggested, at the
outset, that possibly the mucin from the sheath may be different
from the mucin of the strictly tendinous portion. In the previous
work no such discrimination was made, but both parts were ex-
tracted together. A comparison of the results for the nitrogen
content of the mucins, of the first three extracts of both series
from the tendon and its sheath, show that the nitrogen is lower in
the second of each than in the first and third, and highest in the
third. The figures range from 11.69 to 13.27 per cent. The
sulphur content is highest in the first of each, the figures varying
from 1.38 to 2./S per cent. These results indicate that there are
several mucins in white fibrous connective tissue ; just how many
our future work may determine.

Further experiments on the glucosazone-like substance obtain-
able from the reducing bodies gave products melting at 182° C.
Thus far it has not been possible to entirely free the crystals from
the brownish globules that occur with them, so that probably
these figures are still too low.

Before these experiments were started the similarity in the per-
centage composition of Morner's chondromucoid and the tendon
mucin analyzed by Chittenden and Gies four years ago suggested
to us that the two substances are perhaps closely related. This
was further emphasized by the fact that the osazone crystals they
obtained had the same general appearance as the crystals of glu-
cosazone and, therefore, might have arisen from glucosamin, one
of the decomposition products of chondromucoid. Our own re-
sults increase the probability that the two substances are nearly
identical.

We believe that continued investigation will show that the
differences among the mucins, mucoids and chondroproteids are
not as great as their varying physical properties and behavior have
suggested, but that each is a combination of proteid with a gluco-
sulphonic acid, the characters of each compound, just as in the
case of the nucleoproteids, being dependent largely on the pro-
portions of proteid and acid radicals.

Abstracts of Reports. 39

Reprinted from the American Journal of Ptiysiology, 1 902, vi ; Proceedings of the
American Physiological Society, p. xxvii.

h. THE COMPOSITION AND CHEMICAL QUALITIES OF
THE ALBUMOID IN BONE.

By p. B. Hawk and William J. Gies.

In the first report to this society of the discovery of osseo-
mucoid attention was drawn to the fact that the method used for
the preparation of the glucoproteid would also favor a study of
the albuminoid constituents of osseous tissue. The collaginous
residue remaining after extraction of osseomucoid from ossein
yields an insoluble, elastin-like substance on boiling in water.
This substance is neither the elastin of Smith nor the keratin of
Broesicke, but appears to be almost or quite identical with Mor-
ner's chondroalbumoid. Although our product is digestible in
pepsin-hydrochloric acid, it appears to be somewhat more soluble
in dilute acid and alkali than chondroalbumoid. UnHke the latter
body, however, it does not contain loosely bound sulphur.

We have prepared a number of samples of osseoalbumoid
from ossein by the method Morner used for the preparation of
the albumoid substance in cartilage. The chief difficulty in this
work has been the removal of phosphates and the preparation of
ash-free products. Our analyses thus far indicate the average
elementary composition given in the summary below, where com-
parison is also made with keratin and elastin.

C.

H.

N.

s.

0.

Osseoalbumoid .

■ S0.03

6.85

15.93

0.55

26.64

Ligament elastin

. 54- 08

7.20

16.85

0.30

21.57

Hair keratin .

. 50.65

6.36

17.14

5.00

20.85

Osseoalbumoid does not contain phosphorus. Unfortunately,
analytic comparisons with chondroalbumoid are not now possible,
as Morner made no analyses of that body, although he found that
the nitrogen content (three determinations) of albuminates made
from it varied between 15 and 16 per cent. We have obtained
larger proportions of this residual substance from bone than from
cartilage. It is our purpose to study chondroalbumoid in this
connection also.

40

Abstracts of Reports.

Reprinted from the American Journal of Physiology. 1900, iii ; Proceedings of
the .\merican Physiological Society, p. xii.

i. SOME CHEMICAL CH.WGES JN THE DEVELOPING EGG.

By P. A. Levkne.

This work gives the results of an attempt to elucidate the
chemical process of construction of animal tissue. Thus far the
investigation has been limited to the distribution of nitrogen in the
different nitrogenous compounds of the developing egg of differ-
ent ages. All the nitrogenous substances produced on decompo-
sition of proteids may be classified in two di.stinct groups : Those
of acid nature, like the monoamido-acid, and those of basic na-
ture. The following table demonstrates to some extent the part
the same substances play in tissue construction :

Nitrogen in monoamido

compounds.
Nitrogen in form of bjses.
Nitrogen in form of proteids.

Unfertilized 24 Hours After ; 10 Days After
Eggs. Fertilization. Fertilization.

Per Cent. Per Cent Per (Jent.

21.10

12.07
66.00

21.37
25.10

53-57

22.72
12.48
64.79

19 Days After

Fertilization

Per Cent.

O

28.25
71.84

It has also been found that the quantity of the xanthin bases
and of nucleo-compounds increases with the growth of the egg
embryo. The importance of mineral salts for the formation of
tissues was demonstrated by the increasing quantity of mineral
substance in the egg in the course of its growth.

Reprinted from the American Journal of Physiology, 1900, iii ; Proceedings of
the American Physiological Society, p. x.x.

j. THE PHYSIOLOGICAL ACTION OF TELLURIUM
COMPOUNDS.

"By L. D. Mead and William J. Gies.*

Our work with tellurium compounds was begun at the sug-
gestion of Dr. Victor Lenher, who very kindly furnished us with
an abundant supply of chemically pure tellurium, preparations. In
view of the use of potassium and sodium tell urates as antihy-
drotics, to reduce the night sweats of pulmonary consumption, we

*See second footnote, page 13.

Abstracts of Reports. 41

have determined the influence of small quantities of tellurium com-
pounds on the nutritional processes. We find that quantities of
tellurious oxide, sodium tellurite, and tellurium tartrate, not ex-
ceeding o. I gram daily in two doses, do not materially alter pro-
teid metabolism in dogs brought to a state of nitrogenous equilib-
rium, even when the dosage is continued for a week. After the
administration of these non-toxic amounts the feces were fairly
constant in elimination, quantity and character. There was no
appreciable effect on the elimination of water. Digestion did not
appear to be materially hindered. Tellurium was eliminated in the
urine and the odor of methyl telluride in the expired air was very
pronounced.

Larger doses, however, 0.2 to 0.5 gram at a time, cause vio-
lent vomiting and induce disintegration of the gastric mucous
membrane. Our experiments on a dog with gastric fistula show
that there is a very decided interference with the secretion of
hydrochloric acid after the administration of tellurium in these
quantities and, also, that regurgitation of bile is one of the conse-
quences. The action of pepsin and trypsin outside the body is
not materially influenced by quantities of tellurium tartrate and
sodium tellurite under one per cent.

Tellurium is eliminated in the breath, urine and feces of the
dog. Reduction to the metallic state occurs when tellurium com-
pounds come in contact with the tissue cells, though tellurium it-
self is soluble in the body juices and is distributed to the various
organs. Two days after subcutaneous injection of a little more
than I gram of the tartrate, 38 milligrams of tellurium were re-
covered from the tissue about the point of injection, i 2 from the
liver, 9 from the kidneys, 7 from the bile, and 3 from the brain.
Additional experiments will be made with sodium and potassium
tellurates.

42 Abstracts of Rki-orts.

Reprinted from the American Journal of Physiology, 1900, iii ; Proceedings of the
American Physiological Society, p. xix.

k. THK INFLUENCE OF PROTOPLASMIC POISONS ON THE

FORMATION OF LYMPH. (WTTH A NOTE ON

LYMPH FORMATION AFTER DEATH.)

By Leon Asher and Wii.iiam J. Gies.*

The work reported upon here very briefly was done in the
Physiological Institute at Bern. An attempt was made in this in-
vestigation to ascertain, as far as possible, the changes which may
occur in lymph after the administration of protoplasmic poisons,
by studying the influence of such poisons on the phenomena
usually produced by well-known lymphagogues. In this way we
attempted to distinguish between the so-called physiological and
the physical factors participating in the production of lymph. Our
experiments were on dogs, and with quinine and arsenic. The
usual methods of lymph collection and analysis afforded the data
for our conclusions.

Quinine did not interfere with the usual influence of dextrose,
although it did suppress the action of leech extract. Our results
with dextrose, therefore, indicate that the increase in the quantity
of lymph following its injection in large quantity is due mainly to
physical factors. In the case of leech extract, on the other hand,
we conclude there has been an interference with the action of the
physiological factors that appear to be responsible for the changes
usually brought about by this lymphagogue.

That the increase in the amount of lymph after large quantities
of dextrose have been injected is not due specifically to increased
capillary pressure, as is held by Cohnstcin and Starling, was shown
in one of our experiments in no uncertain way. After an injection
of I gram of quinine, 25 grams of dextrose and 0.5 gram more of
quinine followed ten minutes later, and 35 c.c. of blood was drawn
off Almost immediately the usual effect of dextrose became evi-
dent. In a few minutes, however, the dog died, yet, for more
than three hours thereafter, the flow continued, and that, too,
without artificial respiration or any mechanical assistance whatso-
ever. The rate of flow gradually increased for more than an hour,

* See second footnote on page 20.

Abstracts of Reports. 43

when it slowly fell back to, and below, the rate of the first period.
During the three and a half hours of the experiment the total flow
of lymph was 140 c.c. During the first half hour, when the nor-
mal conditions prevailed, the flow was only 12.8 c.c. The amount
of total solids at the start was 5.02 per cent., at the end 5.9 per
cent. The sugar rose from 0.19 per cent, to 2.2 per cent. This
experiment seems to emphasize Heidenhain's view that the increase
of lymph following injections of large quantities of dextrose is due
to changes of osmotic pressure in the tissue spaces.

Following injections of arsenic, which is said to very greatly
increase the permeability of the blood vessels, especially those of
the portal system, there was little in the flow and character of the
lymph resembling the usual effects of lymphagogues. We con-
clude, .therefore, that Starling's hypothesis does not fully account
for the action of lymphagogues, and that the mechanical theory of
lymph formation fails so long as it does not explain the most
striking phenomena of the process — those following the injection
of Heidenhain's lymphagogues or Asher's "liver stimulants."
The physiological theories of Heidenhain and of Asher and
Barbera would explain them.

Reprinted from the Proceedings of the Section of Biology of the New York
Academy of Sciences : Annals of the New York Academy of Sciences, 1 900-1901 ;
xiii, p. 434; also, Science, February 16, 1900, xi, p. 269.

1. THE INFLUENCE OF PROTOPLASMIC POISONS ON THE
FORMATION OF LYMPH. (SECOND REPORT.)

By William J. Gies.

The author reported upon the changes which may occur in
lymph after the administration of protoplasmic poisons. Quinine
did not interfere with the usual influence of dextrose, although it
did suppress the action of leech extract. The results with dex-
trose indicate, therefore, that the increase in the quantity of lymph
following its injection in large quantity is due mainly to physical
factors. In the case of leech extract, on the other hand, there is
an interference with the action of the physiological factors that
appear to be responsible for the changes usually brought about by
this lymphagogue.

44 Abstracts of Reports.

That the increase in the amount of lymph after large quantities
of dextrose have been injected is not due primarily to increased
capillary pressure, as is held by Cohnstein and Starling, was shown
in one of the experiments in which quinin caused the death of the
animal, and yet from which the lymph continued to flow for three
hours. After injecting arsenic, which is said very greatly to in-
crease the permeability of the blood v^essels, especially those of the
portal system, there was little in the flow and character of the
lymph resembling the usual effects of lymphagogues.

It appears, therefore, that Starling's hypothesis of increased
capillary permeability does not fully account for the action of
lymphagogues and that the mechanical theory of lymph forma-
tion fails so long as it does not explain the most striking phenom-
ena of the process — those following the injection of Heidenhain's
lymphagogues or Asher's " liver stimulants." The physiological
theories of Heidenhain and Asher would explain them.

Reprinted from the Report of the Meeting of the British Association for the Ad-
vancement of Science, 1900, p. 897.

m. ON STIMULATION AND EXCITABILITY OF THE
ANEMIC BRAIN.

By William J. Gies.

[From the Physiological Institute of the University of Bern.]

The research indfcated by this subject was conducted in the
Physiological Institute at Bern, upon the suggestion and under the
constant direction of Professor Kronecker. Our aim was to de-
termine definitely the sequence of events during perfusion of various
so-called indifferent solutions through the brain, the data thus
obtained to afford a starting-point for future research with such
liquids as may be found to exert specific and characteristic influences.

In this report I shall present only the briefest outline of the
experiments and the results obtained.

The animals employed were toads, frogs, rabbits and dogs.

The solutions used were various strengths of pure sodium
chloride. Ringer's solution and Howell's modification of it ;
Schiicking's solution, both of calcium and sodium saccharate, and
serum.

Abstracts of Reports. 45

The perfusion in the cold-blooded animals was conducted with
the least possible pressure through the abdominal vein. All of
the various solutions already enumerated, except the serum, were
used. We made thirteen experiments (seven Avith toads and six
with frogs), each of which continued for a period of two to eight
hours, with a total transfusate of 250 to 1,600 c.c.

During the period of perfusion the following functions grad-
ually weakened and then usually disappeared in this order : (a)
Respiration ; (d) skin reflex ; (<:) lid reflex ; (d) nose reflex ; (^e)
heart beat.

The times of disappearance of these functions varied with the
total length of the experiments, and apparently also with the
amount of fluid transfused.

Convulsive extension of the limbs occurred in all of the ex-
periments in the earlier stages, but toward the close of each, and
before the reflex movements of the eyelids ceased, no such mani-
festations could be induced.

In passing it should be noted that :

((^) All of the animals became edematous ; even those in which
perfusion took place at the lowest possible pressures and for the
shortest periods.

[d) Also, that it was impossible to entirely remove the blood
corpuscles, even when the perfusion continued uninterruptedly for
eight hours, and as much as 1,600 c.c. of fluid had slowly passed
through the body. In all cases the fluid flowing from the cannula,
and particularly that pressed from the heart and brain, contained
quite an appreciable number of red and white corpuscles.

We carried out thirteen experiments with rabbits and two with
dogs, all of the previously mentioned fluids having been used..

Considerable difficulty was encountered in the attempt to find
a method which would prevent almost instant death of the animals,
and yet which would speedily result in pronounced anaemia.

Ligaturing, either in the neck or in the chest, the arteries to
the brain, before or simultaneously with the beginning of the
perfusion, brought on convulsions immediately. Even when the
perfusion had been begun shortly before the arterial blood was
completely shut off, it remained impossible to prevent convulsions
and quickly ensuing death.

46 Abstracts of Reports.

Finally, instead of closing the arteries to tiic brain, the abdom-
inal aorta, vena cava and vena porta were tied off and the heart's
action utilized to pump the liquid through the brain, the perfused
fluid going into the heart by one jugular and from the brain through
the other. By this method anremia could also be induced, convul-
sions entirely prevented, and life considerably prolonged.

As in the experiments with the cold-blooded animals, there
was in these also a fairly regular disappearance of functions, the
intervals appearing to vary with the total time of perfusion. With
all of the solutions, including serum, both in the rabbits and in
the dogs, the order of cessation usually was : (a) Respiration ; (/?)
lid reflex ; (c) nose reflex ; (V/) heart beat.

In some of the experiments, it should be noted, the nose and
lid reflexes ceased at practically the same instant. In a few, also,
it was impossible to determine the sequence of termination of these
two and respiration.

In a single special experiment with a small dog (5 kilos),
200 c.c. of blood was taken, and an equal quantity of horse .serum
immediately afterwards was transfused to take its place. This
process was repeated three times at intervals of half an hour.
After the fourth withdrawal of fluid, the dog ceased to breathe and
did not recover when the serum was transfused. Aside from
variations in heart action and respiration, there were no special
functional changes until the end, when respiration suddenly ceased,
and the other functions quickly disappeared in the order of the
other experiments. Death was neither preceded nor accompanied
by convulsions.

The more important conclusions of this preliminary research
are :

1 . When the brain is subjected to acute anaemia produced by
the ligature of its arteries, or by the transfusion of indifferent solu-
tions such as physiological saline. Ringer's, Schi'icking's and also
serum, its functions are not mamtained and convulsions ensue ;
but these may be prevented by producing gradual instead of
acute anaemia.

2. In gradual ancemia of the brain, as induced in these experi-
ments, the following functions cease, usually in this order: (a)
Respiration ; {b) lid reflex ; {c) nose reflex ; {d) heart beat.

Abstracts of Reports. 47

Reprinted from the American Journal of Physiology, 1903, viii ; Proceedings of
the American Physiological Society, p. xiv.

n. FURTHER STUDIES OF THE TOXIC AND ANTITOXIC
EFFECTS OF IONS.

By Jacques Loeb and William J. Gies.

This research was conducted at Wood's HoU during the past
summer. It confirmed Loeb's original observation that each elec-
trolyte in solution at a certain concentration is able to prevent the
development of the Fiuiduhis ^g'g after fertilization, and also to de-
stroy the egg. Our experiments further confirmed the fact that
this poisonous action can, in general, be wholly or partly inhibited
by the addition of a proper amount of another electrolyte.

We also obtained results emphasizing the fact first observed by
Loeb, and furnishing new evidence to show that the degree of
antitoxic influence exerted by the second electrolyte increases with
the valency of the cation. The antitoxic action of bivalent cations
was found to be very much greater than that of univalent cations ; the
antagonistic power of trivalent cations is considerably greater than
that of the bivalent. This rule does not hold with all cations,
however ; such cations as Cu, Hg and Cd are exceptions.

Our experiments made it very apparent that the antitoxic ac-
tion of the salts employed was not due to slight amounts of H or
OH ions in their dissociated solutions, since neither solutions of
pure acids nor of pure alkalies were able to exert such an-
tagonism.

It was found, finally, that solutions of non-electrolytes, e. g.,
urea, cane-sugar, glycerin, alcohol, have no antitoxic influence ex-
cept under conditions which favor the formation of less soluble or
less dissociable compounds with the electrolyte (such as saccharate),
whereby the concentration of the toxic ion is considerably
reduced.

Koch's recent investigations on the influence of ions on leci-
thin solutions emphasize the possibility previousl}' suggested by
Loeb, that the observed antagonistic effects of ions may be re-
ferred, in part at least, to changes induced in the physical and per-
haps chemical conditions of substances such as lecithin in the
cell.

48 AliSTKACTS OF REPORTS.

Reprinted from the American Journal of Physiolog)', 1 902, vi ; Proceedings of
the American Physiological Society, p. xxix.

o. ARE PROTEIDS WHICH ARE PREPARED BY THE USUAL
METHODS COMBINED WITH FAT OR FATTY ACID?

BV E. R. POSNER AND WlI.LIAM J. GlK.S.

Chemical analysis of the glucoproteids has resulted in wide
variations in the figures for elementary composition, not onl}- for
bodies from different sources, but for products of similar origin.
Such variation has been attributed to admixture of impurities, par-
ticularly of non-nitrogenous character. Nerking's recent experi-
ments with mucins, ovomucoid and various simple animal and
vegetable proteids indicate that possibly the mucin substances, and
other proteids as they are commonly prepared, are admixed or
combined with fat or fatty acid.

In order thoroughly to test this matter we have analyzed nu-
merous samples of "chemically pure" connective tissue mucoids
and albuminoids. Using Dormeyer's method on quantities of pro-
teid from 2 to 13 grams in weight, and following Nerking's pro-
cedure, our extractive results were always entirely negative.

We are convinced, therefore, that the mucoids and albuminoids
as they are prepared to-day are not " fat-proteid compounds."

Repiinted from the Proceedings of the Section of Biology of the New York
Academy of Sciences: Annals of the New York Academy of Sciences, 1900-1901,
xiii, p. 489; also Science, June 15, 1900, xi, p. 951.

p. SOME CHEMICAL NOTES ON THE COMPOSITION OF
THE COCOANUT.

By J. E. KiKKWooD and \Yili.iam J. Gies.

The authors carried out qualitative work on the ungerminated
nut, preparatory to a study of the digestive processes during ger-
mination. The chief constituents of the endosperm are cellulose
and fat. Some soluble carbohydrate is present, besides globulin
and proteose, but no albumin or pepton. Only amylolytic fer-
ment has so far been found.

The milk of the nut is normally acid ; probably due to acid
phosphate. It contains earthy phosphate, reduces Fehling's solu-

Abstracts of Reports. 49

tion, sours on standing and acquires much of the odor and phys-
ical appearance of soured cows' milk. It shows only small
quantities of proteid and fat.

The " meat" of the average nut contains from 2 to 3 gms. of
globulin, which may be obtained in crystalline form. We have
made three preparations by the usual methods. The nitrogen
averages for these were 17.91 per cent, 17.81 per cent., 17.68
per cent. The ash for the same was o. 1 3 per cent., 0.41 per cent.,
1.05 per cent.

From the "meat" of 12 nuts it was possible to separate a
little more than 3 gms. of proteose by the usual method. The
average of three closely agreeing determinations of nitrogen was
18.57 psr cent. ; of the ash it was 1.7 1 per cent.

The quantitative relationships of these and other constituents
will be subjects of continued investigation.

Dr. Custis drew attention to the irritation of the mucous
membrane of the bladder and urethra caused by drinking too
freely of cocoanut milk. Dr. Gies, in answer to a question,
stated that the content of proteid food-stuff is small.*

Reprinted from the Proceedings (of the Section of Botany) of the American As-
sociation for the Advancement of Science, 1900, p. 275. Also Science, October 19,
1900, xii, p. 585.

q. THE COMPOSITION OF THE ENDOSPERM AND MILK
OF THE COCOANUT.

By J. E. KiRKwooD and William J. Gies.

The analyses reported by the authors are intended to prepare
the way for exact study of the nutritional changes in the germinated
nut.

The milk of the fresh nut is acid to litmus (acid phosphates)
and its specific gravity averages about 1022. It quickly sours on
standing, acidity increasing as fermentation progresses. Its chief
constituents are water, carbohydrates and saline matters. It con-
tains only traces of proteid and fat. General analysis of the milk
gave the following average data : Water, 95.3 per cent.; solids,

* In the abstracts in Science and in the "Annals " this answer was quoted incor-
rectly. See footnote No. i, on page 324 of reprinted paper No. 36.

50 Abstracts of Reports.

4.7 per cent. Of the latter, 88.5 per cent, i.s organic, 11.5 per
cent, is inorganic.

The main bulk of the solid matter in the endosperm consists
of fat and cellulose ("crude fiber"). There is some soluble car-
bohydrate ; a small proportion of globulin and proteose ; at most
only a slight quantity of albumin ; no pepton. The globulin has
been separated in crystalline form (octahedra and hexagonal plates
mostly), and in reactions and composition corresponds closely with
edestin. Its coagulation temperature varies from 66° to 79° C,
with different conditions. The nitrogen content of the purest
preparation made was 17.91 per cent.; the ash, 0.13 per cent.
The proteose we analyzed contained 18.57 P^*" cent, of nitrogen and
1. 7 1 per cent. ash. The fresh endosperm contains 0.75 per cent,
of nitrogen, which, multiplied by the usual factor (6.25), would
correspond to 4.7 per cent. " albuminoid." Some of this nitrogen,
however, is undoubtedly closely associated with the non-proteid
fibrous elements ; much of it, probably, is in the form of nitrogen-
ous extractive. Very active amylolytic ferment is contained in the
endosperm ; no others have yet been found. These and various
other points are .still under investigation. The following figures
represent the average general composition of the endosperm :
Water, 46 per cent.; solids, 54 per cent. Of the latter 98.1 per
cent, is organic and 1.9 per cent, inorganic ; 43.4 per cent, is fat
and 4.3 per cent, is "crude fiber" (cellulose).*

While this work was in progress we accumulated considerable
data on the gross relationships of the main parts. Three
dozen determinations gave the following average weights and
percentages :

Weight of whole nut, 610 grams.

Integument, 170 grams = 27.9 per cent.

Endosperm, 233 grams = 54.5 per cent.

Milk, 107 grams ^ 17.6 per cent.

The volume of the milk averaged 105 c.c.

*The figures given for "crude fiber" in the original abstract were by mistake
those we then had for " carbohydrate " — 12.9 per cent. By a typographical error this
mistake was further emphasized by the figures " 21.9 per cent "

Abstracts of Reports.

5r

Reprinted from the American Journal of Physiology, 1901, v ; Proceedings of the
American Physiological Society, p. xiv.

r. CHANGES IN THE COMPOSITION OF THE COCOANUT
DURING GERMINATION.

By J. E. KiRKWooD and William J. Gies.

The fresh nuts in the husk were placed on earth kept con-
stantly moist at a tropical temperature. After a period of about
four months the shoots appeared through the husk. At the end
of a year of germination chemical examination was begun. At
this time the milk cavity of the ovule was completely filled with
the fully developed cotyledon, which had almost entirely absorbed
the endosperm at the "stem end," and considerably thinned it
posteriorly.

The cotyledon, particularly the central, more vascular portion,
contains considerable diastatic ferment, and apparently, also, .a
trace of proteolytic enzyme. Cellulose-dissolving and fat-splitting
enzymes have, however, not yet been detected. The appended
table presents a few of our analytic results in percentage figures,
showing the distribution of water, solids, inorganic matter, and
nitrogen, from which numerous deductions as to general growth
may be readily drawn :

A.

D.

Roots.

Tips . .

Tips to husk. .

Very near husk.

Inside of husk

Stem. '

' Root crown " .

Petioles ....

Leaves.

Young . .
Old

Cotyledon. '

'Neck"
Cortex . .

'

'Heart" .

Endosperm.

Anterior. .
Posterior .

Ungerminated nut

Endosperm.

Milk

Water.
Per Cent.

89.89
86.41
82.79
77.92

86.21

83-63
74.66
71.99
78.98
80.83
88.99
23.42
46.08
46.00
95-30

Solids. Inorganic

Percent. ! Matter.
; Per Cent.

10. II

13-59

17.21

22.08

13-79

I.

16.37

25-34

28.01

21.02

19.17

^-

II. 01

0.

76.58

0.

53-92

0.

54.00

I.

4.70

0.

Nitrogen.
Per Cent.

0.27

0-53
0.29

0.45

0.31
0.14

0.65
0.75

52 Abstracts of Reports.

B. ABSTRACTS OF REPORTS OF RESEARCHES WHICH

HAVE NOT VET BEEN PUBLISHED IN

GREATER DP:TAIL, PAGES 52-63;

aa — mm.

Reprinted from the American Journal of Physiology, 1901, v ; Proceedings of the
American Physiological Society, p. x.

aa. DOES MUSCLE CONTALX MUCIN?

By G. a. Fried and William J. Gies.

With a view of testing the work which led to disagreement
between Schepilewsky and Goodman, the connective tissue resi-
dues from 3-5 lbs. of beef and veal, prepared by Schepilewsky's
method, were extracted in the usual manner in half saturated lime-
or baryta-water. (Muscle fibers could never be completely re-
moved before the extraction.) Seven such extractions were made
with as many samples of fresh muscle in appropriate quantities
of dilute alkali. On neutralization, and weak acidification, with
0.2 per cent. HCl, a heavy precipitate was obtained in each ex-
tract, but the substance so precipitated quickly dissolved each
time in slight excess of acid (alkali albuminate ?). In this respect
its behavior was very different from that of connective tissue glu-
coproteid. Onl}- a faint turbidity suggested traces of mucin. In
one experiment, in which Goodman's procedure was somewhat
altered, the connective tissue residue obtained by Schepilewsky's
method was treated first with half saturated lime-water, and later
with 5 percent. KOH. On rendering the extract only very faintly
acid a proteid precipitate was obtained in each case. This was
filtered off, purified and analyzed. With another portion of tis-
sue half saturated baryta-water and subsequently 5 per cent.
NaOH were used with the same result. The average nitrogen
content of the ash-free substance obtained from each extract was
as follows :

1. Ca(OH).„ 16. -,9%. KOH, 15.12%.

2. Ba(0H)2, i6.6q%. NaOH, 14.84%.

None of these preparations yielded reducing substance on de-
composition with acid. We are strongly inclined to the belief
that these products are alkali albuminate, or at least are admixed
w'ith the same. They are neither the "stroma substance" of

Abstracts of Reports. 53

Goodman nor the mucin of Schepilewsky. Schepilewsky's method
will not detect very small quantities of mucin.

Reprinted from the American Journal of Physiology, 1902, vi ; Proceedings of the
American Physiological Society, p. xxviii.

bb. A COMPARATIVE STUDY OF THE REACTIONS OF
VARIOUS MUCOIDS.

By L. D. Mead and William J- Gies.

Comparative studies of many of the precipitation reactions of
osseomucoid, chondromucoid and tendomucoid have shown thus
far a very striking sameness in result. Each of these glucopro-
teids also is digested in pepsin-hydrochloric acid, with a forma-
tion of proteoses and peptones, and the separation of nitrogen-
containing substance rich in reducing material, probably chon-
droitin-sulphuric acid or essentially the same body in each case.
The microscopic appearance of the phenylosazone bodies obtained
from each is the same as that of dextrosazone, indicating glucosa-
mine among the products of acid hydration.

All these compound proteids contain sulphur obtainable as
sulphate and as sulphide. They are acid to litmus, neutralize al-
kali, have essentially the same elementary composition and yield
practically the same amount of heat on combustion. In physical
appearance the substances whether dry, freshly precipitated, or in
solution, are practically identical. Attempts to obtain crystalline
mucoid, by the methods which recently have given such fruitful
results in other connections, have thus far been without success.
When the electric current is passed through neutral or alkaline
mucoid solutions (consisting of sodium or calcium salts of mucoids)
turbidity results within a short time and flocks eventually form
and can be filtered off.

Our studies in this general connection have not been com-
pleted. We are convinced, however, that the connective tissue
mucoids are practically identical substances.

54 Abstracts of Reports.

Reprinted from ihe American Journal of Physiology, 1903, viii ; Proceedings of
the American Physiological Society, p. xiii.

cc. FURTHER MUCOID STUDIES.
P)Y William J- Gies.

I. Investigations into the distribution of osseomucoid indicate
that glucoproteid is a normal constituent of all bones. It has
thus far been found in the large bones of wild and domestic mam-
mals and birds, and of reptiles.

II. Connective tissue mucoid shows a tendency to combine
with other proteids. Thus, for example, an alkaline solution of
potassio-mucoid and gelatin yields a precipitate with acid more
promptly than a solution of the equivalent amount of the mucoid
salt alone. Furthermore, the compound precipitate is different
physically. In the case of the gelatin product the precipitate
possesses semi-gelatinous qualities. The compound precipitates
of mucoid obtained from proteid solutions weigh more than the
control mucoid precipitates. This added weight rises, within cer-
tain limits, as the proportion of associated proteid in the solution
increases.

III. Acidification of tissue extracts is not sufficient for com-
plete precipitation of the mucoid. Even with a fifth alkaline ex-
tract of the same tendon pieces, the water-clear acid filtrate from
the precipitated mucoid contains additional glucoproteid.

IV. Precipitated mucoid shows'practically no combining power
with acids. In the hydration of mucoid by pepsin-acid, however,
acid combines with the dissolved proteid products formed in the
process.

V. The blood serum of a rabbit, which had been treated with
several subcutaneous and intraperitoneal injections of neutral solu-
tion of potassio-mucoid, produced precipitates in neutral and very
slightly acid solutions of the latter proteid compound.

These researches are still in progress with the cooperation of
Messrs. E. R. Posner, C. Seifert and H. G. Baumgard.

Abstracts of Reports. 55

Reprinted from the Proceedings of the Society for Experimental Biology and
Medicine: Science, 1903, xvii, p. 469 ; also American Medicine, 1903, v, p, 70S.

dd. PROPERTIES OF "PENCE JONES' PODY."
By William J. Gies.

Through the kindness of Dr. Meltzer a patient's urine contain-
ing this substance had been placed at our disposal for chemical
study. Some of the results of this investigation were presented
and various properties of the body demonstrated. - Special atten-
tion was drawn to a test of Boston's new method of detecting
" Bence Jones' body" in the urine.

Reprinted from the Proceedings of the Society for Experimental Biology and
Medicine : American Medicine, 1903, v, p. 709 ; also Science, 1903, xvii, p. 742.
ee. A MODIFIED ECK FISTULA, WITH A NOTE ON
ADRENALIN GLYC/EMIA.

Bv A. N. Richards.

A method devised by Vosburgh and Richards for extablishing
communication between the portal vein and the inferior vena cava
of the dog was described and demonstrated. In this method two
cannulas are employed. They are constructed on the same prin-
ciple as the one used by Vosburgh and Richards in collecting blood
from the hepatic and portal veins without interfering with the
normal circulation in those vessels.* After suitable incision through
the abdominal wall a cannula of that type, i cm. long, was inserted
into the portal vein about 2 cm. below the entrance of the pancre-
atico-duodenalis. A second cannula of similar design was intro-
duced into the vena cava at a corresponding point. By connecting
the cannulas with a rubber tube, communication was established
between the two vessels. On ligating the hepatic arteries and the
portal vein at the hilum of the liver, circulation through the liver
ceased and the gland was extirpated.

By the successful use of this method Vosburgh and Richards
have found that the application of adrenalin to the surface of the
pancreas brings about a slight rise in the sugar content of the

* American Journal of Physiology, 1903, ix, p. 43. See Reprint No. 25, p. 43.

56 Abstracts of Reports.

blood even after extirpation of the liver. Their experiments thus
far have covered periods of from two to three hours, no systematic
attempts having }-et been made to get the animals to survive the
operation.

Reprinted from the Medical Record, 1899, Ivi, p. 942. ( I'roceedings of the New
\'ork Pathological Society. )

ff. REPORT OF A CHEMICAL EXAMINATION OF A
KNIFE-GRINDER'S LUNG.

By Eugenk Hodeni'yl,
assisted by allan c. eustis and a. x. richards.

The subject of this report was a knife-grinder, thirty-five years
of age, who had died of pernicious anaemia. The history was
that he had worked at his trade for fifteen years. For the first ten
years he was employed as a grinder and worked in a large room
with some forty others ; for about five years previous to his death
he had worked in a very small and ill-ventilated room at the same
occupation with some seven others similarly employed. The lungs
presented a maximum degree of pigmentation, and it had, therefore,
occurred to the speaker that it might be instructive to determine the
amount of carbon contained in the lungs and, if possible, the amount
of emery and iron also. Such an investigation seemed especially de-
sirable, since the speaker had been at the time studying the litera-
ture of " Staubinhalation " without finding a single ca.se in which
the amount of carbon had been determined in similar cases of an- •
thracosis, and, moreover, upon inquiring among his colleagues, he
had found that none had the slightest idea as to the amount of
carbon which might reasonably be expected to be obtained from
such a lung. There were many reports in literature, notably those
by Arnold, in which gold and silver had been extracted from the
lungs of artisans working with these metals, but no case had been
observed in which the amount of carbon, emery and iron had been
determined in the lungs of knife-grinders.

The technique employed was to digest the lung, which
weighed 900 gms., and then obtain the charcoal, emery and
iron by precipitation. The lung was cut into small pieces, placed

Abstracts of Reports. 57

in a little water, to which was added two ounces of Johnson's
preparation of papoid and enough hydrochloric acid to give a
reaction of free acid in the solution. This mixture was kept at
a temperature of 40° C. for ten days, when the lung became
completely liquefied. It was then necessary, on account of the
viscidity of the mass, to add large quantities of water, in order to
secure precipitation. About sixty gallons of water was added,
and this mixture was allowed to stand in a number of tall jars
for many days until precipitation was complete. The precipi-
tate was then repeatedly washed in water until it was believed
that all of the substance soluble in water had been removed. It
was then evaporated to dryness and powdered. At this juncture,
Mr. Allan C. Eustis and Mr. A. N. Richards, assistants in the
department of physiological chemistry of the Columbia University,
kindly undertook the chemical examination, and the speaker took
this opportunity of extending his thanks to these gentlemen for
the very complete analysis which they had made.

Analysis of lung taken from the body of a knife-grinder :

Total weight of lung dried and powdered, 48.1009 gms. Total
solids, 44.7986 gms. ; water, 3.3023 gms.

Soluble in ether, 14.6017 gms. ; insoluble in ether, 30. 1969 gms.

Composition of the portion which was soluble in ether : Free
fatty acids, 7.498 gms. ; neutral fats, 4.044 gms. ; cholesterin, 3.037
gms. (lecithins ?).

Composition of portion insoluble in ether : Proteids, melanins,
etc. (total nitrogen x 6.25), 15.4759 g"^s. ; charcoal (total carbon —
proteid carbon), 7.1989 gms. ; ash, 4.2909 gms.

Composition of ash : K^O, 0.2167 gm. ; Na.,0, 0.3523 gm. •
CaO, 0.0965 gm. ; Fe.Og, 0.0879 g'^-', AiPg, 1.4628 gm. ; SO3,
0.0704 gm. ; pp., 0.9565 gm. ; SiO^, 1.20434 gm.

Dr. Hodenpyl said that, on first receiving this report, he had
been somewhat disappointed that the amount of carbon was not
greater, but since then he had made some simple experiments which
demonstrated that, after all, 7 gm. + of this particular charcoal was
really an enormous amount to be obtained in a lung. It is to be
remembered that this charcoal was in an exceedingly fine state of
subdivision. Thus, on mixing o. i gm. of very finely powdered
animal charcoal in 500 c.c. of water, the fluid was only very slightly

58 Abstracts of Reports.

darkened. One tenth of a gram of the precipitate from the lung,
dissolved in 500 c.c. of water, made the fluid almost jet black in
color, even though of this precipitate, o. i gm. represented only
about ^^ gm. of carbon. Again, it will be seen that about one fourth
of the ash was in the form of an oxide of iron. The amount of
emer>' was represented by oxide of aluminium and oxide of silicon.
These two together made up about 2.5 + gm., so that considerably
over one-half of the ash was in the form of emery, and the emery
and iron together made up more than three fourths of the total
amount of the ash.

Dr. Prudden remarked that more than a barrel of water had
been made as black as ink by the pigment contained in the lungs
of this person. The investigation had an obvious and important
bearing on infection through the lung, because it showed how
many particles might pass all the safeguards which the air pas.sages
present.

Reprinted from the American Journal of Physiology, 1902, vi ; Proceedings of the
American Physiological Society, p. xxix.

gg. OX THE TOXICOLOGY OF SELENIUM AND ITS
COMPOUNDS.

By I. O. Woodruff and William J. GiEs.

The researches of Tunniclifie and Rosenheim indicate that the
numerous cases of "arsenical poisoning " in England recently may
have been due in part to selenium. Through the kindness of Pro-
fessor Victor Lenher our studies are being made with absolutely
chemically pure preparations. Thus far our results on dogs con-
firm most of the general observations of Rabuteau, and of Czapek
and Weil. We are unable, however, to discover Rabuteau 's
crystals in the blood of the heart after death, or to agree with him
that death results from mechanical interference with the circulation.

Selenium is very much more toxic than tellurium, although its
poisonous effects are qualitatively much the same. The expired
methyl compound of selenium is produced in much less quantity
than that of tellurium under similar conditions. Injection of four
milligrams of selenite or selenate per kilo under the skin of dogs
usually results in death in a few minutes. Speedy death follows

Abstracts of Reports. 59

the introduction of like amounts per os or rectum. Four grams
of the finely powdered metal, when taken into the stomach, mani-
fested no toxicity whatever, and passed out in the faeces. The
introduction of soluble salts is quickly followed by elimination of
selenium in the urine and the breath. After subcutaneous injec-
tions, the distribution of selenium to the organs is similar to that
found by us recently for tellurium. Selenium, although chem-
ically related to sulphur, is very much like arsenic in its toxic
properties.

Reprinted from the American Journal of Physiology, 1903, ix ; Proceedings of the
American Physiological Society, p. xvi.

hh. THE INFLUENCE OF CHINTC ACID ON THE ELTMINA-
TION OF URIC ACID.

Bv W. A. Taltavall and William J. Gies.
Our work thus far has shown that the uric acid output in the
urine of dogs is not materially affected by the administration of
chinic acid. We observed only a slight lowering of the small
amounts of uric acid present in the urine to begin with. This re-
sult was obtained when the animal was in approximate nitrogenous
equilibrium on a mixed diet consisting of hashed meat, cracker
meal, lard, bone ash and water, and after daily doses, for ten days,
of chinic acid in amounts var}''ing from i to 20 grams. These re-
sults were obtained before the recent publication of the data of
Hupfer's experiments on himself. They agree with this observer's
conclusions that the therapeutic deductions of Weiss, Blumenthal
and others, in this connection, are without foundation.

Reprinted from the American Journal of Physiology, 1900, iii ; Proceedings of
the American Physiological Society, p. xxxi.

ii. THE PROPORTION OF BASIC NITROGEN YIELDED BY
ELASTIN ON DECOMPOSITION WITH HYDRO-
CHLORIC ACID.

Bv R. H. Chittenden (for Allan C. Eustis).

The lack of agreement between Bergh and Hedin, and Kos-
sel and Kutscher in their study of the basic cleavage products of
elastin led us to a study of the proportion of basic nitrogen split

6o Abstracts of Reports.

off from pure elastin by boiling for lOO hours with 20 per cent.
HCl and stannous chloride. Following the method adopted by
E. Schulze, and determining the total nitrogen in the solution, the
nitrogen in the form of ammonia, and the nitrogen in the phos-
photungstic acid-precipitate, we have obtained very divergent re-
sults. In all, five distinct experiments were tried with the follow-
ing results :

Percentage uf Nitrogen in
Experiment. form of organic bases.

1 0.86

II 17.69

111 15.57

IV 6.50

V 15-14

Our results led us to the conclusion that the method now in
use for the separation of the hexone bases by phosphotungstic
acid, and determination of the nitrogen therein, is unreliable for
quantitative purpo.ses, and that consequently results hitherto ob-
tained by this method must be accepted with caution.

Reprinted from the American Journal of Physiology, 1903, viii ; Proceedings of
the .\merican Physiological Society, p. xv.

jj. A PROTEID RE.\CTION INVOLVING THE USE OF

CHROMATE.

Bv William J. Gif.s.

Several years ago, during a comparative study of the reactions
of various gelatins, the results of which have not yet been pub-
lished, it was observed by Dr. D. H. M. Gillespie and myself that
dilute solutions of potassium chromate did not precipitate gelatin
solutions, but that when such prpteid chromate mi.xtures were
further treated with acid, a fine yellow flocculent precipitate formed
at once. Acids as " weak " as acetic, and also the common min-
eral acids, effected the result, the latter acids more promptly, how-
ever, even in smaller amount.

At intervals I have returned to this reaction, and lately have
made a more careful study of it. Solutions of chromates of mono-
and divalent cations (the only ones thus far employed) cause no
precipitates in neutral or alkaline proteid fluids, but on further

Abstracts of Reports. 6 1

treatment with small amounts of dilute acids — strongly dissoci-
able ones particularly — flocculent precipitation of a proteid-chro-
mate compound occurs in every case. The reaction is especially
striking with such bodies as gelatin and proteose (the precipitates
with these disappearing on warming and reappearing on cooling),
and it seems to be more' delicate than the acetic acid and potas-
sium ferrocyanide test. Salts containing dichromion or trichrom-
ion behave differently.

Since bichromate is formed from chromate on the addition of
acid, it might be supposed that such production is responsible for
the precipitation observed. But bichromate solutions are as inert
as the chromate. When, however, acid is added to a mixture of
proteid and bichromate, precipitation occurs, as in the case with
chromate. Hydroxidion prevents the reaction in all cases. Pos-
sibly the precipitation is due to the formation of dichromic acid,
just as in the acetic acid and potassium ferrocyanide test it is de-
pendent on the formation of hydroferrocyanic acid.

Further study is expected to determine exactly the character
of the ions responsible for the reaction. The results thus far
point to dichromanion in the presence of hydrion.

Reprinted from the American Journal of Physiology, 1 903, viii ; Proceedings of
the American Physiological- Society, p. xxxiv.

kk. THE INFLUENCE OF THE H ION IN PEPTIC
PROTEOLYSIS.

By William J. Gies.

The fact that pepsin shows digestive power only when acid is
present implies the dependence of the enzyme upon hydrion for
its activity. It has frequently been observed that various acids are
efficacious in this connection, though in different degrees.

In some recent experiments on the influence of acidity, I have
used purified fibrin, edestin and elastin as the indicators. Undi-
gested residue, neutralization precipitate and uncoagulable prod-
ucts were determined quantitatively in each digestive mixture.
Various common mineral and organic acids were employed. Vary-
ing proportions of pepsin and acid were taken in uniform volumes
(100 c.c), with the same amount of proteid (i gm.). In cquiper-

62 Abstracts of Repcikts.

ccutagc solutions of acids whose anions have no precipitative effect
on proteid, the relative proteolysis is very different, being greatest
in " strong " acids such as HCl and least in "weak" acids, such
as CH;,.COOH. Eqiiinwlar solutions of the same acids gave
more concordant results in some respects, although the differences
between the effects in such acids as HQ and CH3.COOH were
still very wide. With cquihydnc solutions, the results showed
greater harmony, though there were still striking divergences.
H3PO,, HCl, HNO3, HCIO3, H^AsO^ and (COOH).,, in strengths
equivalent to decinormal KOH (with 50 mgm. of pepsin prepara-
tion, in 100 c.c. at 40°C., four hours), showed practically the same
ability to assist pepsin in the digestion of i gm. of fibrin.

Additional experiments, especially with cqiiidissociatcd solutions
of the acids referred to above, are expected to show the influence
not only of hydrion, but also of the anions, if the influence of the
latter in the acids referred to be appreciable. Similar experiments
are about to be extended to other enzymes.

Reprinted from the Proceedings of the Society for Experimental Biology and
Medicine : Science, 1903, xvii, p. 469 ; also, American Medicine, 1903, v, p. 708.

11. AN IMPROVED CAGE FOR METABOLISM
EXPERIMENTS.

Bv \Vl 1,1,1AM J. GlES.

A cage specially designed for experiments on dogs was shown.
The parts are so adjusted as to favor the collection and separation
of feces, urine and hair. The improvements consist mainly of me-
chanical devices suggested by experimental experiences of the
past few years in metabolism work, all of which are designed to
ensure quantitative accuracy as well as comparative convenience
in the collection of excreta.

Reprinted from the American Journal of Physiology, 1903, ix ; Proceedings of the
American Physiological Society, p. xvii.

mm. PEPTIC PROTEOLYSIS IN ACID SOLUTIONS
OF EQUAL CONDUCTIVITY.

Bv William J. Gies.

Numerous digestive experiments with various equidissociated
acids, and with fibrin as the indicator, have invariably given re-

Abstracts of Reports. 63

suits lacking quantitative agreement. Undigested residue, neu-
tralization precipitate, and uncoagulable products were determined
gravimetrically. With all conditions exactly the same for each
mixture in a series, except the character of the acid, the digestive
products differed not only in the rate of their formation, but also
in their amounts. The digestive results were particularly discor-
dant in mixtures containing relatively small amounts of pepsin act-
ing for comparatively short periods of time. That the anions
greatly modified the action of the common cation seems certain,
the SO^ anion being especially antagonistic in its influence.

The temperature of the digestive mixtures in each experiment
was kept steadily at 25 °C. The acids used thus far were of the
same conductivity as a 0.2 per cent, solution of hydrochloric acid.

I am much indebted to Mr. C. W. Kanolt, of the Department
of Physical Chemistry of Columbia University, not only for the
acid solutions already used, but for others about to be employed
in additional experiments.

PAPERS

REPRINTED FROM THE FOLLOWING JOURNALS:

Journal of Experimental Medicine, 1896 — 13.*
American Journal of Physiology, 1898- 1903 — i, 3, 4, 5, 6, 7,
5, 9, II, 16, 21, 24, 25, 26, 30, 34, 35.
Yale Scientific Monthly, 1898 — 39.

Archives of Neurology and Psychopathology, 1899 — 12, 15.
Zeitschrift fiir Biologic, 1900 — 19.

American Medicine, 1901-1903 — 2, 17, 18, 28, 32, 33.
Medical News, 1901-1902 — 10, 23.

Reference Handbook of the Medical Sciences, [901 — 14.
Philadelphia Medical Journal, 1901 — 20.
Medical Record, 1901 — 27, 31.
Therapeutic Monthly, 1902 — 22.
Archiv fiir die gesammte Physiologic, 1902 — 29.
Bulletin of the Torrey Botanical Club, 1 902-1903 — 36, 37.
Journal of the New York Botanical Garden, 1902— 1903 — 38, 40.

* The numerals following the titles of the journals correspond with those before the
titles of the papers listed on pages 26, 27 and 28.

A. CHEMICAL INVESTIGATIONS OF ANIMAL TIS-
SUES AND TISSUE CONSTITUENTS.

Reprints, Nos. 1-15.

67

Reprinted from the American Journal of Physiology. I

Vol. V. — May i, 1901. — No. IV.

AN IMPROVED METHOD OF PREPARING AND

PRESERVING MEAT FOR USE IN

METABOLISM EXPERIMENTS.

By WILLIAM J. GIES.

[From the Laboratory of Physiological Chemistry, of Columbia University, at the College

of Physicians and Surgeons, Netv York.]

THE chemical problems in metabolism experiments are as diffi-
cult as they are numerous. Not only must the excreta be
analyzed in detail, but, in work of the highest value, the composition
of the food must also be definitely ascertained. Usually, the purely
analytic labor involved in studies of this character is so great that
important phases of the experiments have to be ignored or left for
subsequent special investigation. Methods of the greatest simplicity,
which are easily carried out in the shortest time and with the highest
degree of accuracy, are naturally the first to be selected. Conse-
quently, any improvements of acceptable methods, which increase their
adaptability in any one of these particulars, are to be welcomed.

The process the author has lately been em.ploying to prepare pro-
teid food in bulk for experiments on dogs includes a few improve-
ments which make it perfectly adapted to metabolism work, and
which, besides, lessen considerably the analytic and mechanical labor
involved.

In the method referred to, the fresh lean beef, after all loose fat
and connective tissue has been removed, and tendonous layers excised,
is put through a meat-chopper. The hash thus obtained is then
divided into portions of convenient bulk, and each portion is enclosed
in cheese-cloth bags, and submitted to increasing pressure^ as long
as bloody fluid accumulates. Three to four hours are usually suffi-
cient for getting rid of all fluid that can be separated. The com-
pressed masses thus obtained may be kept under moistened cloth to
prevent the surfaces from drying during the pressing of the remaining
portions. Too much hash in the press makes thorough removal of
surplus fluid impossible. In preparing about 50 kilos of the meat,

1 The ordinary " meat press," employed for various purposes, such as the pre-
paration of tinctures from herbs, etc., serves very well.

23s

236 William J. Gics.

the author has found it convenient to press 6 to 10 kilos at a time.
The size of the press in use would, however, naturally determine the
amount of the hash to be pressed at one time.

The compact cakes are next broken in a large dish, intimately
mixed by thorough kneading, and then very small quantities, picked
out here and there all through the mass, are transferred directly to
capacious tubes, weighed and analyzed.^ Thus far we have not had
occasion to make other than nitrogen determinations in the meat pre-
pared in this manner. Excellent results were obtained with 2 to
3 grams for each analysis, although larger quantities may readily
be utilized, perhaps with even greater accuracy.

Simultaneously with the sampling of the hash for analysis it should
be quickly rolled between the hands into balls weighing about 50 to
100 grams. These are dropped lightly into wide-mouthed bottles of a
capacity sufficient to hold five or six of the balls. The latter are not to
be pressed together, but ought to rest very lightly on each other.
The bottles are then promptly sealed and placed in a cold-storage
room, where the temperature is maintained at or below 0° C. The
meat-balls quickly solidify, and in the frozen condition can, of course,
be kept indefinitely. After the balls are frozen there is usually a very
light and delicate film of frost on the inside walls of the bottle, in
places, indicating naturally that only a very slight quantity of water
leaves the meat during the interval before the frozen state is reached.
Under these conditions there is never sufficient movement of fluid
to result in the formation of ice at the bottom. If, however, the
frozen condition is not reached within a few hours, and maintained,
bloody fluid is certain to trickle slowly to the bottom, in spite of the
preliminary removal by pressure, thus changing the composition of
the substance throughout the entire mass.

The hash prepared and kept in this way retains its normal appear-
ance, odor, and taste for a very long time. If the bottles are small,
containing little more than enough for one, or at most two days'
feeding, practically no change can take place while material is being
withdrawn, if this be done quickly. The globular form is of particular

1 If the tubes are weiglied after they have been thoroughly dried at room tem-
perature, and before the hash is put into them, any interior condensation of water
from the meat would be included, as it should be, with the weight [by difference]
of prepared substance. This procedure would serve very well for nearly all of the
analyses commonly made. The hash should, of course, be completely removed
from the weighing tube in each determination.

Preparing Meats for use in Metabolism Experiments. 237

advantage, in this connection, because it makes the removal of the
meat, even in the frozen condition, very easy. When it is desired
to take out meat for use, the bottles need to be kept at room tem-
perature for only a few minutes before the delicate icy connections
between the balls have thawed sufficiently to permit of easy with-
drawal. Special thawing of the contents in bulk, in order to take
out a sufficient supply of meat, is unnecessary. The balls remain-
ing after each removal may be speedily returned to the cold-room
without undergoing any change to speak of. The weighing, after
removal, may be made very accurate by shaving off sufficient from
an additional ball to give the desired quantity.

After the weighed meat has been placed in the feeding-dish, the
hash soon softens and is ready for ingestion in a few minutes. Its
treatment after removal from the bottle must naturally depend upon
the requirements of the experiment in which it is to be used. In
the researches in this laboratory on dogs in nitrogenous equilibrium,
the meat has been weighed in a common glass crystallization dish.^
in which were also placed definite quantities of cracker dust and
lard, with subsequent addition of given proportions of water. On
thoroughly stirring this mixture, the balls quickly fall apart, and, if
the quantity of water is not excessive, the fluid finally has the con-
sistency of thick soup. The odor of fresh meat is predominant
when the cracker dust and lard are not too great in amount. Gentle
warming suffices to raise the mixture to the ordinary temperature.
It may be added that dogs eat this mixture very readily for weeks.
Further, it is very digestible and nutritious.

To answer the question whether any important changes in the
chemical composition of the meat take place during prolonged periods
of preservation, the nitrogen content was determined in two samples
of each of several preparations, at intervals of about ten days, for
several weeks, with the results tabulated below.''^

The analytic data obtained not only show the general uniformity in
composition of meat preserved in this way, but demonstrate, like-
wise, that no important chemical alteration takes place at any time

1 In shape the common glass crystallization dish is very well adapted to the
licking up of last portions. Because of its transparency the operator can also
easily bring together to the centre the fine particles which the animal missed at
first, thus favoring final ingestion of the entire meal.

2 The analyses were made by the Kjeldahl method. The quantities of hash
used varied from 2.1362 to 3.3550 grams.

238

William J. Gics. ^

during the period of preservation, if the proper precautions are
observed. The unimportant fluctuations in nitrogen percentages in
the table are all within the limits of unavoidable error of analysis.
The average percentages emphasize the fact of perfect uniformity
throughout.

Percentages of Nitrogen.

Preparation.
No.

Before freezing.

After freezing.

At time of
preparation.

10 days.

21 days.

30 days."

1

3.58
3.49

3.56
3.51

• 3.57
3.45

3.58
3.57

2

3.60

3.55

3.58
3.46

3.69
3.59

....

3

3.58
3.67

3.60
3.59

3.64

3.58

3.59
3.67

4

3.69
3.73

....

3.70
3.75

3.64
3.68

Averages.

1

2
3
4

3.53
3.57
3.62
3.71

3.-53
3.52
3.59

3.51
3.64
3.61

3.72

3.57

3.63
3.66

It may be suggested that the use of this method is impracticable
where special cold-storage facilities are lacking. It can be said, how-
ever, in anticipation of such a conclusion, that practically the same
satisfactory preservative results could be obtained, although with less
convenience, of course, if the bottles were placed in an ordinary
refrigerator and surrounded each day with the common freezing
mixture of crushed ice and salt. Melting of the ice would not be
very rapid, under these conditions, and it could be renewed at
little expense whenever necessary.

The chief advantages gained by the use of nitrogenous food
material prepared by the method just described are: —

Preparing Meats for use in Metabolism Experiments. 239

1. The perfect freshness of the food at the time of its consump-
tion, even weeks after its preparation ; therefore, its similarity in
appearance, odor, and taste to ordinary fresh meat, and its superiority
to forms of nitrogenous food to which the animal is unaccustomed, or
for which it has no desire.

2. The constancy of composition of the food throughout even the
longest experiments, by which circumstance the labor of analysis is
reduced to a minimum.

This method is therefore especially useful in metabolism experi-
ments on dogs.

Reprinted from "American Medicine," Vol. II, No. 21,
page 820, November 23, 1901.

A NEW CONSTITUENT OF BONE.

BY

WILLIAM J. GIES, M.S., Ph.D.,

of New York.

Instructor of Physiologic Chemistry in the College of Physicians and
Surgeons.

Early in the last century (1838) Johannes Miiller was
the first to observe that when hyaline cartilage is boiled
in water a product is formed which closely resembles
gelatin, physically and chemically. Miiller gave the
name " chondrin " to the cartilage jelly formed in this
way. Marchand, a few years later, applied the term
"chondrigen" to the antecedent substance in the tissue
which on boiling was transformed into "chondrin."
For many years "chondrin" and " chondrigen " were
looked upon as distinct and definite chemic substances,
and numerous deductions regarding connective tissue
relationships were based upon this assumption.

About a decade after Miiller' s discovery, Hoppe-
Seyler, in a study of their decomposition products,
showed that these proteid materials were not as nearly
related to gelatin and collagen as had been inferred.
Subsequently, Bodecker and others found that a reducing
substance could be separated from "chondrin." Eich-
wald and Obolensky, about the same time, obtained sim-
ilar reducing bodies from various mucoids.

This coincidence led von Mering in 1873, under
Hoppe-Seyler's direction, to make a search for mucoid
in cartilage. He identified it in aqueous extracts of the
tissue by the acetic acid method. Three years later,
Morochowetz, under Kiihne's direction, made more
extended experiments in this connection and demon-
strated that "chondrin" is a mixture — containing gela-
tin, mucoid and inorganic matter. Morner has lately
shown that cartilage contains collagen, albumoid (elas-
tin ?), chondromucoid and chondroitin sulphuric acid, in
considerable quantity, and that "chondrin" is a mix-
ture of gelatin, chondromucoid, chondroitin sulphuric
acid and soluble salts.

We now know that mucoids are normally present, in

small quantity at least, not only in cartilage, but all
forms of connective tissue, although for a long time
this fact was not appreciated. The author has lately
shown the presence of mucoid in bone, thus establishing
closer chemic relationship between mature bone and
cartilage than had been supposed to exist, and demon-
strating, further, that, as far as mucoid content is con-
cerned, osseous tissue is not an exception among connec-
tive tissues, as previously it seemed to be.

In referring to Morochowetz's discovery that " chon-
drin " is a mixture containing mucoid, Drechsel, in 1883,
wrote as follows : " If chonclrin is in reality gelatin and
mucin the transformation of cartilage into true bone is all
the more easily comprehended, for in that case such devel-
opment would consist essentially in only the elimination
of the mucoid constituent." The deposition of inorganic
matter in addition is, of course, to be understood.

For years it has been said that cartilage would yield
"chondrin," but that true bone would not. The views
of Hofmann, expressed in 1875, are representative of
those held in this connection until very recently. He
stated that " chondrin may be obtained from bone before
ossification, but ossified bone yields only gelatin. . . .
Embryonic bones contain no collagen, but do contain
chondrigen, which is not transformed into the first-
named, but before ossification is displaced by it. Com-
pletely calcified bone does not contain even a trace of
chondrigen." Until the author's work was begun it had
been generally accepted that o&seous tissue does not con-
tain glucoproteid. An examination of the statements in
recent textbooks on the chemic qualities of bone shows
that the pressure of mucoid is either denied or the ques-
tion ignored.

The later and more prominent experimental results
repeatedly given as authority for the statement that
mature compact bone does not contain mucoid, have led
to inaccurate conclusions. Von Ebner, in 1887, indi-
cated that the decussating fibers of Sharpey are similar
to those in fibrous connective tissue in general, and that
they ai-e not calcified, but that the calcareous deposit in
bone is confined to the interfibrillar areas. These obser-
vations led Young ^ to investigate the question whether
the matrix, in which the fibers of the bone structure are
embedded, "is completely calcified or not." He con-

1 Young: The Journal of Physiology (English), 1892, Vol. xill,
p. 803.

eluded that this question could be most readily solved
by ascertaining whether mucin, " the most abundant
constituent of the uncalcifled matrix or ground substance
of connective tissue, is present or absent." Working
under Halliburton's superintendence. Young failed to
extract from bone, with lime-water or dilute baryta-
water, any substance that could be precipitated with
acetic acid. He concluded, because of this seeming
absence of glucoproteid from compact bone, that " in the
process of ossification the connective tissue matrix is
apparently completely calcified."

Unfortunately this important conclusion was brought
about by three very obvious defects of procedure. In
the first place. Young employed too much alkaline
extractive fluid in proportion to the amount of bone
taken in his experiments, thus making it exceedingly
difiicult to detect any existent mucoid. Again, the
absolute quantities of bone extracted were so small that
no positive result could reasonably have been expected.

The chief objection, however, to the method Young
employed was the direct application of dilute lime or
baryta-water to a dense, compact tissue, thoroughly
impregnated with salts which for the most part are insol-
uble in such medium. It is not dilficult to understand
how, in the case of the femur, for example, the stone-
like structure of the compact portion, composed as it is
largely of tribasic earthy phosphates, imposed a serious
obstacle to the usual action of lime-water on contained
mucoid substance, and therefore it is natural to assume
that for this reason, if for no other, no glucoproteid was
detectable in Young's experiments. Certainly, removal
of the salts from bone is the necessary preliminary to
extraction in dilute alkali, if any hope is to be entertained
of finding mucoid in that tissue.

The several diflficulties just alluded to have been
overcome by very ordinary means, and the author has
succeeded in obtaining a surprisingly large yield of
mucoid from both the femur and the rib of the ox by the
following general method :

After the fresh bones had been thoroughly freed of
adherent muscle and connective tissue, they were kept
in 0.2 to 0.5 fo hydrochloric acid for the removal of inor-
ganic matter. In the course of a few hours the dilute
acid took out the salts from the surface of the bones just
as satisfactorily, although not as rapidly as stronger acid
would have removed it. After this treatment the bones
were scraped twice daily with a stout, well-sharpened

scalpel. The superficial decalcified layer was thus easily
removed in long, narrow, thin, elastic shavings, very
soft and pliable. The dilute acid was completely renewed
after each scraping. The ossein obtained in the first two
scrapings was thrown away, for fear it was contaminated
with minute particles of superficial connective tissue
elements belonging to the periosteum, which might not
have been completely removed in the preliminary treat-
ment. While the shavings accumulated they were kept
in dilute alcohol to prevent putrefactive changes. As
much as six to seven kilos of moist shavings were used
at one time. The shavings were next run through a
meat-chopper, and the resultant hash thoroughly washed
free of alcohol and acid by decantation in distilled water.
Finally the bulky ossein hash was transferred to several
large bottles and repeatedly shaken at intervals for about
48 hours, with moderate excess of half-saturated lime-
water. On strongly acidifying the filtered extract with
0.2^ hydrochloric acid, a bulky flocculent precipitate
rapidly separated. This was purified by the process of
washing, reprecipitating, etc., usually employed for final
preparation of pure glucoproteids.

This newly-discovered substance, osseomucoid, is
practically the same as the mucoid in tendon, cartilage
and other connective tissues. It not only responds to the
general proteid tests, but appears to have the same solu-
bilities and precipitative reaction as the other connective
tissue mucoids, and yields the same large proportion of
reducing substance on decomposition with mineral acids.
Furthermore, the combustion equivalents of osseo-
mucoid, chondromucoid and tendomucoid, as shown in
the table below, are practically identical, indicating
close chemic relationship of these glucoproteid products.^

The average composition of four purified preparations
of osseomucoid is given below, where comparison may
also be made with the elementary composition of similar

products :

Combustion

c. H. N. s. o. equivalent.

Osseomucoid 47.07 6.69 11.98 2.41 31.85 4,992c.

Chondromucoid... 47.30 6.42 12..58 2.42 31.28 4,88.3c.

Tendomucoid 47.47 6.68 12.58 2.20 31.07 4,%7c.

Average 47.28 6.60 12.38 2.34 31.40 4,947c.

These variations are quite within the limits of unavoidable errors
of analysis. In the analytic work the author received the able assistr
ance of his colleague, Mr. P. B. Hawk.

1 More detailed reference to the method of preparation and the
chemic qualities of this substance was made in a recent number of the
American Journal of Physiology : 1901, Vol. v, p. 387.

This discovery makes it evident that ordinary com-
pact bone, like the other forms of connective tissue, con-
tains mucin substance, and also, contrary to Young's
deduction, that in the process of ossification, the connec-
tive tissue matrix is not completely removed. Further,
it makes it easier to understand the accumulation of
mucoid in various pathologic formations in osseous tis-
sue which numerous observers, in recent years, have
shown may often be considerable in amount.

The influence of disordered metabolism of this
mucoid substance on the development of various bone
tumors, particularly of the myxomatous type, can only
be guessed, at present, but may prove to be more pro-
nounced than the writer now supposes. Our knowledge
of mucoid degeneration, not only in bone, but also in
other tissues, will doubtless greatly advance as we learn
more definitely the chemic phases of glucoproteid syn-
thesis under normal conditions, and as we come to an
understanding of the functions in the tissues of the various
forms of these peculiar substances.

Reprinted from the American Journal of Physiology.

Vol. V. — July i, 1901. — No. VI.

CHEMICAL STUDIES OF OSSEOMUCOID, WITH DETER-
MINATIONS OF THE HEAT OF COMBUSTION OF
SOME CONNECTIVE TISSUE GLUCOPROTEIDS.

By p. B. hawk and WILLIAM J. GIES.

[Frof?i the Laboratory of Physiological Chemistry, of Columbia University, at the College
of Physicians a?id Surgeons, New Yorh.]

CONTENTS.

Page

I. Preparation of osseomucoid 387

Historical 387

Method of preparation 393

Discussion of modifying factors 396

Properties of osseomucoid 399

II. Composition of osseomucoid 402

Methods of analysis 403

Records of analysis 404

Summaries and discussion of analytic results 412

III. Heat of combustion of osseomucoid, tendon mucin, and chondromucoid . . 417

Historical 417

Method of determination 419

Experimental results 421

Discussion of data 421

IV. Summary of conclusions 424

I. Preparation of Osseomucoid.'

HISTORICAL.

TT seems to have become generally accepted that osseous tissue
■*- does not contain glucoproteid. A study of the statements in
the recent text-books, regarding the composition of bone, reveals the
fact that either the existence of mucoid in bone structure proper
is directly denied or else that nothing whatever is said as to its
possible presence. The marrow of bone, however, has repeatedly
been said to contain mucin, although reference to the sources of the
information usually given in this connection shows that very little

^ GiES : Proceedings of the American Physiological Society (New Haven
meeting, December, 1899); This journal, 1900, iii, p. vii. Also, Gies : Proceed-
ings of the American Association for the Advancement of Science (New York
meeting, June, 1900), [900, p. 131. See foot-note, p. 402, for reference to subse-
quent report.

387

3S8 p. B. Haiuk and William J. Gies.

work has been done to ascertain the facts, and that the results of that
work are anything but conclusive.

Neumeister^ states, in this connection, that "neither mucin, nor
any body belonging to the glucoproteids, has ever been detected in
osseous tissue, although fibrous connective tissue and cartilage do
contain such substance." Referring to ossein, prepared in the usual
manner, Gautier- writes: "It does not yield glucose (reducing
substance) after prolonged boiling in dilute acid." " The absence of
mucin in compact bone is noteworthy," says Halliburton,'^ " showing
that the ground substance is entirely replaced by calcareous matter.
Marrow, however, yields mucin." Hammarsten ^ gives considerable
attention to the composition of bone, but ignores this phase of the
subject altogether."

Morochowetz,'' in 1876, called attention to the fact that the so-
called "chondrin" or "cartilage jelly" of the older writers was in
reality a mixture of substances. Morochowetz stated that it con-
sisted of gelatin and mucin. Drechsel,' referring a few years ago
to Morochowetz's deductions in this regard, wrote as follows: "If
chondrin is in reality gelatin -|- mucin, the transformation of carti-
lage into true bone is all the more easily comprehended, for in that
case such development would consist essentially in only the elimi-
nation of the mucigenous constituent." The deposition of inorganic
matter in addition is, of course, to be understood.

For years it was said that cartilage would yield chondrin, but that
true bone would not. The views of Hofmann^ are representative
of those held for a long time. He stated that "chondrin may be
obtained from bone before ossification, but ossified bone yields only
gelatin." At another place Hofmann writes:^ "Embryonic bones
contain no collagen but do contain chondrigen, which is not trans-
formed into the first-named, but before ossification is displaced by
it. Completely calcified bone does not contain even a trace of

' Neumeister : Lehrbuch der physiologischen Chemie, 1897, p. 453.

- Gautier : Le9ons de chimie biologique normale et pathologique, 1897, p. 108.

8 Halliburton: Schafer's Text-book of Physiology, 1898, i, p. rii.

* Hammarsten : Lehrbuch der physiologischen Chemie, 1899. p. j^zdet seq.

* See note, p. 400.

•■' Morochowetz: Jahresbericht ubcr die P^ortschritte der Thierchemie, 1877,

P- 37-

" Drechsel: Hermann's Handbuch der Fhysiologie, 1883, Bd. v, Th. i, p. 598.
® Hofmann: Lehrbuch der Zoochemie, 1875-78, p. 25.
^ Hofmann : Ibid., p. 32.

Chemical Studies of Osseomucoid. 389

chondrigen." Morner^ finally showed that cartilage contains chon-
dromucoid (" mucin "), chondroitin sulphuric acid, collagen and
albumoid (elastin?), and that chondrin is composed of the first two
of these and gelatin.

Bone marrow. — Hoyer's ^ histological studies led him to assume
that the ground substance of bone marrow is a loose, soft, mucous
tissue. He did nothing in a chemical way to substantiate this view.
Rustizky,-^ some time later, working with Rexlinghausen and under
Hoppe-Seyler's direction, pointed out the incorrectness of this
inference of Hoyer's, but, nevertheless, claimed to have shown the
presence of a water-soluble mucin in the marrow of the bones of the
rabbit. It was found to be absent from the marrow of the ox. Bone
marrow from other animals was not examined.

It may reasonably be doubted, however, whether Rustizky's work
is entirely reliable, for his deductions were based solely on the reduc-
tion test with alkaline copper solution after acid-decomposition of
acetic acid precipitates, and no assurance was given that reducing
substances were removed before the treatment with acid was begun,
nor, indeed, that the precipitate itself had any proteid qualities other
than precipitability with acetic acid. Further, the positive result
with the rabbit tissue is referred by Rustizky and those who quote
him, to marrow alone, although in Rustizky's experiments, after the
adherent muscle had been removed, the whole bone, including the
periosteum, was finely broken up in a mortar and the inixture
extracted for mucin. It might with good reason, therefore, be
assumed that any mucin really detected came from the periosteum,
or the compact portion, instead of the marrow of the bones of the
rabbit, and that a negative result was obtained with the ox marrow
because the latter had been previously removed from the bone and,
as Rustizky states, treated separately.

The question should still be regarded as an open one.. Since
Rustizky's time no results have been reported bearing on this sub-
ject. The author hopes to complete, in the near future, more definite
experiments in this connection.

Compact Bone. — The experimental results repeatedly given as
authority for the statement that mature, compact bone does not con-
tain mucin have led to equally uncertain conclusions. No particularly

^ C. Th. Morner : Skandinavisches Archiv fiir Physiologie, 1889, i, p. 210.

2 HoYER : Centralblatt fiir die medicinischen Wissenschaften, 1S69, p. 257.

3 Rustizky: Ibid., 1872, p. 561.

390 P. D. Hazuk and William J. Gics.

chemical investigations seem to have been made in this connection
until a few years ago. Von Ebner' had shown that the decussating
fibres of Sharpey are similar to those in fibrous connective tissue
in general, and that they are not calcified, but that the calcareous
deposit in bone is confined to the interfibrillar areas. These observa-
tions led Young '^ to investigate the question whether the matrix, in
which the fibres of the bone structure are embedded, " is completely
calcified or not." He concluded that this question could be most
readily solved by ascertaining whether mucin, " the most abundant
constituent of the uncalcified matrix or ground substance of con-
nective tissue, is present or absent." Working under Halliburton's
superintendence, Young failed to extract from bone with lime water
or dilute baryta water any substance that could be precipitated with
acetic acid. He concluded, because of this seeming absence of gluco-
proteid from compact bone, that, " in the process of ossification,
the connective tissue matrix is apparently completely calcified."
Young's results would imply the absence, from bone, not only
of mucin but of chondromucoid as well, deductions which remained
undisputed, so far as the author knows, until this work was begun.

Young's result and his general conclusion did not seem to har-
monize with several well-known facts. Morner's^ researches, for
example, on the proteids of cartilage, which were published in detail
several years before Young's results were announced, showed that
chondromucoid is present in relatively large quantity in that tissue,
and of course suggested, further, that bone derived from cartilage
contains a chondromucoid residue.

Practically all forms of uncalcified fibrous tissue from which the
intercalated material has not entirely disappeared are known to
contain mucin ; yet bone, according to Young, would be regarded as
an exception, although its large quantity of ground substance holds
"bone corpuscles" in great number, and it contains circumferential,
decussating and perforating fibres, as well as the fibrillar tissue of the
Haversian canals and the fibrous structures among the " systems."

Since bone is formed in all cases by an ossification of connective
tissue, and as collagen and other proteids are among the substances
regularly contained in bone, it seems natural to sup{X)se that during
the developmental changes some of the connective tissue glucoproteid

* Von Ebner : Archiv fiir mikroskopische Anatomic, 1887, xxix, p. 213.

2 Young : The journal of physiology (English), 1892, xiii, p. 803.

3 C. Th. Mor>;er: Loc. cit

Chemical Shtdies of Osseomucoid. 391

would remain with the other organic substances. Furthermore, if
glucoproteid has any definite function to perform in the connective
tissues, if its presence there signifies anything, there is certainly reason
to believe that it plays some part, however obscure, in bone metabol-
ism, also. The organic constituents already identified in bone, or, let
us say, the usual connective tissue elements which remain in bone
after ossification is complete, are, according to Halliburton, " collagen,
small quantities of elastin from the lining of the lacunae and canali-
culi, proteids and nuclein from the cells, and a small quantity of fat
even after the removal of all the marrow." ^ Why not mucin or chon-
dromucoid ? Surely, unless the ground substance of the antecedent
tissue is entirely removed as impregnation with inorganic matter
proceeds and permanently replaced in the mature bone — and there
is no histological evidence of any such fact — mucoid substance
ought to be separable, in small proportion at least, from osseous
tissue.

Upon referring to Young's paper the author was impressed with
the inadequacy of the method which had led to only negative results
and conclusions. Young treated hard, compact bone, either in the
form of fine shavings or in powder, for from three to five days with a
" large excess of lime water or dilute baryta water." Just what the
" large excess " was intended to accomplish it is hard to surmise; for,
on the assumption that probably at most only a very small proportion
of mucin could be present in bone, subsequent precipitation would
be favored if the extract were kept concentrated. Even finely divided
tendon is usually treated with only 2 to 4 c.c. of half saturated lime
water for every gram of tissue extracted, when easy separation of its
glucoproteid is desired, and tendon probably contains relatively as
much mucin as any other form of connective tissue. In Young's ex-
periments as much as lOO c.c. of the dilute alkali was taken for each
gram of substance extracted.

Another defect in Young's work that the author regrets to call
attention to was the use of too small quantities of bone. In one
experiment only 2.5 grams of bone powder were used ; in the besi
of them only 1 1 grams were taken. According to Halliburton the
normal adult connective tissues contain 0.5 to 0.8 per cent of mucin. ^

^ Halliburton' : Loc. cit. It is in connection with this statement that Halli-
burton accepts the results of the work of Rustizky and Young, with the comment
already quoted.

2 Halliburton : Te.xt-book of chemical physiology and pathology, 1891, p. 47S.

392 P. B. Hawk aiid William /. Gies.

The largest amount of mucin Halliburton and Stevenson obtained in
their quantitative work was 1.02 percent — from skin.' From the
human Achilles tendon the largest amount obtained by them was
0.77 per cent. Now, if we assume for the moment that bone might
contain as much mucin as was found in the skin analyzed by
Halliburton and Stevenson — roughly i per cent — an assumption far
too liberal, then the 2.5 grams of bone employed in one of Young's
experiments might have yielded 0.025 gram of mucin in the 100 c.c.
of dilute alkali used, or the 1 1 grams in the best of Young's experi-
ments might have given o. 11 gram in 500 c.c. of solution. But these
amounts are the greatest which could have been assumed to occur in
bone and certainly it would have been extremely difficult, if not
impossible, to precipitate smaller quantities than these from extracts
purposely made so dilute. Solutions of pure mucin containing ap-
proximately these minute amounts of the proteid may yield fiocculent
precipitates with concentrated acetic acid after standing some time,^
but tissue extracts, holding other dissolved proteids and saline matters,
act differently.

As has just been indicated, the very small quantities of bone
powder or shavings, used in Young's experiments, were treated for
several days with a large excess of lime or baryta water. At the end
of that time, varying amounts of acetic acid were added and, to use
Young's own phrase, " no precipitate came down in any case."
Nothing is said about turbidity, yet traces of mucin under these con-
ditions certainly could hardly have caused more than cloudiness.

The chief objection, however, to the method Young employed was
the direct application of dilute lime or baryta water to a dense com-
pact tissue, thoroughly impregnated with salts which for the most
part are insoluble in such medium. It is not difficult to understand
how, in the case of the femur, for example, the stone-like structure
of the compact portion, composed as it is largely of tribasic earthy
phosphates, imposed a serious obstacle to the usual action of lime
water on contained mucoid substance, and therefore it is natural to
assume that for this reason, if for no other, no mucin was detectable
in Young's experiments. Minute division of the dense tissue in this
instance could hardly make the conditions more favorable for extrac-

1 Halliburton and Stevenson : Ibid., p. 478.

■■^ This can occur only when the mucin has been dissolved in a verj- small quan-
tity of dilute alkali. The salts formed on acidification tend to keep mucin in
solution.

Chemical Studies of Osseomucoid. 393

tion. The proportion of inorganic matter, and its influence against
extraction of mucoid, would naturally remain almost the same in
every particle, however small.

These obvious defects in the methods heretofore employed led the
present writer to investigate this very simple problem in a way which
seemed more favorable to the separation of mucoid. The several
difficulties just alluded to have been overcome by very ordinary means,
and a substance has been prepared from bone having all the general
characters of the glucoproteids.^

METHOD OF PREPARATION.

In a few preliminary experiments, merely to test the objections
here raised against Young's methods, but with no expectation of more
definite results than he obtained, the author used 200-250 grams of
powdered femur — made from only the compact portion of the shaft,
which had previously been thoroughly scraped with a scalpel for the
removal of all superficial connective tissue. These quantities were
much larger than Young's. The femur powder was extracted for
several days with just enough half-saturated lime water to cover if.
On several occasions a very faint turbidity was obtained upon adding
to the filtered extract 5 per cent acetic acid or 0.2 per cent hydro-
chloric acid until the reaction was distinctly acid. Even after standing
a long time, the turbidity remained diffuse, and, as in Young's experi-
ments, borrowing his phrase again, " no precipitate came down."
But the turbidity was encouraging.

The author next proceeded to remove the salts from the bone as
a necessary preliminary to extraction in dilute alkali, and by the
following method succeeded in obtaining a surprisingly large yield
of glucoproteid from both the femur and the rib of the ox.

The fresh bones, just after removal from the animals, were freed as
thoroughly as possible from adherent muscle and connective tissue.
In order to prevent putrefactive complications, the marrow, in the
case of the femur, was completely cleaned out and the bones then
placed in running water for twenty-four hours. At the end of that

^ The terms mucin, mucoid, and chondromucoid have been used here to refer
to connective tissue glucoproteid. Recent researches seem to indicate that the
particular substances to which these names have been applied are not as different
chemically as had been supposed. See Cutter and Gies : Proceedings of the
American Physiological Society; This journal, 1900, iii, p. vi. Also Panzer:
Zeitschrift fiir physiologische Chemie, 1899, xxviii, p. 363; and Levene: Ibid.^i
1901, xxxi, p. 395.

394 P- ^- Haiuk a7id William J. Gies.

time the closely adherent connective tissue was somewhat swollen
and could easily be completely scraped from the bones with an ordi-
nary heavy scalpel. The inside of the shaft of the femur was again
thoroughly swabbed. After this had been accomplished the bones
were kept in 0.2-0.5 per cent hydrochloric acid. In the course of a few
hours the dilute acid took out the inorganic matter from the surface
of the bones just as satisfactorily, although not so rapidly, as much
stronger acid could have done. It was better adapted for the purpose,
also, because there was no special danger that transformation of mu-
coid would result from its use, — a fact of which there could be little
doubt, because the acidity of the fluid in contact with the bones
was constantly diminishing by reaction with the earthy compounds.'

After this treatment the bones were scraped twice daily with a
stout, well-sharpened scalpel. The superficial decalcified layer was
thus easily removed in long, narrow, thin, elastic shavings, exceed-
ingly soft and pliable. The dilute acid was completely renewed
after each scraping.^ The ossein obtained in the first two scrapings
was thrown away, for fear it was contaminated with minute particles

^ This fact was observed repeatedly. The following results of one experiment
in this connection show how rapid is the decrease of total acidity. In several
preliminary titrations 100 c.c. of a special 0.5 per cent HCl solution was found
to be exactly neutralized by 38.2 c.c. of a convenient dilute solution of ammonia;
Congo red was used as the indicator. A perfectly fresh femur of the usual size,
after it had been thoroughly cleaned, was placed in 1000 c.c. of this particular
solution of 0.5 per cent HCl. At intervals, after the fluid had been thoroughly
stirred, total acidity was determined, with the same alkaline solution, in portions
that had been boiled, for a few minutes, for elimination of carbon dioxide:

.S.45 p. M. (femur first placed in acid) : 100 c.c. neutralized by 38.2 c.c. NH4OH.

• 8.00 P. M : 100 c.c. neutralized by 18.2 c.c. NH^OH.

11.15 P. M : 100 c.c. neutralized by 8.1 c.c. NII4OII.

10.30 A. M : 100 c.c. neutralized by 1.3 c.c. NII^OH.

All determinations were made in triplicate, with varying volumes and the figures
obtained agreed closely. These relative results show that at least 50 per cent
of the total free acid was neutralized during the first three hours of contact
with the bones.

2 The quantity of dilute acid used for decalcification was about a litre for each
portion of femur 6-8 inches in length ; only the diaphysis was employed. When
placed for a few hours in hydrochloric acid as dilute as 0.05 per cent, very thin,
delicate shavings, so light that they float in water and dilute alcohol, may be
obtained. Treatment with 0.5 per cent hydrochloric acid permits much more
rapid decalcification, however, and makes the scraping process much easier. One
half per cent hydrochloric acid was used in most of the experiments described in
the .second section, p. 402.

Chemical Studies of Osseomucoid. 395

of superficial connective tissue elements belonging to the periosteum,
which, perhaps, had not been completely removed in the preliminary
treatment. The scraping process was continued until only a very
thin, translucent layer inclosed the marrow cavity. While the shav-
ings accumulated they were kept in 0.2 per cent hydrochloric acid for
thorough decalcification, and for such gelatinization of collagenous
elements as might be helpful to disintegration of the tissue and
more complete liberation of " cement substance " during subsequent
extraction. This treatment also prevented putrefactive changes.^ At
the end of two weeks two scrapings a day of two dozen sections of
ox femur a little more than half a foot in length gave 1700 grams
of moist ossein. The surplus moisture had been eliminated by
cumulative pressure in a meat press.

The shavings were next run through a meat-chopper,^ and then
placed in running water until they were washed free from chloride.
Finally the bulky ossein hash was transferred to several stoppered
bottles and repeatedly shaken with half-saturated lime water in the
proportion of from 2 to 5 c.c. of extractive fluid for every gram of
the moist hash. Within ten minutes after the lime water treatment
began, the extractive fluid became very frothy on shaking, and with
excess of dilute acid a flocculent precipitate was obtained in a small
portion. The extraction was continued for forty-eight hours, by
the end of which time, it was subsequently found, almost all of the
soluble substance had been removed. The filtered extract was then
treated with 0.2 per cent hydrochloric acid.-^ The first addition
produced heavy turbidity, and, after neutralization, a bulky flocculent
precipitate separated at once in moderate excess of 0.2 per cent
hydrochloric acid and fell rapidly to the bottom under a water-clear
fluid.4

From this point the usual method for the purification of mucin was

1 Subsequent experiments indicated that this acid treatment of the shavings,
favoring gelatinization, is not particularly advantageous, perhaps is undesirable.
Dilute alcohol (10 per cent) has been found to serve very well for preservative
purposes during this preliminary period. See methods, p. 404 et seq.

2 This can be done quite easily before the acid is washed out of the shavings,
but is very difficult thereafter.

3 Preferred to acetic acid as precipitant, because of its greater solvent action
on non-glucoproteid material and because former experience has shown that con-
nective tissue mucin is more easily thrown down with it.

* The precipitate closely resembled, in appearance and behavior, tendon mucin
and chondromucoid.

396 p. B. Haiok and William J. Gics.

pursued. The precipitate was several limes washed, by dccantation,
in water made slightly acid with hytlrochloric acid, then freed from
acid by washing in water, filtered ofif, later dissolved in half-saturated
lime water, reprecipitated with 0.2 per cent hydrochloric acid, re-
peatedly washed in acidified water, in water, and in alcohol, and
lastly treated with boiling anhydrous alcohol-ether (50 per cent) as
long as anything dissolved out. The alcohol was washed out with
anhydrous ether. The purified substance dried quickly in the air to
a very light, white, or faintly cream-colored powder devoid of hygro-
scopic qualities. Seventeen hundred grams of moist femur ossein
yielded a trifle more than 7 grams of the substance ; 875 grams of
rib shavings gave 3.5 grams. In each case the amount of prepared
substance was equal to approximately 0.4 per cent of the moist
ossein.^

The acid filtrate from the substance thus prepared contains gelatin
and a body closely related to, if not identical with, the separated
mucoid. Possibly chondroitin sulphuric acid and gelatin combina-
tions, such as Schmiedeberg^ recognized, are in solution. The
author is not sure that nucleoproteid is not contained in it. These
matters are under investigration.

DISCUSSION OF MODIFYING FACTORS.

It will be seen from the analytic results given on page 402
that the substance which has been isolated by the method just
described is typical glucoproteid. In considering its preparation by
this method the author would not ignore the possibility that chon-
droitin sulphuric acid has combined with some of the gelatin, result-
ing from the action of the acid on the collagen, to form an artificial
glucoproteid. It is well known that such combination of these
substances may occur after prolonged contact at body temperature
or more quickly in the presence of free acid, and it might be assumed
that such syntheses took place in these experiments. Morner found
that chondroitin sulphuric acid has strong affinity for gelatin, in
acidified solution, and made use of this tendency to detect the

^ Various minor improvements of the method of preparation suggested them-
selves as the work progressed. Notes of these are made in the second section,
p. 404 et seq.

■^ ScHMiEDEBERG : Archiv fiir experimentelle Pathologic und Pharmakologie,
1891, xxviii, p. 355.

Chemical Studies of Osseojnucoid. 397

ethereal compound. ^ Schmiedeberg ^ has given the names " pepto-
chondrin " and glutinchondrin " to the insoluble intermediate com-
binations of gelatin pepton and chondroitin sulphuric acid, and
" chondralbumin " or " chondralbuminoid " to the soluble products,
formed in his process of isolating chondroitin sulphuric acid from
cartilage. His experiments clearly indicate that various substances
containing chondroitin sulphuric acid, similar to chondromucoid, are
present in cartilage, probably all of them loose compounds of the
acid with simple proteid. Morner^ has shown that chondroitin sul-
phuric acid may combine with simple proteid in the urine, which
compound, on acidification, separates as an insoluble substance having
most of the qualities of uromucoid. Krawkow* has also called atten-
tion to the fact that various combinations of chondroitin sulphuric
acid may be induced with different proteids.

It has frequently been said that bone contains a trace of chondroitin
sulphuric acid, but if any is present as such in osseous tissue, or as a
simple alkali salt, it would seem that the author's preliminary treat-
ment in these experiments should have entirely extracted it from
the ossein, unless, perhaps, the hydrochloric acid, used to remove
inorganic matter, fixed it in situ by quickly furnishing it with the
requisite amount of gelatin before its solution from the decalcifying
tissue. Morner,^ it will be recalled, used essentially this same acid
treatment to gelatinize the collagen of cartilage in order to extract
chondromucoid more completely and easily. After preliminary
treatment with distilled water he digested the cartilage shavings in
0.1-0.2 per cent hydrochloric acid at 40°C. to transform insoluble
collagen into soluble gelatin, thus disintegrating the tissue some-
what and favoring subsequent extraction of the glucoproteid from
the residue with 0.05-0.1 per cent potassium hydroxide. Although
it would be expected that this preliminary treatment with water should

1 C. Th. Morner : Loc. cit. The precipitate of gelatin and chondroitin sul-
phuric acid is readily soluble in excess of mineral acids. Salts interfere with
precipitation of the compound by 0.2 per cent hydrochloric acid. Chondroitin
sulphuric acid itself interferes to a certain extent with precipitation of chondro-
mucoid by dilute acid at room temperature. See also, Zeitschrift fiir physiologische
Chemie, 1894, xx, p. 357, and K. A. H. Morner, cited in note below.

2 SCHMIEDEBERG : LoC. cit.

^ K. A. H. Morner : Skandinavisches Archiv fiir Physiologie, 1895, vi, p. 332.
* Krawkow : Archiv fiir experimentelle Pathologie and Pharmakologie, 1897,
xl, p. 195.

5 C. Th. Morner : Loc. cit.

398 /'. D. Haiuk a7id Williaui J. Gies.

suffice to dissolve out all of the preformed or loosely combined chon-
droitin sulphuric acid, it is possible that some of it may have remained
in the cartilage in Mdrner's experiments, just as some might have
remained in the decalcified tissue in the present experiments. Mt3r-
ner has ignored the matter entirely, and no one else has called atten-
tion to such possibility. The question raised in this connection is
now being studied. The author inclines to the belief that artificial
glucoproteid was not formed in the ossein in the manner just
discussed.

It should not be forgotten, of course, in any consideration of this
matter, that no one has ever shown definitely the existence of pre-
formed, free chondroitin sulphuric acid in normal bones. Morner's^
first researches on the distribution of chondroitin sulphuric acid in
the bones of the ox did not disclose its presence. Unlike Schmiede-
berg,^ however, he was able to prepare it from some pathological
human cartilaginous and osseous structures — in six cases of enchon-
droma, in one of chondroma osteoides mucosum tibiae and one of
exostosis cartilaginea humeri. Morner's method of detecting chon-
droitin sulphuric acid in these investigations, consisting, as it did in
part, of treatment with 2 per cent potassium hydroxide, makes it
uncertain whether this complex ethereal sulphuric acid existed as
such in the bones he analyzed or whether it was derived from pre-
existent glucoproteid in the extraction process.'^ The present writer
thinks the latter view more probable.

Later, Morner's ^ studies of the content of sulphuric acid in the
ash of the bones of the ox, as well as in the acid extract obtained by
treatment of bones from the same animal with boiling hydrochloric
acid (25 i)er cent), led to the deduction that the constant trace of SO3
found, 0.01-0.04 pc'' cent, came from a very slight quantity of chon-
droitin sulphuric acid, and Morner assumed that these indirect
methods gave positive proof of the presence of this substance in
bone, contrary to the former negative results, because of the "greater
delicacy" they possessed over his original direct estimations. His
methods of detection do not warrant the belief, however, that the SO3

1 C. Th. Morner : Zeitschrift fiir physiologische Chemie, 1895, xx, p. 357.

- .SCH.MIEDEBERG : LoC cit.

•^ Levexe has separated a substance similar to chondroitin sulphuric acid from
tendon mucin and other mucoids. Cleavage was accomplished by essentially the
same treatment — with 2 percent sodium hydroxide: Zeitschrift fiir physiologische
Chemie, 1901, xxxi, p. 395. See also Schmiedeberg, loc. cit., for similar facts.

•* C. Th. jMorner : Zeitschrift fiir physiologische Chemie, 1897, xxiii, p. 311.

Chemical Studies of Osseomucoid, 399

came directly from preformed chondroitin sulphuric acid or an alkali
salt. It might have come indirectly from glucoproteid, which, if
present, would have been decomposed into simple proteid and SO3
combinations during the treatment in each of the processes used.^
Bielfeld '-^ recently found as much as 0.076 per cent of SO3 in the ash
of foetal bones and attributed this increase over Morner's figures to a
greater amount of chondroitin sulphuric acid in the embryonic tissue.
It is quite as reasonable to assume, however, that the SO3 detected by
Bielfeld was originally a part of chondroitin sulphuric acid in constitu-
ent glucoproteid. Krawkow'^ also states that he found chondroitin
sulphuric acid in the diaphysis of the femur of the horse, sheep, and
ox. He decalcified with hydrochloric acid ; he does not state the
strength of the acid employed, but it may have been sufficient to
decompose mucoid. Subsequently the prepared ossein was digested
in artificial gastric juice (with probable formation of " peptochondrin,"
etc.), and chondroitin sulphuric acid was extracted from the un-
digested residue, after treatment with potassium hydroxide (amount
and strength not stated), in continuation of Schmiedeberg's process.
The methods Krawkow employed make it probable that the ethereal
compound was derived from antecedent complex material, and his
results prove nothing regarding preformed chondroitin sulphuric acid,
or the presence in bone of a simple salt of the same.

PROPERTIES OF OSSEOMUCOID.

The substance prepared by the method previously outlined has the
general qualities of the glucoproteids, and for the sake of convenient
reference the author proposes for it the name osseomucoid, although
he believes that it is quite as nearly related to the mucins of tendon
and ligament^ as is chondromucoid of cartilage.^

^ See Vandegrift and Gies : This journal, 1901, v, p. 287, for similar facts
connected with SO3 in the ash of ligament and for related points. Krawkow has
separated chondroitin sulphuric acid by destructive method from ligamentum
nuchs as well as from bone.

2 Bielfeld: Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 350.

3 Krawkow : Loc. cit.

* Richards and Gies : Proceedings of the American Physiological Society ;
This journal, 1901, v, p. xi. Also, Cutter and Gies : Loc. cit.

^ Long after the completion of the experiments described under this head, and
shortly before this paper was sent to the editor, the author received Cohnheim's
Chemie der Eiweisskorper (1900) and was surprised to find, on page 285, the
following : " The ground-work of bone, apart from a very slight quantity of mucoid

400 P. B. Hawk and Williafii J. Gics.

Osseomucoid dissolves readily in 0.05 per cent sodium carbonate
and in 5 per cent sodium chloride, from which solutions it may be
precipitated with mineral or organic acids. It appears to dissolve
only slightly in cold 0.2 per cent hydrochloric acid. The moist
substance is acid to litmus, lacmoid, and congo red. When the pure
product, which had been precipitated with hydrochloric acid, was
thoroughly decomposed in dilute nitric acid no chlorine reaction could
be obtained in the fluid with silver nitrate. Like tendon and liga-

(mucin) and cliondroitin sulphuric acid luhich perhaps are not contained in true
bone, consists of collagen, etc." Cohnheim bases this statement regarding possible
presence of mucoid on the authority of some observations of Morochowetz (Ver-
handl. d. Heidelberger naturh.-med. Vereins, N. F., i, p. 480, 1S76), whose opinion
in this particular cqnnection seems to have received no attention at the time (the
text-books of his day do not refer to it), and appears to have been entirely over-
looked until Cohnheim brought it to light again (see historical review, p. 387). The
only other reference to .Morochowetz's work the author has had access to, in the ab-
sence of the original paper, is the abstract in the Jahresbericht iiber die Fortschritte
der Thierchemie, 1877, p. 37, where, it may be seen, the article was entitled : ''Zur
Histochemie des Bindegewebes." Unfortunately, the abstract fails to mention
bone among the tissues examined, which suggests, of course, that Morochowetz's
result or statement in connection with it was a minor one. From the title of the
paper it may be inferred that if any work was done on bone it was purely histo-
chemical in nature and that no mucoid substance was really separated or accu-
rately identified. Besides — and this is a point of considerable significance in
this connection — the body which Morochowetz identified in the various other
tissues under examination and which he called mucin, did not, he says, contain
sulphur, a statement clearly indicating inaccurate chemical observation, since all
of the connective tissue mucins contain a relatively large proportion of sulphur.
From Colmheim's statement it may also be judged that the mucoid to which
Morochowetz referred was not definitely ascertained to be a part of true osseous
tissue. On discovering the statement in Cohnheim's book, the author wrote at
once to his colleague. Dr. H. C. Jackson, lately in Professor Hofmeister's labora-
tory, for detailed information as to the contents of .Morochowetz's paper. Dr.
Jackson consulted the original in the Strassburg library and, thanks to his kind-
ness, the author is able to say that Morochowetz claimed to have obtained mucin
(a sulphur-free glucoproteid I) from several forms of connective tissue, such as
cornea and cartilage. The only form of bone studied was embryonic in structure
and consequently contained much pure cartilage. Morochowetz states he obtained
the same substance from fcetal bone that he had previously identified in various
forms of cartilage. His deductions are to be referred rather to cartilage, therefore,
than to true bone.

Since the above was given to the printer the author received, through the
courtesy of Dr. Leon Asher, of Bern University, a reprint of Morochowetz's
paper in the Heidelberg Verhandluneen. A study of the same confirms all that
has been said here reyarding it.

Chemical Studies of Osseomucoid. 401

ment mucins, and chondromucoid, it dissolves in dilute alkali, and
when sufficient substance is suspended in the liquid, neutralization of
the latter results with formation of an alkali salt of the proteid, which
is soluble in neutral fluid. Osseomucoid gives the biuret, Millon's, and
the xanthoproteic reactions very distinctly. Neutral solutions of its
salts are not coagulated on boiling. It gives only a slight sulphide
reaction with lead acetate after decomposition in hot potassium
hydroxide. The fluid containing the products of its decomposition by
boiling 2 per cent hydrochloric acid, however, gives a heavy precipi-
tate of barium sulphate with barium chloride in the presence of free
hydrochloric acid, and strong reduction of Fehling's and Nylander's
solutions may be obtained after neutralization. This carbohydrate
substance yields osazone crystals with phenylhydrazin. Osseomucoid
is partly digested in " pepsin-hydrochloric acid ; " the anti-albumid-
like residue probably contains substance similar to peptochondrin.
On hydration in boiling mineral acid, anti-albumid, albuminate, pro-
teose and pepton are formed and have been identified.

The original preparations, one from the rib, the other from the
femur, of the ox, were partially analyzed, with the results shown in
the table on page 402.^

The discovery of a mucoid constituent of bone naturally suggests
numerous lines of investigation, some of which have already been
indicated. In what quantity, for example, does osseomucoid exist in
bone at various stages of development .-* Is it peculiar to some bones
or is it found in all } How has it affected previous analyses of bone
gelatin, of bone ash, etc. ? What is its biological significance ; its
relation, if any, to pathological formations, its exact place in the
glucoproteid classification ; its inner make-up, composition reactions,
etc. These and other related problems are under investigation and
the author hopes to present detailed results of these studies in the
near future. The following sections, on composition and heat of
combustion, give complete results of some of the work in this general
plan.

1 The analyses were incomplete, only because the bulk of each preparation was
used for the qualitative determinations which were necessary for ascertaining the
general properties of the substance. The methods employed were the same as
those outlined on p. 403 of the following section. Customary quantities were used.
Sulphur was not determined in the ash because bone contains merely traces of
sulphate and the reagents were free from it. Probably only that derived, on
oxidation, from the proteid itself, would be found in the ash. Complete analytic
results are given in the succeeding section.

40:

P. B. Hawk a? id Williavi J. Gics.

In concluding this section, the author wishes to acknowledge his
indebtedness to Mr. Christian Seifert, assistant in this laboratory, for
much valuable help. Mr. Seifert carefully prepared, under the
author's supervision, all of the bone shavings used in these experi-
ments and cheerfully accomplislied that arduous task at the cost of
considerable personal inconvenience.

PKRfKNT.XGE CoM r<->SlTION.

Preparation.

Nitrogen.

Total
sulphur.

Sulphur

combined

as SU3.

Total
phosphorus.

Ash
phosphorus.

Ash.

A. Rib.

12.78
12.99
12.80
12.91

1.68
1.75

0.98
0.91

0.086
0.031

0.051
0.039

2.28
2.19

1j. Femur

13.38
13.41
13.45

1.89
1.87

1.04
1.11

0.108
0.054

0.057
0.061

2.62

2.57

Calculated for ash-free substance.^

A.

13.17

1.76

0.97

0.013

B.

13.77

1.93

1.11

0.022

II. Composition of Osseomucoid.^

The results of the preliminary analyses seemed to establish beyond
doubt the general glucoproteid nature of osseomucoid. Complete
elementary analysis was necessary, however, to determine definitely
its chemical relationships. We have made such analyses of a number
of additional products from the femur of the ox, which were prepared

^ Reference to phosphorus content, and other deductions as to chemical rela-
tionship, are deferred to the succeeding section, where more complete analyses
are given. See p. 412.

2 Hawk and Gies : Proceedings of the American Physiological Society (Bal-
timore meeting, December, 1900). This journal, 1901, v, p. xv. Previous reports
noted on p. 387.

Chemical Studies of Osseomucoid. 403

and purified, with several variations, as will be indicated, by the
method already given .1 The results obtained in this work harmonize,
it will be seen, with the original deductions.

METHODS OF ANALYSIS.

Carbon and hydrogen. — Estimations were made, with all due precau-
tions, by the method of oxidation in properly arranged combustion
tubes, the gaseous products formed in the process passing through a
layer of granulated copper oxide and over a reduced copper spiral.
The absorbing apparatus consisted of three U-tubes of suitable size,
containing concentrated sulphuric acid in the first, for the absorption
of water, soda lime in the second and soda lime, with pumice stone
moistened by sulphuric acid, in the third, for the absorption of carbon
dioxide.^ The soda lime was prepared as recommended by Benedict.^
The tubes of the absorbing apparatus were wiped with cloth, in all
cases, before weighing, and finally weighed upon a counterpoised
balance until constant figures were obtained.'^

Nitrogen. — Nitrogen was determined by the Kjeldahl process.
Digestion of the substance in concentrated sulphuric acid was com-
pleted with small quantities of metallic mercury. Before distillation
with excess of caustic soda, the mercury was precipitated with
potassium sulphide. In the titrations, congo red was used as the
indicator.

Total sulphur and phosphorus. — These elements were determined
by the well known fusion methods. Fusion was made in silver
crucibles (over alcohol flames in the sulphur determinations), with
solid potassium hydroxide and potassium nitrate, each free from
phosphorus and sulphur.^

Sulphur combined as SO3. — Sulphur in the form of ethereal sul-
phuric acid was determined as follows : The substance was digested
with about 175 c.c. of 2 per cent hydrochloric acid over an alcohol
flame for six hours in a flask connected with a reflux condenser.
At the end of the boiling process, when cleavage was complete, the

1 See p. 393.

2 Benedict: Elementary organic analysis, 1900, p. 34.

^ Benedict: Journal of the American Chemical Society, 1899, xxi, p. 393.

^ An important precaution. Considerable variation in the results may occur
when it is not observed.

^ When traces of these elements were present in the reagents, their quantities
were carefully determined and corrections made accordingly.

404 P- B. Hawk and William J. Gies.

acidity of the fluid was reduced somewhat with pure ammonium
hydroxide, although the mixture was left distinctly acid. It was
then filtered for the separation of antialbumid-like substance which
had formed in small proportion during the process. The sulphuric
acid in the hot filtrate and washings finally was precipitated with
barium chloride, and the figures for sulphur obtained from the barium
sulphate in the usual manner.^

Ash. — Inorganic matter was estimated by direct incineration of the
substance in a platinum crucible over a very low flame. Phosphorus
of the ash was determined in nitric acid solution of the same by the
customary method involving the use of " molybdic solution " and
" magnesia mixture." ^

RECORDS OF ANALYSIS.^

Preparation No. 1. — Bones in 0.3 per cent HCl. 2,700 grams moist shav-
ings accumulated in 0.2 per cent HCl. Before extraction in lime water, acid
was removed by washing in large volumes of water. When decanted fluid no
onger gave acid reaction to litmus, ossein hash was extracted in half-saturated lime
water, 4 c.c. of dilute alkali per gram of substance, for forty-eight hours. End
of that time, extract neutral ; gave only slight precipitate on acidification with
0.2 per cent HCl. Acid had not been completely washed out by decantation
method. Hash placed in half-saturated lime water again ; same quantity for
same time. Second extract gave excellent precipitate on acidification with
0.2 per cent HCl. Slight precipitate of first extract discarded, only second
purified. Dissolved in half-saturated lime water, filtrate opalescent. Repre-
cipitated once with 0.2 per cent HCl. Washed in water, alcohol, ether, etc.
Purified product snow-white, very light, amorphous powder. 6.5 grams.
Dried to^ constant weight at 100-110° C. and analyzed with following results :

Carbon and Hydrogen} 0.1520 gram substance gave 0.2667 gram CO2 =
47.85 per cent C, and 0.0952 gram H2O = 7.01 per cent H; 0.1728
gram substance gave 0.3046 gram CO2 = 48.08 per cent C, and 0.1078
gram HjO = 6.98 per cent H.

1 Great care was taken to prevent introduction of sulphate during the method
of preparation of the osseomucoid analyzed. The reagents used were entirely free
from SO .

'^ Sulphur of the ash was not determined. See note, p. 401.

3 Very brief reference to the more important details of preparation precedes the
analytic data of each particular sample of osseomucoid. The method given on
p. 393 is followed in a general way for each preparation.

* Osseomucoid is so light and bulky that larger quantities of substance could
hardly be used conveniently in these determinations. Special care was exercised,
herefore, in all the analyses.

Chemical Studies of Osseojnucoid. 405

Nitrogen. 0.2606 gram substance gave 0.0369 gram N = 14.15 per cent N ;
0.2557 gram substance gave 0.0361 gram N = 14.11 per cent N ; 0.2520
gram substance gave 0.0354 gram N = 14.06 per cent N.

Total Sulphur. 0.2518 gram substance gave 0.0204 gram BaS04 = 1.12 per
cent ( ?) S ; 0.2530 gram substance gave 0.0249 gram BaS04 = 1.36 per
cent S; 0.2510 gram substance gave 0.0252 gram BaS04 = 1.38 per
cent S.

Sulphur combined as SOs- 0.2390 gram substance, after boiling in HCl, gave
0.0103 gi'ani BaSOi = 0.59 percent S ; 0.2418 gram substance, after boil-
ing in HCl, gave 0.0085 gram BaSO^ = 0.49 per cent S.

Ash. 0.3134 gram substance gave 0.0070 gram Ash = 2.24 per cent Ash ;
0.2560 gram substance gave 0.0054 gram Ash = 2. 11 per cent Ash;
0.2572 gram substance gave 0.0064 gram Ash = 2.49 per cent Ash.

Total Phosphorus. 0.2509 gram substance gave 0.0009 gram MgaPoOT =
0.099 per cent P; 0.2516 gram substance gave 0.0007 gram MgoPoOv =
0.078 per cent P.

Ash Phosphorus. 0.8266 gram substance left 0.0187 P^^ cent Ash, which gave
0.0008 gram MgaP^Oy = 0.029 P^^ Q-^ViX. P.

Percentage Composition of the Ash-free Substance. ^

Average.
C 4S.97 49.20 49.08

H 7.17 7.14 7.16

N 14.48 14.44 14.39 14.44

S 1.39 1.41 1.40

O 27.92

Preparation No. 2. — Preliminary treatment same as in Prep. No. i, except
that bones were decalcified in 0.5 per cent HCl. 1,900 grams moist shavings.
Profiting by previous experience, however, acid was washed out in running
water. Extraction made in 10 c.c. half-saturated lime water for each gram of
ossein ; continued twenty hours. 2.5 per cent acetic acid used to precipitate.
Substance separated in large flocks and settled out more slowly than when
thrown down by dilute HCl. Dissolved in half-saturated lime water. Filtrate
slightly turbid or opalescent in spite of repeated filtration. Reprecipitated once
with 2.5 per cent acetic acid in moderate excess. Washed in water, alcohol,
etc. Partly gummy on drying. 5.7 grams dried at 100-110° C. and analyzed,
with appended results :

Carbon a7id Hydrogen. 0.1273 gram substance gave 0.2216 gram CO.2 =
47.48 per cent C, and 0.0815 gram H20 = 7.i6 per cent H; 0.1306
gram substance gave 0.2276 gram CO2 — 47-53 per cent C, and 0.0777

^ Reference to phosphorus content is made on p. 412.

4o6 P. B. Hawk and William J. Gics.

gram HoO = 6.66 per cent ( ?) H ; 0.1280 gram substance gave 0.2242
gram COo — 47.77 percent C,an(l 0.0834 gram H.^O = 7.29 per cent H.

Nitrogen. 0.2522 gram substance gave 0.0348 gram N — 13.79 P^'" '^^"^ ^ J
0.2188 gram substance gave 0.0305 gram N = 13.94 per cent N ; 0.2484
gram substance gave 0.0349 gram N = 14.02 per cent N.

Total Sulphur. 0.2037 gram substance gave 0.0210 gram BaS04 = 1.42 per
cent S; 0.2035 S'''^""' substance gave 0.0202 gram BaS04 = 1.37 per
cent S.

Sulphur combined as SO^. 0.2021 gram substance, after boiling in HCI, gave
0.0089 gram BaSOi = 0.61 per centS; 0.2035 gram substance, after boil-
ing in HCI, gave 0.0105 gra"i BaS04 = 0.71 per cent S.

Ash. 0.2556 gram substance gave 0.0066 gram Ash = 2.58 per cent Ash ;
0.2528 gram substance gave 0.0064 gram Ash = 2.53 per cent .Ash.

Total Phosphorus. 0.2012 gram substance gave 0.0006 gram Mg.jPoO^ =
0.083 PS^ '^^"t Pj 0-3127 gram substance gave 0.0005 gram MgoP.iOy =
0.045 P^'' ^^^^ ^•

Ash Phosphorus . 0.5084 gram substance left 0.0130 gram Ash, which gave
0.0007 gram Mg^PoO; = 0.038 per cent P.

Percentage Composition of the Ash-free Substance.^

Average.

C 48.72 48.77 49.01 48.83

H 7.35 .... 7.48 7.42

N 14.15 14.30 14.38 14.27

S 1.46 1.41 1.43

O 28.05

Preparation No. 3. — Preliminary treatment same as for Prep. No. 2. 2,900
grams moist shavings. Two extractions made ; first for twenty hours, second
for seventy-two hours. Osseomucoid precipitated by 0.2 per cent HCI.
Much less substance precipitated from second extract than from first. Com-
bined and dissolved in 0.05 per cent KOH. Filtrate slightly opalescent.
Thrice reprecipitated by 0.2 per cent HCI.- Then washed once in o.i per
cent HCI, lastly in H.jO, etc. 11. 2 grams light cream colored powder. Dried,
etc., with following analytic results:

Carbon and Hydrogen. 0.1106 gram substance gave 0.1858 gram C02 =
45.82 per cent C, and 0.0681 gram H.20 = 6.89 per cent H; 0.1143
gram substance gave 0.1946 gram CO2 = 46.43 per cent (?) C, and

^ It will be observed that the composition of the product precipitated by acetic
acid (Prep. No. 2) is essentially the same as that prepared with 0.2 per cent hydro-
chloric acid (Prep. No. i).

■■^ Extra reprecipitation seems to have resulted in lowering of the percentage of
carbon and nitrogen, and raising that of sulphur and oxygen. See p. 407.

Chemical Studies of Osseomucoid. 407

0.0698 gram HoO = 6.83 per cent H ; 0.0970 gram substance gave
0.1627 gram CO.2 = 45-75 per cent C, and 0.0620 gram H2O = 7.15 per
cent H ; 0.1075 gram substance gave 0.1810 gram COo = 45.92 per cent
C, and 0.0680 gram HoO = 7.08 per cent H.

Nitrogen. 0.2790 gram substance gave 0.0366 gram N = 13-13 per cent N ;
0.3281 gram substance gave 0.0433 gram N = 13.20 per cent N ; 0.2651
gram substance gave 0.0348 gram N — 13- 12 per cent N.

Total Sulphicr. 0.2526 gram substance gave 0.0336 gram BaS04 = 1.83
per cent S; 0.2516 gram substance gave 0.0332 gram BaS04 = 1.82 per
cent S.

Sulphur Combined as SO^. 0.2434 gram substance, after boiling in HCl, gave
0.0183 gram BaS04 = ^.03 per cent S; 0.2438 gram substance, after
boiling in HCl, gave 0-0181 gram BaS04 =: 1.02 per cent S.

Ash. 0.2602 gram substance gave 0.0039 gram Ash = 1.50 per cent Ash;
0.2589 gram substance gave 0.0040 gram Ash = 1.54 per cent Ash.

Total Phosphorus. 0.2504 gram substance gave 0.0009 gram Mg.2P207 =
o.ioo per cent P ; 0.2506 gram substance gave 0.0004 gram Mg2P207 =
0.045 P^"" ^^^'^ P ; 0.2874 gram substance gave 0.0005 gram Mg2P20v —
0.048 per cent P.

Ash Phosphorus. 0.5 191 gram substance left 0.0079 gram Ash, which gave
0.0003 gram Mg2P207 = 0.016 per cent P.

Percentage CoMPOsrrioN of the Ash-free Substance.

C 46.53 .... 46.46 46.63

H 7.00 6.94 7.26 7.19

N' 13.33 13.40 13.32

S 1.86 1.85 1.85

O

Average.
46.54

7.10

1335

31.16

Preparation No. 4. — Same preliminaries as for Prep. No. 2. 3,950 grams
moist shavings. Extraction in 10 c.c. half-saturated hme water for each
gram of ossein ; continued seventy-two hours. Osseomucoid precipitated
with 0.2 per cent HCl. Dissolved in slight excess of 0.05 per cent NaOH
and reprecipitated five times ; each solution filtered. Filtrate at first tur-
bid or opalescent as each time heretofore. After the pores of the filter
paper became clogged, however, the filtrate was collected more slowly, but
came through as clear as water, though yellowish in color.^ About three-

1 Possibly the observed differences in analytic results between this and the
previous preparations were due to the presence of bone corpuscles, etc., in the
latter, which had not been completely removed in the process of filtration. See
also foot-note, p. 406.

4o8 P. B. Hawk atid Willia?n J. Gics.

fourths of final solution obtained water clear ; turbid portion discarded. After
fifth reprecipitation substance was thoroughly stirred up in 8 litres of 0.2 per
cent HCl. There was no particular diminution in quantity, although the flocks
seemed to shrink somewhat and become heavier and more granular. Acid
washed out with H.jO. Final purification same as heretofore. In spite of
losses, 7.7 grams osseomucoid obtained; very light, cream colored. Analyzed
in the usual way, the appended results were obtained :
Carbon and Hydrogen. 0.1124 gram substance gave 0.1906 gram €0-2 =

46.25 per cent C, and 0.0669 gram H.^O = 6.66 per cent H; 0.1311

gram substance gave 0.2216 gram COo = 46.14 per cent C, and 0.0797

gram HoO = 6.81 per cent H.
Nitrogen. 0.2670 gram substance gave 0.0320 gram N = ii-97 per cent N ;

0.2810 gram substance gave 0.0339 gram N = 12.06 per cent N.
Total Sulphur. 0.2526 gram substance gave 0.0406 gram BaSOi = 2.21 per

cent S; 0.2534 gram substance gave 0.0373 gram RaS04 = 2.03 per cent

S ; 0.3032 gram substance gave 0.0406 gram BaSOi = 1-84 per cent (?)

S; 0.3290 gram substance gave 0.0503 gram BaSOi =2.10 per cent S.
Sulphur Combined as SO^^. 0.3227 gram substance, after boiling in HCl, gave

0.0259 gram BaS04 = i.io per cent S; 0.3237 gram substance, after

boiling in HCl, gave 0.0251 gram BaSOi = 1-04 percent S.
Ash. 0.2662 gram substance gave 0.0012 gram Ash = 0.45 per cent Ash ;

0.2656 gram substance gave 0.0012 gram Ash = 0.45 per cent Ash.
lotal Phosphorus. 0.3022 gram substance gave 0.0004 gram MgoP.jO^ =

0.044 per cent P; 0.3028 gram substance gave 0.0002 gram MgoPo07 =

0.018 per cent P.
Ash Phosphorus. 0.5318 gram substance left 0.0024 gram Ash, which gave

0.0003 gram MgoP-^Ov = 0.016 per cent P.

Percentage Composition of the Ash-free Substance.

Average.

C 46.46 46.35 46.40

H 6.69 6.84 6.77

N 12.02 12.11 12.06

•S 2.22 2.04 2.11 2.12

O 32.65

Preparation No. 5. Bones decalcified in 0.5 per cent HCl. In sixteen days
4,410 grams moist shavings obtained. Shavings each day were placed in o. i
per cent HCl ; on the following day, and thereafter until used, in 25 per cent
alcohol. Latter was acid from acid in shavings. Acid washed out with water
by decantation until pieces of the ossein hash no longer reacted acid to litmus.
6 c.c. half- saturated lime water used to extract, for each gram of ossein. After
two hours, extract was nearly neutral ; showing that acid in interior of pieces

Chemical Studies of Osseomucoid. 409

had not been completely washed out.^ Sufficient 10 per cent KOH was then
added, drop by drop with thorough shaking, to make approximately 0.05 per
cent KOH in the fluid. After twelve hours the alkalinity had again perceptibly
diminished; 2 c.c. half-saturated hme water for each gram of ossein finally
added. Extractive period, from beginning, was fifty-two hours. Extract in the
end very frothy. Was diluted with equal volume of water, and osseomucoid
pre<:ipitated from diluted solution with 0.2 per cent HCl. Reaction was made
only very slightly acid ; precipitation purposely left incomplete, the turbid por-
tion yielding small amount of flocculent precipitate on further acidification.
This was discarded. Main precipitate dissolved in half-saturated Hme water
and reprecipitated eight times. Just before final precipitation with 0.2 per cent
HCl, the filtrate, after passing through the same filter paper repeatedly, was
obtained as clear as water. In the end poured into 0.2 per cent HCl drop
by drop, with instantaneous precipitation. Substance finally washed in sixteen
litres 0.2 per cent HCl and twenty-four litres o.i per cent HCl, with thorough
stirring ; eventually in water, alcohol, etc. During the washing in water, some
of the product persisted in floating, as had been the case in all previous
preparations. In this particular case the floating portion was finally skimmed
off" and discarded. 17.8 grams of cream colored fluffy powder were obtained.
Dried and analyzed :

Carbon and Hydrogen. 0.1247 gram substance gave 0.2180 gram CO2 =■
47.68 per cent C, and 0.0718 gram HoO = 6.44 per cent H; 0.1492
gram substance gave 0.2615 gram COj = 47.80 per cent C, and 0.0877
gram H2O = 6.58 per cent H ; 0.1615 gram substance gave 0.2809 §i^3,m
CO2 = 47-44 per cent C, and 0.0938 gram H2O = 6.50 per cent H.

Nitrogen. 0.3026 gram substance gave 0.0355 ^^"^ N = ii-75 per cent N ;
0.3022 gram substance gave 0.0352 gram N = 11.64 per cent N.

Total Sulphur. 0.5674 gram substance gave 0.1020 gram BaSO^ = 2.47 per
cent S ; 0.5306 gram substance gave 0.0969 gram BaSO^ = 2.51 per cent S.

Siilphur combined as SO^- 0.4026 gram substance, after boihng in HCl, gave
0.0452 gram BaS04 — 1.54 per cent S; 0.4018 gram substance, after boil-
ing in HCl, gave 0.0572 gram BaS04 =1.96 per cent(?) S; 0.3512 gram
substance, after boiling in HCl, gave 0.0382 gram BaSO^ =1.50 per
cent S.

Ash. 0.3542 gram substance gave o.ooio Ash = 0.28 per cent Ash; 0.3518
gram substance gave 0.0009 gram Ash = 0.26 per cent Ash ; 1.329 gram
substance gave 0.0043 gram Ash = 0.32 per cent Ash.

Total Phosphorus. 0.6371 gram substance gave 0.0002 gram Mg2P20- =
0.008 per cent P; 0.9381 gram substance gave 0.0007 ^^""^ Mg2P207 =
0.021 per cent P.

Ash Phosphorus. 1.329 gram substance left 0.0043 gi"^"^ Ash, which gave
0.0007 gram Mg2P207 = 0.015 P^r cent P.

^ See foot-note, p. 410.

4IO F. B. Hawk and VVilliatu J. Gies.

Percentage Composition of the Ash-free Suhstance.i

Average.
C 47.82 47.94 47.58 47.78

M 6.46 6.60 6 52 6.53

N 11.78 11.67 11.72

S 2.48 2.52 2.50

O 31.47

Preparation No. 6. Bones in 0.5 per cent HCl eighteen days. 6,680
grams moist shavings obtained by end of that time. As tliey were made they
were placed in 10 per cent alcohol, repeatedly renewed to remove acid during
period of accumulation. Alcohol washed out later in water by decantation.
Extraction in half-saturated lime water, 8 c.c. per gram of ossein. End of four
hours, extract nearly neutral. 10 per cent KOH added as before to make
0.05 per cent KOH in extract. After eighteen hours, extract again nearly
neutral. 10 per cent KOH added to make total of o.i per cent KOH. Alka-
linity gradually decreased ; due not only to combining power of osseomucoid
but also, probably, to failure to completely wash out HCl." Ossein in dilute
alkali for ten days. Powdered thymol prevented putrefactive change. Extract
finally obtained as perfectly clear filtrate. Diluted with four volumes water and
this treated with equal volume 0.4 per cent HCl. Immediate precipitation in
large flocks, which became smaller and more granular after thorough stirring,
and quickly settled out. Precipitate dissolved in fifth-saturated baryta water and
reprecipitated with 0.4 per cent HCl nine times. Tenth reprecipitation made
by filtering the 3I litres of the baryta solution of substance into twenty litres of
0.2 per cent acid. Each drop solidified on contact and fell quickly to the
bottom in globular form. Globules were broken up on stirring. Thoroughly
washed in 0.3, 0.2 and o. i per cent HCl, later in water, etc., as usual. Final
product very light, snow-white powder: 29.75 grams. Following results of
analysis were obtained :

Carbon and Hydrogen. 0.1862 gram substance gave 0.3176 gram COo =
46.52 per cent C, and 0.1114 gram H.,0 = 6.65 per cent H; 0.1877
gram substance gave 0.3190 gram CO2 = 46.36 per cent C, and 0.1128
gram HjO^ 6.68 per cent H ; 0.1449 gram substance gave 0.2469 gram
CO.j = 46.47 per cent C, and 0.0906 gram H.jO = 7 per cent H ;
0.1649 gram substance gave 0.2802 gram CO.j = 46.34 per cent C, and
o. 1013 gram H.2O = 6.87 per cent H.

^ See foot-notes, pp. 406 and 407.

^ It is evident that the decantation process must be repeated very frequently
if all acid is to be washed out. Filtered running water serves best for this
purpose.

Chemical Studies of Osseomucoid. 4 1 1

Nitrogen. 0.3000 gram substance gave 0.0360 gram N = 12 per cent N ;
o 3000 gram substance gave 0.0357 gram N = 11.90 per cent N ; 0.3000
gram substance gave 0.0360 gram N = 12 per cent N.

Total Sulphur. 0.3887 gram substance gave 0.0734 gram BaSOi = 2.59 per
cent S; 0.2761 gram substance gave 0.0502 gram BaS04 =: 2.50 per
cent S.

Sulphur combined as SO3. 0.3045 gram substance, after boiling in HCl, gave
0.0344 gram BaS04 = 1.55 per cent S ; 0.3355 gram substance, after boil-
ing in HCl, gave 0.0379 gram BaS04 = i-55 per cent S.

Ash. 0.2658 gram substance gave 0.0006 gram Ash = 0.23 per cent Ash;
0.2650 gram substance gave 0.0006 gram Ash = 0.23 per cent Ash;
1. 3781 gram substance gave 0.0036 gram Ash = 0.26 per cent Ash.

Tc^tal Phosphorus. 0.6840 gram substance gave 0.0002 gram Mg.2PoO^ =
0.008 per cent P.

Ash Phospho?-us. 1.3781 gram substance left 0.0036 gram Ash, which gave
0.0009 gram MgaP-^OT = 0.018 per cent P.

Percentage Composition of the Ash-free Substaxce.i

Average.
C 46.63 46.47 46 5S 46.45 46.53

H 6.67 6.69 7-01 6.89 6.81

N .... 12.03 11.93 12 03 1199

S 2.60 2.51 2.55

O 32.12

Preparation No. 7. Fifty sections of femur decalcified in particularly dilute
HCl — 0.05 per cent." Scraped twice daily. Shavings, as they were col-
lected, were placed directly into 3-5 litres of water, 12-24 hours, and then in
10 per cent alcohol until sufficient quantity accumulated. At end of three
weeks 2,500 grams very thin, narrow, elastic shavings obtained. After hashing,
the finely divided ossein was extracted in half-saturated lime water, 20 c.c. per
gram of hash, for seventy-two hours. Alkalinity had perceptibly diminished
by end of that time. Water clear filtrate obtained. With 0.2 per cent HCl in
excess finely flocculent precipitate at once. Same purification process as for
Prep. No. 6. Reprecipitated only five times. Final product very fight, white
powder; 5.2 grams. Analytic results as follows :

Carbon and Hydrogen. 0.2470 gram substance gave 0.4304 gram CO2 =
47.51 per cent C, and 0.1487 gram H2O = 6.69 per cent H; 0.1952

^ See foot-notes, pp. 406 and 407.

^ The analytic results of this preparation agree very well with those for prepa-
tions Nos. 5 and 6, and indicate that the acid used in decalcifying has had no
particular influence on the products separated.

412

p. B. Hawk and William J. Gies.

gram substance gave 0.3389 gram CO., = 47.35 per cent C, and 0.1158
gram H,.0 = 6.59 per cent H.

Nitrogen. 0.1754 gram substance gave 0.0212 gram N = 12.05 P^^ ^^"^ ^ j
0.2431 gram substance gave 0.0296 gram N = 12.18 per cent N.

Total Sulphur. 0.4482 gram substance gave 0.0783 gram BaS04 — 2.40 per
cent S; 0.6320 gram substance gave 0.1158 gram BaS04 = 2.52 per
cent S.

Sulphur combined as SO a- 0.617 i gram substance, after boiling in HCl, gave
0.0678 gram BaSO^ = 1.5 1 per cent S ; 0.5009 gram substance, after boil-
ing in HCl, gave 0.0501 gram BaS04 = 1.37 per cent S.

Ash. 0.7256 gram substance gave 0.0022 gram Ash = 0.30 per cent Ash ;
0.2891 gram substance gave 0.0008 gram Ash = 0.28 per cent Ash.

Total Phosphorus. 0.5661 gram substance gave 0.0005 gram Mg.2P207 =

0.025 P^^ ^^'"'^ P-
Ash Phosphorus. 1.0147 gram substance left 0.0030 gram Ash, which gave
0.00 10 gram Mg.2P207 = 0.027 P^^ ^^"t P.

Percentage Composition of the Ash-free Substance.

Average.
C 47.65 47.49 47.57

6.71

6.61

12.09

12.22

2.41

2.53

6.66
12.15

2.47
31.15

Summaries and Discussion of Analytic Results.

Content of phosphorus. — Before reviewing the general results of
the analyses of the seven preparations we have carefully studied,
special attention should be directed to the data on phosphorus
content. The averages of our figures for percentage amount are
here summarized : —

Substance.

Preliminary
preparations.

Preparations completely analyzed.

Aver-
ages.

Rib.

Femur.

1.

2.

3.

4.

5.

6.

7.

All.

Dry.

0.058

0.081

0.088

0.06+

0.046

0.031

0.014

0.008

C.025

0.046

Ash.

0.045

0.059

0.029

0.038

0.016

0.016

0.015

0.018

0.027

0.029

Ash free.

0013

0.022

0 049

0.026

0.030

0.015

0.017

Chemical Studies of Osseomucoid.

413

It is very evident, from these results, that osseomucoid is a sub-
stance free from phosphorus. Not only are the above quantities
entirely too small to have any particular significance, but all of them
are within the ordinary variations in accuracy of the method of deter-
mination itself, and fluctuations may be due to unavoidable analytic
error. Such traces as are indicated by the very painstaking determi-
nations we have made undoubtedly are a part of the ash and not of
the organic substance itself. The higher figures for the earlier
preparations might be interpreted to mean phosphorized-proteid
impurity. The differences are too slight, however, to warrant any
such conclusion.^

Sulphur combined as SO3, — We have not yet attempted to separate
chondroitin sulphuric acid from osseomucoid, but the large proportion
of combined SO3 detected in, and separated from osseomucoid strongly
indicates the presence of such a radicle in its molecule, particularly
also because of the acid reaction of the proteid itself. The recent
results obtained by Panzer,^ on ovarial mucoid (paramucin), and
Levene,^ on various connective tissue and glandular glucoproteids,
further suggest the probability that such an acid radicle will eventu-
ally be separated from osseomucoid. The percentage quantities of
sulphur combined as SO3 in all our preparations are here summarized,
for ash-free substance, and the general averages contrasted with
the amounts in chondromucoid and the mucins of ligament and
tendon : — *

Preliminary
prepara-
tions.

Preparations completely analyzed.

Averages.

Chon-
dromu-
coid.

Tendon
mucin.

Liga-
ment
mucin.

Rib.

Fe-
mur.

1.

2.

3.

4.

5.

6.

7.

All.

4-7.

Averages.

0.97

1.11

0.55

0.68

1.05

1.08

1.53

1.55

1.44

1.11

1.40

1.76

1.43

1.07

^ The tendon mucins analyzed by Dr. Gies, several years ago, contained 0.17
per cent P (average), which was also found to equal the percentage of phosphate
in the ash. This observation has since been verified by Mr. Cutter, and identical
results obtained for ligament mucin by Dr. Richards, in this laboratory. See also
Krawkow's figures for percentage of amyloid : Krawkow, loc. cit.

2 Panzer : Loc. cit.

3 Levene : Loc. cit.

^ C. Th. Morner, Cutter and Gies, Richards and Gies : Loc. cit.

414

P. B. Hawk a?id William J. Gies.

General Review. — The appended table summarizes the results for
average percentage composition of osseomucoid (ash-free substance)
and gives average composition of preparations 1-7; also of prepara-
tions 4-7, inclusive, the latter having been specially grouped together
because of the greater attention given to their purification, by repeated
reprecipitation, as has already been indicated : —

Indh id

Lial preparations.

Aver

age.s.

Ele-
ments.

1.

2.

3.

4.

5.

6.

7.

1-7

4-7

C

49.08

48.83

46.54

46.40

47.78

46.53

47.57

47.53

47.07

H

7.16

7.42

7.10

6.77

6.53

6.81

6.66

6.92

6.69

N

14.44

14.27

13.35

12.06

11.72

11.99

12.15

12.85

11.98

S

1.40

. 1.43

1.85

2.12

2.50

2.55

2.47

2.05

2.41

0

27.92

28.05

31.16

32.65

31.47

32.12

31.15

30.65

31.85

The above results emphasize the glucoproteid character of osseo-
mucoid, for, like practically all of these compound proteids, osseo-
mucoid has a relatively low content of carbon and nitrogen, with a
comparatively large proportion of sulphur and oxygen — due to the
content of carbohydrate (probably polysaccharide) and sulphuric
acid radicles; both rich in oxygen, the latter in sulphur.

Lack of particular uniformity in percentage composition, however,
is evident on comparing the analytic results for the individual prepara-
tions. This want of analytic harmony cannot be due to nucleoproteid
impurity, — our results for content of phosphorus show that conclu-
sively,^— nor does it seem probable that admixture of other soluble
proteid can be the cause, for bone contains too little such material to
warrant that belief. We have already considered the possibility of
chondroitin sulphuric acid combining with any gelatin made during
the process of decalcifying, to form different products of varying solu-
bilities, but, as has already been suggested, there is no reason to
believe that bone contains sufficient chondroitin sulphuric acid to

^ The content of phosphorus is too low for an assumption that either nucleo-
albumin (0.4-0.8 per cent P) or phosphoglucoproteid (0.45 per cent P) was
admixed. Comparatively large quantities of tlie substance contained the merest
trace of iron. Undoubtedly this minute amount is to be recognized as inorganic
impurity.

Chemical Studies of Osseomucoid. 415

effect such a result. ^ We varied our method of preparation some-
what each time a new product was made for analysis, as may be seen
in the records of analytic results, but, unless it be assumed that osseo-
mucoid is very unstable, like submaxillary mucin, for example, and
therefore easily influenced by the mild chemical treatment to which
it was subjected, these changed conditions would not account for
altered composition. We have seen, however, that osseomucoid
behaves like tendon mucin and chondromucoid. We have every
confidence in the accuracy of our methods of analysis and their
manipulation.

Hammarsten,^ it will be remembered, found that frequent precipi-
tation of submaxillary mucin resulted in a lowering of the percentage
of carbon and nitrogen of the purified product because of fractional
elimination of nucleoalbumin. Our preparations 4-7 were given
particular attention in this regard, with general results similar to
those obtained by Hammarsten, and it may be that we have had to
deal with unsuspected proteid impurity, which could only be, and per-
haps was finally, eliminated by repeated reprecipitation. In the absence
of direct evidence of such impurity, however, — and every condition
seems to be against its occurrence, — we think our results justify the
conclusion that the mucin substance of bone varies in composition
just as the glucoproteid from other sources does, and that the figures
in our analyses represent the make-up of several of these very closely
related bodies. Such a conclusion not only accords with our analytic
results but harmonizes also with the deductions drawn, under simi-
lar conditions for other tissues and products, by various observers.-^

There appear to be many forms of glucoproteid. In all probability
the acid and carbohydrate radicles of the mucoids have the power of
uniting with various proteids in varying proportions to form different
compounds, and while they can easily be arranged into general groups
as we classify them to-day, in inner make-up they are doubtless mul-
tifarious. Such a conception of the chemical nature of the mucin
substances would account for the wide variations that have been
observed in the elementary composition not only of apparently the
same substance, but also of very nearly related products from differ-

1 See p. 396.

2 Hammarsten: Zeitschrift fiir physiologische Chemie, 1888, xii, p 163.

3 ChittenD-EN and Gies : The journal of experimental medicine, 1896, i,

p. 186. Also, SCHMIEDEBERG, K. A. H. MORNER, CUTTER and GlES, KrAWKOW,

Richards and Gies : Loc. cit.

4i6

P. B. Hawk and William J. Gies.

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Chemical Studies of Osseomucoid. 417

ent tissues. Until we know more about the inner nature of simple
proteid, and of such complex substances as chondroitin sulphuric acid
which readily unite with proteid in the normal and pathological met-
abolic changes in the tissues, it will be difficult to reach, from analytic
results, conclusions more definite regarding various glucoproteids than
those we have been able to draw fi'om our analyses of osseomucoid.

Compared results. — In the general summary, on page 416, of ana-
lytic figures for tissue mucoids, direct comparison may be made with
the osseomucoid averages. The figures for crystallized serum albumin
are also given for convenient comparison of the collated analytic data
with similar results for simple proteid.

III. Heat of Combustion of Osseomucoid, Tendon Mucin and

Chondromucoid.

historical.

In any consideration of the metabolism of energy in the body, the
combustion equivalents of the food and excreta are factors of funda-
mental importance. It is now generally agreed, we believe, by all
who have given special attention to such studies, that careful deter-
minations of the potential energy, as expressed in calories, of all the
constituents of the tissues should be made, if various important
phases of metabolism are to be more thoroughly comprehended.

Although the "fuel values " of numerous albuminous mixtures, and
some proteid substances, taken from the animal body have been very
carefully estimated, no attention appears to have been paid, in this
connection, to the glucoproteids, members of which group of bodies
constitute so large a proportion of the interfibrillar or intercellular
substance of various forms of connective tissue. We considered it
desirable, therefore, to determine the combustion equivalent of osseo-
mucoid and also of related glucoproteid, not only for the general
thermochemical interest such results would have, but in the belief,
also, that the caloric values obtained would throw further light on
the chemical relationships of these tissue proteids, and ultimately
be of worth in any metabolic study of their syntheses and trans-
formations.

The researches of Stohmann, B. Danilewsky, Rubner, Berthelot
and Atwater, and their pupils, have shown that the combustion equi-
valents of the chemically pure animal proteids thus far studied vary
from averages of 5270 calories for gelatin and 5298.8 calories for

4i8 P. B. Hinok aiid Williaiu J. Gics.

pepton, to 5961.3 calories for elastin ; with egg albumin, at 5735.2
calories, representing about the mean value.' The work of these
observers also indicates in a general way that the higher the percen-
tage of carbon in the proteid, the greater its combustion equivalent;
the greater the proportion of oxygen, on the other hand, the lower
the heat of combustion. Thus elastin, which, we have seen, has the
highest equivalent, contains about 55 per cent of carbon and 20 per
cent of oxygen ; pepton, with a much lower equivalent, contains
roughly 50 per cent of carbon and 26 per cent of oxygen ; albumin,
having an average combustion equivalent, contains approximately 52.5
per cent of carbon and 23 per cent of oxygen.

Considerable variation is to be noted on comparing the figures for
calories obtained for the same compound by different observers. This
fact may be attributed, however, to different degrees of purity of the
products burned, as well as to variations in the accuracy of the
methods employed. Thus the caloric value of "ossein" is 5039.9
according to Stohman and Langbein ^ and 5410.4 according to
Kerthelot and Andre ^ — a difference of 370.5 calories. But as
" ossein " is in strictness a tissue residue, not a pure chemical sub-
stance, these variations are not at all surprising.

The only strictly compound proteid investigated by combustion
methods thus far is haemoglobin. Its potential energy appears to be
relatively high, varying from 5885.1- to 5914^^ calories. The com-

^ The first of these figures was obtained by Atwater (see foot-note, p. 419).
The rest were determined by Stohmann and Langbein, with the improved Ber-
thelot method, and are taken from the table in the Centralblatt fiir Physiologic for
1892 (vi), p. 157. B. Danilewsky obtained somewhat lower figures for pepton,
an average of 4900 calories (Centralblatt fiir die medicinischen VVissenschaften,
1885, xxiii, p. 678), but as these were derived by the older Thompson-Stohmann
process, which was not as accurate as the Berthelot method, the values given by
Stohmann and Langbein are probably more trustworthy. Fibroin is the only
native proteid thus far studied which has a combustion equivalent lower than
that of pepton. According to Stohmann and Langbein it is 4979.6 calories.
Berthelot and Andre found it to be 5095.7 (Centralblatt fiir Physiologic, 1890,
iv, p. 609). An excellent resume of combustion methods and results is given by
Atwater : Methods and results of investigations on the chemistry and economy
of food (Bulletin No. 21, Ofiice of Experiment Stations, U. S. Department of
Agriculture), 1895, p. 113: also by BuNGE : Lehrbuch der physiologischen und
pathologischen Chemie, 1894, p. 62, and by Gautier : Le9ons de chimie bio-
logique normalc et pathologique, 1897, p. 788.

- Stohmann und Langbein : Centralblatt fiir Physiologic, 1892, vi, p. 156.

^ Berthelot et Andre: Ibid., 1890, iv, p. 609

Chemical Studies of Osseomticoid. 419

bustion equivalent of milk casein, classified, by some, as pseudonu-
cleoproteid, varies from 5629.2^ to 5858.3 ^ calories.

Of the results thus far obtained in calorimetric experiments the
most important for us in this particular connection are those for
" chondrin." Stohmann and Langbein have found the combustion
equivalent of "chondrin" to be 5130.6 calories ; ^ Berthelot and
Andre^ place it at 5345.8 calories.^ This difference of 21 1.8 calories
may be attributed to variations in the composition of the product
burned, for " chondrin," with approximately 50 per cent of carbon
and 28 per cent of oxygen, is a mixture consisting mostly of cartilage
gelatin, chondromucoid and chondroitin sulphuric acid. It is almost
impossible to make two preparations of the mixture having the same
composition and in which the proportions of the components are alike.
It is to be observed, however, that, even if the higher figures be
accounted more correct, the value expressed by them is still about as
low as any thus far determined for animal proteid— even for the
hydratcd forms such as pepton. The lowered potential energy of
" chondrin," as well as its lowered percentage of carbon and the raised
proportion of oxygen, may be reasonably attributed in great part to
the carbohydrate portions of the contained chondroitin sulphuric
acid and chondromucoid.*

METHOD OF DETERMINATION.

The determinations of heat of combustion in our own experiments
were made in a Berthelot bomb calorimeter as modified and improved
by Atwater and Blakeslee. Most of the experimental work in this
connection was done by Mr. Hawk, in the chemical laboratories of
Wesleyan University, the privileges of which were very kindly
extended for the purpose by Professor Atwater, to whom we are

. 1 Stohmann und Langbein : Loc. cit.

2 Berthelot et Andre : Loc. cit.

^ B. Danilewski, working with the older and less accurate method, found it
to be 4909 calories : Centralblatt fiir die medicinischen Wissenschaften, 1885,
xxiii, p. 678.

* The values for heat of combustion of connective tissue collagens have never
been determined. For the hydration product of mixed collagens, commercial
gelatin, the value is 5,270 calories. Atwater : Report of the Storrs (Conn.)
Agricultural Experiment Station, 1899, p. 92 (Fish gelatin = 5493 calories :
B. Danilewsky, loc. cit.). Cartilage gelatin has not been studied, in this con-
nection. The combustion equivalent of disaccharides averages about 3900 calo-
ries ; of polysaccharides about 4200 calories.

420

p. B. Haivk mid William J. Gies.

greatly indebted, also, for many courtesies and much valuable

assistance.

Combustions of pure substances of known calorific power were

thermometer is graduated to hniiiire<ith^
thousandths with a magnifying lens.^

Figure 1. — Atwater-HIakeslee
bomb calorimeter and acces-
sory apparatus as arranged
for combustions. — The plati-
num lined bomb of steel,
holding oxygen and the sub-
stance to be burned, is im-
mersed in water contained in
a metal cylinder (Q) ; the
latter is surrounded by con-
centric covered cylinders
(T, U) of indurated fibre.
Air spaces between the outer
cylinders favor retention of
heat in the water. The water
is kept in motion with the
aid of a stirrer (SS) driven
by a small electric motor,
thus equalizing temperature.
Oxygen is forced into the
empty bomb through the side
passage (G) in the neck (D).
Perfect closure of this pas-
sage is made by the valve
screw (F). The electric cur-
rent, for fusing the iron wire
over the substance to be
burned in the capsule (O), is
conveyed by the insulated
wires (W, V), one of which
(W) is connected with the
valve screw (F) and thus
with one of the platinum
wires inside the bomb (I),
and the other (V) with the
insulated platinum wire (H)
which passes through the
cover of the bomb. The

)t a degree, and is capable of being read to

made at intervals to test the apparatus and manipulations. The cus-
tomary method of ignition, by means of iron wire, was used, and the

1 For full description .see Atwater and Blakeslee : Report of the Storrs
(Conn.) .Agricultural Experiment Station, 1897, p. 199.

Chemical Studies of Osseomucoid. 421

necessary correction made for its heat of combustion. Proper correc-
tion was also made for the thermal changes due to oxidation of the
nitrogen of the proteid to nitric acid. The quantities of proteid
employed in each determination varied from 0.6 to i.o gram. Each
sample burned completely without special difificulty.

Two of the best of our completely analyzed preparations of osseo-
mucoid were burned in the bomb. Samples of preparations No. 5
and No. 6 (see preceding section) were selected for the purpose. All
but one of the tendon mucins employed for the same purpose were
prepared and analyzed by Cutter and Gies,^ and represent the gluco-
proteids, made by fractional precipitation methods, from both the
sheath and the shaft of the tendo Achillis of the ox. The mucin of
preparation, " c 8 " was made and analyzed several years ago by
Chittenden and Gies.'^ The preparations of chondromucoid which we
oxidized in the calorimeter were made by Morner's ^ method, especially
for this work. Preparation "a 9 " represents the mixed mucoid from
three successive extractions of cartilage from the nasal septum of
the ox; preparation "b 10" only the glucoproteid in the second
extract of a separate portion of cartilage from the same source.
Elementary analyses, in duplicate, were made by the methods given
on page 403.

EXPERIMENTAL RESULTS.

In the summary on page 422 the figures in duplicate determinations,
under " heat of combustion," represent small calories at constant vol-
ume per gram of substance dried at 100-110° C. to constant weight;
the analytic figures represent elementary composition of perfectly
anhydrous substance; complete averages and other data are also
included.

DISCUSSION OF DATA.

The striking feature of the results for heat of combustion is the
fact that they are uniformly low. The general averages fall far below
the figures for potential energy of all the common proteids, including
the hydrated forms, and even beneath the smallest equivalent recorded
for fibroin (see page 418). This result was naturally to be expected,

1 Cutter and Gies : Loc. cit. The complete analytic data given here for
these preparations anticipate the detailed publication of the results obtained.

2 Chittenden and Gies : Loc. cit.

^ C. Th. Morner : Skandinavisches Archiv fiir Physiologie, 1889, i, p. 210.

422

P. B. Hawk mid William J. Gies.

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Chemical Studies of Osseomucoid.

423

however, because of the decreased proportion of carbon and nitrogen,
and the raised percentage of sulphur and oxygen produced in these
compound substances by the union of proteid with carbohydrate and
sulphuric acid radicles in tlieir construction. The general average
equivalent falls about midway between the figures for calorific value
of polysaccharide and albumin.

Very little stress can be laid on the differences shown in the above
table for the separate groups, because they are entirely too slight,
and quite within the limits of unavoidable experimental error. On
the other hand, the group agreement is so decided in the main that
further experimental evidence is furnished, we think, of the chemical
similarity and close relationship of the three substances, or groups of
substances, under examination. It is interesting, also, to find that
such differences as are expressed in the group averages run parallel
with the fluctuations in amount of carbon and oxygen, the equiva-
lents increasing as the percentage of carbon rises, and falling as the
oxygen goes up in proportion.

The above average figures for composition and combustion equiva-
lent are brought into direct comparison below with a similar average
given by Stohman and Langbein : — ^

Investigators.

Substances.

Average percentage
composition.

Combustion

equivalent.

Small

calories.

Stohmann and
Langbein.

Numerous animal and
vegetable proteids ;
not including mu-
coids.

C H N S 0
52.64 7.08 16.00 1.03 23.20

5711

Hawk and Gies.

Connective tissue glu-
coproteids only.

C H N S 0
47.43 6.63 12.22 2.32 31.40

4981

The general relation of our results to those obtained for other com-
mon proteids and albuminous mixtures is so clearly shown in the
table 2 of averages on page 424 that further comment is unnecessary.

^ Stohmann and Langbein : Loc. cit.

^ Results not our own are selected from those for many substances burned and
analyzed by Berthelot and Andre : Loc. cit.

424

P. B. Hawk and IVilliatn J. Gies.

.Substance.

Combustion

equivalent.

Small calories.

Conibusti(3n

equivalent.

Large calories.

Percentage

of

carbon.

Percentage

of

oxygen.

For substance

Per gram.

containing 1 gm.
of carbon.

Chondromucoid.

4883

10.65

45.87

32.90

Tendon mucin.

5009

10.43

48.(H

30.75

Osseomucoid.

4992

10.59

47.16

31.79

Hsemoglobin.

5914

10.62

55.51

17.62

Egg albumin.

5691

10.99

5177

24.15

" Ossein."

5414

10.81

.50.10

24.60

"Chondrin."

5346

10.54

50.89

23.03

Fish gelatin.

5242

10.80

48.53

25.54

Fibroin.

^o*':

10.60

48.09

27.41

IV. Summary of Co\clu.sions.

1. A substance, designated as osseomucoid, having the chemical
and physical qualities of mucin and chondromucoid, may be extracted
from the rib and femur of the ox with lime water. Such extraction
may be made most satisfactorily from ossein prepared, in the form of
shavings, from bones which have previously been partly decalcified
with very dilute acid (0.05-0.5 per cent HCl).

This discovery makes it evident that ordinary compact bone, like
the other forms of connective tissue, does contain mucin substance,
and further, contrary to Young's deduction, that in the process of
ossification the connective tissue matrix is not completely removed.

2. The percentage composition of seven preparations of osseo-
mucoid varied between the following extremes, with the subjoined
general averages for the seven, also for the four agreeing quite
closely and to which particular attention was given in the process
of purification : —

c H N .s o

E.xtremes: 4908-4640 7.42-6.53 14.44-11.72 1.40-2.55 2792-32.65

Average 1-7 : 47 53 6.92 12.85 2.05 30.65

Average 4-7: 47.07 669 11.98 2.41 31.85

Chemical Studies of Osseomucoid. 425

It is probable that there are two or more glucoproteids in bone,
judging from the variations noted in the results for percentage
composition.

Osseomucoid does not contain phosphorus. Between i and 1.6
per cent of its sulphur may be split off as SO3 on boiling in dilute
hydrochloric acid.

3. The energy liberated on oxidation of the mucin substances, as
represented by osseomucoid, tendon mucin, and chondromucoid, is
less than that for any other form of proteid except fibroin. The
average of twenty duplicate determinations for ash-free substance
is 4981 small calories per gram, just midway between the average
equivalents for albumin and polysaccharide.

The average potential energy of osseomucoid (4992), tendon mucin
(5009), and chondromucoid (4883) is found to be so nearly the same
for each substance that additional experimental evidence is furnished
of the very close chemical relationship of these connective tissue
glucoproteids. Slight and variable differences in the content of
carbon and oxygen in these substances appear to account for the
minor fluctuations in the figures for combustion equivalent.

The average elementary ash-free percentage composition of the ten
samples of typical glucoproteid studied by the combustion method is :

c

H

N

S

0

47.43

6.63

12.22

2.32

31.40

The figures for elementary ash-free composition of the preparations
of tendon mucin and chondromucoid studied in this connection agree
quite well with those for similar products analyzed several years
ago by Morner and by Chittenden and Gies. The observed analytic
variations are comparatively slight, but suggest that tendon and
cartilage each contains several closely related mucin substances.

Reprinted from the American Journal of Physiology.

Vol. VII. — April i, 1902. — No. I.

CHEMICAL STUDIES OF ELASTIN, MUCOID, AND
OTHER PROTEIDS IN ELASTIC TISSUE, WITH
SOME NOTES ON LIGAMENT EXTRACTIVES.^

By a. N. RICHARDS and WILLIAM J. GIES.

\Froni the Laboratory of Physiological Chemistry of Columbia University, at the College
of Physicians and Stirgeotts, N'ew Vorh.]

CONTENTS.

Page

I. Elastin 94

Preparation 94

Historical 94

Improved method 98

Elementary composition, preparations 1-8 99

General summary 104

Reactions 104

Sulphur content 105

Distribution of nitrogen 107

Is elastin a "fat-proteid compound " ? 110

Digestibility HI

Heat of combustion 114

II. Mucoid 116

III. Coagulable proteids 118

IV. Nucleo-proteid 125

V. Collagen (gelatin) 127

VI. Crystalline extractives 130

VII. Summary of conclusions 133

COMPREHENSION of function is dependent on knowledge of
structure and composition. The influence of any tissue on
the other parts of the body is more easily understood as our appre-
ciation of the varieties and relations of its constituent elements in-
creases. Elastic tissues have received little analytic attention. They
have been overlooked by reason, apparently, of their seeming meta-
bolic passivity and because they serve mainly mechanical functions.

The earlier observers regarded the cervical ligament as an extra-
vascular tissue, for the most part, with practically no special chemical

1 Some of the results of this research have already been given in the Proceed-
ings of the American Physiological Society: This journal, 1900, iii, p. v. ; 1901,
V, p. xi.

93

94 ■^- ^' Richards a7id Willia'tn J. Gies.

activity and believed that it consisted almost wholly of the albumi-
noid elastin. Recently, however, it has been found in this laboratory ^
that the ligamentum nuchre of the ox contains not only the large
percentage of water and elastin, and the slight amounts of inorganic
matter, collagen, and fat assumed to be present by the earlier investi-
gators, but also appreciable quantities of mucoid,- coagulable proteid
and crystalline extractives. These later results indicate that the
production of elastin is the feature of ligament metabolism, and
they indicate, further, that the chemical changes normally occurring
in yellow elastic tissue are greater than had been supposed.

We have recently subjected the various constituents of elastic
tissue to a somewhat detailed study. The particular form of tissue
from which the constituents were prepared in all our experiments
was the ligamentum nuchas of the ox.

I. Ligament Elastin.

Preparation. Historical. — Tilanus ^ was probably the first to
analyze elastic tissue. In his earlier preparations of "pure tissue"
small pieces of the cervical ligament of the cow were first extracted
in cold water to remove traces of blood and inorganic matter, and
then dehydrated (and fat eliminated) with alcohol and ether. This
product was hardly anything better than "prepared" ligament. In
a second preparation he extracted in boiling dilute acetic acid in
addition. Extraction with the acid doubtless removed all of the
coagulable proteid and most of the collagen, but probably left behind
most, or at least much, of the mucoid. The residue prepared in this
way (after thorough removal of acid by washing in water and then
dehydrating), unlike the product obtained by the first method, was
said to be free of sulphur. Tilanus assumed it to be a pure chemical
substance — elastin — and gave it the formula Q.^Hg^iN^^Oi^. In
both of the methods used by Tilanus the tissue extractives were
doubtless completely eliminated.

W. Miiller'* improved Tilanus's methods by adding treatment in
boiling dilute alkali and cold dilute mineral acid to the preparation

^ Vandegrift and Gies : This journal, 1901, v, p. 287.

'^ We use the word " mucoid " in the sense first suggested by CoHXHEnr. See
Cutter and Gies : This journal, igof, vi, p. 155 (foot-note).

3 Tilanus : See Mulder, Versuch einer allgemeinen physiologischen Chemie,
Zweite Halfte, 1844-51, p. 595.

* VV. MuLLER : Zeitschrift fiir rationelle ]\Iedicin, dritte Reihe, i86i,x, p. 173.

Elastin, Mtccoid, and Other Proteids in Elastic Tisstte. 95

process. He alternately boiled finely divided ligamentum nuchae
from the horse and ox in dilute acetic acid and in dilute potassium
hydroxide, and then extracted in cold dilute hydrochloric acid.^
Such treatment tended to remove the residual collagen and all of the
mucoid, but also favored decomposition of the elastin. Miiller states
that his purified product was fibrous in microscopic appearance and
seemed to be unaffected by the alkali treatment.

Horbaczevvski 2 made still further modification of the method used
by Miiller by introducing repeated extraction of the cervical liga-
ment of the ox in boiling water. The treatment in boiling water
thoroughly transformed insoluble collagen into soluble gelatin al-
though it made subsequent extraction of coagulated proteid more
difficult. Horbaczewski continued all of his extractions for longer
periods than any of his predecessors. Subsequently, Chittenden and
Hart,^ commenting on Horbaczewski's work and the method of
elastin preparation used by him wrote as follows : " So vigorous is
the method of treatment, that it appears almost questionable whether
a body belonging to a group noted for ease of decomposition might
not suffer some change in such a long process of preparation."

Chittenden and Hart* compared elastin made from the ligamentum
nuchge of the ox by Horbaczewski's method with that obtained in
their own process, which was the same except that the substance
was not extracted in alkali. The chief difference noted was that the
elastin which had been treated with potassium hydroxide contained
no sulphur, whereas that which had not been extracted with alkali
contained 0.3 per cent. For the first time the danger in the use
of hot alkali was appreciated and pointed out.^ At the same time
the probable presence of mucoid was overlooked. There is no
reason for believing that the mucoid could have been completely
removed from the tissue pieces without the aid of alkali.

Bergh^ recently obtained elastin from the cervical ligament by
Horbaczewski's method, but added, also, digestion in pepsin-hydro-

^ This was the method then commonly used for the preparation of resistant
tissue elements like cellulose and chitin.

2 Horbaczewski: Zeitschrift fiir physiologische Chemie, 1882, vi, p. 330.

^ Chittenden and Hart : Studies from the Laboratory of Physiological
Chemistry, Yale University, 1887-88, iii, p. 19.

* Chittenden and Hart: Loc. cit.

^ Objections had also been raised from another standpoint by Zollikofer :
Annalen der Chemie und Pharmacie, 1852, Ixxxii, p. 169.

^ Bergh : Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 337.

96 A.N. Richards and William J. Gics.

chloric acid. Elastin is readily digestible in gastric juice,^ however,
so that this modification of treatment could hardly have dissolved
very much that the acid and alkali did not remove, except elastin
itself. Aside from determining the presence of sulphur in his own
preparation of elastin and in Griibler's (a commercial product from
the same source and prepared by Horbaczewski's method), Bergh
made no attempt to ascertain the elementary composition of ligament
elastin.

The ligament elastins made in various studies of this albuminoid
by other observers (whose analytic results are referred to below),
were all from the same source — ligamentum nuchce of the ox.
Morochowetz''^ made his products by Miiller's method. Stohmann
and Langbein ■* obtained theirs by the Horbaczewski process. Zoja,*
Mann,^ and Eustis ^ each used the method of Chittenden and Hart.

The following summary gives the average analytic results for
percentage composition of the ash-free products prepared from liga-
ment by the above methods."

TiLANUS:

Method of preparation. C H N S O

(<7) Not extracted with acid 54.65 7.26 17.41 0.34 20.34

(-!-) Extracted with acid 55.65 7.41 17.74 .... 19.20

MiJLLER* Extracted with hot alkali .... 55.46 7.41 16.19 .... 20.94

Horbaczewski 3 Extracted with hot alkali .... 54.32 6.99 16.75 21.94

^ See page 1 1 1 .

2 MOROCHOWETZ : St. Petersburger medicinische Wochenschrift, neue Folge,
1886, iii, p. 135.

8 Stohmann und Langrein : Journal fi.ir praktische Chemie, neue Folge,
1891, Ixiv, p. 353.

* Zoja: Zeitschrift fiir physiologische Chemie, 1897, xxiii, p. 236.

^ Mann: Archiv fiir Hygiene, 1899, xxxvi, p. 166.

^ Chittenden (for Eustis) : Proceedings of the American Physiological
Society, 1899, This journal 1900, iii, p. xxxi.

" For the composition of elastins from other sources than ligament see Vande-
GRIFT and Gibs, loc. cit., also Cohnheim : Chemie der Eiweisskorper, 1900,
p. 293. We have no occasion in this review to refer to elastins which were not
analyzed. Various observers have engaged in chemical studies of elastin without
satisfying themselves of the purity of their products.

8 MiJLLER found 0.08 per cent of sulphur in his elastin, but assumed it to be
due to accidental impurity.

9 The analytic results credited to Etzinger by Charles in his " Elements of
Physiological and Pathological Chemistry" (1884, p. 129), were those obtained by
Horbaczewski. Etzinger made no analyses of ligament elastin. See Zeit-
schrift fiir Eiologie, 1874, x, p. 84.

c

H

N

S

O

?

?

?

0.63

p

54.24
54.08

7.27
7.20

16.70
16.85

0.30

21.79
21.57

55.03

?
?

7.20

?

16.91
16.96
16.52

0.18

0.28

?

20.68

?
?

Elastin, Mucoid, and Other Proteids in Elastic Tissue. 97

Method of preparation
MoROGHOWETzi Extracted with hot alkali ....
Chittenden and Hart:

(«) Prepared by Horbaczewski's method

(3) Their own, without extraction in alkali
Stohmann and Langbein :

Extracted with hot alkali ....
ZojA Not extracted with hot alkali . . .

Mann Not extracted with hot alkali . . .

EusTis2 Not extracted with hot alkali . . . 54.42 7.40 16.65 0.14 21.39

It will be observed, from the preceding statements and summary,
that as a general rule extraction with hot alkali resulted in the prep-
aration of a sulphur-free product. On the other hand, methods which
did not include alkali extraction gave elastins containing sulphur.
The exceptions resulted, probably, when the alkali treatment was
not as prolonged or thorough as customarily.

That treatment in hot alkali is apt to cause decomposition is now
almost self-evident. Referring to this subject, Chittenden and Hart
stated that " treatment with acid of the alkaline solution obtained in
preparing elastin by Horbaczewski's method, plainly showed the
presence of hydrogen sulphide." Did this sulphur come from the
elastin and is elastin a sulphur-containing body, or did it arise from
another substance originally in the ligament?^

The only constituents of elastic tissue which seem to require

^ It has been shown by Chittenden and Hart that in elastoses there is a
diminution of the content of carbon, and an increase in the content of oxygen, pro-
portional to the extent of proteolysis. In spite of this fact, Morochowetz's analy-
ses of elastose gave the following results :

c

H

N

S

O

55.90

7.29

16.68

0.62

19.50

He did not completely analyze the original elastin — only sulphur was determined
as given above. It seems necessary to conclude that the elastin used by MoRO-
CHOWETz was an impure product and that the figures above for sulphur are
inaccurate.

^ EusTis made only a partial analysis. We are greatly indebted to him for a
sample of his product, from which we obtained the remaining results. The
individual ash-free determinations, by the methods we used farther on, were as
follows :

C H N S

54.52 7.47 16.64 0.12

54.32 7.32 16.66 0.15

^ See page 105.

98 A. N. Richai'ds and Williain J. Gies.

treatment with alkali, in addition to acid, in order to effect their
complete solution are mucoid, and traces of nucleoproteid. These
bodies as they are situated, resist the action of acids, the former
particularly, and their removal from compact tissue particles is easy
only when alkali is used. They are readily soluble in cold dilute
lime-water, which has no effect on the elastin.^

Iviproved inetJiod. — Our improved method of preparing ligament
elastin includes extraction in cold lime-water instead of destructive
treatment with boiling potassium hydroxide, and may be given briefly
as follows : Ligamentum nuchae was cut into strips, these very finely
minced in a meat chopper- and the resultant hash thoroughly washed
in cold running water for from twenty-four to forty-eight hours. Traces
of blood, lymph, and much coagulable proteid, with extractives, were
removed in this process. The finely divided tissue was then thor-
oughly extracted for from forty-eight to seventy-two hours in large
excess of cold half-saturated lime-water, renewed occasionally, for
complete removal of residual simple proteid,^ and also mucoid and
nucleoproteid. After the alkali had been thoroughly remo\'^d by
washing in water, the minced substance was boiled in water, with
repeated renewals, until only traces of dissolved proteid (elastoses)
could be detected in the washings. The tissue was then boiled in
ten per cent acetic acid for a few hours, treated with five per cent
hydrochloric acid at room temperature for a similar period, again
extracted in hot acetic acid and in cold hydrochloric acid, finally
washed free of acid in water, and then kept in boiling alcohol and

^ It will be remembered that half-saturated lime-water and very weak alkaline
fluids of approximately the same strength have been repeatedly used for extractive
purposes in the past because they manifest no destructive action on compound
proteids and albuminoids at room temperature.

^ The ordinary hashing machine can be very advantageously used for this pur-
pose. It not only finely divides the tissue but also tends to loosen the fibres in all
of the particles, and thus greatly favors extraction of interfibrillar substance.
Previous observers make no mention of the use of special mincing apparatus. In
some of the preceding work the ligament was merely cut into small pieces with a
knife.

* Our results with the simple proteids of ligament (page 118) suggest that in the
preparation of elastin due regard must be paid to the fact that the fresh ligament
contains at least 0.6 per cent of soluble and coagulable proteid. It certainly can-
not be an easy matter to remove all this from the fibrous meshwork, particularly
after the tissue has been boiled in water, and possibly some of the variations in
the figures reported for the composition of elastin and the nature of its decom-
position products may be due to such impurity not completely eliminated.

Elastin, Mucoid, a7id Other Proteids in Elastic Tissue. 99

ether until dehydration was complete, and all fat and extractive
substance had been removed.^

The elastin particles prepared in this way were soft and porous
and could easily be ground in a mortar to a cream-colored, very light
powder.^ Under the microscope the particles were seen to consist
uniformly of typical elastic fibres. No extraneous matter was held
in the meshes of these.

In order to study the effect of the above modified method of prep-
aration, as well as to obtain further information on the composition
of elastin, we made several samples of elastin both by the Chittenden
and Hart method and our own and subjected the products to com-
parative analysis. The analytic methods employed were the same as
those recently described in detail in a paper from this laboratory.^

Elementary composition. Preparation No. 1. — Preparations I— 4 were
made by the Chittenden and Hart process as follows : Finely minced
tissue (ipo grams) was boiled in water until practically nothing more
dissolved.* This process required about ten changes of i litre of
water and a total of seventy-five hours for completion. The sub-
stance was next warmed in i litre of ten per cent acetic acid for one
and one-half hours on a water bath. It was kept in the same fluid
eighteen hours longer at room temperature and then boiled for four
hours directly over a flame. The acid was then thoroughly washed
out and the substance kept in five per cent hydrochloric acid for
eighteen hours at room temperature. After the mineral acid had
been thoroughly removed the treatment in the acids, with appropri-
ate washing, was repeated. Finally, dehydration and removal of fat
and extractive matter were effected in boiling alcohol-ether in the
usual manner. The analytic results follow :

Carboti and Hydrogen. 0.2909 gm. substance gave 0.5752 gm. CO2 and
0.1906 gm. HoO = 53.93 per cent C and 7.28 per cent H; 0.2538 gm.
substance gave 0.5078 gm. CO2 and 0.1659 S^^- H2O = 54-56 (?) per

1 Further details will be found with each preparation under records of analysis,
pages 99 and loi. See also page in.

2 Compare with the experience of Horbaczewski and of Chittenden and
Hart, who evidently had not succeeded in completely dehydrating.

^ Hawk and Gies : This journal, 1901, v, p. 387.

^ One variation here from the Chittenden and Hart process is to be noted.
We put the cleaned ligament through a hashing machine. The tissue used by
Chittenden and Hart was "chopped quite fine." The more finely divided the
tissue the easier and more complete the extraction, of course. See pages 98 and 104.

lOO A. N. Richards and Willia??i J. Gits.

cent C and 7.26 per cent H; 0.2603 gm. substance gave 0.5159 gm.

CO2 and 0.1703 gm. HgO = 54-05 per cent C and 7.27 percent H;

0.2591 gm. substance gave 0.5 118 gm. CO.^ and 0.1681 gm. HoO = 53-87

per cent C and 7.21 per cent H.
Nitrogen. 0.2909 gm. substance gave 0.0469 gm. N — 16.12 per cent N ;

0.3527 gm. substance gave 0.0565 gm. N ■= 16.01 per cent N.
Sulphur. 1.2540 gm. substance gave 0.0153 gm. BaS04 = 0.17 per cent S;

0.9790 gm. substance gave 0.0141 gm. BaS04 — 0.20 per cent S; 0.6661

gm. substance gave 0.0119 gm. BaS04 = 0-25 per cent S.
Ash. 0.4504 gm. substance gave 0.0038 gm. Ash = 0.84 per cent Ash ;

0.3424 gm. substance gave 0.0025 gm. Ash = 0.73 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.
C 54.35 .... 54.48 54.30 54.38

II 7.34 7.32 7.33 7.27 7.32

N 16.25 16.13 16.19

S 0.17 0.20 0.25 0.21

O 21.90

Preparation No. 2.

Carbon and Hydrogen. 0.2572 gm. substance gave 0.5067 gm. CO2 and
0.1702 gm. H.2O = 53-73 per cent C and 7.35 per cent H ; 0.3730 gm.
substance gave 0.7383 gm. CO.2 and 0.2408 gm. H-.O = 53-98 per cent
C and 7.17 per cent H ; 0.4186 gm. substance gave 0.27 11 gm. HoO =
7.22 per cent H ; 0.4614 gm. substance gave 0.9096 gm. CO2 and 0.2968
gm. H2O = 53-77 per cent C and 7.15 per cent H.

Nitrogen. 0.4863 gm. substance gave 0.081 1 gm. N = 16.67 P^^ ^ent N;
0.2892 gm. substance gave 0.0481 gm. N = 16.62 per cent N; 0.2521
gm. substance gave 0.0426 gm. N = 16.88 per cent N.

Sulphur. 0.5535 ?P^- substance gave 0.0089 g"^- BaS04 = 0.22 per cent S ;
0.7942 gm. substance gave 0.0112 gm. BaS04 — 0.19 per cent S.

Ash. 0.5009 gm. substance gave 0.0030 gm. Ash = 0.60 per cent Ash;
0.5364 gm. substance gave 0.0031 gm. Ash = 0.58 per cent Ash.

Percentage Composition of the Ash-free .Substance.

Average.

c

54.05

54.30

54.09

....

54.15

H

7.39

7.21

7.26

7.19

....

7.26

N

16.77

16.71

16.98

16.82

S

0.22

0.20

0.21

0

« • . •

21.56

Elastin, Mucoid, and Other Proteids in Elastic Tissue. loi

Preparation No. 3.

Carbon and Hydrogen. 0.2562 gm. substance gave 0.5 loi gm. CO2 and

0.1694 gm. HoO = 54-30 per cent C and 7.35 per cent H.
Nitrogen. 0.3305 gm. substance gave 0.0550 gm. N = 16.64 P^^^ cent N ;

0.3577 gm. substance gave 0.0596 gm. N = 16.67 P^^ cent N.
Sulphur. 1. 1549 gm. substance gave 0.0128 gm. BaS04 = 0.15 per cent S;

0.7953 gm. substance gave o.oioo gm. BaSO^ = 0.17 per cent S.
Ash. 0.6690 gm. substance gave 0.0045 gm. Ash = 0.67 per cent Ash;

0.5782 gm. substance gave 0.0038 gm. Ash = 0.66 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.
C 54.67 .... .... .... .... 54.67

H 7.40 .... .... .... .... 7.40

N .... 16.75 16.78 .... .... 16.76

S .... .... .... 0.15 0.17 0.16

O .... .... .... .... .... 21.01

Preparation No. 4.

Carbon and Hydrogen. 0.2571 gm. substance gave 0.5084 gm. CO2 and 0.1671

gm. H2O = 53-93 per cent C and 7.22 per cent H.
Nitrogen. 0.3386 gm. substance gave 0.0562 gm. N = 16.59 P^r cent N;

0.2545 gm. substance gave 0.0426 gm. N = 16.72 per cent N.
Sulphur. 0.9068 gm. substance gave 0.0163 S™- BaS04 = 0.25 per cent S;

1.0077 g'^''- substance gave 0.0163 g^^- BaS04 = 0.22 per cent S.
Ash. 0.4931 gm. substance gave 0.0052 gm. Ash = 1.05 per cent Ash;

0.4412 gm. substance gave 0.0050 gm. Ash =1.13 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.
C 54.52 .... .... .... .... 54.52

H 7.30 .... .... .... .... 7.30

N .... 16.77 16.90 .... .... 16.83

S .... .... .... 0.25 0.22 0.24

O .... .... .... .... .... 21.11

Preparation No. 5. — Preparations 5-8 were made by our own
method. 100 grams of ligament strips were washed in cold running
water 24-48 hours. The strips were next run through a hashing
machine and the hash thoroughly extracted several times (for 3 days)
in half-saturated lime-water. The last extract did not become turbid
on acidification. The alkali was completely washed out of the hash

102

A. N. Richards and William J. Gies.

with water. The rest of the process — boiling in water, etc., was the
same in time, order, and character as that for preparations 1-4.

Carbon and Hydrogen. 0.2448 gm. substance gave 0.4819 gm. COo and
0.1648 gm. H.jO = 5369 per cent C and 7.48 per cent H; 0.2627 g"^-
substance gave 0.5142 gm. CO2 and 0.1776 gm. HoO = 53.38 per cent
C and 7.51 jDer cent H ; 0.4568 gm. substance gave 0.8922 gm. COo and
0.2916 gm. HoO =■ 53.27 per cent C and 7.09 percent H.

Nitrogen. 0.3735 gm. substance gave 0.0620 gm. N = 16.59 per cent N;
0.2420 gm. substance gave 0.0400 gm. N = 16.51 per cent N; 0.2498
gm. substance gave 0.0417 gm. N = 16.69 P^^ ^^"^ ^•

Sulphur. 1.0358 gm. substance gave 0.0119 S™- BaS04 = 0.16 per cent S;
0.5907 gm. substance gave 0.0075 S""*- B^SOi = 0.17 per cent S.

Ash. 0.3943 gm. substance gave 0.0029 S"^* ^^'^ = °-74 P^'' ^^"'- '^^^^ ''
0.3907 gm. substance gave 0.0036 gm. Ash = 0.92 per cent Ash.

Percent.'Vge CoMPOsr

C
H

N
S

o

54.14
7.54

53.83

7.57

53.72
7.15

16

rioN OF THE Ash-free Substance.

16.65

16.83

0.16

0.17

Average.
53.90

7.42
16.74

0.16
21.78

Preparation No. 6.

Carbon and Hydrogen. 0.3285 gm. substance gave 0.6550 gm. C0.> and
0.2161 gm. H2O == 54-38 per cent C and 7.31 per cent H ; 0.2539 gm.
substance gave 0.5036 gm. CO2 and 0.1654 gm. HoO = 54.09 per cent
C and 7.24 per cent H ; 0.3343 gm. substance gave 0.6662 gm. CO.2 and
0.2202 HoO = 54-35 per cent C and 7.32 per cent H.

Nitrogen. 0.4117 gm. substance gave 0.0701 gm. N= 17.02 per cent N;
0.2965 gm. substance gave 0.0510 gm. N = 17.18 per cent N; 0.2797
gm. substance gave 0.0478 gm. N = 17.08 per cent N.

Sulphur. 1.3763 gm. substance gave 0.0128 gm. BaSO^ = 0.13 per cent S ;
1.1255 g™- substance gave 0.0121 gm. BaSOi = 0.15 per cent S.

Ash. 0.9620 gm. substance gave 0.0008 gm. Ash = 0.08 per cent Ash;
1.0230 gm. substance gave 0.0009 S"^- ■^sh = 0.09 per cent Ash.
Percentage Composition of the Ash-free Substance.

C
H

N
S

o

54.43
7.32

54.14

7.25

54.40
7.33

17.03 17.20 17.09

13

0.15

Average.
54.32

7.30
17.11

0.14
21.13

Elastin, Mticoid, and Other Proteids in Elastic Tissue. 103

Preparation No. 7.

Carbofi afid Hydroge7i. 0.2584 gm. substance gave 0.5120 gm. CO2 and

0.1685 g"^^- ^S^ = 54-04 per cent C and 7.25 per cent H.
Nitroge7i. 0.4656 gm. substance gave 0.0764 gm. N= 16.42 per cent N;

0.4482 gm. substance gave 0.0744 gm. N = 16.60 per cent N.
Sulphtir. 0.8678 gm. substance gave 0.0096 gm. BaSO^ = 0.15 per cent S ;

0.8896 gm. substance gave 0.0080 gm. BaS04 = 0.12 per cent S.
Ash. 0.5082 gm. substance gave 0.0038 gm. Ash = 0.75 per cent Ash;

0.3540 gm. substance gave 0.0030 gm. Ash = 0.85 per cent Ash.

Percentage Composition of the Ash-free Substance.

c

54.47

Average.

54.47

H

7.30

7.30

N

16.55 16.73

16.64

S

0.15 0.12

0.14

0

..•• •.*. ....

21.45

Preparation No. 8.

Carbon afid Hydrogen. 0.2552 gm. substance gave 0.5000 gm. CO2 and

0.1666 gm. H.2O = 53.43 per cent C and 7.25 per cent H.
Nitrogen. 0.3169 gm. substance gave 0.0536 gm. N= 16.90 per cent N;

0.4482 gm. substance gave 0.0431 gm. N — 16.84 per cent N.
SulpJiur. 0.8235 S™- substance gave 0.0087 g^^- BaS04 = 0-15 per cent S ;

0.5679 gm. substance gave 0.0059 &^- BaS04 = 0.14 per cent S.
Ash. 0.4533 g"^- substance gave 0.0032 gm. Ash = 0.71 per cent Ash ;

0.3851 gm. substance gave 0.0031 gm. Ash = 0.81 per cent Ash.

Percentage Composition of the Ashfree Substance.

Average.
53.84

7.31

17.03 16.96 .... .... 17.00

0.15 0.14 0.14

21.71

The results for elementary composition of our eight preparations
are brought together in the appended general summary. No great
differences in the average composition of the preparations of each
group are to be found. In fact the general analytic harmony is very
striking and rather unexpected. The significant feature is to be seen
in the figures for sulphur. The quantity is slight throughout, with

c

53.84

H

7.31

N

S

0

104

A. N. Richards and Williavi J. Gies.

the content of sulphur in preparations 5-8 regularly lower than that
of preparations 1-4.^

General Summ.\ry of Elkmentary Composition.

Ele-
ments.

Preparations 1-4.
Made by the method of Chitten-
den and Hart.

Made 1

Preparations 5-S.
by the method of Richards
and Gies.

Gen'l
av.

1

2

3

4

Av.

5

6

7

8

Av.

C

54.38

54.15

54.67

54.52

54.43

53.90

54.32

54.47

53.84

54.14

54.29

II

7.32

7.26

7.40

7.30

7.32

7.42

7.30

7.30

7.31

7.33

7.33

N

16.19

16.82

16.76

16.83

16.65

16.74

17.11

16.64

17.00

16.87

16.76

S

0.21

0.21

0.16

0.24

0.21

0.16

0.14

0.14

0.14

0.14

0.18

0

21.90

21.56

21.01

21.11

21.39

21.78

21.13

21.45

21.71

21.52

21.44

The following summary affords ready comparison in this connection
with related results for average elementary composition : —

Ligament elastin : C H N S O

HoRBACZEWSKi 54.32 6.99 16.75 .... 21.94

Chittenden and Hart . . 54.08 7.20 16.85 0.30 21.57

Richards and Gies .... 54.14 7.33 16.87 0.14 21.52

Aorta elastin :

ScHWARz2 54.34 7.08 16.79 0.38 21.41

Bergh 5399 7.54 15.20 0.60 22.67

Reactions. — We have little to add in this connection to what has
already been noted. We have found, however, that elastin is not as
resistant to acids and alkalies as it is generally considered to be.
When the original tissue is very finely and thoroughly divided with a
meat chopper, as was the case for the first time in our experiments,
the particles undergo some solution in the acids used in the extrac-
tion process. The purified poivdcred substance is slightly soluble
even in cold 0.2 per cent hydrochloric acid on long standing and
dissolves very quickly and completely in i per cent potassium
hydroxide on warming. These results suggest that the state of
division of the tissue in preparation of elastin greatly influences
solubility and thereby also purification. We believe that the agree-
ment in composition between the two groups of our products was

^ See references under "Sulphur content," page 105.

- SCHWARZ : Zeitschrift fiir physiologische Chemie, 1894, xviii, p. 487.

Elastin, Mucoid, and Other Proteids in Elastic Tisstie. 105

dependent largely on the particularly fine division of the tissue
employed. The acids used for extractive purposes were given an
excellent opportunity to decompose and completely dissolve inter-
fibrillar extraneous matter.

Sulphur content. — It will be recalled that in the older methods of
elastin preparation extraction of the elastic tissue by boiling in dilute
alkali for several hours was a part of the process and that, although
the resultant substance varied somewhat in composition, it was free
from sulphur in a majority of cases, Chittenden and Hart were the
first, as we have already pointed out, to call attention to the proba-
bility that sulphur is really an integral part of elastin, and that on
boiling with alkali the constituent sulphur is removed. By avoiding
the use of alkali Chittenden and Hart prepared elastin with a content
of sulphur amounting to 0.3 per cent. They said in this connection,
" Whether pure elastin does contain sulphur or whether the 0.3 per
cent present in preparation B (made by their own method) is a con-
stituent of some adhering proteid, removable by alkali, we are not at
present prepared to say, but deem it probable that elastin does con-
tain a small amount of sulphur."

Zoja and Eustis have recently confirmed the Chittenden and Hart
result. Schwarz lately found about the same amount of sulphur in
elastin from the aorta, but states that all was removable on boiling
with I per cent potassium hydroxide and that the residual product
was identical with the original body. Bergh has also obtained as
much as 0.55 per cent of sulphur in aorta elastin prepared by the
old alkali extraction method.

The results for sulphur content of all our preparations are given
on page 106.

It will be seen that the average sulphur content of the five prepa-
rations made according to the older method was 0.20 per cent, whereas
the elastin made by our own process, from which we had positively
excluded the presence of mucoid and coagulable proteid, shows a per-
centage of sulphur amounting to 0.15 per cent, an average difference
of 0.05 per cent in favor of the improved method. This difference,
slight though it is, is fairly constant throughout. The analyses were
made with the very greatest care. Our results seem to show conclu-
sively that sulphur, in minute quantity at least, is a component part
of pure ligament elastin.

Schwarz, it will be remembered, found that after treatment of aorta
elastin with boiling one per cent potassium hydroxide for four hours

io6

A. N. Richards and William J. Gics.

all of the sulphur (0.38 per cent) was split off in a form which could
be precipitated as lead sulphide, leaving a sulphur-free, insoluble
elastin having all of the properties of the original substance. Liga-
ment elastin seems to be a different substance. On decomposing
samples of our eight preparations in one per cent potassium hydroxide
as Schwarz did, no sulphur in the form of sulphide could be detected

Elastin made Ijy the
Chittenden and Hart method.

Elastin ni.ide by the
Richards and Gies method.

Number
of prep-
aration.

Percentage of sulphur.^

Number
of prep-
aration.

Percentage of sulphur. ^

Direct deter-
minations.

Average.

Direct deter-
minations.

Average.

1

2
3
4
92

0.17
0.25
0.20

0.22
0.20

0.15
0.17

0.25
0.22

0.16
0.18

0.21
0.21
0.16
0.24
0.17

5
6

7
8

0.16
0.17

0.13
0.15

0.15
0.12

0.15
0.14

0.16
0.14
0.14
0.15

General average . . 0.20

General average . . 0.15

1 The ash of each preparation was slight in amount. The ash
contained an appreciable proportion of sulphur — an average of 0.11
per cent of the proteid of each group of preparations. This was doubt-
less derived in great part from the organic sulphur during incineration.

2 This preparation was not completely analyzed, and therefore was
not included in the series under elementary composition, page 104. It
contained only 0.54 per cent ash.

in any of them, even when the whole volume of alkaline fluid was
used for the test. A sample of the elastin prepared by Eustis, by
the older method, however, which did not include preliminary treat-
ment with lime-water for removal of mucoids, etc., gave decided
sulphide reaction under similar circumstances. Our preparations com-
pletely dissolved in the warm alkali.

These facts indicate that the small amount of sulphur contained in

E las tin, Mucoid, and Other Proteids i^i Elastic Tisstte. 107

pure elastin is held in a form of combination not convertible into
sulphide by treatment with boiling alkali.

Distribution of nitrogen. — The nitrogen of the proteids appears to
exist in various amino forms, none of it being in nitro or nitroso
combination. Some of it is easily split off in the form of ammonia
by acid and by alkali. Usually, however, the largest quantity is
obtainable on decomposition in the form of monamido acids and a con-
siderable proportion is frequently separable in diamido combination.

No attempts to ascertain the distribution of nitrogen in the elastin
molecule were made until very recently.^ Soon after KosseP had
stated his belief that all proteids would yield hexone bases on decom-
position Bergh ^ attempted to isolate lysin and arginin from among
the cleavage products obtained from elastins of the cervical ligament
and the aorta. His attempts resulted negatively.^

Hedin^ by essentially the same methods came to the same negative
result. He was unable, also, to identify histidin. These results
would imply that elastin does not contain a protamin radicle.

Kossel and Kutscher,^ however, by an improved method, subse-
quently isolated arginin from among the decomposition products of
ligament elastin and thus directly contradicted the conclusions of
Bergh and Hedin. The quantity of arginin isolated by them was
unusually small — much less than that for most of the other proteids.
Not long ago these same observers''' were able to separate and identify
lysin also among the bases obtainable from elastin.

The lack of agreement between Bergh and Hedin on the one side
and Schwarz and Kossel and Kutscher on the other led to the study
made by Eustis,*^ under Chittenden's direction, of the proportion of
basic nitrogen split off from elastin on decomposition with hydro-
chloric acid and stannous chloride. Following the method adopted

^ HoRBACZEWSKi Studied some of the decomposition products from a different
standpoint: Jahresbericlit der Tliier-Cliemie, 1885, xv, p. 37. Schwarz made a
study of aorta elastin similar in this respect to that of Horbaczewski : Schwarz,
loc. cit.

^ Kossel : Zeitschrift fiir physiologische Chemie, 1896-97, xxii, p. 176.

3 Bergh : Loc. cit.

* Schwarz had previously found " lysatinin " (lysin + arginin) among the
cleavage products of aorta elastin. Loc. cit., p. 497.

^ Hedin: Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 344.

^ Kossel and Kutscher: Zeitschrift fiir physiologische Chemie, /(5/</., p. 551.

■^ Kossel and Kutscher: Ibid., 1900-0T, xxxi, p. 165.

^ Chittenden (for Eustis) : Loc. cit.

io8 A.N. Richards and William J. Gies.

by Schulze ^ and numerous other investigators, Eustis, in five experi-
ments on the same preparation of elastin, obtained the following
divergent results for percentage of nitrogen in the form of organic

bases :

12 3 4 5

0.86 17.69 15.57 6.50 15.14

These discordant data, while they indicated that elastin does yield
hexone bases, led to the conclusion that the method then in use for
the separation of the hexone bases by phosphotungstic acid and
determination of the nitrogen therein, was unreliable for quantitative
purposes.

The divergence of the results obtained by Eustis made it seem
desirable for us in this work to repeat his experiments, with elastin
made by the method of preparation he used and also with products
purified by our own method.

The preparations used for this particular purpose were Nos. i, 3,
and 7. (See page 104). The method of cleavage was the same as
that used by Eustis. Decomposition took place (in the presence
of one gram of stannous chloride) in 20 per cent hydrochloric acid
in quantity equal to 4 c.c. per gram of substance used. The mixture
was boiled each time in a reflux condenser for ninety-six hours.
Separation of tin, precipitation with phosphotungstic acid and the
other steps to quantitative determination were the same in detail
as those taken in this connection by Schulze. The substance which
remained in the acid mixture as an insoluble residue, very slight in
quantity (possibly melanin), contained a mere trace of nitrogen.

In addition to a study by Schulze's procedure, we also made a
similar decomposition of preparation No. 6 by the method of Kossel
and Kutscher.'-^ 100 grams of substance was boiled in a solution of
300 grams of pure concentrated sulphuric acid in 600 grams of water
for fourteen hours in a reflux condenser. Further details of the
separations were the same as those in the experiments of Kossel and
Kutscher.

In the following table we give the essential data obtained by both
methods, the figures expressing averages of closely agreeing results :

^ Schulze: Zeitschrift fiir physiologische Chemie, 189S, xxiv, p. 276.

2 Kossel und Kutscher: Zeitschrift fiir ph3'siologische Chemie, 1900-01,
xxxi, p. 165. The authors show that this method gives more abundant yield of
hexone bases than any other.

Elastin^ Mucoid^ and Other Proteids in Elastic Tissue. 109

Number
of prep-
aration.

Weight

of ash-free

elastin

used.

Nitrogen.

Grams.

Percentage of total.

Grams.

Total.

Ammo-
nia.

Mon-
amido
acids.

Bases.

Ammo-
nia.

Mon-
amido
acids.

Bases.

A-3

1
7

13.4361
11.4472
11.5549

2.2371
1.8533
1.9227

0.0507
0.0434
0.0333

2.1351
1.8238
1.8311

0.0666
0.0420
0.0593

2.26
2.34
1.73

95.44
98.42
95.23

2.98
2.26
3.08

B-6

105.2000

18.1012

0.2572

17.0081

0.9153

1.42

93.96

5.06

1.94

95.76

3.34

It will be noted that although there is some variation in the per-
centage figures, there is yet a striking agreement among them. The
latter fact is true even though two methods were employed and very
different quantities of elastin were taken for each series of determin-
ations. It is to be noticed also that the nitrogen in the bases was
greatest for preparation No. 6 as determined by the Kossel and
Kutscher method, a result in harmony with the claim of these ob-
servers that their process furnishes the most abundant supply of
hexone bases. The uniformity of our results is in striking contrast
to the disagreement of those obtained by Eustis.

Although the strictest quantitative accuracy cannot be claimed
for the methods employed,^ it does seem warrantable to conclude
from our results in this connection that elastin contains an appre-
ciable amount of nitrogen which on proper decomposition may be
identified as nitrogen in the form of hexone bases.

In addition to the above results somewhat more specific data as
to hexone bases were obtained with preparation No. 6 in a continua-
tion of the Kossel and Kutscher method previously used. These
afford the direct comparison made with similar results obtained by

^ See papers in the Zeitschrift fiir physiologische Chemie, 1 898-1901 (vols,
xxv-xxxi) by Hedin, Gulewitsch, Henderson, Friedmann, Kutscher, and
ScHULZE and Winterstein.

I lO

A. JV. Richards a7id William J. Gies.

Kossel and Kutscher on histon, salmin, zein and gelatin, among other
products,^ in the appended summary:

Substance.

Percentage of total
nitrogen.

Percentage of weight of
original substance.

His-
tidin.

Arginin.

Lysin.

Ammo-
nia.

His-
tidin.

Arginin.

Lysin.

Ammo-
nia.

Ligament elastin

Commercial

gelatin
Zein

Histon (thymus)

Salmin ....

0.170
?

1.410

1.790

0

1.380
16.600

3.760
25.170
87.800

1

>

0

8.040

0

1.375

1.400

13.530

7.460

0

0.027

P

0.810

1.210

0

0.197

9.300

1.820

14.360

84.300

1

5-6'!

0
7.700

0

0.287
0.300
2.560
1.660
0

J Unusual difficulty was experienced in our attempts to separate lysin quanti-
tatively. By difference our figures for nitrogen of lysin were 6.65 per cent of the
total. We do not include them in the above table, because we have no confidence
in their accuracy. The microscopic appearance of the histidin dichloride and
arginin nitrate prepared by us was typical. The quantities obtained were too
slight for satisfactory analysis.

" Approximate value.

Elastin appears to be characterized by containing a comparatively
small quantity of hexone radicles. Our results indicate that histidin
as well as the lysin and arginin found by Kossel and Kutscher may
be split off from this albuminoid on appropriate treatment.

Is elastdn a " fat-proteid compound?" — Nerking^ has very recently
found that various proteids as they are commonly prepared, contain
fatty or fatty acid radicles. He did not examine elastin in this con-
nection. We have done so, with entirely negative result.

Samples of preparations Nos. 5 and 6 were used for the purpose.
The amounts of substance taken were 5.6747 gms. of No. 5 and
8.7429 gms. of No. 6. After two weeks' continuous extraction in
anhydrous ether in a Soxhlet extractor, only 0.0015 g"^- of extractive
substance (dried in vacuo) could be obtained from No. 5 ; only
0.0013 gm. from No. 6. After digesting each preparation in pepsin-

^ Their work ort elastin in this connection was only qualitative. Lysin was
isolated and identified. Loc. cit., p. 205.

^ Nerking: Archiv fiir die gesammte Physiologic, 1901, Ixxxv, p. 330.

Elastin, Mucoid, and Other Proteids hi Elastic Tissue. 1 1 1

hydrochloric acid, in continuation of Dormeyer's method, as was
done by Nerking in his woric, and then thoroughly extracting the
digestive mixture in the customary manner with ether, only 0.0017
gm. of ether-soluble matter was obtained from No. 5, only 0.0013
gm. from No. 6. Thus, in the double extraction process only 0.0032
gm. of ether-soluble matter (0.056 per cent) was obtained from No.
5 ; only 0.0026 gm. (0.03 per cent) from No. 6. These amounts are
too minute, however, to mean anything positive — are, in fact, within
the limits of unavoidable errors of extractive work. The pepsin used
in the digestive process contained 0.5 to i mgm. of ether-soluble
matter, which should be subtracted from the above totals in each
case. At most the merest trace of soluble matter could have existed
in either of the preparations. Thus it is certain that elastin as pre-
pared by the method we employed does not contain fat or fatty acid,
either in ordinary molecular combination or as an admixture.^ These
experiments have nothing to do, of course, with the question of fatty
radicles contained within the proteid molecule.

Digestibility. — In the preceding determinations of possible fat ad-
mixture it was necessary to digest the elastin. Our preparations
were readily digested in gastric juice, a result quite in harmony with
the earliest observations of Etzinger.^ Thus samples of preparations
Nos. 5 and 6, weighing respectively 5.6747 gms. and 8.7429 gms., com-
pletely dissolved, in twenty-four hours, in mixtures of 300 c.c. of
0.2 per cent hydrochloric acid and 0.2 gm. of commercial pepsin
scales (very active preparation) kept at 40° C. Cumulative power
of combining with the acid was manifested by the products as is the
case with other proteoses and peptones.^ At the end of twenty-four
hours only the merest turbidity remained in the fluid, showing that
only a very slight amount of antialbumid had formed.

In the work of Chittenden and Hart on elastin and elastoses,
elastin peptone could not be detected among the products of pepsin
and trypsin proteolysis, even though zymolysis continued under

^ PosNER and GiES : Proceedings of the American Physiological Society, 1901,
This journal, vi, p. xxix. This result indicates that the difficulty experienced by
HoRBACZEWSKi and Chittenden and Hart in completely removing " fat-like
matter" from their elastins was due to the compact character of the pieces of their
preparations. Dehydration was complete in our own {page 99), with the result
that fat extraction in purifying was comparatively easy. See Chittenden and
Hart, Loc. cit., p. 21.

^ Etzinger: Zeitschrift fiir Biologie, 1874, x, p. 84.

^ Chittenden ; Digestive proteolysis, 1894, p. 52.

112 A. N. Richards and William J. Gies.

favorable conditions for several days. Peptone was absent also from
the products formed on hydration of their elastin with very dilute
acid. Although they were unable to detect peptone among the
proteolytic products of elastin, Chittenden and Hart seem to have
assumed its probable formation under favorable conditions, however,
for toward the end of their paper they say : " Under the conditions
of our experiments, no appreciable amount of true peptone was formed
in any of the digestions; at least, nothing approaching a peptone
in reactions was to be found in any of the digestive fluids, after satu-
ration with ammonium sulphate. We propose, later, to attempt a
study of the elastin peptone, using for this purpose the elastoses just
described as well as elastin itself, and more vigorous digestive fluids,
both peptic and tryptic." ^ Chittenden and Hart found that Hor-
baczewski's " elastin peptone " was in reality deutero-elastose and
that his " hemi-elastin " is the same as proto-elastose.

After our digestive mixtures had been duly extracted with ether,
in accordance with the original aim of the experiments immediately
preceding, we allowed proteolysis to continue for about six weeks.
Ether was added to the acid mixture occasionally to prevent bac-
terial changes. At the end of that period the elastose precipitate
obtained on saturating the boiling neutral fluid with ammonium sul-
phate was surprisingly large. The filtrate was also made alternately
acid and alkaline and thoroughly boiled each time while saturated
with ammonium sulphate.^ Only very slight additional proteose
precipitates were obtained in this way. Ammonium sulphate was
removed from the filtrate with alcohol and barium carbonate in the
customary manner. The final filtrate gave a strong biuret reaction
with a slight amount of cupric sulphate and an excess of potassium
hydroxide. The amount of peptone precipitable by absolute alcohol
was comparatively slight, although more than could have arisen,
directly or by auto-digestion, from the pepsin preparation used at
the outset.

In a special experiment in this connection 8.15 gms. of preparation
No. 6 were digested in a solution of 900 c.c. of 0.4 per cent hydro-
chloric acid and 2 gms. of the very active commercial pepsin used
above. Toluol was added to the mixture as a preservative, although
the acid would have prevented bacterial action. Complete solution

1 Chittenden and Hart : Loc. cit., p. 36. See also Chittenden : Digestive
proteolysis, 1894, p. 72.

2 KiJHNE : Zeitschrift fiir Biologie, 1892, xxix, p. i.

Elastin^ Mucoid, and Other Proteids in Elastic Tissue. 113

of the elastin occurred within twenty-four hours. At the end of
seventeen days a large proportion of elastose was separated by satu-
ration of the neutral, acid and alkaline fluid with ammonium sulphate.
Separated quantitatively in absolute alcohol containing ether the ash-
free substance recovered as elastose (albuminate and antialbumid
were absent at this stage of the digestion) was 7.43 gms., showing
that at least 0.7 gm. of the original elastin had been transformed into
peptone.^ The final ammonium sulphate filtrate gave a strong biuret
reaction when large excess of potassium hydroxide was present in
the fluid. Some of the peptone contributing to this biuret reaction
must, however, have arisen from the pepsin preparation.

These experiments show that elastoses are particularly resist-
ant to progressive proteolysis through the action of pepsin, although
they demonstrate that a small proportion of true peptone is formed
from them during prolonged periods of favorable contact with the
enzyme.^

The precipitate obtained from the above digestive mixtures on
saturation with ammonium sulphate contained both primary and
secondary elastoses. It retained the color of the original elastin.
Judging from the reactions of solutions of the mixed proteoses, the
amount of proto-elastose was relatively large. Such solutions, when
concentrated, became heavily turbid on warming, as Horbaczewski^
and subsequently Chittenden and Hart observed. Turbidity was
decided even when tubes containing the clear concentrated fluid were
immersed in water at 38° C or held under the tongue. Such turbid
solutions cleared up again on cooling. The clear concentrated solution
gave heavy precipitates with small quantities of concentrated nitric
acid, picric acid, potassio-mercuric iodide, and other proteid precipi-
tants, but such precipitates were only partially, if at all, soluble on
warming. When these reagents were added to diljite solutions,
however, the precipitates which were formed at once dissolved on

1 All weights were made of substance dried to constant weight at ioo°-i05° C.

2 In similar experiments, concluded after this paper had gone to the editor,,
8 grams of elastin yielded only 1.38 gram of crude elastose when the digestion
had proceeded for forty-six days. After digesting for seventy days 10 grams of
elastin yielded less than i gram of elastose. A small proportion of proto-
elastose was contained in the latter mixture. Large proportions of peptone were
formed. These results harmonize with, and emphasize the conclusions above.

^ HoRBACZEWSKi, CHITTENDEN, and Hart : Loc. cit. See also, MoRO-

CHOWETZ, SCHWARZ : LoC. cit.

114 A. N. Richards a7id William J. Gies.

warming and reappeared on cooling, just as in the case of other pro-
teoses. Addition of excess of concentrated sodium hydroxide to the
concentrated proteose solution was followed by heavy precipitation
of some of the proteid, the precipitate persisting even when the solu-
tion was boiled.

The above reactions appear to have been due to proto-elastose,
which seems to be a peculiar member of the proteose family.

Heat of combustion. — The potential energy of the proteids, ex-
pressed in calories, varies from about 5,000 to 6,000 small calories per
gram of substance. Proteids such as peptone and osseomucoid, with
comparatively small content of carbon, have the lowest combustion
equivalents, whereas bodies like haemoglobin, with relatively large
proportion of carbon, have the very highest. The heat of combustion
of any albuminous substance depends largely on the amounts and
combinations of carbon and oxygen contained in it. The figures for
composition of elastin suggest that its heat of combustion is rela-
tively great.

The only previous observations on elastin made in this connection
were those published by Stohmann and Langbein.^ These observers
worked with elastin made by Horbaczewski's method. The combus-
tion equivalent was determined by the improved Berthelot method,
and averaged 5,961.3 small calories per gram of ash-free substance —
the highest equivalent for animal proteid.

Last June, while enjoying the freedom of Professor Atwater's
laboratory, we made a thermochemical study of some of our products.^
We wish here to express our thanks to Professor Atwater for his help
and encouragement in this work and to acknowledge, also, our in-
debtedness to his assistants, Messrs. E. M. Swett and Emil Osterberg,
for experimental aid.

The following table summarizes our results for the preparation of
ligament elastin made by Mr. Eustis by the Chittenden and Hart
process, for one made by us by the same method (No. 2), and for
two preparations made by our own method (Nos. 5 and 6) ; it also
includes the results obtained by Stohmann and Langbein :

^ Stohmann and Langbein : Loc. cit.

2 The apparatus used and method employed were the same as those previously
described in This journal : 1901, v, p. 419. Quantities of 0.7-0.8 gram were burned
at a time. The figures in the table are for substance dried to constant weight at
105°-! 10° C.

Elastin^ Mucoid^ and Other Proteids in Elastic Tissue. 115

Preparation.

Direct deter-
minations.

Averages : Calculated for ash-free substance.^

Heat of combus-
tion. Small ca-
lories per gram.

Percentage composition.

Heat of combustion.
Small calories.

i

II

Av.

C

H

N

S

O

Per
gm.

For substance
containing 1
gm. of carbon.

EUSTIS . . .

Richards
and GiES
Prep. No. 2.

Prep. No. 5.

Prep. No. 6.

5933

5849
5840
5923

5947

5821
5871
5909

5940

5835
5855
5916

54.42

54.15
.53.90
54.32

7.40

7.26
7.42
7.30

16.65

16.82
16.74
17.11

0.14

0.21
0.16
0.14

21.39

21.56
21.78
21.13

5%0

5870
5904
5967

10952

10840
109S4
10985

Average ....

5886

54.20

7.34

16.83

0.16

21.47

5925

10933

Stohmann and Langbein . .

55.03

7.20

16.91

0.18

20.68

5961

10832

1 The percentage of ash in EusTis' preparation was 0.34; in our own it varied
between 0.08 and 0.83. The Stohmann and Langbein preparation contained 0.07
per cent ash.

The general relation of the above results to those for other proteids,
is seen at a glance in the following summary:

Substance.

Average percentage
composition.

Heat of combustion.
Small calories.

g

H

N

S

O

Per
gram.

For substance

containing 1

gram of carbon.

Ligament elastin^ . . .

Various animal and veg-
etable proteids, not in-
cluding glucoproteids^ .

Connective tissue mucoids^

54.36

52.64
47.43

7.32

7.08
6.63

16.85

16.00
12.22

0.17

1.03
2.32

21.31

23.20
31.40

5932

5711
4981

10912

10849
10505

1 The figures for ligament elastin are the averages of the results obtained by
Stohmann and Langbein and in our own experiments.

2 Averages obtained by Stohmann and Langbein.

3 Averages obtained by Hawk and GiES : This journal, 1901, v, p. 423.

ii6 A. N'. Richards and William J. Gies.

II. Mucoid.

Although a few indefinite statements regarding mucoid in liga-
ment^ were made shortly after Rollett's detection of this substance
among the proteids of tendon, no attempts to separate and identify
such a substance in elastic tissue were recorded before this work was
begun. It seems that its presence had been inferred, not shown,
Vandegrift and Gies have lately found that the quantity of mucoid in
the ligamentum nuchae of the ox averages 0.525 per cent of the
fresh and 1.237 P^^ ^^^^ of the dry tissue.^ The quantity of mucoid
in ligament is considerably less than in tendon.^

Our mucoid preparations were made by the method used by Chit-
tenden and Gies.^ Quantities of ligament hash varying from three to
nine kilos were employed at a time. Much of the mucoid was lost
mechanically in the purification process. Special care was taken to
reprecipitate from solution in potassium hydroxide (0.05 per cent) or
half-saturated lime-water several times ; also, to wash thoroughly and
to dehydrate and purify in boiling alcohol-ether.

We have not made an extended analytic study of ligament mucoid,
but the following facts show its near relationship to the other connec-
tive tissue mucoids.^

In physical appearance the purified product is practically the same
as tendomucoid or osseomucoid, although the latter substances can be
dehydrated more easily. It gives the proteid color reactions very
distinctly. It yields reducing substance and ethereal sulphate on
decomposition with two per cent hydrochloric acid. The reducing
substance forms dextrosazone-like crystals with phenylhydrazine, a
fact indicating the presence of glucosamine among the hydration
products. Among the other products resulting from its hydration in
dilute acid are an antialbumid-like body, acid albuminate, proteoses
and peptone. It is digestible in pepsin-hydrochloric acid and leaves
a residue containing considerable reducing substance. Its sulphur
may be obtained both as sulphate and sulphide.

^ KtJHNE : Lehrbuch der physiologischen Cliemie, 1868, p. 363.
'^ Vandegrift and Gies : Loc. cit.
8 Buerger and Gies : This journal, 1901, vi, p. 219.

* Chittenden and Gies : Journal of experimental medicine, 1896, i, p. r86.
' Mead and Gies: Proceedings of the American Physiological Society, 1901,
This journal, 1902, vi, p. xxviii.

Elastin, Mucoid, and Other Proteids in Elastic Tissue. 117

Ligament mucoid is soluble in 0.05 per cent solution of sodium
carbonate, half-saturated lime-water and 5 per cent sodium chloride.
It is insoluble in o.i per cent hydrochloric acid, but is somewhat
soluble in 0.2 per cent solution of the same. It is less resistant to
acid than the mucoid from tendon or bone and somewhat more diffi-
cult to precipitate completely from its solution. The pure substance
does not contain phosphorus. It is acid to litmus, neutralizes dilute
alkali and has the same general precipitation reactions as the other
connective tissue mucoids. None of our preparations contained
chlorine.

The percentage amounts of nitrogen and sulphur in mucoids fur-
nish favorable data for general comparisons of composition. The
summary below gives our results in this connection, together
with the proportion of ethereal sulphur. In the analyses the usual
amounts of substance, dried to constant weight at ioo°-iio° C were
taken. The quantity of ash in the preparations varied between 1.04
per cent and 1.90 per cent. The ash consisted mostly of calcium and
of phosphoric acid. The quantity of total phosphorus in preparation B
(the only one analyzed in this connection) was 0.18 per cent. The
phosphorus of the ash of preparation B amounted to 0.16 per cent of
the proteid.

Preparations.

A

B

C

D

E

General
Averages.

Nitrogen

12.80
13.01

13.40
13.64

13.74
1366

13.90
13.82

13.27
13.22

-

12.90

13.52

13.70

13.86

13.25

13.44

Total sulphur ....

2.05
2.09

1.77
1.68

1.49

1.37
1.27

1.45
1.40

2.07

1.73

1.49

1.32

1.42

1.61

Sulphur as SO3 . . .

1.32
1.17

1.02

0.90

1.25

1.02

0.90

....

....

1.06

The percentage content of nitrogen appears to be uniformly higher
in ligament mucoid than in related connective tissue glucoproteids.
The content of sulphur is somewhat lower. It is to be noted, how-

ii8 A. N. Richards and William J. Gies.

ever, that experiments recently completed in this laboratory ^ indicate
that there is more than one mucoid in tendon and bone, some of the
glucoproteid separable from these tissues having as much as fourteen
to fifteen per cent of nitrogen. We are inclined to believe, from the
above results, that the same deduction regarding variability of general
composition may be made with respect to mucoid substance in liga-
ment also. It is possible, of course, that our preparations have been
contaminated somewhat with coagulable proteid or other impurity we
failed to remove. At the same time we used every precaution to
prevent admixture.

III. Coagulable Proteids.

The simple proteids of the connective tissues have received very
little attention. Those who have worked with the albuminoid con-
stituents have usually confined their studies to those particular sub-
stances, and the various papers on the mucoids have made only
incidental reference to the albumins and globulins.

We were surprised at the outset of these studies by the compara-
tively large amount of coagulable proteid present in ligament. In
two preliminary quantitative determinations with the ligamentum
nuchae of the ox we found that the coagulable proteid was equal on
an average to 0.64 per cent of the fresh tissue.^ The quantities of
coagulable proteid in tendon and cartilage, we found, were much
less, and, moreover, were very difficult to separate and determine
satisfactorily.^

^ Cutter and Gies, Hawk and Gies : Loc. cit.

* Additional results are given bj* Vandegrift and Gies: Loc. cit.

* Using the methods employed with ligament (to be described on page 119),
we found that aqueous extracts of the tendo Achillis of the ox contained only two
coagulable proteids — one separated at S4°-S7° C., corresponding to "(2)" in
ligament: the other at 73° C, apparently the same as "(4)" in ligament. (See
page 120). Loebisch, touching on this matter incidentally in his preparation of
tendomucoid, referred to what he called serum globulin and a proteid coagulating
at 78° C. He took no special pains, however, to remove the blood completely before
making the extraction in water. See, Zeitschrift fiir physiologische Chemie, 1886,
X, p. 43, foot-note.

Extracts of hyaline cartilage, in the few experiments we tried, gave negative
results. On boiling, the extracts became opalescent. Flocks did not form, even
with a fairly strong acidity. Chondromucoid and chondroitin sulphuric acid were
present, of course. These bodies doubtless interfered with coagulation of such
albumin or globulin as mav have been contained. Von Merino obtained merelv

Elastin, Mtuoid, and Other Proteids hi Elastic Tissue. 1 1 9

In order to determine, if possible, the number and character of the
simple proteids present in ligament we made use of various common
methods, among them the process of fractional coagulation. For
this purpose several extracts were made — aqueous and saline. Five
per cent solution of magnesium sulphate was used generally for the
latter type.

In the preparation of these extracts only such ligaments were used
as seemed to be free from blood in all parts. The tissue was freed of
extraneous matter and at first cut into narrow strips, which were kept
in running water for from twelve to twenty-four hours. This treatment
removed blood and lymph. The strips were then run through a meat
chopper and the finely minced substance treated with enough extrac-
tive fluid to just cover it. At the end of from twelve to twenty-four
hours, after repeated stirring, the fluid was strained through cloth and
filtered. Each extract obtained in this way was always free from
haemoglobin, as examination with the spectroscope demonstrated, —
a result implying also the absence of most, if not all, lymph pro-
teids as well. Such extracts were either practically neutral in reac-
tion or weakly alkaline to litmus. On heating, the solutions became
very turbid and after addition of a trace of acid, flocculent separation
in a water-clear fluid took place. All extracts contained such saline
matter in solution as was found by us previously in ligament ash.

In determining the temperatures of coagulation the apparatus
recommended by Gamgee^ and commonly used in such work was
employed, and 20-40 c.c. of the extract, made very faintly acid with
acetic acid, was taken for each series of observations. The tempera-
ture was raised very gradually, and as soon as turbidity ensued the
flame was removed and the solution kept at that temperature, or raised
very slowly until the precipitate became flocculent. At this point the
temperature was kept constant for from one-half to three-quarters of
an hour, and then the solution filtered. The filtrates in each case were
as clear as water. Upon raising the temperature beyond the previous
maximal point the fluid remained clear until it had reached a temper-
ature several degrees higher, when suddenly the next turbidity ensued.

Working in this way we obtained separations at the following
temperatures :

the same opalescence on boiling. See, Ein Beitrag zur Chemie des Knorpels,
1873, P- 7- (Inaugural-Dissertation, Strassburg.)

1 Gamgee: Text-book of the physiological chemistry of the animal body,
1880, i, p. 15.

Extremes of

Average

temperature. 1

temperature.

310.490 (J

40° C.

51°-61° C.

56° C.

60°-70° C.

65° C.

740.750 Q

75° C.

77°-85°C.

82° C.

120 A. N. Richards and William J. Gics.

No.

1.
2.
3.
4.
5.

All of these were obtained from each of the above types of extracts ;
(i), (4), and (5) were comparatively slight in amount.

The question naturally arose whether the precipitates separating
at the above temperatures represented individual proteids in the
tissue. Direct elementary chemical analysis would not have sufficed
to answer this question definitely, for only very minor differences in
composition exist among the albumins and globulins. Nor would a
study of the decomposition products of these coagula have afforded
any more definite conclusions. (See pages 126 and 127.)

We have sought the solution of the problem in fractional separa-
tion experiments by the methods repeatedly used by Hofmeister,
Kauder, and others, particularly for the differentiation of albumins and
globulins. Our results in this connection, on extracts made by the
method previously detailed, are briefly summarized below :

A, Aqueous extracts treated 7i>ith {^NH^-^SO^ in substance.

(a) When the aqueous solutions were half-saturated with (N 1^4)2804,
a fairly heavy precipitate was obtained, which consisted theoretically in
whole or for the most part of globulin, albumin not being precipitated by
this proportion of (NH4)2S04 (see page 124). The MgSOi solution of
this precipitate contained bodies (i), (2), and (4) in the table above.

ib) In the aqueous solution of this same precipitate {a), bodies (i),
(3), '(4), and (5) were thrown down on heating. Precipitates (i) and (3)
were comparatively heavy, the others were slight. Diluted with an equal
volume of water, this aqueous solution of precipitate {a) gave bodies (i),
(3), (4), and (5).

{c) The filtrate from precipitate {a) was saturated with (N 114)2804.
The substance thrown out of solution in this way was dissolved in water
and the solution heated. It gave precipitates (2), (3), (4), and (5).

B. MgSOi extracts treated with MgSO^ in substance.

When the MgS04 extracts were saturated with MgSOi, a heavy pre-

1 The extremes represent the limits of all our observations. As a rule the
separations occurred at or about the mean temperature, with comparatively long
intervals.

Elastin^ Mucoid, and Other Proteids in Elastic Tissue. 121

cipitate was obtained, which, dissolved in 5 % MgSOi solution, contained
products (i) and (2). The filtrate from the MgSOi precipitate, on heat-
ing, gave bodies (2), (3), (4), and (5).

Comparison of the figures for coagulated products under A and B
will show that of the total number of bodies in the aqueous and saline
extracts of ligament only one can be completely separated by satura-
tion with MgSO^ or by half-saturation with (NH4)2S04, viz. — the
one which separates at or about 40° C. (i). All the other substances
are to be found in the filtrates from the precipitates formed on
addition of MgSO^ to saturation or of (NH4)2S04 to half-saturation.

C. Contimious fractional precipitation of aqueous a?id MgSOi extracts with
MgSOi and {NJI^zSOi in substance, and with saturated solution of

We have attempted to make a closer differentiation of the coagulable
proteids contained in ligament extracts than was possible by the methods
under A and B. The extracts for these experiments were made by the
method outlined on page 119. The extract to be tested was accurately
neutralized. To a measured portion of it was added, a few grams at a
time, the salt used for precipitative purposes. As soon as a flocculent
precipitate had formed it was filtered off and washed with a solution of
the precipitating substance of a strength equivalent to that of the mother
liquid. To the filtrate, plus enough of the washings to make it up to the
original volume, were again added weighed quantities of the salt. When
a second precipitate had appeared it was treated in a manner exactly
similar to that to which the first was subjected. This process was con-
tinued till the solution was saturated or until all proteid had been
removed. The precipitates were then dissolved in a small quantity of
water with the aid of the saline matter adhering to them, and subjected
to fractional coagulation in the usual manner. The results for the
globulins are appended :

(a) 5% MgS04 extract. Volume 100 c.c. Solid substance used to
precipitate was MgSO^.

Results: Precip. I. 5 gms. = turbidity ; 25 gms. = heavy floccu-
lent precipitate.
Precip. II. 35 gms. = turbidity; 53 gms. to saturation =

flocks.
Coagulations : Solution of Precip. I. 44°-47° C. (i)
Solution of Precip. II. 64° C. (3)

Nothing more from either I or II on boiling.
{b) Aqueous extract was treated with an equal volume of saturated
solution of (NH4)2S04. The resultant precipitate (globulin?) was dis-

12 2

A. N. Richards and William J. Gies.

solved in water and treated with a very slight amount of dilute acetic acid.
A precipitate corresponding to separation No. i in the coagulation series
formed as a result of this treatment. The same was filtered off and the
filtrate carefully neutralized. This neutral filtrate was used below in (c)
and {d).

(c) Neutral filtrate obtained in (d). Volume loo c.c
salt MgSO^.

Results: Precip. I. 20 gms. = turbidity ; 42 gms. = flocculent
precipitate.
Precip. II. 43 gms. = turbidity ;

precipitate.
Precip. III. 56 gms. = turbidity ;

precipitate.
Precip. IV. 73 gms. = turbidity ; saturation + acid =

final precipitate.
Coagulations: Solution of Precip. I.

Precipitating

50 gms. = flocculent
63 gms. = flocculent

Solution of Precip. II.
Solution of Precip. III.
Solution of Precip. IV.

5i°-58°C. (2);
65°-67° C. (3).
68° -69° C. (3).
66°-67° C. (3).
54°-56° C. (2) ;
67°-7o° C. (3).
If this method gives evidence of the presence of distinct proteids in a
solution, as various observers believe, we seem to have dealt in this in-
stance with at least two substances.

id) Neutral filtrate obtained in (^), previously used in {c). Volume
100 c.c. For precipitation purposes, instead of MgSO^ in substance,
saturated solution of (NH4)2S04 was used.^

Results: Precip. I. 100 c.c. of the original filtrate + 65 c.c. sat-
urated solution of (N 04)2804 = turbidity ;
on standing, flocks separate.
100 c.c. original filtrate + 82 c.c. saturated

solution (NH4)oS04 — precipitate.
100 c.c. original filtrate -|- 91 c.c. saturated

solution (NH4)2S04 = precipitate.
100 c.c. original filtrate + 100 c.c. saturated
solution (NH4)2S04 = precipitate.

Precip. II.
Precip. III.
Precip. IV.

1 In this series addition of (NH4)2S04 solution was made cautiously until tur-
bidity began. On standing, the precipitate became flocculent. This was filtered
off and the total volume made up to the original amount with an appropriate quan-
tity of (NH4)2S04 solution of equal strength. This fluid was then treated care-
fully with more saturated solution until further precipitation occurred. The
intervals between initial turbidities were quite marked, though less so than in
the experiments under (a) and {c).

Elastin, Mucoid, and Other Proteids in Elastic Tissue. 123

At this point, according to the theoretical differences between albumins
and globulins, all the globulin-like substance ought to have been removed
from the solution (half-saturated with (NH4)2S04). The addition of
larger proportions of (NH4)2S04 to the solution gave further precipitates
as follows :

Results (continued) :

Precip-. V. 100 c.c. original filtrate + 125 c c. saturated

solution (NH4)2S04 = precipitate.
Precip. VI. 100 c.c. original filtrate +142 c.c. saturated

solution (N 114)2804 = precipitate.
Precip. VII. 100 c.c. original filtrate + 150 c.c. saturated

solution (N 114)2304 ~ precipitate.
No further precipitation was obtainable, either with more
(N 114)2804, by the addition of acid or
on boiling.
Coagulations: 8olution of Precip. I. 6i°-63° C. (3)
8olution of Precip. II. 66°-67° C. (3)
Solution of Precip. III. 66°-67° C. (3)
Solution of Precip. IV. 56°-s8° C (2)
Solution of Precip. V. 53°-59° C. (2)
Solution of Precip. VI. 56°-57° C. (2);

64°-68° C. (3)
Solution of Precip. VII. 58°-6o° C. (2) ;
67°-7o° C. (3)

A study of the results under C shows that among .the substances
extractable from ligament by MgSO^ solution or water is one which
is precipitable from MgSO^ extract by addition of 25 gms. of MgS04
to 100 c.c. of extract, or from a dilute saline solution by trace of acid
at about 40° C. (i), or by larger amount of acid at room temperature.

A second substance, presumably a globulin, was precipitated by 53
gms. of MgSO^ from MgSO^ extracts and coagulated at about 65° C.
(3). This substance, apparently, may also be separated from the
aqueous solution of the precipitate obtained on half-saturation of
aqueous extract with (NH4)2S04 or by the addition of MgSO^ in
quantities varying from 20 gms. per 100 c.c. of extract to the satura-
tion quantity for the same volume. It was also obtained from such
solution by additions of from 65 to 150 c.c. of saturated solution of
(NH4)2S04 per 100 c.c. of proteid solution.^

1 This substance appears to be comparable to fibrinoglobulin, also to serum
albumin. See Cohnheim, Loc. cit., pp. 143 and 161.

124 A. N. Richards and William J. Gies.

There is apparently another substance, separating at about 56° C.
(2) and precipitable from solution in water by 42 gms. of MgSO^ per
100 c.c. of proteid solution ; also by from 73 gms. of MgSO^ to the
saturation equivalent for the same volume of proteid fluid. It is pre-
cipitated also by 100-150 c.c. of saturated solution of (NH4)2S04 per
100 c.c. of proteid extract. From its coagulation temperature it
would seem to be comparable to fibrinogen.^

The two other proteids in the extracts of A and B coagulated at
about 75° C. (4) and 82° C. (5). Like (i) they occurred in only
very small amounts. They correspond to the albumins ("serins")
found in ox-serum, by Halliburton, coagulating at yy^ C. and 84° C.
respectively.^

Of these five products the one separating at the lowest temperature
is not a coagulum. (See page 125). The proteid which separates
at about 65° C. is also peculiar. It begins to separate from its solu-
tion when 82 c.c. of saturated solution of (NH4).,S04 are added per
100 c.c. of its own, and is not completely precipitated till the amount
of admixed saturated (NH4)2S04 solution reaches 150 c.c. per 100 c.c.
of proteid fluid. According to the generally accepted observations
of Hofmeister, Kauder, and others on the proteids of serum, globulins
are precipitated by the addition of 92 c.c. of saturated (NH4)2S04
solution to 100 c.c. of proteid fluid, whereas the precipitation of
albumins does not begin until more than 128 c.c. have been added.
This substance, in respect to its behavior toward ammonium sulphate
partakes, therefore, of the characteristics of both globulin and albu-
min.^ The fractional precipitation and coagulation methods are not
of sufficient definiteness in result for us to contend that the precipi-
tates we have obtained are not mixtures of albumins and globulins.*

1 Compare with the serum albumins studied by Michel: Jahresbericht der
Thier-Chemie, 1895, xxv, p. 11. See also Hammarsten: Lehrbuch der physiolo-
gischen Chemie, 1899, p. 132.

2 Halliburton: Jahresbericht der Thier-Chemie, 1884, xiv, p. 126; 1886, xvi,
p. 344. The first of these also corresponds to serum globulin in coagulation
temperature, but serum globulin is precipitated on half-saturation with ammonium
sulphate, the above bodies were not.

8 This solution was completely saturated. Our analytic results showed the
presence of 53.67 per cent of (NH4)2S04. Kauder's results for the same were
52.42 per cent. See, Archiv fiir experimentelle Pathologic und Pharmakologie,
1886, XX, p. 411.

* The boundary line between albumins and globulins, never very definitely
marked, has been growing less and less distinct. See Starke : Zeitschrift fiir
Biologie, 1900, xl, p. 494.

Elastin, Mucoid, aiid Other Proteids in Elastic Tissue. 125

These various proteids do not appear to come wholly from residues
of serum — the quantity in which they may be obtained seems to be
too great to permit of such an assumption. We believe, however,
that it is impossible to remove every trace of serum from such a tis-
sue without modifying the chemical character of the contents.

We are not unmindful, in considering the character of these
products, of the known influences exerted on the coagulation tem-
perature of proteids by the reaction of the fluid, its degree of acidity,
the proportion and character of saline matter in solution, rapidity of
heating, presence of foreign soluble organic bodies, concentration,
etc. All of these conditioning factors were carefully governed, how-
ever, to prevent erroneous deductions.

IV. NUCLEOPROTEID.

We believe that the substance separating at 40° C. (i) in nearly
all of the preceding coagulation experiments is, in great part at least,
nucleoproteid. That the substance was directly precipitated at that
temperature, not coagulated, was apparent from the fact that when
the various extracts employed were treated with a slight amount of
acetic acid and then allowed to stand over night, a light flocculent
precipitate settled out. After its removal only precipitate (2) and
the higher bodies previously obtained separated from the filtrate on
heating. That this acid precipitate was not a true coagulum was
further evidenced by the fact that it dissolved readily in 0.5 per cent
sodium carbonate, from which solution it was easily precipitated by
slight excess of dilute acid.

When 100 c.c. of the aqueous extract of ligamentum nuchae was
treated with 0.5 c.c. of 36 per cent acetic acid, a bulky flocculent pre-
cipitate was obtained which dissolved easily in dilute alkali. This
precipitate was not coagulable either in acid or alkaline fluid and
after fusion with alkali gave a good phosphate reaction with molybdic
solution. Further, after a very large quantity of the aqueous extract
of the tissue had been evaporated to a small bulk on the water bath
and the heavy precipitate of coagulated proteid filtered off, the viscid
filtrate gave an abundant precipitate on the addition of but a few
drops of 36 per cent acetic acid. This precipitate dissolved readily
in 5 per cent sodium chloride and was reprecipitated on saturation
with the same substance. Its solutions would not coagulate in any

126 A. jV. Richards and William J. Gies.

medium. The substance so obtained contained phosphorus in organic
combination.

Various proteids are precipitable from their solutions on acidifica-
tion. Those of special interest for us in this connection are gluco-
proteids, nucleoproteids and globulins. When carefully tested as to
its solubility in dilute acid the substance obtained in these experi-
ments was found to be precipitated by moderate excess of 0.2 per
cent acetic or hydrochloric acid. Serum globulin and fibrinogen
may be precipitated from their solutions by minute quantities of acids.
They are readily soluble, however, in moderate excess of the acids just
mentioned — in the proportion which was favorable to the precipita-
tion of the substance above. The same would be true of small quan-
tities of albuminate. Furthermore, as has already been pointed out,
our acid precipitate, unlike the other bodies just mentioned, contains
phosphorus and was non-coagulable.

Connective tissue mucoid has much the same characteristics as
this substance. Mucoid, however, is a phosphorus-free glucoproteid,
and on boiling with acids yields reducing substances. When our
acid-precipitated product was boiled for several hours with 2 per cent
hydrochloric acid, the fluid neutralized, and tested with Fehling's
solution, only a trace of a reduction occurred. Our substance could
not, therefore, be mucoid, although the slight reduction suggests that
a trace of mucoid might have been admixed with it.^

A special preparation of this acid precipitate was made as follows:
Aqueous extract of 8 kilos of ligamentum nuchae was obtained as in
the method given on page 119, and to it was added 0.5 c.c. of 36 per
cent acetic acid per 100 c.c. of extract. The flocculent precipitate
which formed on standing was dissolved in 0.3 per cent solution of
sodium carbonate. This fluid was neutralized and then acetic acid
added until precipitation occurred, i to 1.3 c.c. of 36 per cent acetic
acid was required per 100 c.c. of fluid to effect the same — a total
acidity which would have dissolved globulins readily. This precip-
itate was again dissolved and was reprecipitated in the same manner,
after which it was washed free of acid and dehydrated, and purified
as usual in alcohol and ether. 4.5 gms. (0.056 per cent of the fresh
tissue) were obtained.

^ Aqueous extracts of the tissue are in reality extracts in dilute saline solution,
the salts of the tissue contributing their solvent power. Mucoid is somewhat
soluble in such extracts. Possibly, however, the reducing substance was derived
from the nucleoproteid.

Elastin, Mucoid, and Other Proteids in Elastic Tissue. 127

Analysis of this product gave the following results for percentage
content of phosphorus in the ash-free substance: ^ (i) 0.49, (2) 0.45 ;
average, 0.47.

These figures for phosphorus content are somewhat lower than they
are for most nucleoproteids. Mucoid impurity, as we have already
suggested, may have partially accounted for this lowering of phos-
phorus content.

That the substance was not a " cell nucleo-albumin "^ was shown
by the results of the following experiment : About 2 gms. of the sub-
stance was decomposed with acid in the usual way and a test made
for nuclein bases among the cleavage products, with positive result.
" Ammoniacal silver solution " gave the typical fiocculent brown pre-
cipitate. No precipitate formed, on cooling, in the solution of this
precipitate in nitric acid (i.i specific gravity). On neutralizing
however, and rendering slightly alkaline with ammonia, xanthin silver
in quantity practically equal to the original precipitate was obtained.
Tested with Fischer's modification of VVeidel's reaction this precip-
itate gave positive results for xanthin.^

That the substance is nucleoproteid, or at least contains a large
proportion of this compound albuminous substance, we feel confident.
Although we are not accustomed to associate nucleoproteids with any
but glandular tissues, the fact remains that nucleoproteids are to be
found in every cell, and therefore must exist in every tissue. Pekel-
haring* has lately found that 0.37 per cent of fresh muscle — a com-
parable tissue in this connection — consists of a nucleoproteid con-
taining 0.7 per cent of phosphorus.

V. Collagen (Gelatin).

All forms of connective tissues contain collaginous fibres. Eulen-
berg^ first demonstrated the presence of collagen in ligamentum
nuchae by obtaining gelatin from it. Recently the quantity was

1 The merest trace of phosphorus was present in the ash, 4-6 per cent of the
total quantity. This was deducted from the figures for total phosphorus. The
ash amounted to 0.75 and 0.89 per cent — average, 0.82. 0.5-0.6 gram of sub-
stance was used in each of the determinations by the usual methods.

2 See Cohnheim: Loc. cit., pp. 181-183.

^ Fischer: Berichte der deutschen chemischen Gesellschaft, 1897, xxx, p. 2236.

* Pekelharing: Zeitschrift fiir physiologische Chemie, 1896-97, xxii, p. 245.
See also, Kossel, Ibid., 1882-83, vii, p. 7.

5 Eulenberg: See Schultze, Annalen der Chemie und Pharmacie, 1894,
Ixxi, p. 277.

128 A. N. Richards and William J. Gies.

accurately determined and was found to be 7.23 per cent of the fresh
and 17.04 per cent of the dry tissue — equal, roughly, to one-fourth
the amount of contained elastin.^

The presence of so much elastin in ligament makes it impracticable
to separate the collagen as such, by the Ewald and Kiihne process of
digestion with trypsin in alkaline medium.^ In order to obtain some
idea of its character, however, we transformed it into gelatin and then
separated and studied the latter.

Preparation of ligament gelatin. — After the cleaned ligament had
been put through a meat chopper the hash was thoroughly washed
in running water and later thoroughly extracted in half-saturated
lime-water. After the alkali had been completely removed with
water, the residual tissue was boiled for a short time in distilled
water. The filtrate was concentrated somewhat on the water bath
and then the gelatin precipitated from it by pouring it into a large
excess of alcohol. The typical fibrous precipitate of gelatin was
obtained in this way. This was redissolved in water and reprecipi-
tated in alcohol several times, then dehydrated and completely puri-
fied in alcohol-ether.

Elementary composition. — The following data were obtained in ele-
mentary analysis of one preparation by the methods previously used
in this connection for elastin.

Carbon and Hydrogen. 0.2324 gm. substance gave 0.1372 gm. H2O = 6.56

per cent H ; 0.3773 gm. substance gave 0.6860 gm. CO2 and 0.2250 gm.

H.jO = 49-59 per cent C and 6.63 per cent H; 0.3681 gm. substance

gave 0.6705 gm. CO2 and 0.2194 gm. HoO = 49.68 per cent C and 6.62

per cent H,
Nitrogen. 0.2867 gm. substance gave 0.0501 gm. N. = 17.47 percent N;

0.3578 gm. substance gave 0.0634 gm. N = 17.72 per cent N.
Sulphur. 0.7370 gm. substance gave 0.03050 gm. 63804 = 0.568 per cent

S; 0.9417 gm. substance gave 0.03734 gm. BaSOi — 0.544 per cent S.
Ash. 0.3503 gm. substance gave 0.0058 .gm. Ash = 1.65 per cent Ash;

0.2746 gm. substance gave 0.0047 g'^^- ^^ =1.71 per cent Ash.

1 Vandegrift and Gies : Loc. cit.

■^ Ewald and Kuhne : Jahresbericht der Thier-Chemie, 1877, vii, p. 281.

Elastin, Mucoid^ and Other Proteids in Elastic Tissue. 129

Percentage Composition of the Ash-free Substance.^

Average.

C .... 50.44 50.53 50,49

H 6.67 6.74 6.73 6.7]

N 17.77 18.02 17.90

S 0.58 0.56 0.57

O 24.33

The following summary of percentage elementary composition
shows the relation of ligament gelatin to bone and tendon gelatin
and to purified commercial gelatin, the latter consisting of a mixture
of gelatins from bone and other connective tissues:

C

H

N

S

O

Ligament gelatin . .

. 50.49

6.71

17.90

0.57

24.33

Tendon gelatin^ . .

. 50.11

6.56

17.81

0.26

25.24

Commercial gelatin-^ .

. 49.38

6.81

17.97

0.71

25.13

Bone gelatin* . . .

. 50.40

6.64

18.34

24.64

Recent studies of the composition of connective tissues indicate
that there are perhaps three groups of collagens. These appear to
be characterized by appreciable differences in elementary composi-
tion. Thus the collagens in tendon^ and bone^ yield gelatins con-
taining approximately i8 per cent of nitrogen. Corneal collagen'''
contains about 17 per cent of nitrogen. Cartilage collagen yields a
gelatin containing little more than 16 per cent of nitrogen.^ Our
results in this connection indicate that the collagen of ligamentum
nuchae is essentially the same as that in tendon and bone.

Heat of combustion. — In two determinations of the heat of com-
bustion of ligament gelatin we obtained an average of 5276 small
calories (5261, 5291) as the combustion equivalent. These figures
accord very well with those previously obtained by other observers
for different gelatins, as will be seen from the following summary,

^ The sulphur of the ash amounted to 0.17 per cent of the dry proteid. This
was not subtracted from the above figures — much of it doubtless arose during
incineration.

^ Van Name : Journal of experimental medicine, 1897, ii, p. 124.

^ Chittenden and Solley : See Chittenden, Digestive proteolvsis, 1894,
p. 32.

* Mulder: See Hoppe-Seyler, Physiologische Chemie, iSSr, p. 100.
s Van Name : Loc. cit.

* Hoppe-Seyler: Physiologische Chemie, 1881, p. 100.

"^ C. Th. Morner : Zeitschrift fiir physiologische Chemie, 1894, xviii, p. 224.
® C. Th. Morner : Jahresbericht der Thier-Chemie, 1888, xviii, p. 221.

I^O

A. yV. RicJiards and William /. Gics.

which gives also the combustion equivalents of two proteids having
equivalents among the very lowest for albuminous substances:

Substance.
Dried at 100°-110° C.

Heat of combustion.
Small calories.

Percentage composition.

Per gram.

For substance

containing 1

gm. of carbon.

Carbon.

O.xygen.

Ligament gelatin . .
Fish gelatin i ...
Commercial gelatin- .
Fibrin pepton •' . . .
Tendomucoid* . . .

5276
5242
5270
5299
5003

10450

10800

10577
1(H15

50.49

48.53

5010
48.04

24.33

25.54

25.79
30.62

1 Rkrthelot kt Andrf. : Centralblatt fiir Physiologic, 1890, iv, p. 611.

- Atwater: Report of the Storrs (Conn.) Agricultural E.xperinient Station, 1899,
p. 92.

8 SroifM.vxN und L.anghei.n : Journal fiir praktische Chemie, neue Folge, 1891,
xliii, p. 375.

•* Cutter and Gies: Loc.cit.

Crystalline Extractives.

In our first report of this work ^ we called attention to the fact that
ox ligament contains an appreciable quantity of crystalline extractives.
The only crystalline substance whose identity we had definitely deter-
mined at that time was creatin, although the general method of
detecting nuclein bases had shown the presence of one or more of
these bodies also. A continuation of this work has given us more
definite results.

Preparation of extract. — The " extract " containing the crystalline
substances was obtained in the following manner: 15-20 pounds of
ligamenta nuchcX, which were perfectly fresh and which had only
mere traces of blood in them, were finely minced in a meat-chopper.
The hash was extracted in a moderate amountof water at 40° C. for
12-24 hours — ether or powdered thymol preventing putrefaction.
The extract was strained through cloth, then heated to boiling, after
which sufficient acid was added to completely separate coagulable

^ Richards and Gies : Loc. cit.

Elastin, Mucoid, and Other Proteids in Elastic Tissue. 131

proteid and contained mucoid.^ That practically no haemoglobin
was present was shown by the fact that the precipitate at this point
was entirely white.

The slightly acid filtrate was then neutralized and evaporated on a
water bath to a thin syrup. This concentrated extract had all of the
physical properties of ordinary " meat extract." It contained traces
of proteid (derived gelatin and albuminate probably) but no reducing
substance could be detected in it.^ Chloride and phosphate of sodium
and calcium were present in comparative abundance. Sulphate was
also detected.

Creatin. — The concentrated extract was diluted with several
volumes of water and treated with lead acetate for the removal of in-
organic radicles. The excess of lead was precipitated with hydrogen
sulphide and the filtrate evaporated to a thin syrup on the water bath.
On standing thirty-six hours, typical crystals of creatin formed in
good quantity. After filtering and evaporating to greater concen-
tration occasionally a new but smaller crop of crystals was obtained
each time.

The fluid concentrated in this way was treated with moderate ex-
cess of 90 per cent alcohol and the solid matter tested, together with
the separated crystals, for creatin. The crystals and the alcohol pre-
cipitate were readily soluble in water. On hydration with acid in
the usual way, the fluid gave the typical crystals of creatinin zinc
chloride with an alcoholic solution of zinc chloride, and also responded
to Weyl's reaction.

Hypoxanthin. — The alcoholic filtrate from the precipitated creatin
was next evaporated nearly to dryness to get rid of alcohol, a little
water added, the fluid made alkaline, filtered, and then treated with an
appropriate quantity of " ammoniacal silver solution." The resultant
heavy brown precipitate of nuclein bases, on decomposition with hot
nitric acid of i.i specific gravity, gave a yellowish filtrate, which, on
cooling, deposited a large proportion of crystalline substance, mostly
needles of hypoxanthin silver nitrate. The mixture was allowed to

1 A slight amount of mucoid is always contained in the aqueous extract of liga-
ment. The salts present in the extract exert solvent action on it.

2 Leucin and tyrosin were delected at this point in microscopic examination of
one sample of our extracts. We have assumed that these were formed from proteid
by hydration in the process of heating to boihng and subsequent evaporation.
Some creatinin was also detected several times. This probably arose from the
creatin by hydration.

132 A. N. Richards and Williajn J. Gies.

stand for twenty-four hours for complete precipitation of the crystal-
line matter.

The filtrate from the crystals still contained nuclein base (doubtless
xanthin, which may have been formed from the hypoxanthin), as was
shown by the brown precipitate which appeared in small quantity
when the fluid was again rendered slightly alkaline.

The crystalline precipitate containing hypoxanthin silver nitrate
was decomposed in a warm mixture of water and ammonium sulphide
on the water bath, the mixture filtered hot, concentrated on a water
bath, there saturated with ammonia and again filtered hot. A com-
paratively large amount of hypoxanthin could be detected in this
filtrate.

Guanin. — The substance insoluble in the ammoniacal fluid yielded
beautiful crystals of guanin. These were obtained by Horbaczewski's ^
method of solution in alkali, and treatment with alcohol and acetic
acid. The crystals were large and they very closely resembled those
of creatinin zinc chloride.

The bulk of the crystalline extractives consisted of creatin, hypo-
xanthin and guanin. We were unable to prove the presence of adenin
and carnin, although we occasionally obtained results by the cus-
tomary qualitative methods indicating the presence of these sub-
stances. No tests were mide for other extractives.-

It is interesting to note in this connection that guanin has been
found to occur in the ligaments of pigs with guanin gout.^

The amount of nuclein bases found in these extracts was too great
to allow of the assumption that they were derived from the small
quantity of blood and lymph remaining in the tissue when the separ-
ation was begun. Normal blood contains only traces of nuclein
bases* and the tissue itself contained at the outset only traces of
blood. In tissues, such as muscle, which contain relatively few
nuclei, nuclein bases are found in the uncombined state, and in this
condition undoubtedly represent late stages in the catabolism of
nuclear proteids. Our data show a similar catabolism in ligament,
thus leading us to a conclusion which would hardly be suggested by
the " passive mechanical functions " of the tissue — a conclusion

1 HORBACZEWSKi : Zeitschrift fiir pliysiologische Chemie, 1897, xxiii, p. 229.

2 We obtained essentially the same results as those above in continuance of the
work on tendon extract already referred to by Buerger and Gies : Loc. cit.

8 Hammarstex : Lehrbuch der physiologischen Chemie, 1899, p. 119.
* Kossel : Zeitschrift fiir physiologische Chemie, 1882-83, vii, p. 22.

E las tin. Mucoid, and Other Proteids in Elastic Tissue. 133

which harmonizes, however, with the fact that this tissue contains a
variety of substances which represent intermediate stages of chemical
differentiation.

Summary of Conclusions.

I. By improved method of preparation several samples of ligament
elastin were made, having the following average percentage com-
position :

c

H

N

S

0

54.14

7.33

16.87

0.14

21.52

All of these preparations contained sulphur. None of it could be
split off as sulphide on boiling with caustic alkali.

Only very small proportions of elastin nitrogen could be split off in
the form of ammonia and hexone bases on decomposition with acid,
Arginin, lysin, and histidin have been identified among the basic
bodies separated in this way.

Elastin is not a "fat-proteid compound." No extractive material
could be separated from our analyzed preparations by Nerking's
process.

Our purified powdered elastin readily digested in pepsin-hydro-
chloric acid. Elastoses and true peptone were formed, proto-elastose
predominating in quantity. The amount of true peptone formed was
comparatively small even after long periods of favorable contact of
the elastin and elastoses with the enzyme in acid solution, showing
that elastoses are particularly resistant to progressive zymolysis.

The average combustion equivalent of four preparations of elastin,
determinations in duplicate, was 5925 small calories.

2. Ligament contains mucoid having the general qualities of other
connective tissue glucoproteids. Analysis of five preparations gave
the following average percentage results :

N S S as SO3

13.44 1.61 1.06

3. Extracts of ligament contain proteid coagulating at 56° C,
65° C, 75° C, and 82° C. Although these figures indicate identity
with some of the albuminous substances of the blood, the coagulable
proteids of our extracts do not appear to have arisen wholly from
contained serum.

1 34 A. N. Richards and IVilliam J. Gies.

4. A slight amount of nucleoproteid is contained in ligament and
was detected in aqueous and saline extracts.

5. The gelatin obtained from ligament had the following percent-
age composition :

C H N so

50.49 6.71 17.90 0.57 24.33

These results indicate that the collagen of ligament is identical
with that of bone and tendon.

The heat of combustion of ligament gelatin was found to be equal
to 5276 small calories.

6. Among the crystalline extractives obtainable from ligamentum
nuchae were creatin, hypoxanthin, and guanin.

Reprinted from the American Journal of Physiology.
Vol. VI. — Novp:mber i, 1901. — No. III.

THE COMPOSITION OF TENDON MUCOID.i

By W. D. cutter and WILLIAM J. GIES.

[Fi-oi?i the Laboratory of Physiological Chemistry, of Coliivibia University, at the College
of Physicians and Surgeons, New Vorh.}

CONTENTS.

Page

I. Percentage content of sulphur and nitrogen 156

Preparation of fractional products 157

Analytic results 160

II. Complete elementary composition 163

Records of analysis 163

Discussion of results 166

III. Relation to other connective tissue glucoproteids 170

Composition 170

Heat of combustion 171

IV. Summary of conclusions 172

TN their paper on the glucoproteid of white fibrous connective tissue
■^ Chittenden and Gies - stated that the average amount of sulphur
in three analyzed preparations of tendon mucoid^ was 2.33 per cent.
Loebisch,* who previously had been the only one to analyze this
substance completely, found in it an average of but 0.81 per cent of
sulphur, and ascribed to it the formula C^goH^j^Ng^S^Ogo with a
molecular weight of 3936. Referring to the unexpectedly high results
of their sulphur determinations, as compared with those obtained by
Loebisch, Chittenden and Gies wrote : " We present these figures

^ Some of the results of this work were reported before the American Physio-
logical Society. See the Proceedings, Cutter and Gies : This journal, 1900, iii^
p. vi.

2 CnrrTENDEN and Gies : Journal of experimental medicine, 1896, i, p. 186.

3 Following Cohnheim's suggestion (Chemie der Eiweisskorper, 1900, p. 259)
we use the term " mucoid," instead of the previously accepted " mucin," to desig-
nate this substance. We agree with Cohnheim that, for the sake of definiteness,
the term "mucin" may be best applied to the glucoproteids elaborated by true
secretory cells, and the term "mucoid" to similar substances in the tissues. In
the present unsettled state of our chemical knowledge regarding these bodies, such
a distinction is at best of only temporary convenience. The original differences
have little importance in the light of the results of recent researches.

^ Loebisch: Zeitschrift fiir physiologische Chemie, 1886, x, p. 40.

155

156 /F. D. Cutter and William J. Gies.

with some doubt in our own minds, but, having obtained them as
the result of most careful work, we see no possible explanation other
than that this amount of sulphur is actually present in the mucin
molecule." ^

The divergent results of these two investigations naturally throw
some doubt on the question of the elementary composition of tendon
mucoid. We have attempted not only to ascertain definitely the
amount of sulphur in tendon mucoid, but also to explain the previous
discrepancy in experimental data relating to sulphur content. In
addition to the results in this particular connection, certain others
of significance obtained by us may be appropriately given with them.

I. CON'TENT OF SULPIIUR AND NiTROGEN.

Historical. — Rollett"- was the first to show that tendon contains
mucin-like material. He described some of the qualities of the sub-
stance, but made no elementary analyses of it. Eichwald'^ merely
verified Rollett's qualitative results, in this connection.

Loebisch used Rollett's method to prepare sufificient quantities of
tendon mucoid for analysis. Only three preparations were analyzed
by Loebisch. But one sulphur determination was made on each,
with the following results: (a) 0.82 per cent; (b) 0.80 per cent;
(c) 0.82 per cent. Chittenden and Gies, who were the next to ana-
lyze this particular glucoproteid material, used improved methods of
preparation and purification and, in sulphur analysis, obtained seven
concordant results on three purified products, with the following
averages: (a) 2.34 per cent; (b) 2.35 per cent; (c) 2.31 per cent.
The difference is very striking.

With respect to the amount of nitrogen in tendon mucoid, a sim-
ilar though not so decided analytic difference was established in these
two investigations. Loebisch made only four determinations of nitro-
gen in his three purified preparations. The average of two closely
agreeing results for his first preparation was 1 1.80 per cent ; for the
second the single result was 11.84 per cent and for the third it was
11.59 P^r cent. Chittenden and Gies made ten determinations in
three preparations with the following averages of results in close

1 Chittenden and Gies: Loc. cit., p. 197.

^ RoLLETT : Untersuchungen zur Naturlehre des Menschen und der Thiere
(Moleschott), 1859, vi, p. i. Also, Ibid., i860, vii, p. 190.

3 EiCHWALD : Annalen der Chemie und Pliarmacie, 1865, cxxxiv, p. 177.

The Composition of Tendon Mucoid. 157

agreement: (a) 11.94 per cent; (b) 11.80 per cent; (c) 11. 51 per
cent. They found, further, that the nitrogen content of a series of
very carefully prepared fractional products varied between 11.51 per
cent and 12.26 per cent, data which seem to suggest, though they do
not establish, the existence of several related mucoids as components
of ordinary tendinous tissue.

Preparation of Fractional Products. — At the outset of these
experiments we assumed that tendon contains more than one gluco-
proteid. This seemed probable for several reasons. Among the
latter is the fact that the larger tendons show considerable variation
in texture throughout their length. Thus the tendo Achillis of the ox,
from which the previously analyzed tendon mucoids were extracted,
is comparatively soft and very tough in the main shaft, but toward
its connections with the bones becomes more compact, and outwardly
somewhat resembles cartilage. The superficial qualities of the thick
sheaths enveloping the two large branches of the Achilles tendon in
this animal also resemble those of cartilage to a certain extent.

These physical modifications within the tendinous tissue naturally
suggest chemical differentiation of the constituents. Previous an-
alytic variations respecting tendon mucoid may have been dependent
on extraction of different mixtures of distinct though closely related
bodies. Loebisch does not state which portions of the tendons were
employed in his work. Chittenden and Gies used sections of the
main shaft, together with portions of the two branches and the
sheaths of the latter. In our own experiments these parts were
extracted separately.

General Method. — In the preparation of mucoid for use in these
experiments the Achilles tendon of the ox was employed. Follow-
ing the usual method, the tissue, immediately after removal from
the animals, was thoroughly freed of extraneous matter and cut into
very thin cross sections. These pieces were washed in water and
then extracted in half-saturated calcium hydroxide. The mixtures
were shaken at regular intervals. The mucoid was precipitated from
the filtered extract with dilute hydrochloric acid.^ The precipitated
substance was repeatedly washed ; first in dilute hydrochloric acid, to

1 We Lave always found that mucoids may be precipitated from lime-water or
sodium carbonate solution much more satisfactorily with dilute HCl than with any-
other acid. The substance seems to separate much more quickly and completely
in the presence of slight excess of this acid. Chlorides have comparatively slight
solvent action on mucoids in the presence of free HCl, unless admixed in excess.

158 //'. D. Ctittcr and IVilliain J. Gics.

remove all traces of adherent proteid impurity, then in water until it
was free of acid. It was next redissolved in dilute alkali and repre-
cipitated once with dilute hydrochloric acid. The washing process
was repeated. Finally the acid-free substance was dehydrated and
purified by long-continued treatment with large quantities of boiling
alcohol-ether; then dried iti, vacuo and weighed.

First Experiment. Series A and B. — In this experiment two parallel series
of fractional extractions were made and the mucoid in each separated and ana-
lyzed. 4600 gms. of the main shaft of the tendon about five inches in length,
with from two to three inches of its bifurcations, were employed in Series A.
In Series B only the sheaths of the branches, weighing 1900 gms., were used.
Both lots of finely divided tissue were given identical treatment at each stage
of the experiment. .-Ml extractions were made with 2 c.c. of half-saturated
lime-water per gm. of moist tissue. After the extracts had been strained
through cloth, the tendon pieces were thoroughly washed in water to prevent
adherent dissolved mucoid from becoming part of the succeeding extract.
The first extracts in each series were readily precipitated and brought to the
flocculent condition with very slight excess of 0.2 per cent hydrochloric acid.
Subsequent extracts, however, became only turbid with large e.xcess of 0.2 per
cent HCl — even with an equal volume. It was necessary, therefore, to add
stronger acid (1.5% HCl) to separate the mucoid in flocks.^ In purifying, the
substance was redissolved in half-saturated lime-water. Powdered thymol,
used in the second experiment also, entirely prevented bacterial action.

The summary, Table I, on page 159, gives additional significant
facts relating to these fractional preparations.

A striking feature of these preparations was the fact that precipita-
tion became more and more difificult with each extraction. More acid
was required in each successive extract (except the fourth of Series B)
to bring the mucoid to the flocculent condition. It will be seen from
the data in Tables I and II that this was entirely independent of the
proportion of contained mucoid. The alkali could not have effected
decomposition, and thereby possible variations, because it was too

^ In each instance the acid was added slowly in small quantities. The mix-
tures were thoroughly stirred and allowed to stand for flocks to form. After
waiting a sufficient time, more acid was added if separation had not taken place.
At first 0.2 per cent HCl was used. If after an equal volume of the acid had
been stirred in. flocks failed to separate, 1.5 per cent HCl was added little by little.
Separation took place instantly upon reaching the proper amount of acid. On
reprecipitating, the same procedure was followed. The proportion of acid required
was not recorded in the latter case, but great variations were observed. This
method was employed in the second experiment also.

The Composition of Tendon Mucoid.

159

weak. Further, the volumes of fluid in each series were kept con-
stant and the temperature was always about the same, so that the
salts formed on acidification of the alkali of the extracts had essen-

TABLE I.

Extract.

Time of
extraction.

Amount of

HCl present to

completely

precipitate. 1

Weight of puri-
fied product.^

Number.

Volume c.c.

Hours.

Per cent.

Grams.

Series A.
First
Second
Third
Fourth

9200
9200
9200
9200

24
24
24

48

0.04
0.18
0.26
0.32

6.52
9.79
3.55
3.13

Series B.
First
Second
Third
Fourth

3800
3800
3800
3800

24
24
24
48

0.03
0.17
0.46
0.37

4.23
1.65

\ 0.93

1 The figures for per cent of HCl necessarily present to precipitate in flocks
express approximate values. The precise amount of acid neutralized by the
Ca(OH)2 was not directly determined. It was the same of course throughout each
series. Greater exactness would have emphasized the facts made significant by the
above data.

■^ These weights are for substance dried iji vacuo. The amount of each prepara-
tion could not be exactly quantitative, of course, because of slight losses during
their purification. The mucoids are very difficult substances to handle and their
preparation is decidedly laborious. Every effort was made to reduce inevitable loss
to a minimum, however, and, as the loss was relatively the same in each preparation,
the weights are entirely reliable for the intended comparisons.

tially the same influence throughout. The extracts were strained
quickly at practically the same time and were promptly treated with
acid, so that no changes could have occurred by reason of delay in
final treatment.

i6o W. D. Cutter and IVilliam J. Gics.

The figures for weights of substance in each extract suggest varia7
ble resistance, on the part of the mucoid, to the solvent action of the
dihite alkali. None of the extracts were ever saturated and all were
distinctly alkaline. The peculiar behavior of these preparations
harmonizes with the view that the tissue contains two or more gluco-
proteids, and that the products separated by the usual method of
mucoid extraction are mixtures of different bodies.

(<•) Second Experiment. Series C and D. — A second set of preparations
was made in essentially the same way as in the first experiment. 6600 gms. of
the main shaft of the tendon and its branches, of the same size as heretofore,
were extracted in Series C ; 4200 gms. of sheath in Series D. The periods of
extraction were shorter at the beginning and longer at the close of this
experiment than previously. In purifying, the substance was redissolved in
0.5 per cent sodium carbonate.

The summary of results given in Table II, page 161, connected with
preparation, is directly comparable with Table I.

In this experiment, also, successive increase in proportion of acid
was necessary for precipitation, the results harmonizing in detail with
those of the first experiment. Variations in the quantities of separ-
ated mucoid again pointed to variable resistance to the action of the
extractive. Fractions of a single substance would hardly act so
differently at successive intervals under essentially the same con-
ditions.

Analytic results. — Although the differences in the action of our
several products indicated the existence of two or more mucoids in
tendinous tissue, more direct evidence than qualitative variation was
necessary to justify such a conclusion. We very carefully analyzed
these products, with results that confirm the original deduction.

The amounts of nitrogen and sulphur in mucoids furnish excellent
data for general comparisons of composition. Table III, on page
162, summarizes our results for percentage content of nitrogen and
sulphur in the a.sh-free substance dried at 105-110° C. to constant
weight.^ The analyses were made by the customary methods —
Kjeldahl for the nitrogen; fusion with NaOH over alcohol flame,
and precipitation with BaClj, for sulphur.

1 The proportion of asli in these preparations was usually much less than i per
cent. In only four was it more than that, and in none of these did it exceed 1.78
per cent. It consisted mostly of phosphate and chloride ; only a trace of sulphate
was present.

The Composition of Tendon Mticoid.

i6i

These results seem to prove that more than one substance has
been extracted — that mixtures have been obtained. The results
for every member of each series differ decidedly in one respect or

TABLE II.

Extract.

Time of
extraction.

Amount of

HCl present to

completely

precipitate.!

Weight of puri-
fied product. 1

Number.

Volume cc.

Hours.

Per cent.

Grams.

Series C.

First

13200

17

0.03

14.56

Second

13200

20

0.15

24.88

Third

13200

26

0.17

17.26

Fourth

13200

30

0.38

2.04

Fifth 2

13200

65

0.45

4.09

Series D.

First

8400

17

. 0.02

11.85

Second

8400

20

0.15

13.41

Third

8400

26

0.45

3.19

Fourth

8400

30

0.39

0.29

Fifth

8400

65

0.35

0.59

1 See notes under Table I.

2 A sixth extraction lasting 124 hours was mad

; in Series C. A

trifle more than

a gram of unpurified substance was obtained. Th
HCl was necessary in order to bring it to the floe

e presence of nea
culent condition.

rly 1 per cent of

This substance

was true mucoid — on decomposition it yielded a
from these results that it is very difficult to com

reducing substance
pletely extract gh

;. It is evident
icoproteid from

tendinous tissue.

another from the rest m the group, and this, too, in spite of the fact
that the analyses of all were conducted by uniform methods and
under conditions as nearly the same as it is possible to attain. The
extremes in percentage content are too far apart to be due to un-
avoidable analytic errors.

If. D. Cutler and William. J. Gics.

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The Composition of Tendon Mucoid. i'63

It will be noticed that the nitrogen of the mucoids of the first
extracts is greater in amount than in the second — just as was found
in the single similar experiment by Chittenden and Gies. With one
exception the nitrogen of the mucoid in the second extract is much
less in each series than in any of the others of the group but be-
comes greater with each succeeding extraction. The sulphur, on the
other hand, shows gradual decrease in Series A and C, but remains
much the same in the other two. The average content of sulphur
in the mucoids of Series B and D (prepared from the sheaths) is
appreciably higher than in the others. The nitrogen average is
practically the same in all.^

II. Complete Elementary Composition.

We made complete analysis of several of our preparations in order
to obtain additional evidence in the connections just discussed, and
to add if possible to our knowledge of general composition.

Closely related members of Series C and D of our preparations
were selected for this purpose. The methods of analysis were those
commonly in use. We followed those outlined in detail in a recent
paper on a similar subject from this laboratory,- so that their descrip-
tion may be omitted here. Special care was taken to keep as nearly
uniform as possible all conditions known to affect analysis, so that
the results would be directly comparable.

No. 1. Mucoid of first extract of Series C.

Carbon and Hydrogen. 0.3550 gm. substance gave 0.6120 gm. CO2 and

0.2100 gm. HoO = 47.02 per cent C and 6.57 per cent H ; 0.4120 gm.

substance gave 0.7140 gm. CO, and 0.2480 gm. H2O = 47.26 per cent

C and 6.69 per cent H.
Nitrogen. 0.2275 S™' substance gave 0.0282 gm. N= 12.40 per cent N;

0.1484 gm. substance gave 0.0187 %^^- ^ = 12.61 per cent N ; 0.1894 gm.

substance gave 0.0236 gm. N = 12.46 per cent N.
Total SidpJmr. 0.5665 gm. substance gave 0.0905 gm. BaSO^ = 2.19 per

cent S ; 0.6547 gm. substance gave 0.1045 S™- BaSOi = 2.19 per cent S.
Sulphur combined as SOz- 0.4210 gm. substance, after boiling in HCl, gave

0.0413 gm. BaSOi = 1.33 per cent S; 0.2880 gm. substance, after boiling

in HCl, gave 0.0286 gm. BaSO^ = 1.35 per cent S.
Ash. 0.1727 gm. substance gave 0.0012 gm. Ash = 0.69 per cent Ash.

1 Compare with results for carbon and ox3'gen, also, in Table IV, page 168.

2 Hawk and Gies: This journal, 1901, v, p. 403.

c

47.34

H

6.63

N

S

o

....

164 IF. D. Cutter and William J. Gies.

Percentaok Compositio.n ok the Ash-free Suh.stan( e.^

Average.
47.59 47.47

6.74 6.68

12.49 12.70 12.55 12..58 •

2.20 2.20 2.20

31.07

No. 2. Mucoid of second extract of Series C.

Carbon and Hydrogen. 0.1252 gm. substance gave 0.7320 gm. H.jO — 6.50
per cent H ; 0.1903 gin. substance gave 0.3292 gm. CO... and o. 1122 gm.
H..0 = 47.18 per cent C and 6.55 per cent H; 0.1303 gm. substance
gave 0.2245 gm. CCj and 0.0760 gm. H.jO = 46.99 per cent C and 6.48
per cent H.

Nitrogen. 0.2523 gm. substance gave 0.0295 gm. N = TI.70 per cent N;
03037 gm. substance ga\e 0.0355 Z^^- ^ — ii-68 per cent N.

Total Sulphur. 0.6541 gm. substance gave 0.0830 gm. BaS04 = 1.74 per
cent S ; 0.7209 gm. substance gave 0.0974 gm. BaSO^ = 1.85 per
cent S.

Sulphur combined as SO?,. 0.4798 gm. substance, after boiling in HCl, gave
0.0567 gm. BaS04 = 1.62 per cent S; 0.3760 gm. substance, after boil-
ing in HCl, gave 0043 7 gm. BaSOi = 1.59 per cent S.

Ash. 0.1989 gm. substance gave 0.0017 S"^- ^^^ = °-^S P^'' *^^"'^ Ash;
0.1200 gm. substance gave 0.0009 »'"'''• ^^''^ — °-75 P^'' '^^"'^ Ash.

Percentage Compositiox of the Ash-free Substance.

Average.
C .... 47.56 47.36 47.46

H 6.56 6.60 6.53 6.56

N 11.79 11.77 11.78

S 1.75 1.86 1.81

O 32.39

No. 3. Mucoid of third extract of Series C.

Carbon and Hydrogen. 0.1194 gm. substance gave 0.2063 g"^- ^^-i ^"d

0.0709 gm. H.,0 =47.12 per cent C and 6.60 per cent H ; 0.0973 gm.

substance gave 0.1694 gm. COo and 0.0566 gm. HoO = 47.48 per cent

C and 6.46 per cent H.

1 Only traces of phosphorus were present, equal in amount to the phosphorus
in the ash. This was ascertained for each preparation. The quantity was
greatest in this particular product— 0.26 per cent and 0.24 per cent in two
determinations.

The Composition of Tendon Mucoid.

165

Nitrogen. 0.2181 gm. substance gave 0.0275 *g™- N= 12.61 per cent N;

0.3675 gm. substance gave 0.0462 gm. N = 12.57 per cent N j 0.2831

gm. substance gave 0.0351 gm. N = 12.41 per cent N.
Total Sulphur. 0.7412 gm. substance gave 0.0982 gm. BaS04 = 1.82 per

cent S; 0.6574 gm. substance gave 0.0887 S™- BaSO^ = 1.85 per cent S.
Sulphur combined as SO^. 0.6686 gm. substance, after boiling in HCl, gave

0.0653 gm. BaSOi = i-34 per cent S.
Ash. 0.1720 gm. substance gave 0.0018 gm. Ash ^ 1.04 per cent Ash.

Percentage Composition of the Ash-free Substance.

c

47.62

47.98

H

6.66

6.53

N

S

0

12.74 12.70 12.5

1.84

1.87

Average.
47.80

6.60
12.66

1.85
31.09

No. 4. Mucoid of first extract of Series D.

Carbon and Hydrogen. 0.0770 gm. substance gave 0.1372 gm. CO.i and

0.0480 gm. HoO = 48.60 per cent C and 6.93 per cent H ; 0.0968 gm.

substance gave 0.1721 gm. CO.. and 0.0578 gm. H.,0 =- 48.48 per cent

C and 6.63 per cent H.
JS/itrogen. 0.3946 gm. substance gave 0.0495 %^^- ^ ^^ ^2.55 per cent N ;

0.3154 gm. substance gave 0.0396 gm. N = 12.55 per cent N.
Sulphur. 0.5967 gm. substance gave o. 1159 gm. BaSO^ = 2.68 per cent S ;

0.7591 gm. substance gave 0.1603 gm. BaSOi = 2.89 per cent S.
Sulphur combined as SO^. 0.8904 gm. substance, after boihng in HCl, gave

0.0886 gm. BaSOj = 1.36 per cent S.
Ash. 0.1983 gm. substance gave 0.0015 gm. Ash = 0.75 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.
48.97 48.87

6.98

6.68

12.64

12.64

2.70

2.91

48.92
6.83

12.64
2.80

28.81

No. 5. Mucoid of second extract of Series D.

Carbon and Hydrogen. 0.1779 gm. substance gave 0.3101 gm. COo and 0.1028
gm. H2O = 47.54 per cent C and 6.42 per cent H ; 0.0608 gm. substance
gave 0.1066 gm. CO., and 0.0365 gm. H2O = 47.82 per cent C and 6.69
per cent H.

1 66 IF. D. Cutter a7id IVilliavi J. Gics.

Nitrogen. 0.3046 gm. substance gave 0.0380 gm. N = 12.48 per cent N ;

0.2545 gm. substance gave 0.0316 gm. N = 12.45 P^'' ^^"^ ^•
Sulphur. 0.7143 gm. substance gave 0.1226 gm. BaS04 = 2.35 per cent S ;

0.9S41 gm. substance gave 0.1608 gm. KaS04 = 2.24 per cent S.
Sulphur combined as SO^. 0.7130 gm. substance, after boiling in HCl, gave

0.0805 gm. BaS04 =1-55 per cent S.
Ash. 0.3477 gm. substance gave 0.0059 gm. Ash = 1.69 per cent Ash;

0.1665 gm. substance gave 0.0031 gm. Ash = 1.86 per cent Ash.

Perckntagk Composition of the Ash-free Substance.

Average.
C 48.40 48.67 48.54

II 6.54 6.81 6.68

X 12.70 12.68 12 69

S 2.39 2.28 2.34

O 29.75

Discussion of results. — The general summary of our results for
complete elementary composition, Table IV, may be compared with
similar data obtained in the previous investigations. It will be ob-
served that although there is some variation within each series — very
slight in Loebisch's, quite marked in our own — the three group aver-
ages are very nearly the same. This is particularly significant in this
connection. It suggests that mixtures of generally uniform composi-
tion resulted in each of the previous studies. Leobisch varied his
method very little and obtained practically uniform products ; Chitten-
den and Gies varied theirs more decidedly, and the result was distinct
variation in composition of substance extracted. By the fractional
method in our own experiments, still greater differentiation was
effected.

We do not mean to suggest that our own products are chemical
individuals. They are mixtures, just as all the previously described
tendon mucoids have doubtless been. Further research, with more
elaborate methods, and particularly with reference to inner groupings
of the elements, will be necessary for definite differentiation, if such
is possible while we remain in our present profound ignorance of the
structure and peculiarities of proteid molecules.^

The amounts of nitrogen in our preparations appear to be slightly
greater than those previously determined, although the nitrogen con-

^ Hawk and Gies : Loc. cit., p. 414 ei seq.

The Composition of Tendon Mticoid. 167

tent of preparation No. 2 (Second extract, Series C), which was the
largest in quantity of all our products,^ conforms closely with the
generally accepted figures for content of this element.

The only particularly discordant results in the general averages are
those for content of sulphur and oxygen (by difference) obtained by
Loebisch. We had hoped that this low figure would be explained by
our results, but none of our products contained so little sulphur. Our
figures in this connection accord very well with those given by Chit-
tenden and Gies. As has already been stated, Loebisch made only a
few analyses — only one determination of sulphur in each of his three
preparations. He duplicated results in only half of the analyses he
reported.

In referring to the differences in composition observed among their
products, Chittenden and Gies stated: "Our results seemingly jus-
tify the assumption that white fibrous connective tissue contains more
than one mucin, or else that the mucin obtainable from this tissue is
prone to carry with it a certain amount of some other form of proteid
matter which the ordinary methods of purification are not wholly
adequate to remove. . . . There is at the present time no standard
of purity with regard to this body, and it is quite as probable
that fibrous connective tissue contains two or more mucins as
that there is only one mucin in the tissue, and that any devia-
tion from the figures obtained by Loebisch or by us in preparation
No. 3 is due to the presence of a larger or smaller amount of proteid
impurity." ^

We can no longer believe that proteid impurity is responsible for
the observed variations. In the first place the quantity of soluble
proteid in tendon, other than mucoid, is very slight. Experiments
in progress in this laboratory indicate that it is less than 0.3 per
cent. If, however, it were possible for all of this small quantity to
combine permanently with the precipitated mucoids, it could not ac-
count for the regular ri^e and fall of nitrogen content observed in
each series of our experiments.^ Although it is conceivable that the
mucoid of the first extract could be so affected, such an assumption
would not explain the rise of nitrogen in the third and subsequent
extracts, particularly in view of the marked fall of the same in the
second. Then, too, each product was so thoroughly washed in excess

^ See table on page 161.

2 Chittenden and Gies: Loc. cit., p. 194.

2 See the table on page 162.

i68

JV. D. Cutter and William J. Gics.

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The Composition of Tendon Miicoid 169

of 0.2 per cent hydrochloric acid, that unless very intimate and unusual
chemical union resulted, lymph proteids must have been quickly and
completely dissolved from the precipitates. We know of no other
substance in tendon which would resist the washing treatment and,
by mechanical admixture or chemical combination, account for the
orderly variations observed in the analytic series.^

It is much more probable, we think, that an answer to these con-
siderations will be found in the fact that the mucoids are labile bodies
of great variety in the tissues and with more than one function to
perform. Their acid radicles doubtless make them prone to enter
into numerous ion combinations. The very complexity of these sub-
stances makes it natural to assume that exactly the same proportions
of the constituent radicles would in metabolic changes be the excep-
tion rather than the rule.

All of the products separated in these experiments were true gluco-
proteids, responding to each of the well known reactions and yielding
reducing substance in abundance.

We have repeated the experiments of Chittenden and Gies on the
osazone substance obtainable with the reducing body and, working
with a larger quantity of mixed mucoid products by the same and also
improved methods, obtained a crystalline product melting at 182° Q?
In microscopic appearance the crystals are identical with those of
glucosazone. We have not yet been able to free the substance en-
tirely from the brownish globules that occur with it and which persist

^ Since this paper went to the printer we have seen Nerking's recent note on
fat proteid compounds, in the Archiv fiir die gesammte Physiologic, 1901, Ixxxv,
p. 330. His results indicate that various proteid products, which have been puri-
fied by the usual methods, contain fat or fatty acid in close combination ; further,
that this fatty radicle may be broken off, and extracted, by Dormeyer's method.
No such combination with ovomucoid was shown, but about three per cent of
extractive matter was found to be combined with submaxillary mucin. Nerking
does not state, however, that the mucin was thoroughly extracted in hot alcohol
ether during the preliminary process of purification, in the customary manner. No
results are presented for tendon mucoid ; but Loebisch, and Chittenden and
GiES have already called attention to the fact that tendon mucoid when freshly
precipitated is admixed with extractive matter that is removable only after long
continued extraction. All our preparations were given careful and extended treat-
ment in boiling alcohol-ether, and we do not believe that the variations in com-
position noted are due to such fat combination. We hope that studies which have
lately been in progress in this laboratory, will soon furnish direct evidence con-
cerning this and related questions.

^ The product obtained by Chittenden and Gies melted at 160° C.

I 70 JV. D. Cutter aiid William J. Gics.

in spite of all our attempts to purify the crystals. It seems certain
that glycuronic acid and glucosamin, or very closely related bodies,
are formed together in the decomposition of tendon mucoid with hot
dilute mineral acid.

III. Relation to other Connective Tissue Glucoproteids.

Composition. — It appears to be definitely established by the
numerous results of these and the preceding experiments that the
amount of sulphur in tendon mucoid is relatively high — almost the
same as in chondromucoid and osseomucoid — and that Loebisch's
data in this particular connection can no longer be accepted as
correct. We have never been able to prepare a tendon mucoid having
less than 1.3 per cent of sulphur.^

The sulphur is present in at least two combinations, as in the case
of chondromucoid and osseomucoid. After boiling with alkali, lead
sulphide may be obtained on addition of lead acetate. The amount
combined in the form of SO3 is relatively large, varying as the analytic
data for each preparation show, between 1.33 and 1.62 percent of the
whole molecule. The average amount of SOa sulphur in chondro-
mucoid is 1.76 per cent. In osseomucoid it equals 1.40 per cent.
Levene- has lately separated from tendon mucoid a substance very
similar to chondroitin sulphuric acid. The quantity of this substance
separable from the mucoid has not been estimated.

Two years ago, in our preliminary report, we made the following
statement: ^ " Before these experiments were started, the similarity
in the percentage composition of Morner's chondromucoid and the
tendon mucin analyzed by Chittenden and Gies suggested to us that
the two substances are perhaps closely related. This was further
emphasized by the fact that the osazone crystals they obtained had the
same general appearance as the crystals of glucosazone, and, therefore,
might have arisen from glucosamin, one of the decomposition products
of chondromucoid." Levene's results and our own increase the prob-
ability that the two substances are very much the same.

The following summary of average elementary composition shows
the general relationship of very nearly identical products:

^ See table, page 162.

2 Levene: Zeitschrift fiir physiologische Chemie, 1901, xxxi. p. 395.

* Cutter and Gies : Loc. cit.

The Composition of Tendon Mncoid.

171

C H N S O

Chondromucoid Morxer 47.30 6.42 12.58 2.42 31.28

Tendomucoid ((7) Chittenden and Gies 48.76 6.53 11.75 2.33 30.63

{b) Cutter and Gies (1) . 47.47 6.68 12.58 2.20 31.07

Osseomucoid Hawk and Gies . . 47.07 6.69 11.98 2.41 31.85

Average . . 47.65 6.58 12.22 2.34 31.21

Heat of Combustion. — Heat of combustion furnishes important
means of estimating chemical relationships, though its indications are
not, perhaps, so delicate as those of elementary analysis. The deter-
minations in these experiments were made by the method described
by Hawk and Gies. In Table V we give the heat of combustion of
our five completely analyzed preparations, together with comparative

TABLE V.

Combustion Equivalents.

Direct determinations.

Averages

for ash-free substance.

Heat of com

bustion.

Percentage

Heat of combustion.

Preparation.

Small cal

Dries.

content.

Small calories.

Per gram of s

abstance.

Car-
bon.

Oxy-

Per gm. of
substance.

For sub-
stance con-
taining 1 gm.

1

gen.

I

II

Average.

of carbon.

I. Tendomucoid.

No. 1

4925

4940

4933

47.47

31.07

4967

10463

No. 2

4963

4930

4947

47.46

32.39

4986

10506

No. 3

4921

4934

4928

47.80

31.09

4979

10416

No. 4

4908

4920

4914

48.92

28.81

4951

10121

No. 5

5044

5036

5040

48.54

29.75

5131

10571

Average.

4952

4952

4952

48.04

30.62

5003

10415

II. Osseomucoid.

Average of two

4972

4985

4979

47.16

31.79

4992

10589

preparations.

III. Chondromucoid.

Average of two

4865

4869

4867

45.87

32.90

4883

10647

preparations.

172 jr. /J. Cutter and Williaiii J. Gics.

data from the summary in a recent paper from this laboratory.' The
figures show only imperfectly the differences among the tendon
mucoids. They are valuable chiefly for the indication they furnish
that the various glucoproteid products referred to are essentially the
same compounds.

W'e still believe " continued investigation will show that the differ-
ences among the mucins, mucoids, and chondroproteids are not as great
as their varying physical properties and behavior have suggested, but
that each is a combination of proteid with a glucosulphonic acid, the
qualities of each compound, just as in the case of the nucleoproteids,
being dependent largely on the proportions and character of the pro-
teid and compound acid radicles." -

IV, Summary of Conclusions.

The more important conclusions to be drawn from the results of
this research are :

1. Tendon contains more than one glucoproteid. The average

percentage composition of five preparations of mixed mucoid was as

follows :

C H N S O

48.04 6.67 12.47 2.20 30.62

These figures agree very closely with those published by Chittenden

and Gies.

2. The composition of mucoid from the shaft and from the sheath:

C

H

N

S

0

Shaft (3)

47.56

6.61

12.34

1.95

31.52

Sheath (2)

48.73

6.75

12.66

2.57

29.28

3. Tendon mucoids contain an average amount of sulphur equal to
that found by Chittenden and Gies — approximately 2.30 per cent.
Not a single product had the very low content of sulphur ascribed to
this substance by Loebisch.

4. The average composition of mucoid separated from white fibrous
connective tissue by the customary methods is very nearly the same
as that of chondromucoid and osseomucoid.

5. Thermochemical studies of the mucoids in tendon, cartilage, and
bone emphasize the probability that these bodies are very intimately

related.

^ Hawk and Gies : Loc. cit., p. 422.
2 Cutter and Gies: Loc. cit.

Reprinted from the American Journal of Physiology.

Vol. VII. — July i, 1902. — No. IV.

ON THE COMPOSITION AND CHEMICAL PROPERTIES
OF OSSEOALBUMOID, WITH A COMPARATIVE
STUDY OF THE ALBUMOID OF CARTILAGE.i

By p. B. hawk and WILLIAM J. GIES.

[^From the Laboratory of Physiological Chemistry of Columbia University, at the College
of Physicians and Surgeons, N'ew Vorh.]

CONTENTS.

Page

I. Osseoalbumoid 341

Historical 341

General method of preparation 342

Preparations 1-9, with analytic results 343

Conclusions from analytic data 351

II. Chondroalbumoid 354

Historical 354

Method of preparation 355

Records of analysis, preparations A and B 355

Conclusions from the data of analysis . . ' 357

III. Summary of conclusions 358

A T the time of the first announcement of the writer's discovery of
•^ ■^ glucoproteid among the constituents of osseous tissue,^ it was
pointed out that the method of preparing osseoniucoid furnishes
residual material particularly well adapted to the study of other or-
ganic substances in bone. This method, it may be recalled, consisted,
briefly, in preliminary softening of superficial layers of bone by re-
moving inorganic matter with dilute acid (e.g:, 0.2 per cent HCl). The
softer tissue was next transformed into thin shavings by scraping it
with a scalpel, and finally, after hashing the material and washing it
free of acid in water, was extracted with dilute alkali, such as half-
saturated lime-water. The residual product thus obtained naturally
contained collagen, also any other albuminoid constituent possibly
present in the tissue; other soluble proteid substances, such as lymph
proteids or nucleo-compounds, having been eliminated. The weak-
ness of the acid and alkali used in the process of preparing the ossein

^ A preliminary account is given in the Proceedings of the American Physio-
logical Society: This journal, 1902, vi, p. xxvii.
^ GiES : Proceedings, /i!^/^., 1900, iii, p. vii.

340

Compositio7i and Properties of Osseoalbumoid. 341

makes it very probable, further, that any albuminoid constituents other
than collagen are entirely unaffected chemically by such extraction
process.

These observations induced us to study the elastin-like substance
of bone. This constituent has been referred to by several investiga-
tors. They have given us anything but a clear idea of its qualities,
however. In this connection it was found desirable, also, to make a
comparative study of the albumoid of cartilage, which has been referred
to by various observers quite as indefinitely.

Osseoalbumoid.

Historical. — Numerous investigators have made chemical studies
of osseous tissue. In their researches, the organic, proteid residue
left behind after solution of the salts in acid, the so-called ossein, has
usually been regarded as consisting entirely of collagen. Lymph
proteids and nucleo-compounds have been recognized, however, and
elastic fibres are admittedly present in normal bone and in ossein,
though in comparatively small number.^

Broesike^ some years ago reviewed the data of microchemical
study of osseous tissue, and published, also, the results of several ex-
periments by himself, which led him to believe that keratin is among
the normal bone constituents. The substance he called keratin was
evidently located, in part at least, in the lining of the lacunae and
canaliculi. His conclusion that this substance was keratin was de-
pendent on its seeming indigestibility, and, further, on its lack of
solubility in various reagents in which keratin, also, is unaffected
chemically.

Smith ^ soon after, under Kiihne's supervision, made it very
evident that Broesike had fallen into a number of experimental errors,
and that, as a consequence, the latter observer's chief chemical de-
ductions were fallacious. Instead of finding ossein indigestible in
pepsin-hydrochloric acid, for example, Smith clearly demonstrated, as
.several others seem also to have done before him, that very little solid
matter remains after treatment of the organic elements of bone with
an active enzyme solution. He observed, further, that such residual
substance as is resistant either completely disappears, when subjected

1 Halliburton: Schafer's Text-book of physiology, 1898, i, p. in.
- Broesike : Archiv fiir mikroskopische Anatomic, 1882, xxi, p. 695.
^ Smith; Zeitschrift fiir Biologic, 1883, xix, p. 469.

342 P. B. Hawk and William J. Gies.

to the influence of a new pepsin-acid solution, or is converted into a
slight proportion of nuclein-Iike material entirely different from the
keratins.^ Although Smith did not establish the identity of the sub-
stance which Broesike called keratin, his work suggested that the
material was elastin.^

With nothing very definite on the point of chemical identity we
therefore proceeded with our attempts to isolate sufficient material
for analysis.

General method of preparation. — All our preparations were made
from the femur of the ox. We have already indicated that the pre-
liminary part of the preparation process consisted first in transform-
ing bone into ossein shavings, then putting the shavings through a
hashing machine and extracting the mucoid, nucleoproteids, etc.,
from the finely divided tissue.^

After this treatment, the alkali remaining in the shavings was
removed by repeated washing in water. When this process had been
completed the hash was heated in water in a large, agate-ware kettle
until gelatinization of the collagenous elements was complete. In the
later preparations the kettle was kept covered so as to elevate the
temperature of the mixture to the highest point possible under the cir-
cumstances. When it was desired to renew the hydrating fluid, the
mixture was at first strained through fine cloth or a sieve. When it
became more finely divided, toward the later stages of the disinte-
gration, filtration on a hard filter sufficed for ready separation of the
solid matter. Conclusion of the gelatinization process was determined
not only by the almost complete disappearance of fibrous structure
from the residual flocks, but also by the nearly negative reaction of
the filtered fluid with picric acid. Such slight reaction with this
reagent as persisted after a few days' boiling was due undoubtedly to
proteoses formed from the residual matter.

^ The results of Smith's experiments are obviously in harmony with the fact
that large quantities of bone are ordinarily digested in the alimentary tract of
carnivora. We ourselves have witnessed the complete digestion of small pieces
of fresh bone in a large proportion of normal gastric juice taken from a fistula in
a dog, only a small proportion of nuclein-like material remaining undissolved-

^ This has since generally been taken for granted. See Text-books of physi-
ological chemistry by Halliburton (1891, p. 493), Gautier (1897, p. 107),
Neumeister (1897, p. 454), and Hammarsten (1899, P- 326).

^ This method was given in detail in the second contribution from this labora-
tory on the subject of the preparation of osseomuc-oid. This journal, 1901, v,
P- 393-

Composition a7id Properties of Osseoalbumoid. 343

The resultant product contained the elastin-like substance, which
was purified in boiling alcohol-ether in the customary manner.

Modifications of, and additions to this method are noted below under
each preparation.

Preparation No. 1. — Our first product was made by the general
method just outlined. In this case the ossein shavings were boiled
in water for 12 hours and the residue heated continuously in a flask
over the boiling water of a bath for 328 hours — as long as the substance
appeared to diminish in bulk. The final product was dehydrated, and
extraneous matter removed, by treatment in alcohol and ether in the
usual process of proteid purification.

The material thus obtained was light and fluffy, and grayish brown
in color. The moist substance was lightly flocculent, dark brown,
granular for the most part, but consisting in small degree of fibrous
fragments — probably elastic material.' To our great surprise the
supposedly pure product contained 76.32 per cent of ash, mostly cal-
cium phosphate.^ The ash-free substance contained the following:""*

c H

49.81% 6.68%

In pepsin-hydrochloric acid, samples of this product digested very
readily, proteoses forming in good proportion.

It was very evident from these results that the soft ossein shavings,
obtained after treatment with dilute acid as above, still contained
considerable inorganic matter, which remained in part in the organic
residue even after its complete disintegration in hot water.

Preparation No. 2. — The remaining substance of preparation No. i,
about 4 grams, was washed in 0.025 P^^ cent hydrochloric acid re-

^ The reader need hardly be reminded of the great diflficulty in the way of
absolute purification of residual tissue constituents, particularly when such prod-
ucts form a comparatively small proportion of the original structure. Products
of the kind before us here, which are never dissolved, filtered, and precipitated, are
very apt to accumulate dust particles, fragments of various extraneous matters,
etc. The greatest precaution is insufficient to entirely prevent such adventitious
admixture. In all of these preparations the greatest care was constantly taken to
diminish such accidental adulteration, and before analysis was begun, each product
was very thoroughly looked over for particles of foreign material.

^ The ash was brick-red in color. The same color characterized the ash from
all of these products — both from bone and cartilage. A fairly large proportion
of iron was detectable in these inorganic residues.

* The methods of elementary analysis used throughout this work were those
in general employment already described by us: This journal, 1901, v. p. 403.

344 P- B- Hawk and William J. Gies.

peatedly for a week, until only slight quantities of phosphate could be
detected in the washings. After dehydration, etc., this product still
contained 46.25 per cent of ash.^ The physical condition of the pre-
viously dried material was doubtless unfavorable to complete elimi-
nation of the saline matter in the very weak acid used.

This product was found to be entirely insoluble in cold dilute
potassium hydroxide, even when as strong as i per cent. No biuret
reaction could be obtained in the filtrate after the substance had been
frequently stirred with the alkali for about a day.

In dilute hydrochloric acid — 0.2 per cent or less — the substance
diminished in quantity by reason of the solvent action on the admixed
phosphate, but no biuret reaction could be obtained with the acid
extract even after it had been in contact with the substance for
twenty-four hours.

The preparation itself gave the Millon's, xanthoproteic, and biuret

reactions very distinctly. The composition of the ash-free substance

was as follows :

C H N

49.71% 6.62% 1611%

Preparation No. 3. — This was obtained from several pounds of
shavings which had been made in 0.5 per cent hydrochloric acid^
and preserved during their accumulation in 10 per cent alcohol.
After the removal of the mucoid the shavings had been kept extract-
ing in large excess of 0.25 per cent potassium hydroxide for four
months, for complete elimination of traces of mucoid and nucleo-
compounds.

When the alkali had been washed out, the ossein hash was kept in
boiling water ten hours daily for thirteen days. At first the hot
water became faintly alkaline each time it was renewed, because of

^ The persistently high proportion of ash in these two preparations brought to
mind the old question of possible chemical combination between some of the
inorganic and organic substances of bone. (Consult the discussion of this matter
by Drechsel in Hermann's Handbuch der Physiologic, 1883, v, (i), p. 609).
Our later results, however, as will be seen, do not offer the same indications as
those of the first two preparations. From our later data it appears that there are
only mechanical obstacles to the ready removal of the inorganic matter, and that,
when these are overcome by more thorough acid treatment, the amount of ash is
not much above that found associated with the average proteid from other sources.

2 Shavings for the preceding preparations were made from bones treated with
0.2 per cent HCl. The shavings had been kept in 25 per cent alcohol before
extraction of the mucoid.

Composition and Properties of Osseoalbumoid. 345

liberation of mechanically held alkali on disintegration of the tissue
pieces. This alkali had persisted in spite of the previous thorough
washing. Finally, however, the warmed mixture was entirely neutral.
The boiling process was continued much longer than appeared to be
necessary merely to make certain that all collagenous matter had
been transformed into soluble material.

In order to remove more throughly inorganic matter from the sub-
stance remaining after the boiling process, the product was repeatedly
washed for ten days in cold hydrochloric acid of a strength increas-
ing at first from 0.05 per cent to 0.2 per cent, and later decreasing to
0.05 per cent. Much phosphate was taken out in this way. A
slight biuret reaction was obtainable in the washings with the 0.2 per
cent hydrochloric acid. This was not obtained with the o. i per cent
acid at first, although as the phosphate content diminished the
residual proteid became more susceptible to the action of the acid
and slight solution in o.i per cent acid finally occurred.^

After purification in alcohol-ether, etc., 1.36 gram of substance re-
mained. This preparation, in spite of the long-continued washing in
acid just before dehydration, contained 5.85 per cent of ash. Samples
of this substance gave the usual proteid color reactions and digested
easily in artificial gastric juice. The digestive product was mostly
proteose, after twenty-four hours at 40° C.

The analytic results for this preparation were as follows :

Carbon and Hydrogen. 0.1021 gm. substance gave 0.0576 gm. HgO = 6.31

per cent H; 0.1030 gm. substance gave 0.1764 gm. CO., = 46.71 per

cent C, and 0.0580 gm. HoO = 6.30 per cent H.
Nitrogen. 0.1599 8"^- substance gave 0.02413 gm. N = 15-09 per cent N.
Total Sulphur. 0.6440 gm. substance gave 0.0490 gm. BaS04 = 1-05 pcr

cent S.
Ash. 0.1213 gm. substance gave 0.0071 gm. Ash = 5.8'; per cent Ash;

0.2580 gm. substance gave 0.015 1 gm. Ash = 5.85 per cent Ash.
Sulphur of the Ash. 0.2580 gm. substance left 0.0151 gm. Ash, which gave

0.0046 gm. BaSOi = 0.16 per cent S.'^

1 Note remarks on solubility, etc., of ligament elastin by Richards and GiES :
This journal, 1902, vii, p. 104.

2 This amount of sulphur is not deducted from the quantity calculated for ash-
free substance. The large amount of sulphur in the substance makes it probable
that the SO4 of the ash was derived by oxidation of organic sulphur. This applies
equally well to all of our preparations, both from bone and cartilage.

346 p. B. Hawk and William J. Gies.

Percentage Composition of the Ash-free Substance.^

Average.
C .... 49.61 .... .... 49.61

H 6.70 6.69 .... .... 6.70

N .... .... 16.03 .... 16.03

S .... .... .... 1.11 1.11

O .... .... .... 26.55

Preparation No. 4. — This product was obtained from shavings made
about six months previously from bones treated with 0.5 per cent
hydrochloric acid. The shavings were washed once in o.i per cent
hydrochloric acid and thereafter kept in acidified 25 per cent alcohol
until several pounds of material had been obtained. During the six
months after removal of the mucoid the ossein hash was repeatedly
washed in 0.3 per cent potassium hydroxide. Finally, after the alkali
had been removed as usual, hydration was effected in boiling water,
repeatedly renewed and made faintly acid with acetic acid. From
this point the process of treatment was identical with that for prepa-
ration No. 3.

A marked physical difference between this and the former products
was observed. The residual material, although quite resistant to the
action of the boiling water, was somewhat gelatinous in appearance.
Though divided into minute flocks, these were somewhat adherent,
and tended to collect at the top of the hot water in a semi-gelatinous
layer. This was easily broken up into flocks on stirrring. The prod-
uct was finally much diminished in bulk and appeared more soluble
in dilute acids than any of the preceding preparations. We did not
obtain sufficient for quantitative analysis. The residual substance
gave the proteid color reactions. It appeared to be a transformation
product resulting from the action of the acid in the Jjoiling fluid
during the process of hydrating the collagen, although, aside from
differences in physical form and solubility, it was identical with the
other products. It contained loosely-bound sulphur, was digestible,
and did not yield reducing substance on decomposition with acid.

Preparation No. 5. — Ossein shavings were freshly prepared after
treatment of the bones with 0.2 per cent hydrochloric acid. Several
kilos of the material were made. While they were accumulating, the

^ The substance was found to be entirely free from phosphorus in organic
combination. Phosphate was the chief constituent of the ash.

Compositio7i and Properties of Osseoalbumoid. 347

shavings were kept in o.i per cent hydrochloric acid. This was fre-
quently renewed. After elimination of the mucoid with lime-water,
the shavings were washed free of alkali with very dilute acetic acid.
The rest of the process was essentially the same as that for prepar-
ation No. 3.

The fluid poured off at first, after the hydration had been begun,
was very faintly alkaline, showing, as in previous instances, that, in
spite of the acid treatment, some of the lime-water was held un-
affected in the tissue. This product appeared to be somewhat more
soluble in 0.2 per cent hydrochloric acid than preparation No. 3.
About one gram of purified substance was obtained.^

The ash of this preparation amounted to 5.88 per cent. The ana-
lytic data obtained for ash-free substance were :^

CHS
50.57 7.17 1.17

Preparation No. 6. — The results of the ash analysis of our previous
preparations made it very evident that more attention was necessary
to the removal of phosphates. Although treatment of the residual
substance with dilute acid was effective in removing most of the phos-
phate held in it, it was impossible to use sufficiently strong acid for
the purpose at that point because of the solvent and transforming
action of the same on the remaining proteid. It seemed desirable,
therefore, to give still more attention to the removal of inorganic
matter from the shavings in the first place.

A large quantity of hashed ossein made with 0.5 per cent hydro-
chloric acid, from which the osseomucoid had been removed and
which had been under 0.25 per cent potassium hydroxide for eight
months, was washed free of alkali in water and then thoroughly
stirred with 0.8 per cent hydrochloric acid at intervals for a day.
Much phosphate was removed in this process. The hash was given
similar treatment in 0.6 per cent hydrochloric acid, with the same
result. A third washing was made in 0.4 per cent hydrochloric acid.
Thereafter the hash was washed for several days in 0.2 per cent acid

1 It should be kept in mind, of course, that the quantities of substance finally
obtained do not represent fully the amounts of osseoalbumoid in the tissue. A
considerable proportion is transformed into soluble products with the collagen in
the hydration process, as well as lost mechanically in purifying.

2 Our determinations of phosphorus of this and subsequent preparations showed
that there is none present in organic combination.

348 p. B. Hawk and Williaiit J. Gies.

until only traces of phosphate were being removed. At this point
the washings did not yield a biuret reaction.

After the acid had been thoroughly washed out, leaving in a
readily soluble form ^ such traces of phosphate as might still be in
the tissue, the usual hydration process was carried out. The final
residual substance had a somewhat gelatinous appearance, just as in
the case of preparation No. 4. In this instance, also, the initial
hydration was made in the presence of a trace of acid which had not
been thoroughly washed out. Only about 0.6 gram of substance was
obtained in the process.

The amount of ash in this preparation had been reduced by the
improved method to 3.07 per cent. Analytic percentage data
obtained for the ash-free substance were :

C H

50.45 7.24

A microscopic study was made in this connection of the changes
in the ossein during the heating process. Samples were taken each
day during the ten days that the boiling was continued. Each
sample was placed in 70 per cent alcohol after it had been washed in
water.

At the end of the first day in the boiling water the fibrous structure
of the material still remaining undissolved was but little modified,
but much granular matter was present in the hydration fluid. The
fibrous structure gradually disappeared, however, and long before the
completion of the hydration process practically nothing but small
collections of granular matter represented the original structures.
An occasional fragment of what appeared to be an elastic fibre could
be detected, however. ^

Preparation No, 7. — Shavings, which had been made seven months
previously from ossein obtained in 0.5 per cent hydrochloric acid, were
kept in 0.25 per cent potassium hydroxide until ready for use in
these experiments. After most of the alkali had been removed with
water the hash was washed for several days in hydrochloric acid

1 This method of concluding the preliminary extractive process with acid had
the special advantage, over the previous methods, of transforming tri-basic earthy
phosphate into acid modifications. The washing with alkali alone naturally had
little or no extractive action on the earthy phosphates, but, on the contrary, tended
to convert residual phosphates of calcium and magnesium into fixed forms.

■^ See foot-note, page 343.

Co7nposition and Properties of Osscoalbumoid. 349

increasing in strength to 0.2 per cent. When the acid appeared to
be removed by subsequent washing in water the hydration process
was begun. The fluid soon acquired an acid reaction, however.
This reaction persisted in several of the first warm washings.

The product soon became quite gelatinous. It was very resistant
to the further action of the boiling water. Eventually nearly all of
the substance went into solution, although the renewed fluids re-
mained neutral. At the end of a week's boiling, daily for about ten
hours, too little remained for quantitative analysis.

Preparation No. 8. — The results obtained with preparations Nos.
4, 6, and 7 indicated that the presence of acid, however little it might
be in the fluid during hydration, tended to effect transformation into
somewhat gelatinous material. It was evident that this substance
was not gelatin. At the same time it was clear that it was different
from the residue obtained in the absence of acid or in the presence
of alkali. That the difference was mainly physical was indicated by
the fact that the analytic results for the semi-gelatinous form were
essentially the same as for that obtained without the influence of
acid in the hydration process. It seemed best to avoid this un-
necessary complication, and in this preparation it was accomplished.

About 3 kilos of shavings were freshly prepared from bones treated
with 0.5 per cent hydrochloric acid. After removal of the mucoid
with lime-water, as usual, the shavings were kept in 0.3 per cent
hydrochloric acid for three weeks to remove inorganic matter. The
acid was frequently renewed. At the end of this time only a trace
of phosphate reaction was obtainable in the acid washings. The
acid was very thoroughly removed by repeated washing in cold and
warm water. The boiling process in large volumes of frequently
renewed water continued for 112 hours. The moist material was
flocculent, granular, cream colored, and had no gelatinous qualities.

The usual treatment with acid before boiling in alcohol-ether was
omitted. 16.6 grams of purified product were obtained. The ash
amounted to only 2.08 per cent. It had the usual brick-red color.

The analytic results for this preparation were as follows :

Carbon and Hydrogen. 0.2032 gni. substance gave 0.3640 gm. COo = 48.86
per cent C, and 0.1251 gm. H.2O = 6.89 per cent H; 0.2035 S™-
substance gave 0.3683 gm. CO., = 49-36 per cent C, and 0.1254 gm.
HoO = 6.90 per cent H.

Nitrogen. 0.4184 gm. substance gave 0.06573 gm. N = 15.71 per cent N;
0.2420 gm. substance gave 0.03803 gm. N = 15-71 per cent N.

350 P. B. Hawk and William J. Gies.

Total Sulphur. 0.5012 gm. substance gave 0.0406 gm. BaS04 = 1.12 per

cent S; 0.5050 gm. substance gave 0.0421 gm. BaS04=i.i5 per

cent S.
Total Phosphorus. 0.4008 gm. substance gave 0.0078 gm. MgoP.jOT = 0.54

per cent P.
Phosphorus of the Ash. 0.0174 gm. Ash gave o.oioo gm. Mg2P207 = 0.33

per cent P.
Ash. 0.4850 gm. substance gave 0.0102 gm. Ash = 2.10 per cent Ash \ 0.4838

gm. substance gave 0.0099 g""*- -^^^ ~ 2-06 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.
C 49.90 50.41 .... .... .... .... 50.16

H 7.04 7.04 .... .... .... .... 7.04

N .... .... 16.04 16 04 .... .... 16.04

S .... .... .... .... 1.14 1.17 1.16

O .... .... .... .... .... .... 25.60

This preparation, after purification and drying, vi^as found to be
entirely insoluble in water, 10 per cent sodium chloride, 0.2 per cent
hydrochloric acid, and 0.5 per cent sodium carbonate ; but slowly
soluble in 10 per cent hydrochloric acid and 10 per cent potassium
hydroxide. Solution was more rapid in the alkali than in the acid. In
all of the reagents except water and sodium chloride, complete solu-
tion took place speedily on boiling. Albuminates were formed in
this treatment and could be precipitated on neutralization. Part of the
sulphur in the substance could be split off on heating with potassium
hydroxide and detected as sulphide with lead acetate. The larger
proportion of the sulphur was closely united, however.

The substance gave the typical proteid color reactions distinctly
and digested in pepsin hydrochloric acid, with a formation of
albuminate and proteoses. A small proportion of an albumid-like
residue remained undissolved. This was soluble in dilute alkali and
insoluble in dilute acid. Peptone could not be detected — probably
only traces had been formed from the small quantity of substance
used in the test.^ On decomposition with 2 per cent hydrochloric
acid the product failed to yield reducing substance.

Preparation No. 9. — This was made by essentially the same process

^ Compare the similar results obtained with ligament elastin by Richards and
GiES: This journal, 1902, vii, p. 11 1.

Composition a?td Properties of Osscoalbumoid. 351

as that for preparation No. 8. The original shavings, about 2 kilos,
were washed in acid for about three weeks longer than those of the
previous preparation, even after practically no more phosphate could
be detected in the extracts. The acid was very completely washed
out in cold and warm water before hydration was begun. The boiling
process was discontinued at the end of eighty-two hours.

The physical properties of the product were identical with those of
preparation No. 8.^ Between 5 and 6 grams of purified substance
were obtained. The ash amounted to only 2.76 per cent. It had the
usual brick-red color.

This product was found to be identical, in qualitative chemical
characteristics, with preparation No. 8. The results of its quantita-
tive analysis are appended :

Carbon and Hydrogen. 0.15 10 gm. substance gave 0.2710 gm. 002 = 48.95
per cent C, and 0.0944 gm. H.2O =■ 6.99 per cent H ; 0.1520 gm. sub-
stance gave 0.2709 gm. COo = 48.61 per cent C, and 0.0900 gm. H.iO
= 6.63 per cent H.

Nitrogen. 0.2435 gm. substance gave 0.03847 gm. N = 15-80 per cent N ;
0.2715 gm. substance gave 0.04317 gm. N = 15-90 per cent N.

Total Sulphur. 0.5042 gm. substance gave 0.0418 gm. BaS04 =1.14 per
cent S ; 0.5050 gm. substance gave 0.0437 gm. BaS04 =: 1.19 per
cent S.

Ash. 0.4007 gm. substance gave 0.0108 gm. Ash = 2.69 per cent Ash;
0.4014 gm. substance gave 0.0114 gm. Ash = 2.84 per cent Ash.

I'KRCKNTAC.E COMPOSITION OF ASH-FREE SUHSTANCE.

Average.
C 50.34 50.00 .... .... .... .... .50.17

H 7.19 6.82 .... .... .... .... 7 01

N .... .... 16.25 1635 .... .... 16.30

S .... .... .... .... 1.17 1.22 1.19

O 25.33

Conclusions from analytic data. — The summary on the next page
shows at a glance the average results of all our elementary analyses.
It also brings into comparison the figures for composition of typical
preparations of keratin, elastin, collagen, and albumoid.

^ Preparations Nos. 8 and 9 at this stage very closely resembled the similar
products from cartilage to be described farther on.

352

P. B. Hawk and William J. Gies.

Summary of Analytical Results for Percentage Composition of
osseoalbumoid.

Preparation.

Ash-free Substance.

Ash.

No.

C

H

N

S

O

1

49.81

6.68

....

76.31

2

49.71

6.62

16.11

46 25

3

49.61

6.70

16.03

1.11

26.55

5.85

4

5

50.57

7.17

1.17

5.88

6

50.45

7.24

....

3.07

7

8

50.16

7.04

16.04

1.16

25.60

2.08

9

50.17

7.01

16.30

1.19

25.33

2.76

Average.

1-7

50.03

6.88

16.07

1.14

25.88

8-9

50.16

7.03

16.17

1.18

25.46

1-9

50.07

6.92

16.12

1.16

25.73

Albumoid ^ .

50.46

7.05

14.95

1.86

25.68

Albumoid ^

53.12

6.80

16.62

0.79

22.67

Collagen 3 . .

50.75

6.47

17.86

24.92

Keratin* . .

49.45

6.52

16.81

4.02

23.20

Elastin^ . .

54.14

7.33

16.87

0.14

21.52

1 From cartilage. See page 357 of this paper.

2 From the crystalline lens. Morner: Zeitschrift fiir physiologische Chemie,
1894, xviii, p. 78.

3 From gelatin. Hofmeister : Ibid., 1879, ii, p. 322.

* From white hair. KiJHNE and Chttfenden : Zeitschrift fiir Biologie, 1890,
xxvi, p. 291.

5 From ligamentum nuchae. Richards and Gies : This journal, 1902, vii, p. 104.

Composition and Properties of Osseoalbumoid. 353

The chemical qualities of the albumoid product separated from
bone in these experiments indicate that the substance is neither a
collagen, a keratin, nor an elastin. This may also be seen from the
analytic figures. Unlike the collagens, it does not yield gelatin. It
is readily digestible, whereas the keratins are indigestible. It con-
tains an abundance of loosely united sulphur ; elastins contain only
slight quantities of sulphur, — some of them, no loosely bound sulphur
at all. The properties of our product, while somewhat different, as
we have said, approach to a certain extent those of the elastins of
ligamentum nuchs^ or the aorta.^ They appear to be identical for
the most part with those of the albumoid of cartilage.^

Since all the albumoids are residual tissue constituents of vari-
able qualities and composition, though of typical resistance to the
action of solvents,* it seems proper to classify the product we have
obtained from bone as an elastin-like albumoid and to refer to it,
therefore, as osseoalbumoid. We freely admit that, while our
chemical knowledge of the albuminoids remains as slight as at
present, such classification has the virtue of only temporary
convenience.

No attempt has been made in these experiments to ascertain the
exact location of osseoalbumoid in the tissue. It appears probable,
however, that the substance is the same as that regarded as keratin
by Broesike and which was found by him in the lining of the lacunae
and canaliculi. We are inclined to believe, also, that the elastic
fibres of the bone, perhaps also elastic portions of blood-vessels in
the Haversian canals, have contributed substance to our prepara-
tions.^ It is possible, of course, that the residual matter prepared by
the method we have employed is composed of more than one sub-
stance, although the harmony in our analyses, of preparations made
by a changeable process, indicates that the products obtained are not
admixed to any appreciable extent with variable constituents.

The proportionate amount of osseoalbumoid in bone is small. It

1 Richards and Gies : Loc. cit.

2 SCHWARZ: Zeitschrift fiir physiologisclie Chemie, 1894, xviii, p. 487.

8 M5RNER : Skandinavisches Archiv fiir Physiologic, 1889, i, p. 234. See also
page 357 of this paper.

* COHNHEIM : Chemie der Eivveisskorper, 1900, p. 299.

^ Recent staining methods show that bone contains very little elastic material.
See Abstract of Melnikow-Raswedenkow's paper, in American Medicine, 1901,
ii, p. 466.

354 P' ^- Hawk and William J. Gies.

appeared somewhat greater, however, than the quantity of the corre-
sponding constituent of cartilage.^

Chondroalbumoid.

The qualities of the albumoid obtained from bone were found to be
so nearly the same as those ascribed to the albumoid in cartilage that
a comparative study of the latter body appeared to be particularly
desirable in this connection.

Historical. — It will be recalled that in his classical researches on
the constituents of hyaline cartilage, Morner^ separated a product
which he considered an albumoid. This body was a residual sub-
stance obtained from the tracheal cartilages of the ox after complete
hydration of the collagenous elements in boiling water in a Papin's
digestor at 110-120° C.

The substance obtained in this way was entirely insoluble in i per
cent potassium hydroxide, but slightly soluble in 5 per cent solution
of the same reagent. It was readily soluble in boiling o.i per cent
alkali. It digested completely, with a formation of albuminate, pro-
teose, and peptone. It contained considerable loosely united sulphur,
but did not yield reducing substance on decomposition with acid.^
Its resemblance to keratin and elastin in some respects, and its dif-
ference from them in others, made it necessary for Morner to con-
sider it a proteid of the indefinite albumoid type.

The quantities of albumoid obtained in Morner's experiments were
too small to offer favorable opportunity for elementary analysis.
He transformed into albuminate such material as was available, how-
ever, for the sake of removing insoluble extraneous matter, and then
determined the nitrogen content of the derived products. In two
determinations the alkali albuminate made with boiling o. i per cent
potassium hydroxide contained 15.87 per cent nitrogen; that made
with boiling 0.5 per cent potassium hydroxide had 16.02 per cent.
Neither of these results was for ash-free substance, the ash not having
been determined. The nitrogen content, also not ash-free, of one
preparation, made in boiling 0.5 per cent hydrochloric acid, was 15.43
per cent. Morner concluded that the albumoid itself has a content
of nitrogen ranging between 15 and 16 per cent.

1 Further reference to osseoalbumoid is made on page 357.

2 Morner: Loc. cit.

** Compare with the results of our analysis of osseoalbumoid, page 353.

Compositio7i aud Properties of Osseoalbtimoid. 355

Nothing further has been done to determine the characters of
chrondroalbumoid. When we recall that albuminates are products
in which the proportion of nitrogen is usually different from its
proportion in the substance from which the albuminates are derived,
particularly when obtained with boilvig reagents, it is obvious that
Morner's analytic results tell us very little about the composition of
the original body.

The substance identified by Morner was absent from the tracheal
cartilages (the only ones examined) of calves. Morner concludes,
from this fact, that immature cartilage is essentially different from
the mature form of the tissue in its lack of the albumoid constituent.
This conclusion is based on only a few observations. If, however, it
is found later to be correct, the fact that osseoalbumoid appears to
be present in bone in greater proportion than in cartilage from the
same animal would suggest that, in the development of bone from
cartilage, the proportion of the albumoid constituent increases.

Method of preparation. — In these experiments we used the carti-
laginous portion of the nasal septum of the ox. Several pounds of
these pieces of typical cartilage, about ten inches long and three
inches wide, were used. The outer membranes were removed, the
pure cartilage put through a hashing machine, the resultant hash
thoroughly washed in running water; mucoid, nucleo-proteid, etc.,
thoroughly eliminated in several extractions with dilute alkali after
preliminary treatment with 0.1-O.2 per cent hydrochloric acid; and
the alkali-free residue thoroughly hydrated in boiling water for sev-
eral days under conditions identical with those for the preparation of
osseoalbumoid. The final product was also extracted with o. i per
cent sodium carbonate and 0.5 per cent hydrochloric acid in which
the substance seemed to be entirely insoluble.

The physical appearance of the final products was practically iden-
tical with that of preparations Nos. 8 and 9 of the albumoid from
bone. It accorded also with the appearance of the material described
by Morner.

Records of analysis. — After purification in boiling alcohol-ether,
as usual, the following analytic results were obtained for the two prep-
arations made bv us :

Preparation A.

Carbon and Hydrogen. 0.1998 gm. substance gave 0.3542 gm. CO2 = 4835
per cent C, nnd 0.1200 gm. HoO = 6.72 per cent H ; 0.2008 gm. sub-

356 p. B. Hawk and William J. Gies.

stance gave 0.3538 gm. CO2 = 48.06 per cent C, and 0.1202 gm. HoO

= 6.70 per cent H.
Nitrogen. 0.1929 gm. substance gave 0.02786 gm. N — 14.44 per cent N;

0.2365 gm. substance gave 0.03396 gm. N = 14.36 per cent N.
Total Sulphur. 0.3028 gm. substance gave 0.0393 gm. BaSOi = i-79 per

cent S.
Total Phosphorus. 0.2821 gm. substance gave o.ooio gm. Mg2P207 == o.io

per cent P.
Phosphorus of the Ash. 0.0295' gm. Ash gave 0.0012 gm. MggPsOy = 0.06

per cent P.
Ash. 0.1998 gm. substance gave 0.0076 gm. Ash = 3.80 per cent Ash;

0.2008 gm. substance gave 0.0070 gm. Ash = 3.44 per cent Ash.

Percentage Composition of the Ash-free Substance.

Average.

c

50.16 49.87

50.02

H

699 6.95

6.97

N

14.98 14.90

14.94

S

1.85

0

....

26.22

Preparation B.

Carbon and Ifydroge?i. 0.2019 gm. substance gave 0.3644 gm. CO2 = 49.22

per cent C, and 0.1254 gm. H2O = 6.95 per cent H ; 0.2027 S"^- ^^^'

stance gave 0.3679 gm. CO2 = 49-50 per cent C, and 0.1250 gm. H2O

= 6.90 per cent H.
JVitrogen. 0.4331 gm. substance gave 0.06276 gm. N = 14.49 P^'^ cenX. N;

0.4343 gm. substance gave 0.06307 gm. N = 14.52 per cent N.
Total Sulphur. 0.5028 gm. substance gave 0.0661 gm. BaS04 = 1.81 per

cent S; 0.5034 gm. substance gave 0.0665 S'^^- BaS04 = 1.82 per

cent S.
Ash. 0.4000 gm. substance gave 0.0120 gm. Ash = 3.02 per cent Ash;

0.4009 gm. substance gave 0.012 1 gm. Ash = 3.02 per cent Ash.

Percentage Composition of the Ash-free Substance.

C 50.76 51.04

H 7.17 7.12

N .... .... 14.94 14.97

Average.
50.90

7.14
14.96

86 1.87 1.86
25.14

Composition and Properties of Osseoalbumoid. 357

These preparations possessed the same reactions as those sum-
marized by us on page 354 from Morner's paper, and also those re-
ferred to in some detail in connection with preparations Nos. 8 and 9
of our osseoalbumoid. The reactions for loosely bound sulphur were,
however, very much stronger for the cartilage preparations than for
those prepared from the femur. On the other hand, sulphur obtain-
able from chondroalbumoid, on boiling with 2 per cent hydrochloric
acid, appeared to be less in comparative tests than for the bone
products.

The following summary brings into contrast the analytic averages
for the albumoid products from both sources :

Percentage Composition of Albumoids from Cartilage and Bone.

Elements.

Chondroalbumoid.

Osseoalbumoid.

Preparation A.

Preparation B.

Average A-B.

Average.
Preparations 8-9.

C

50.02

50.90

50.46

50.16

\\

6.97

7.14

705

7.03

N

14.94

14.96

14.95

16.17

S

1.S5

1.86

186

1.18

0

26.22

25.14

25.68

25.46

Conclusions from the data of analysis. — The properties of this sub-
stance are found to be those ascribed to it by Morner. That it is not
exactly the same as osseoalbumoid is indicated by its higher content
of sulphur and its considerably lower content of nitrogen. The larger
proportion of sulphur obtainable from it on cleavage with alkali has
already been referred to.

These differences are not sufficient, however, to prevent the con-
clusion that the two substances are closely related members of the
same class of proteids.

The relative amount of the substance in cartilage appears to be
less, as we have already said, than the proportion of osseoalbumoid
in bone.^

^ For facts regarding location of albumoid in cartilage see Morner's paper,
Loc. cit.

35^ P. B. Hawk and Williafn J. Gies.

Summary of Conclusions.

I. Osseous tissue contains a residual proteid substance, obtainable
after hydration of the collagen, which is neither keratin nor typical
elastin, although it resembles the latter body.

This substance is present in bone in only comparatively small pro-
portion, though apparently in greater relative quantity than the cor-
responding constituent of cartilage.

The average percentage elementary composition of the purest prod-
ucts was found to be as follows, calculated for ash-free substance : '

c

H

N

S

O

50.16

7.03

16.17

1.18

25.46

The analyzed products were free from organic phosphorus.

The substance appears to be very similar to some of the albumoids,
particularly to that from cartilage. It has therefore been termed
osseoalbumoid.

No attempts have been made to ascertain its location in the tissue,
but it appears to be identical with the substance referred to errone-
ously by Broesike as keratin and found by him in the lining of the
lacunae and canaliculi. It is possible, also, that the elastic fibres of
the bone have contributed substance to the preparations.

2. Further investigation of the qualities of chrondroalbumoid con-
firmed most of Morner's conclusions regarding it.

In addition, its elementary composition has been determined, with
the following percentage results for ash-free substance :

C H N s O

50.46 7.05 14.95 1.86 25.68

This product is likewise devoid of phosphorus in organic combi-
nation.

1 Average of preparations Nos. 8 and 9, our purest products. See page 349.

Reprinted from the American Journal of Physiology.

Vol. V. — June i, 1901. — No. V.

THE COMPOSITION OF YELLOW FIBROUS CONNECTIVE

TISSUE.i

By G. W. VANDEGRIFT and WILLIAM J. GIES.

\_Froin the Laboratory of Physiological Chemistry, of Columbia University, at the College
pf Physicians and Sitrgeons, Neio Vorh.]

Historical.

MOST of the animal tissues have been carefully analyzed and their
general composition determined. We have not been able
to find any record of such chemical study of ligament, however.
Gorup-Besanez^ mentions the fact that a few determinations of the
composition of the middle coat of arteries, and several other forms
of connective tissue containing elastic fibres, have been made,
according to which the percentage of water varies between 57.5 per
cent and 75.9 per cent. He doubtless refers to such incomplete
analyses as those of the tunica intima and tunica media of the carotid
artery, made by Schultze and quoted by Gautier,^ as follows :

Per cent.

Water 69.30

Elastin (including collagenous and cellular elements) . 18.65

Other albuminoids 8.72

Extract in water-alcohol 2.27

Soluble salts 0.74

Insoluble salts 0.34

The functions of elastic tissues appear to be mainly of a mechanical
nature, and there has been little to suggest that such forms of con-
nective tissue as ligament contribute anything important in substance
or effect to metabolism. Probably the seeming passivity, in the
metabolic sense, of ligament and allied structures accounts for the
lack of chemical attention they have received.

During Liebig's time, when elementary analysis was expected to
throw much light on those transformations in the body which we now

^ Reported, in part, before the American Association for the Advancement of
Science, June, 1900: Proceedings, 1900, p. 123.

'^ Gorup-Besanez : Lehrbuch der physiologischen Chemie, 1878, p. 649.
3 Gautier: Lecons de chimie biologique normale et pathologique, 1S97, p. 297.

\S7

28S G. IV. Vandcgrift ami Williavi J. Gies.

speak of as anabolic and catabolic, many of the tissues were given
extended study. ^ Liebig, Scherer, Mulder, and many others, in those
days, determined the elementary composition of muscle, blood, hair,
cartilage, bone, tendon, and practically all of the other body parts
(after desiccation), and gave empirical formulae to these tissues just
as they did to pure chemical substances. They deduced from these
formulae relationships and differences which were not particularly in
harmony with observed functions, and which have not been borne out
by subsequent research.

Scherer 2 determined the elementary composition of the dried
middle coat of arteries. To this elastic tissue he ascribed the formula
C48H-6Ni20ifi. Bergh'^and Schwarz'* have since made and analyzed
several pure preparations of elastin from the aorta. The latter's
studies of the composition and reactions of aorta elastin have led him
to conclude that it is identical with the elastin of ligamentum nuchce.
The averages of the analytic percentage results obtained by these
observers are here brought in contrast:

c

Scherer." Tunica media .... 53.49
SCHW.\KZ.5 Purified aorta elastin . . .54.,34
Bergh. Purified aorta elastin . . ^ZW

These results are sufficiently close in agreement to indicate chemi-
cally, as has been found histologically, that the tunica media of the
main arteries is largely composed of elastin.

The earliest results of similar analysis which relate to ligament are,
so far as we have been able to find, those obtained by Tilanus" and
Miiller'* for ligamentum nuchae, after extraction with water, alcohol,
and ether by the first observer and with acetic acid, in addition, by
the second. Tilanus gave his prepared tissue the formula C.52Hgj,Ni40i4.
Numerous investigators have since analyzed elastin from the cervical

1 Liebig: Die organische Chemie in ilirer Anwendung auf Physiologic und
Pathologie. 1842, p. 320 et seq.

^ Scherer: Annalen der Chemie und Pharniacie, 1841, xl, p. i.

* Bergh: Zeitschrift fiir pinsiologische Chemie, 1898, xxv, p. 337.
^ ScHWARZ : Ibid., 1894, xviii, p. 487.

* Phosphorus and sulphur were not determined, but included (by dihference) in
the figures for oxygen.

* Compare with the analyses by Chittenden and Hart, p. 289.

"^ Tilanus : See Mulder, Versuch einer allgemeincn physiologischen Chemie,
zweite Halfte, 1844-51, p. 595.

* MiJLLER: See Gorup-Besanez, loc. cit., p. 140.

H

N

S

0

7.03

15.36

2404

708

16.79

0.38

21.41

7.54

15.20

0.60

22.67

Composition of Yellow Fibrous Connective Tissiie. 289

ligament, prepared by essentially the same process, but with more
elaborate extractions. Comparison is made, in the following summary,
of the latest analyses with Tilanus's and Muller's average results :

c

H

N

S

0

54.98

7.31

17.52

0.33

19.86

55.46

7.41

16.19

20.94

54.08

7.20

16.85

0.30

21.57

TiLANUS.i Prepared ligament .

MuLLER. Crude elastin . -

Chittenden and Hart.^ Pure elastin . . ,

Analyses of Ligamentum NucHiE.

In the analyses here to be described the results were obtained with
ligamentum nuchae, — a ligament composed in great part of yellow
fibres and representing, perhaps better than any other part of the
body, true elastic connective tissue.

Proportions of water, solids, organic and inorganic matter. — Metliod
of determination. Perfectly fresh bloodless ligaments, taken from the
animals immediately after their slaughter, were used. Within a few
hours after removal from the body all adherent connective tissue was
carefully cut off. The cleaned ligament was then divided into
strips and very thin particles cut, from only the deeper portions of
these, with scissors into weighed porcelain crucibles. This division
of the tissue was made as minute as possible, and the process was
carried out with the utmost rapidity to prevent loss of water by
evaporation before the weight of tissue in use was determined. The
weight of fresh tissue taken was determined by difference. The sub-
stance was then dried at 100-110° C. to constant weight, after which
incineration was carefully conducted over a very low flame until all
carbon was burned out and constant weight attained. No special
difficulty was experienced in effecting complete combustion of the
carbon over an ordinary Bunsen burner.

Analytic results. The tables on page 290 summarize the results of
the general analyses of ligamentum nuchse from the ox and calf.

Comparative results. — The data on page 290 show that the ligament
of the full grown animal contains relatively less water and inorganic
matter, and more solid substance and organic matter, than that of
the calf, facts which are in entire agreement with comparative

1 Phosphorus was not determined, but included in the figures for oxygen.

2 Chittenden and Hart : Studies from the laboratory of physiological
chemistry, Yale University, 1887-88, iii, p. 22. Compare with Schwarz's figures,
p. 288.

290

G. Il\ Vaudcgrifl and William J. Gics.

O.x ligament.

1

Ligament
used.

Percentage of fresh tissue.

Percentage of solids.

Numljer.

1

Grams.

W„e,. SoUds. "^,

Inorganic
matter.

Organic
matter.

Inorganic
matter.

1
2
3

4
5
6

7

8

9

10

" 1

5.47
4.34
7.89
8.96
7.64
1 4.49
4.22
3.22
3.29
3 94
3.92

59.34
60.34
58.58
58.46
.56.36
57.37
56.32
55.39
58.10
56.42
56.55 '

40.66
39.66
41.42
41.54
43.64
4263
43.68
44.61
41.90
43.58
43.45

40.26
39.28
40.86
41.11
43.18
42.13
43.17
44.17
41.45
43.05
42.96

0.40
0.38
0.56
0.43
046
0.50
0.51
0.44
0.45
0.53
0.49

99.02
99.06
98.65
98.96
98.94
98.83
98.85
99.01
98.93
98.79
98.89

0 98
0.94
1.35
1.04
1.06
1.17
1.15
0.99
1.07
1.21
1.11

Averages

5.22

57.57
1

42.43

41.96

0.47

98.90

1.10

Calf ligament.

1
2
3
4

5

11.00
8.78
7.49
7.10
7.19

66.24
65.34
64.61
64.72
64.59

33.76
34.66
35.39
35.28
35.41

33.04
33.98
34.71
34.62
34.83

0.72
0.68
0.68
0.66

0.58

97.88
98.04
98.09
98.14
98.36

2.12
1.96
1.91
1.86
1.64

Averages

8.31

65.10

34.90

34.24 0.66

98.10

l.'X)

analytic results for other tissues of growing and mature animals.
The summary on the opposite page contrasts the above average per-
centage figures with those for morphologically related parts :

Cofnposihon of Yellow Fibrous Connective Tissue. 291

Ligament.

Vitreous
humor. 1

Costal
cartilage."-

Bone with
marrow. 3

Adipose

tissue ;

kidney

fat.*

Calf.

Ox.

Fresh tissue.

Water.

65.10

57.57

98.64

67.67

50.00

4.30

Solids.

34.90

42.43

1.36

32.33

50.00

95.70

Organic matter.

34.24

41.%

0.48

30.13

28.15

95.51

Inorganic matter.

0.66

0.47

0.88

2.20

21.85

0.19

Dry tissue.

Organic matter.

9810

98.90

35.29

93.20

56.30

99.80

Inorganic matter.

1.90

1.10

64.71

6.80

43.70

0.20

Inorganic matter. — The ash of ligamentum nuchas contains chloride,
phosphate, carbonate, and sulphate; also, sodium, potassium, calcium,
magnesium and iron, the latter arising in all probability from minute
quantities of blood held in the tissue capillaries.

Sulphate. — The sulphate reaction in our preliminary tests was
decided enough to suggest unusual quantity. In numerous samples
of ash obtained by burning in porcelain crucibles directly over gas
flames we found 8.04 to 9.20 per cent of SO3. Morner'"' has lately
called attention, in connection with the SO3 content of bone ash, to the
well known fact that, during incineration directly over an ordinary
burner, sulphur is introduced in considerable proportion from the
consumed gas. In ash made by incineration in platinum dishes over
alcohol flames, however, we obtained the following results for SO3,
which were determined, in 0.2 to 0.6 gram portions after solution
in hot dilute hydrochloric acid, by the usual barium chloride method :

^ Representing jelly-like connective tissue. Analyses by Lohmeyer, source
of material not specified. See Gorup-Besanez : Loc. cit., p. 401.

2 Human. Analyses by Hoppe-Seyler. See KiJhne : Lehrbuch der physio-
logischen Chemie, 1868, p. 387.

^ Average of many analyses of various human bones before removal of marrow.
Hoppe-Seyler: Physiologische Chemie, 1881, p. 625.

* From the ox. Atwater : Methods and results of investigations on the
chemistry and economy of food, 1895, p. 34.

5 C. Th. Morner : Zeitschrift fiir physiologische Chemie, 1897, xxiii, p. 311.

292

G. W. Vandcgrifi and William J. Gics.

IVrcentage of

SO., in 1

ganicnt

ash.

1

2

3

4

Averages.

A ^ 5.58
1! 5.S0
C 5.71

5.66
5.71
5.50

5.61
5.46
5.79

5.61
5.66

5.62
5.64
5.67

General average . . 5.64

The above results are significant when compared with the following
percentage figures for content of sulphuric acid in the ash of the
tissues and fluids soecified:^

Bone - 0.02
Muscle 3 0.30
Brain 0.75

Liver 0.92
Lungs 1.40
Blood 1.67

Serum 210
Spleen 2.54
Milk 2.64

Bile 6.39

Cartilage * 37. 47

The unusually large proportion of SO;; found in ligament ash un-
doubtedly arises from an organic source. The ash of blood and lymph,
it will be seen, contains much less in proportion, as does also that
of all the other tissues except cartilage. Attention has lately been
called to the fact that mucin is contained in ligament in appreciable
quantity.^ We shall presently show that its percentage amount is
about half that in tendon.^ Mucin contains ethereal sulphuric acid,
in a radicle very similar to, if not identical with, chondroitin sulphuric
acid.' This latter body, and chondromucoid containing it, doubtless
contribute the surprisingly large proportion of SO:i to cartilage ash.^

^ Most of these are taken from Schafek's Text-book of Physiology, 1898, i,

p- n-

- C. Th. M()R.\er: Loc. cit.

8 Weher : Quoted from Hoppe-Seyler, Physiologische Chemie, 1881, p. 651.

* Calculated from Hoi'PE-Seylkr's analyses as given by Kuhxe, Lehrbtich
der physiologischen Chemie, 1868, p. 387.

^ Richards and Gies: Proceedings of the American Physiological Society,
This journal, 1900, iii, p. v; also, Ibid.^ 1901, v, p. xi.

® The greatest amount thus far obtained from normal ox tendon was i per cent.
Chittenden and Gies : The journal of experimental medicine, 1896, i, p. 186.

'' Levexe : Zeitschrift fiir physiologische Chemie, 1901, xxxi. p. 395.

* Bone ash contains only a trace, which has also been attributed to constituent
chondroitin sulphuric acid. See C Th. Mokner : Loc. cit.; also, Bielfeld :
Zeitschrift fUr physiologische Chemie, 1S98, xxv, p. 350.

Composition of Yellow Fibroiis Connective Tisstie. 293

The unusual percentage of SO3 in ligament ash must, it appears to us,
be attributed, in much the greater part, to a similar source — that
is, to the SO3 radicle of the mucin, which, on burning, is trans-
formed, in part at least, to sulphate.

Phosphate and chloride. — In view of the excessive amount of
derived sulphate, determinations of the percentage quantity of other
constituents in ligament ash could not be expected to give exact
figures for proportionate content of inorganic matter in the fresh
tissue. We have, however, determined phosphoric acid and chlorine,
which appear to make up the bulk of the acid radicles. The former
was determined by Mercier's modification of Neubauer's method,^ in
neutralized extracts of 0.5-0.8 gram of ash in 100 c.c. made by pro-
longed treatment with hot dilute hydrochloric acid. The latter was
estimated by Mohr's method,^ in aqueous extracts of 0.4-0.7 gram of
ash in 100 c.c, made by continued heating on the water bath. The
following percentage results were obtained :

12 3 Average.

A. P.2O5 7.46 7.09 7.61 7.39

B. CI 29.16 28.91 28.79 28.95

These figures are all within the customary variations observed for
other tissues. They suggest, of course, that chlorides are the pre-
dominant substances in the ash of ligament.'^

Fat (ether-soluble matter). — - Dormeyer's method* was used in
these determinations. The percentage of water was ascertained for
each sample dried to constant weight, and extraction of fat made from
the pulverized dry material in quantities varying from i8to 35 grams.
The tissue used was taken from only the inner portions of the liga-
ments. The following percentage results were obtained :

12 3 4 5 6 Average.

Fresh tissue. 1.26 0.94 1.03 1.45 0.89 1.17 1.12

The proteid constituents. — The chief organic substance in liga-
mentum nuchae has long been known to be elastin. After Rollett's°

1 Neubauer und Vogel : Analyse des Harns, zehnte Auflage, 1898, p. 731.

2 Ibid., p. 708.

2 Bone contains only traces of chlorine (0.19% in the ash). Cartilage ash con-
tains 3.70% of chlorine. See HaUiburton in Schafer's Text-book of Physiology,
1898, i, pp. 112 and 1 13.

* DoRMEYER : Jahresbericht iiber die Fortschritte der Thier-Chemie, 1896, xxvi,
p. 42.

^ RoLLETT : Untersuchungen zur Naturlehre des Menschen und der Thiere
(Moleschott), 1859, vi, p. I- Also Ibid., i860, vii, p. 190.

294 ^- ^' Vandcgrift and William J. Gies.

researches on the structure of connective tissue, particularly tendon,
it was assumed by various observers ^ that ligament contains repre-
sentatives of the various proteids which Rollett identified. It was
only recently, however, that particular attention was called to the fact
that this representative of yellow fibrous tissue contains appreciable
quantities of coagulable proteid, glucoproteid and extractives.^ The
quantities in which these substances are present make it probable that
they are integral components of the tissue and not merely constituents
of retained blood and lymph. Even after the finely divided tissue has
been well washed in water, a process calculated to remove practically
all lymph, these substances may still be separated from it in relatively
large amount.

Coagulable proteid {albunwi, globulin). The fresh cleaned tissue was
cut into strips and these quickly torn into delicate shreds with forceps.
50-100 grams of the fibrous material were extracted, in each determi-
nation, with 200 c.c. of 1.25-5.0 per cent solution of sodium chloride,
at room temperature for from three to four days. Powdered thymol
prevented putrefactive changes. At the end of that time the extract
was pressed through cloth, filtered, and the tissue thoroughly washed
with water. The extract and washings were then heated to boiling.
The coagulable proteids were completely precipitated on addition of a
very small quantity of dilute acetic acid.-' The precipitate was filtered
on weighed papers, washed free from chloride with water, and the
coagulated proteid determined gravimetrically after drying to con-
stant weight at 100-110° C. The following percentage results were
obtained in six determinations with samples from as many ox
ligaments :

12 3 4 5 6 Average.

Fresh tissue. 0.5SS 0.502 0.59S O.C52 0.652 0.704 0.616

Mucin. — Rapidly shredded ligament, prepared as for the determi-
nations of coagulable proteid, in portions of lOO grams, was extracted,
with repeated shaking, in 250 to 300 c.c. half-saturated lime water
for several days at room temperature. The glucoproteid was com-
pletely precipitated from the extract and washings on acidification
with 0.2 per cent HCl. Its amount was determined, after filtering
on weighed paper and washing free from soluble proteid and chloride,

^ KiJHXE : Loc. cit., p. 363.
'^ Richards and Gies : Loc. cit.

3 The amount of acid added was too slight to precipitate any mucin that may
have been dissolved by the sodium chloride.

Composition of Yellow Fibrotis Connective Tissue. 295

by drying at 110° C. and weighing. The following percentage results
were obtained with ox ligament taken from as many animals :

12 3 4 5 6 7 Average.

Fresh tissue. 0.565 0.429 0.539 0.510 0.490 0.574 0.569 0.525

Elastin. — Finely divided ox ligament from several animals, in
quantities of i6 to 50 grams, after thorough extraction in 5 per cent
sodium chloride solution was boiled in excess of water, with repeated
renewal, until all collagenous fibres were removed by gelatinization
and only very slight turbidity with tannic acid was obtainable in the
cold concentrated filtrate. The undissolved residue was filtered on
weighed papers, thoroughly washed free from traces of dissolved pro-
teid and chloride, dried at 1 10° C. to constant weight and the percent-
age of elastin calculated from the weight obtained, with the following

results : ^

12 3 4 Average.

Fresh tissue. 31.24 32.96 31.51 30.99 31.67

Collagen. — Eulenberg^ observed long ago that ligamentum nuchae
yields gelatin on boiling. In these experiments the percentage con-
tent of collagen, in the form of gelatin, was determined gravimetri-
cally. Weighed quantities, 20-40 grams, of finely divided fresh ox
ligament were thoroughly extracted in half-saturated lime-water for
several days at room temperature, for removal of albumin, globulin,
mucin and extractives. Excess of calcium hydroxide was removed by
washing in water. The tissue was then washed in alcohol and ether to
remove fat, and finally boiled, in fresh portions of water, until only
the merest turbidity could be obtained in small amounts of cold con-
centrated filtrate on addition of tannic acid. This process usually
required six to ten hours. By this time all of the collagen was
gelatinized and very little elastin hydrated. The filtrates were eva-
porated on the water bath in weighed crucibles, the residues dried
at 100-110° C. to constant weight and gelatin determined, after
subtraction of the ash obtained by burning the residue over a low
flame, with the following percentage results : ^

^ This residue consists, strictly, of substances insoluble after such treatment.
Only traces of non-elastin material could still be present, however — • quantities too
small to materially affect the results. Furthermore, a correspondingly small
amount of elastin was probably lost by hydration.

2 EuLENBERG : See Schultze, Annalen der Chemie und Pharmacie, 1849,
Ixxi, p. 277.

^ This method is, of course, open to the objection that possibly hydration pro-

296

G. JV. I'andcgrift and William J. Gics.

Fresh tissue.

?.61

2
6.77

3

7.38

4

6.99

.13

6

7.52

Average.
7.23^"

Extractives. — Crcatin and nuclein bases were detected qualitatively
in aqueous extracts of large quantities of ligaments after removal of
proteids and salts in the usual way, in confirmation of previous obser-
vations in this laboratory,^ but no attempt was made to determine
their quantity nor the character of the individual alloxuric bodies.
In the summary below, extractives are included with the figures for
" undetermined substance," which were obtained by difference.

Average composition. — The results of all our analyses are sum-
marized in the following table, which gives the average percentage
composition of fresh ligamentum nuchae and of the dry solid matter
contained in it, and also the results of partial analysis of the ash :

Fresh 1

gament.

Dry ligament.

Ash.

Percentage composition.

Calf.

Ox.

Calf.

Ox.

Ox.

Water.'^

65.10

57.570

Solids.

34.90

42.430

Inorganic matter.

0.66

0.470

1.90

1.100

SOj.

. . .

0.026

0.062

5.64

P2O5.

0.035

0.081

7.39

CI.

0.136

0.318

28.95

Organic matter.

34.24

41 .%0

98.10

98.900

Fat (ether-soluble matter).

1.120

2.640

Albumin, globulin.

0.616

1.452

Mucin.

0.525

1.237

Elastin.

31.670

74.641

Collagen (gelatin).

7.230

17.(H0

Extractives and undeter-
mined substance.

0.799

1.883

1

ducts of the elastin increased the quantity of gelatin. In reality, however, such
increase is insignificant when the hydration is carefully conducted and is probably

1 Richards and Gies : Loc. cit.

^ The quantity of water in "elastic tissue " given, from Rfaunts' Physiologie

Composition of Yellow Fibro2is Connective Tissue. 297

just about equal in amount to the loss of gelatin in the removal tests with tannic
acid. EwALD and Kuhne (Jahresbericht der Thier-Chemie, 1877, p. 281) found
that collagen is not digested by the proteolytic enzyme of pancreatic juice unless it
has been previously swollen by acid or hot water, whereas most other proteids (in-
cluding those we have found in the ligament), are digested without such prelimi-
nary treatment. We might have determined collagen directly by this process,
perhaps, but we believe the one employed, a modification of Hoppe-Seyler's
method (Handbuch der physiologisch- und pathologisch-chemischen Analyse, 1893,
p. 482), gave results quite as accurate as could be obtained by the former or any
other.

humaine, by Halliburton (A Text-book of chemical physiology and pathology,
1891, p. 58) is 49.6%. The particular source of the tissue is not stated. This
amount is lower than that for any of the connective tissues to which Gorup-
Besanez referred (see page 287), and less than any others we have found
recorded for particular forms of elastic tissue.

Reprinted from the American Journal of Physiology-

Vol. VI. — December i, 1901. — No. IV.

THE CHEMICAL CONSTITUENTS OF TENDINOUS

TISSUE.i

By LEO BUERGER and WILLIAM J. GIES.

[^Fj-otn the Laboratory of Physiological Chemistry, of Columbia University, at the College
of Physicians and Surgeons, N^eio Vor^.]

IN a previous paper from this laboratory ^ the results were given of
some analyses of yellow elastic tissue, represented by the liga-
mentum nuchas of the ox and calf. So little attention has been given
by chemists to structures such as tendon, which possess mainly
mechanical functions, that it seemed to us desirable to investigate in
a similar study the general composition of white fibrous connective
tissue.

Historical.

Early in the last century, when it was assumed that elementary
composition determined not only definite chemical relationships, but
indicated similarities and differences in development as well as func-
tion, the tissues were carefully subjected to elementary analysis.
Like a number of the other parts of the body, tendon, in the fresh con-
dition, was looked upon as consisting of practically a single organic
substance (collagen) holding water mechanically, and admixed with
slight quantities of saline matter and other blood and lymph con-
stituents.^

Scherer* analyzed several forms of gelatin-yielding fibrous tissues.
On the next page we give the results of his elementary analysis of
calf-tendon. The tissue was prepared for analysis by preliminary
maceration and extraction in dilute saline solution. Subsequently the
residue was washed in water and then in boiling alcohol and ether. To
this residue, "collagen," Scherer ascribed the formula C^gHg.^NigO^s-

^ Some of tlrese results were given at the New York meeting of the Ameri-
can Association for the Advancement of Science, June, 1900: Proceedings, 1900,
p. 123.

^ Vandegrift and Gies : This journal, 1901, v, p. 287.

^ See references to collagen content on page 230.

^ Sch?:rer : Annalen der Chemie und Pharmacie, 1841, xl, p. 46.

219

2 20 Leo Buerger and William J. Gies.

Marchand,^ who pointed out a number of defects in Scherer's work,
subjected dried tendons from the foot of the calf to similar analysis.
The results giv^en below for ash-free substance led him to ascribe to
this " collagen " the formula C^i^Hg-^Ni^O^r,. He also calculated its
molecular weight from this formula, expressing it with the figures
5937-5- The composition of the ash-free hydrated tendon ("gelatin"),
taken from the same source, was found by Marchand to accord very
well with the average analytic results of similar products, from bone
and other tissues, obtained by Mulder.'-^ The latter observer gave the
gelatin-yielding tissues (dryj the formula CigH^^N^Oj.

Winkler's ^ analysis of the tendon of the cow, after extraction in cold
water and later in boiling alcohol and ether, led to similar results.

The following summary gives the analytic averages referred to
above : *

C H N O

ScHERER. Crude tendon collagen . . . . 50 51 7.16 1837 23.%

Marchand. Dry calf tendon 50.27 6.77 17.88 25.08

Marchand. Crude tendon gelatin .... 50.02 6.82 18.00 25.16

Mulder. Crude bone gelatin 50.37 6.33 17.95 25.35

Winkler. Crude tendon collagen .... 49.68 6.64 17.94 25.74

Average .... 50.17 6.74 18.03 25-06

These close agreements in analytic figures naturally suggested to
the earlier observers that the chief organic substance of bone, tendon,
and related forms was the same in each ; further, that " gelatin " and
"collagen" were very nearly if not altogether isomeric^ In the light
ofi modern chemical knowledge, however, these analytic harmonies
emphasize the lack of information which elementary analysis of
tissues furnished on the characters and qualities of the various
constituents. Definite separation of the tissue-forming substances,
however, and subsequent detailed analysis of them individually has
increased our appreciation of the important parts the numerous
constituents of the body play in the maintenance of its functions,

1 Marchand: Lehrbuch der pliysiologischen Cliemie, 1844, p. 166.

- Mulder: Versuch einer allgemeinen pliysiologischen Chemie, erste Halfte,
1S44-51, p. 333-

3 Winkler: Quoted by Mulder, he. cit , zweite Halfte, p. 583.

* The small amounts of phosphorus and sulphur detected in these substances
at this time were attributed to inorganic impurity. Oxygen was calculated by
difference, and the figures for it therefore include organic phosphorus and sulphur.

5 HoFMEiSTER has sincc shown, and it is now generally understood, that
gelatin is the hydrate of collagen: Zeitschrift, fiir physiologische Chemie, 1878-79,
ii. p. 299.

The Chemical Constituents of Tendinous Tisstie. 221

Aside from the above elementary analyses, and a few others of
similar character in close agreement with them,^ practically nothing
has been done to determine quantitatively the composition of tendin-
ous tissue. Several observers have determined the proportion of
ash.2 Gorup-Besanez^ states that a few determinations of water and
solid matter in connective tissues, containing collaginous fibres in
abundance, have been made, which show a variable content of water
ranging between 57.5 and 78.9 per cent of the fresh tissue.* Beaunis,
in the table presented by Halliburton,^ gives the average proportion
of water in " connective tissue " as 79.6 per cent ; but this does not
refer to tendon.^

Analyses of Tendo Achillis.

Material and methods of analysis. — In the work described in this
paper the Achilles tendons of the ox and the calf were employed.
The Achilles tendon is easily separated from extraneous matter. It
is more completely collaginous and contains relatively less elastin
than is found in any other tendinous tissue available for such work.
It may be regarded as the best representative of white fibrous con-
nective tissues.

This research followed so closely the plan of our previous study '^
that it is needless to describe in detail the methods of analysis.
The details of procedure not mentioned here may be understood to
correspond with those given by Vandegrift and Gies.

The main shaft of the tendon was used in each experiment. Occa-
sionally small portions of the bifurcations were employed with parts
of the former.*' Only perfectly white tendons were analyzed. Any
tendons showing blood}/ lines superficially or internally were rejected.
Usually the tendons were rapidly cut into very thin cross sections of

^ Gorup-Besanez : Lehrbuch dev physiologischen Chemie, 1878, p. 142.

2 See page 223. Also foot-note, page 225.

^ Gorup-Besanez: Loc. cit., p. 649.

* See Chevreul's results; given by Marchand : Loc. cit., p. 164.

^ Halliburton: Text-book of chemical physiology and pathology, i8gi,
p. 58.

^ Results of analyses of various non-tendinous tissues containing collaginous
fibres, such as the cornea, are not strictly comparable in this connection and are
therefore not given here-

'' Vandegrift and Gies : Loc. cit.

8 See Cutter and Gies: This journal, 1901, vi, p. 157.

222

Leo Buerger and William J. Gies.

GENERAL COMPOSITION.

Ox '

Fen DON.

No.

Tendon
used.

Percentage of fresh tissue.

Percentage

of solids.

Grams.

Water.

Solid matter

Organic
matter.

Inorganic
matter.

Total.

Organic.

Inorganic.

1

5.03

61.55

38.45

37.97

0.48

98.74

1.26

2

7.05

63.20

36.80

36.20

0.60

98.38

1.62

3

5.65

62.34

37.66

37.16

0.50

98.67

1.33

4

5.80

63.58

36.42

35.92

0.50

98.62

1.38

5

5.91

62.02

37.98

37.58

0.40

98.54

1.46

6

4.49

65.05

34.95 .

34.40

0.55

98.43

1.57

7

5.70

62.92

37.08

36.69

0.39 1

98.94

1.06

S

2.69

61.32

38.68

38.27

0.41

98.94

1.06

9

4.02

64.76

35.24

34.76

0.48

98.65

1.35

10

2.54

62.69

37.31

36.83

0.48

98.71

1.29

11

3.82

64.32

35.68

35.25

0.43

98.79

1.21

12

2.72

62.64

37.36

36.%

0.40

98.94

1.06

13

4.21

60.93

39.07

38.64

0.43

98.91

1.09

Aver.

4.59

62.87

37.13

36.66

0.47

98.71

1.29

Calf

Tendon.

1

2.21

65.39

34.61

33.98

0.63

98.18

1.82

2

3.%

66.54

33.46

32.89

0.57

98.30

1.70

3

5.17 [

68.75

31.25

30.60

0.65

97.91

2.0^)

4

4.32

68.32

31.68

31.06

0.62

98.04

1.96

5

4.12

67.23

32.77

32.33

0.44

98.68

1.32

6

2.68

68.84

31.16

30.42

0.74

97.63

2.37

Aver.

3.74

6751

32.49

31. SS

0.61

98.12

1.S8

The Chemical Constituents of Tendinous Tissue. 223

sufificient quantity for the determinations. Sometimes they were cut
into strips with a knife and the strips finely divided with scissors.
All preparations were conducted rapidly and with due regard to the
usual precautions to prevent loss of moisture, etc.

Proportions of -water, solids, organic and inorganic matter. — In these
determinations the finely divided substance was dried at 100-110° C.
to constant weight. Incineration was carefully conducted over a very
low flame until all carbon was burned out and the ash was constant
in weight.

The general summary on the opposite page gives the results of these
determinations for the tendo Achillis from both the ox and the calf.
It will be seen from the general averages that the tendon of the calf
contains relatively more water and inorganic matter than that of the
mature animal. The tissue of the full grown ox on the other hand
contains larger proportions of solid substance and organic matter.

In his determinations of the composition of dry tendon from the
foot of the calf, Marchand ^ also weighed the ash. In three separate
determinations he found the ash to be 1.72, 1.82 and 1.89 per cent —
an average of 1.8 1 per cent of the dry tissue.^ These results accord
very closely with our own, if it be assumed that the tendons of the
calf which Marchand analyzed contained approximately the same
amount of water found in these experiments — 67.5 per cent. At
this rate, the fresh tendons analyzed by him contained 0.59 per cent
of ash.^

The facts brought out by the figures in the table on the opposite page
harmonize with comparative analytic data for other tissues of fully de-
veloped as well as immature animals. On the next page we present a
summary giving percentage figures for the general composition of
morphologically related parts. Attention may be called to the general
similarity in the results for tendon and ligament. Costal cartilage is
somewhat similar to these two in general composition, the analytic
differences being mainly due to its larger content of water and inor-
ganic matter.

Inorganic matter. — Ash in suitable quantity was prepared by
gradual combustion in a nickel crucible over an alcohol burner and
then by complete incineration over a very low flame in a platinum

^ See page 220.

^ See foot-note, page 225 ; also, summary on page 230.

3 The ash of tendons containing ossa sesamoidea would naturally be much
greater than any of the amounts here recorded for the normal tissue.

224

Leo Buerger a7id William J. Gies.

dish. The qualitative characters of the ash of the Achilles tendon
are much the same as those of the inorganic matter in many other
parts of the body. Solutions of the ash were strongly alkaline in
reaction. We detected in it chloride, carbonate, sulphate, and phos-
phate. Of the basic elements sodium, calcium, magnesium, potas-
sium, and iron were particularly prominent. It is probable that the
iron came from traces of haemoglobin in the capillaries. Some of the

COMPARATIVE COMPOSITION.

Tendon.

Ligament.^

1 Vitreous
humor.2

Costal
carti-
lage.8

Bone

with

marrow.*

Adipose

tissue ;

kidney

fat.5

Calf.

Ox.

Calf.

Ox.

P'resh tissue.
Water
Solids
Organic
Inorganic
Dry tissue.
Organic
Inorganic

67.51

32.49

31.88

0.61

98.12
1.88

62.87

37.13

36.66

0.47

98.71
1.29

65.10

34.90

34.24

0.66

98.10
1.90

57.57

42.43

41.%

0.47

98.90
1.10

98.64
1.36
0.48
0.88

35.29
64.71

67.67

32.33

30.13

2.20

93.20
6.80

50.00
50.00
28.15
21.85

56.30
43.70

4.30
95.70
95.51

0.19

99.80
0.20

1 Vandegrift and Gies: Loc. cit.

■^ Representing jelly-like connective tissue. Analyses by Lohmeyer, source of
material not specified. See Gorup-Besa.n'EZ: Loc. cit., p. 401.

2 Human. Analyses by Hoppe-Seyler. See Kuh.ne: Lehrbuch der physi-
ologischen Chemie, 1868, p. 387.

* Average of many analyses of various human bones before removal of marrow.
IIoppe-Skyi.er: Physiologische Chemie, 1881, p. 625.

^ From the ox. Atwater : Methods and results of investigations on the chem-
istry and economy of food, 1895, p. 34.

carbonate doubtless arose from the proteid in the process of oxidation.
Much of the sulphate came from the acid radicle of the tendon mu-
coid. The proportion of ash in tendon, as in ligament, is unusually
small.

Schulz^ has recently detected silicic acid in a number of the forms
of connective tissue. The average amount of silicic acid in i kilo of

1 ScHULZ : Archiv fiir die gesanimte Physiologic, 1901, Ixxxiv, p. 67.

1

2

3

Average,

27.1

27.4

26.6

27.0

72.9

72.6

73.4

73.0

The Chemical Constituents of Tendinous Tissue. 225

dry ox tendon was found to be o. 1086 gram (o.oi per cent of the
solid matter). In the same quantity of dry human tendon silicic acid
amounts on an average to 0.0637 (0.006 per cent of the solid
matter) .1

Soluble ajid insoluble portio7is. Several direct determinations of the
amount of insoluble matter in the ash were made. Ash which had
been reheated in a platinum crucible was cooled in a desiccator.
Quantities of this perfectly anhydrous material, from one to two
grams in weight, were treated with 500 c.c. of distilled water per
gram of substance. The mixture was repeatedly stirred for forty-
eight hours, then filtered on weighed papers and the amount of in-
soluble substance directly determined gravimetrically in the customary
way. The appended percentage results were obtained on three differ-
ent preparations :

Substance itisoliihle in cold water ....
Substance soluble in cold water ....

Similar determinations were made by us on samples of the ligament
ash prepared by Vandegrift and Gies. 24.3 per cent of the same was
found to be insoluble, 75.7 per cent soluble, in cold water. In Pick-
ardt's ^ analyses of the ash of laryngeal cartilage 37.2 per cent was
insoluble in water, 62.8 per cent soluble.

Sulphate. — The ash gave striking sulphate reactions with BaCla in
the presence of free HCl. In some preliminary experiments samples
of ash which had been prepared quickly by incineration in a platinum
dish over a Bunsen gas burner contained from 9.56 to 14.92 per cent
of SOg.^ As these results were obviously affected by sulphur products
in the gas, we next made several preparations of the ash in platinum
dishes over alcohol burners. The following results for SO3 content
in ash prepared in this way were obtained by the usual BaCl2 method,

1 In these determinations Schulz also estimated the percentage of ash in the
dry substance. In tendons of the calf it amounted to 3.19 per cent. In the older
animals it was as low as 2.07 per cent. In human tendon it was as high as 3.88
per cent. The amount of silicic acid in the ash of the tendons from cattle ranged
from 0.23 to 0.66 per cent. In the ash of human tendon it varied between o.ii
and 0.49 per cent. SCHULz's results indicate that the older the animal is the
larger is the percentage of silicic acid in its connective tissues.

2 PiCKARDT : Centralblatt fiir Physiologie, 1892, vi, p. 735.

^ Compare with results for ligament ash, under similar conditions of prepara-
tion, given by Vandegrift and Gies, loc. cit., y>- 291. See also, Bielfeld :
Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 352.

226

Leo Bucrgci' and Williain J. Gics.

in 0.25-0.71 gram portions, after solution in hot dilute HCl and sub-
sequent filtration :

PERCENTAGE OF SO.. IN TENDON .\SH.

1

2

3

4

Average.

A

6.72

6.62

6.68

....

6.67

]}

6.70

6.60

6.65

C

6.60

6.5S

6.63

6.61

6.60

D

6.63

6.S4

6.74

6.69

6.72

E

6.55

6.63

....

....

6.59

General average .

. 6.65

The relation of tendon ash to the ash of other tissues and various
fluids, with respect to SO3 content, may be seen at a glance in the
following summary of SO3 percentages ^ : —

0.92

Serum

. 2.10

Ligament

5.64

1.40

Spleen

. 2.54

Bile . .

6.39

1.67

Milk .

. 2.64

Cartilage

37.47

Bone . . 0.02 Liver .
Muscle . 0.30 Lungs .
Brain . . 0.75 Blood .

There can be little doubt that most of the SO3 in tendon ash arises
from an organic source, just as in the case of bile, cartilage, and lig-
ament. It could not have come from blood or lymph. Bile contains
combined SO3 in salts of taurocholic acid. Cartilage contains salts
of chondroitin sulphuric acid, as well as chondromucoid.^ Ligament
contains mucoid^ and possibly, also, chondroitin sulphuric acid.'^
Tendon contains considerable mucoid, as we shall see, but, according
to Morner,^ no chondroitin sulphuric acid can be separated from the
Achilles tendon. Tendo mucoid, however, contains a radicle similar
to, if not identical with chondroitin sulphuric acid,*^ and it is probabl-e

1 Vandegrift and Gies : Loc. cit., p. 292.

2 C. Th. Morner: Skandinavisches Archiv fiir Physiologic, 1889, i, p. 210.

^ Richards and Gies : Proceedings of the American Physiological Society.
This journal, 1900, iii, p. v ; also. Ibid., 1901, v, p. xi.

•* Krawkow : Archiv fiir experimentelle Pathologie und Pharmakologie, 1897,
xl, p. 195.

5 C. Th. Morner: Zeitschrift fiir physiologische Chemie, 1895, xx, p. 361.

^ Levene: Ibid., 1901, xxxi, p. 395.

The Chemical Coitstihie^its of Tendinous Tissue. 227

that the SO3 liberated during its combustion unites in part with the
basic elements of the ash.^

Phosphate and chloride. — No extended quantitative analysis of the
ash was made because of the large amount of derived sulphate in
it. Figures for the percentage content of other constituents under
the circumstances would afford only approximate values. Phosphate
and chloride, the chief salts in the ash, were present in large propor-
tion, as the following results for percentage content of P.20g and CI
will indicate :

12 3 4 Average

P2O5 . . . 8.3S 8.53 8.30 8.16 8.34

CI ... . 31.73 30.99 31.26 31.52 31.37

The average quantity of chlorine in ligament ash was found by us
to be 7.39 per cent. P2O5 was equal to 28.95 P^r cent of the liga-
ment ash.

Fat (ether-soluble matter). — Although the Achilles tendon does not
appear to hold as much admixed adipose tissue as ligamentum nuchas,
it seems to contain almost as much extractive substance. The
following percentage results in this connection, calculated for fresh
tissue in each case, were obtained by Dormeyer's method :

12 3 4 5 6 7 Average

Fresh tissue . . 0.87 1.10 1.21 1.16 0.98 1.05 0.93 1.04

The proteid constituents. — It has been known for a long time that
tendon consists mostly of collagen. As we have already indicated
the earlier observers considered tendon to be almost pure collagen.
Rollett's^ researches on the structure and composition of connective
tissues demonstrated the presence in tendon not only of such soluble
proteids as might be constituents of contained lymph, but also of
mucoid. Numerous histologists have shown the presence also of
elastic fibres in tendinous tissue.

Coagiilable proteid (albumin, globidiii). — Rollett detected only
traces of coagulable proteid in aqueous extracts of the Achilles
tendon of the horse. Loebisch ^ called attention to the fact that

'^ Levene's result does not harmonize with Morner's. The latter's method
for the detection of chondroitin sulphuric acid in tendon should have revealed the
presence of the acid substance in tendo mucoid identified by Levene. See
Hawk and Gies : This journal, 1901, v, pp. 398-399.

2 Rollett : Untersuchungen zur Naturlehre des Menschen und der Thiere
(Moleschott), 1859, vi, p. i; also. Ibid., i860, vii, p. 190.

^ Loebisch: Zeitschrift fiir physiologische Chemie, 1886, x, p. 43.

2 28 Leo Buerger and William J. Gics.

aqueous extracts of the same tendon of the ox contain slight quan-
tities of coagulable proteid — "serum globulin" and an albumin co-
agulating at 78° C. Richards and Gies ^ recently observed that
aqueous extracts of this tendon from the ox contain minute propor-
tions of two coagulable proteids; one, a globulin, coagulating at
54°-57° C., the other, an albumin, coagulating at "ji^ C.

In this work we experienced great difficulty in making satisfactory
quantitative estimations. The quantity of coagulum for 100-200
grams of tissue was always very slight. Frequently it was impos-
sible to obtain the coagulum in a perfectly clear fluid. The results
were the same in aqueous and in sodium chloride extracts. One or
two indirect methods gave no more satisfactory results. Tendo
mucoid is somewhat soluble in the aqueous and saline extracts of the
tissue, and possibly the observed interference with perfect coagula-
tion of the simple proteids was due to the presence of larger or
smaller amounts of this glucoproteid.

The following percentage results were obtained in extracts from
tissue which had been cut into narrow strips and then very finely
divided with scissors : —

1 2 3 4 5 6 7 Average

Fresh tissue . 0.231 0.1S4 0.191 0.274 0.177 0.219 0.262 0.220

It is possible that not only a small quantity of coagulable proteid
was lost in each determination, but also that a small proportion of
mucoid was admixed with the coagulum as a result of the addition of
the dilute acid ordinarily employed to complete coagulation. We
feel satisfied, however, that the above average amount is very nearly
that contained in this tissue. Much of it doubtless is a part of con-
tained lymph. The average quantity in ligamentum nuchae is 0.616
per cent.

Mucoid? — The proportion of mucoid in tendon is comparatively
large. Halliburton states that the average amount for normal connec-
tive tissues is 0.521 per cent.^ The amount in the human tendo
Achillis he found varied under normal conditions between 0.298 and
0.770 per cent. Chittenden and Gies* obtained as much as i per
cent of chemically pure mucoid from the tendo Achillis of the ox, al-

^ Richards and Gies: Loc. cit.

'^ See Cutter and Gies: Loc. cit., foot-note, p. 155.

* Halliburton: Loc. cit., p. 477.

^ Chittenden and Gies: Journal of experimental medicine, 1896, i, p. 186.

The Chemical Constituents of Tendinous Tissue. 229

though their experiments were not designed for quantitative deter-
minations. The amount in ligamentum nuchas was found by us to
average 0.525 per cent. Our percentage results for the Achilles
tendon of the ox were the following :

12 3 4 5 6 7 Average

Fresh tissue . 1.361 1.420 1.332 1.220 1.043 1.22S 1.380 1.283

In these determinations we profited by the experience of Cutter
and Gies that repeated treatment with excess of dilute alkali is neces-
sary to extract completely mucoid from tendon.^

Halliburton ^ gives a record of determinations of mucoid in human
tissues under abnormal conditions. In one case the Achilles tendon
contained as much as 1.42 per cent. The tendons of the heart under
similar conditions contained 1.65 per cent mucoid.

Elastin. — When tendon pieces are boiled in water they rapidly
diminish in size and only a small quantity of elastin-like material is
left behind. This residual material is not as resistant to the action
of dilute acid and alkali as is the elastin of ligamentum nuchse,
although it appears to be true elastin.'^ The following results for
percentage content were obtained in our quantitative determinations :

12 3 4 5 Average

Fresh tissue .... 1.561 2.130 1.634 1.100 1.740 1.633

Miinz* separated this substance, studied some of its reactions and
decomposition products, and made a few analyses of it. He found its
nitrogen content to vary between 14.31 and 14.48 per cent. The
accuracy of these analytic results has been doubted, since the nitro-
gen content of all elastins has been found to be above 15 per cent.
One of our own specially prepared samples of tendon elastin, after it
had been extracted with alcohol and ether, gave the following percent-
age results on analysis: (a) Nitrogen — by the Kjeldahl method —
15.42, 15.49, 15.45; average, 15-45. (b) Sulphur — by the fusion
method over alcohol burner — 0.48,0.54; average, 0.52. (c) Ash —
1.32, 1.28; average, 1.28. These results agree fairly well with those
for aorta elastin obtained by Bergh^: N, 15.20; S, 0.66; Ash, 0.51.

^ Cutter and Gies : Loc. cit., p. 161.

2 Halliburton : Jahresbericht iiber die Fortschritte der Thier-Chemie, 1888,
xviii, p. 324.

^ KuHNE : Lehrbuch der physiologischen Chemie, 1868, p. 356.
* MiJNZ : Quoted by Gorup-Besanez, loc. cit., pp. 143 and 645.
^ Bergh : Zeitschrift fiir physiologische Chemie, 1898, xxv, p. 341.

2^,0

Leo Buerger and IVilliam J. Gies.

Collagen. — The great bulk of the solid matter of tendon is col-
lagen. We made five quantitative determinations by the indirect
method,^ with the following percentage results :

Fresh tissue

1

50.63

2
52.47

3

30.9S

4
32.27

5 Average
31.59 31.59

The proportion of collagen in the fresh tendo Achillis is almost
exactly the same as that of elastin in ligamentum nuchae.

Recently, in testing his method for the determination of collagen in
connective tissue containing little soluble proteid, Schepilewsky ^

COMPOSITION OF TENDO ACHILLIS.

Fresh

tissue.

Dry

tissue.

Ash.

Constituents.

Calf.

Ox.

Calf.

Ox.

Ox.

Water

67.51

32.49

0.61

62.870

37.130

0.470

1.88

1.266

Solids

Inorganic matter

SO,

0.031
0.039

0.084
0.106

6.65
8.34

P.,Oj

CI

0.147

0.397

31.37

Organic matter

31.88

36660

98.12

98.734

Fat (ether-soluble matter) .

1.040

2.801

Albumin, globulin ....

0.220

0593

Mucoid

1.283

3.455

Elastin

....

1.633

4.398

Collagen (gelatin) ....

31.588

85.074

Extractives and undeter-
mined substance . . .

0.896

2.413

found 80.86 per cent of collagen in dry tendon. The particular ten-
don he used is not mentioned. In the dry Achilles tendons of the
ox analyzed by us the collagen amounted on an average to 85.074 per
cent.

1 See V.\NDEGRIFT and Gies: Loc. cit., foot-note, p. 295.
- ScHEPiLEWSKV : Archiv fiir Hygiene, 1899, xxxiv, p. 351.

The Chemical Constituents of Tendinous Tissue. 231

Crystalline extractives. — Our results for extractives were only
qualitative. Creatin and nuclein bases could readily be detected.
The proportion of extractive matter was small. Our results were
sitnilar to those previously obtained in this laboratory for ligament.
In the table on the opposite page the extractives are included in
" Extractives and undetermined substance," the figures for which were
obtained by difference.

Average Composition. — The data of all our analyses are brought
together in the summary on the opposite page, which gives the average
percentage composition of fresh tendo Achillis and of the dry solid
matter in it, together with the results of partial analysis of the ash.

Reprinted from the American Journal of Physiology.

Vol. VI. ^October i, 1901. — No. II.

DO SPERMATOZOA CONTAIN ENZYME HAVING THE

POWER OF CAUSING DEVELOPMENT OF

MATURE OVA.?

By WILLIAM J. GIES.

[From the Departme?tt of Physiology in the Alarijie Biological Laboratory at Wood's I/oll,

Mass.^

CONTENTS.

Page

Historical 54

Experimental 56

Methods of procedure 56

Results with sperm extracts 59

Results with extracts of fertilized ova . . 70

Discussion of results 72

Summary of conclusions 75

OUR knowledge of the chemical properties of enzymes is very
slight, and our understanding of the part they play in zymolysis
anything but clear. Nevertheless, the great importance in biological
events of these energy-transforming substances is generally recog-
nized. The lack of precise information regarding the essential quali-
ties of enzymes no doubt accounts for the current tendency to
attribute indefinitely to ferment influence various processes of mor-
phological or chemical character which are not satisfactorily compre-
hended through ordinary experimental means, or which, in some
cases, have not even been subjected to such investigation.

A fundamental biological question has lately been put into this cate-
gory. The process of segmentation in the fertilized egg has been
ascribed in part, at least, to enzyme influence.

With the advice and many helpful suggestions of Professor Loeb, I
have attempted to ascertain whether any experimental justification
can be found for recent statements that the spermatozoon carries
substance into the ovum which effects proliferation by zymolysis.

^ I am indebted to the kindness of Professor Curtis for the use of the investi-
gator's room at Wood's HoU, reserved for the Department of Physiology of
Columbia University.

53

54 William J. Gics.

Historical.

Pieri,^ after some observations on Stroiii^^y/oceiitrotiis lividiis and
Echiims esculeutiis in the Marine Laboratory at Roscoff, in August,
1897, reported that he had extracted soluble sperm enzyme having
power to bring about segmentation of the ovum. " Ovulase, " as he
called it, was obtained by merely shaking the spermatozoa of these
Echinoderms for a quarter of an hour in a flask with sea-water, or
with distilled water. ]\Iicroscopic examination of the filtrates showed
that the spermatozoa which passed through the paper were without
tails and immobile ; " that is to say, dead."

The fresh mature ova, well washed in sea-water, were placed in
shallow dishes (size not stated) with the extract, immediately, or
within ten hours, after its preparation. Segmentation proceeded
slowly and reached the morula stage in about ten hours, with the
usual phenomena of karyokinesis. Microscopic examination showed
that there had been no penetration by spermatozoa. The "ovulase"
in distilled water was less effective than that obtained in sea-water;
it produced only a few segmentations (greatest number not
mentioned).

At the end of his paper Fieri himself mentions two "objections"
to his conclusions which it appears to the present writer destroy
their force: (i) Only the spermatozoa in the distilled water (which
extract he has distinctly indicated possessed the lesser, if any, seg-
mental power) were always killed by the shaking process. He sug-
gests that the spermatozoa might be eliminated, and pure " ovulase"
obtained with the aid of the centrifuge or porcelain filter. (2) Some
of the main supply of eggs in sea-water, from which those tested were
taken, segmented (to what stage is not stated), " in spite of the pre-
cautions taken."

Fieri gives few details of his work, and no direct judgment can be
passed on his methods. What proportion of the eggs developed .-'
The few divisions caused by the distilled water extract can hardly be
emphasized, for Fieri found that distilled water alone caused control
eggs to become clear and fragmentary. Is it possible, in microscopic
examination of myriads of such minute bodies as spermatozoa, to be
certain that each individual can be seen .-' Is the apparent lack of

1 Fieri: Archives de zoologie expcrimentale et gdn^rale, 1899, vii: Notes et
revue, ix, p. xxix.

Development of Mature Ova. 55

motility in those actually observed conclusive evidence of the death
of all? Besides, not all of the fluid in use can be examined by means
of the microscope. Further, what effect did boiling have on
"ovulase"? Was it destroyed at that temperature, as all ferments
are ? What means were taken to kill the spermatozoa which may
have been present in the sea-water used to wash the eggs ? These
important points Fieri has not considered.

Shortly after Pieri's communication, Dubois^ presented a brief
note of a similar character. Dubois arrived at the conclusion that
natural fertilization comes about through the action of a fecundative
ferment. He claims that he was able to separate such a body,
" d'une zymase fecundante," from the testicles of Echinus escnlentus,
but no experiments showing its qualities were reported by him.
Dubois named the ferment (.-*) " spermase " and credited it with the
power of modifying a hypothetical substance pre-existent in the ovum,
which he called " ovulose." As long as experimental evidence of the
truth of such a conclusion is wanting, it must continue to remain an
unsatisfying speculation.

Winkler's^ experiments were made on Sphaerechimcs granidans dSid
Arbacia piistiilosa. Every precaution was taken to prevent the action
of live spermatozoa. Winkler made extracts of spermatozoa by shak-
ing them for about half an hour with distilled water (quantities not
stated). In order to prevent destructive action on the part of the
distilled water, a precaution Fieri had not observed, Winkler added
to the extract, before using it on the test ova, a sufficient quantity of
evaporated sea-water to make the concentration of the extract the
same as that of sea-water (" ca. 4%"). Another kind of extract of
sperm was made in the fluid obtained by evaporating 400 c.c. of
sea-water to one fourth its volume.^ The filtered extract was finally
treated with enough distilled water to lower its concentration to
that of normal sea-water.

1 Dubois : Comptes rendus hebdomadaire des seances de la Societe de Biologic,
1900, lii, p. 197. The author has not had access to the original paper and relies
upon the review made of it by Winkler. (Ref. below.)

■■^ Winkler : Nachrichten von der konigliche Gesellschaft der Wissenschaften
zu Gottingen. Mathematisch-physikalische Klasse, 1900, p. 187.

^ Winkler states that the sea-water he used contained " ca. 4% " of saline mat-
ter and that by evaporating 400 c.c. to 100 c.c. he obtained a solution of " ca.
20%."' The author fails to see how anything but a 16% solution was obtained if
the process was conducted as described. Loeb's experiments have shown how
necessary exact knowledge of concentration is in such work.

56 William J. Gics.

In both kinds of extract the eggs showed some tendency to seg-
ment, but only a few divided.' Sometimes with the same extract the
eggs of one individual " reacted," while the eggs of another did not.
Finally, it is decidedly significant that the proliferation went at most
only to the 4-cell stage, and that then separation of the cells occurred
from the absence of retaining membrane, and " abnormal" forms re-
sulted. In the control experiments these manifestations were not
apparent.

W^inkler does not claim that the slight changes he observed were
due to an enzyme. Wo. states that he did not determine the effect of
heat on the power of his extracts. The nature of the active sub-
stance, he says, is completely unknown. It might be reasonable
to assume that dissolved nucleoproteid had stimulated proliferation,
but it seems much more probable that the initial segmentations
Winkler observed were really due to increased concentration and the
consequent osmotic conditions, not to ferment action or extractive
influences. Errors in making up the saline solutions might of them-
selves have accounted for all that was observed. A concentration
very little above that of normal sea-water would produce the results.'-^
Further, it is well known that the eggs of sea-urchins are prone to
divide into a few cells if they are allowed to remain undisturbed in
normal sea-water for about a day.'^

Winkler's results are hardly positive enough, therefore, to permit
of the deduction he draws ; they might, in fact, be used to show
how unwarranted were Pieri's conclusions.

Experimental.

General methods of procedure. — The investigations recently done
under Professor Loeb's supervision in this connection were con-
ducted with Arbacia punctulata. In a few experiments, as will be
pointed out, the testes of Strongylocciitrotus pnrpuratus were used.
Males and females were kept together in a tank in running sea-water
until they were needed. Immediately before they were used all
extraneous matter was carefully washed off in an abundance of
fresh water, which killed any adherent spermatozoa. The various
instruments employed in the work were repeatedly washed in the
same way.

1 " Nur ein nicht sehr grosser Theil."

* LoEB : This journal, 1900, iii, pp. 436 and 437.

3 LoEB : Loc. cU.

Development of Mature Ova. 57

The sea-water in these experiments was collected in a large
stoppered bottle on one day for use upon the next. This insured
the use of the same water for each set of experiments and the cor-
responding controls. Gemmill ^ has shown experimentally that if
free spermatozoa are kept in sea-water (in ''dilute mixture") for
five hours they lose their ability to impregnate the ovum. Conse-
quently our method rendered inert any spermatozoa which may have
been alive in the water at the time of collection and made boiling
unnecessary. Moreover, Loeb ^ has lately called attention to the
fact that sea-urchins have practically died out in the immediate
neighborhood of Wood's Holl, and that for this reason, even at
the height of the spawning season, there is little or no danger that
the supply of sea-water used in this laboratory contains any live
spermatozoa of this animal.

In procuring testes or ovaries the oral surface of the animal was
cut away and the alimentary and vascular membranes carefully
torn out. After thorough flushing in sea-water to eliminate body
fluid and dissolved matter such as digestive enzyme, etc., the glands
were transferred to perfectly clean vessels for appropriate treatment
without delay.

The ovaries, from which the eggs used as indicators were taken,
were transferred directly to a shallow dish with just enough sea-
water to cover them. In most cases the eggs from one animal were
sufficient for a connected series of observations. As a rule the
ovaries were full of eggs and mere shaking sufficed to liberate the
latter into the surrounding fluid, where a comparatively thick layer
quickly formed. A few drops of this sediment, containing thousands
of eggs, were sufficient for each individual test. The ovaries were
never taken from the animal until all other preparations had been
completed, so that the eggs were perfectly fresh when employed.

Only such unfertilized eggs as were found to be normal and mature
were used. In each of the series of experiments to be described
some of the ova were either fertilized directly with spermatozoa or
were first subjected for an hour or two to the influence of solutions
of higher osmotic pressure than sea-water (mixtures of 88 c.c. sea-
water -\- 12 c.c. ^g*^ n KCl were usually made up for the purpose) and
then were placed in sea-water to test their capacity for partheno-
genetic division. In many experiments both methods were used.

^ Gemmill: Journal of anatomy and physiology, 1900, xxxiv, p. 170.
2 Loeb : Loc. cit., p. 450.

58 William J. Gics.

Under these test conditions the eggs employed were always found
to develop into swimming larva: within twenty-four hours. These
facts are not specially noted in the records given below because of
their uniformity throughout. The " control " tests mentioned with
each series refer to the eggs which had been placed only in normal
sea-water for comparison with ova treated by special processes.

In each of the following series of experiments the volume of sea-
water in each test was, as a rule, 100 c.c. (Note exceptions farther
on.) It was increased only by the addition of portions of extract
as specified under each series and by the few drops of sea-water
carrying the eggs, in pipette, from the main supply. The sea-water
was contained in small bowls of uniform size, making the depth of the
fluid (about an inch) practically the same for all of the experiments.
Throughout each series the bowls were kept covered with glass
plates. The air space above the fluid was about an inch in depth,
thus insuring abundant supply of oxygen. Occasionally, as will be
noted, eggs were placed in quantities of the extract alone, held in
smaller vessels. These were also kept covered. The temperature of
the room varied between 18-20'' C. The amount of evaporation, as
indicated by sensible condensation on the under side of the cover-
plates, was comparatively slight during twenty-four hours, so that no
material concentration occurred during the interval.

The extracts of the spermatozoa were made directly from the testes.
It was not thought necessary to attempt separation of the non-
spermatic tissue elements. The testes were always thoroughly
ground to a thick paste in a mortar with dry sand which had been
heated above 100° C. for from fifteen to twenty minutes. Water and
saline extracts were used within a few hours. Fluids containing
preservatives, however, were given more time for extraction, as will
be noted below. The extractions were made in bottles to permit
of frequent and vigorous shaking. Clear filtrates were obtained in
each case without special difficulty.

In each series of experiments carefully measured quantities of ex-
tract were added to sea-water, and the mixtures stirred to prevent
inequalities of concentration. The eggs were distributed after the
mixtures of sea-water and extract had been made. The experiments
were begun in the morning. At intervals of an hour or two until
late at night, samples of eggs were quickly removed with pipettes
from the bowls to watch glasses for observation under the micro-
scope. Hundreds were examined carefully each time. None were

Development of Mature Ova. 59

ever returned to the main supplies. The eggs in each series were
always under observation for from at least twenty to twenty-four
hours, seldom longer than that, and unless otherwise stated the
"results" recorded below are for periods of that length.

Experimental Data.

Our experiments are described here briefly, though in some detail,
so that whatever value they may possess may be accurately estimated.
The first series of extracts were made with spring water.

Fresh water extract. — Fresh testes. — I. The glands from one animal
were extracted in 15 c.c. HoO for i hr., 30 mins. Three tests were made as
follows : —

(i) Control (2) Extract — 4 c.c. (3) Fresh H2O — 4 c.c.^
Result : No segmentation.
11. The glands from one animal were extracted in 15 c.c. H2O for 3 hrs.
(i) Control (2) Extract — 2 c.c. (3) Fresh HoO — 2 c.c.
Result : No segmentation.
III. Glands from two animals in 10 c.c. H2O for 4 hrs.

A. Control. B. Extract : (a) i c.c. (unfiltered), (b) 4 c.c, (c) 0.05
c.c, (d) eggs in 3 c.c. + equal volume of igO n NaCl. C. Some of
(d) into sea-water after 2 hrs.

Result : Irregular parthenogenetic forms in a very small propor-
tion of (a), (b), and (c) after 4 hrs. A few groups of 8 and one or
two of 16 cells from individual eggs, in 24 hrs., in (b). None beyond
the 4-cell stage in (a) and (c). A few parthenogenetic in C as far as the
8-cell stage. No morulae in any. No segmentations in the control.

The results of the third series encouraged the belief that enzyme
action was demonstrable, although we did not lose sight of the fact
that perhaps increased concentrations, induced by unobserved cir-
cumstances, or other unknown conditions, would account for the
proliferations noted. In the fourth and fifth series the effects of
fresh were compared with those of boiled extract.

IV. Five sets of testes extracted in 60 c.c HnO for 3 hrs. One half was
boiled in an Erlenmeyer flask 10-15 niins. An appreciable concentra-

1 It will be understood from what was stated on page 58 that this abbreviated
reference to the three tests means that besides being under normal conditions (in
100 c.c. sea-water alone), eggs were subjected to the influence of both 4 c.c. of
extract in loo c.c. of sea water and 4 c.c. of fresh H.2O in the same large quantity
of sea-water. This system will be adopted throughout for brevity's sake.

6o William J. Gics.

tion resulted, but of course no approximation to the specific gravity of
sea-water was effected.

A. Control. B. Fresh extract : (a) lo c.c, (b) eggs in 8 c.c. ex-
tract alone. C. Boiled extract: (c) lo c.c, (d) eggs in 8 c.c.
extract alone. D. Samples of B and C in loo c.c. sea- water after
I hr., 30 mins.

Result: During the first 12 hrs. there was no segmentation in
any of B and C. An occasional kidney-shaped cell was found in the
control and D after 5 hrs. At the end of 24 hrs. there were a few
4 to 8 cell divisions in the eggs of (a) and (c) which had been trans-
ferred to sea-water. Only a few 2 to 4 cell groups were found in the
control at the end of the same period.
V. Testicles from 15 animals extracted in 85 c.c. H.jO for 3 hrs. One half
was boiled as in the preceding series.

A. Control. B. Fresh extract: (a) 20 c.c, (b) 10 c.c, (c) eggs
in ID c.c extract alone. C. Boiled extract: (d) 10 c.c, (e) 8 c.c
D. Eggs in B and C transferred to normal sea-water after i hr.

Result : Not a single segmentation could be detected. A very
few of the eggs of (d) and (e) which had been transferred to sea-
water were kidney-shaped as though in an initial parthenogenetic stage.

The results of the first five series were indecisive, but, where
positive, they strongly suggested initial osmotic parthenogenesis,
caused probably by conditions beyond control, rather than zymolytic
influences. On the assumption that the concentration of the extracts
was somewhat lower than sea-water in spite of the salts and proteids
dissolved from the testes, and that variations in effects occurred as a
consequence, the sixth series was arranged to overcome this difficulty.

VI. Fourteen sets of glands were extracted in 35 c.c. H.^O for 3 hrs. Just
before the filtered extract was used it was mixed with an equal volume
of normal NaCl, making approximately a | « NaCl mixture (sea-water
is equivalent to about §« NaCl).

A. Control. B. Extract : 20 c.c, 10 c.c, i c.c, eggs in 10 c.c. ex-
tract alone. C. Eggs in each of B transferred to 100 c.c sea-water at
the end of 2 hrs.

Result : No divisions or irregular forms.

The generally negative results of the preceding experiments made
it seem desirable to resort to other means before abandoning the
study of fresh water extracts. Various enzymes are more easily
extracted after the containing cells have been dried and thoroughly
broken up. This expedient was tried, therefore.

Development of Mature Ova. 6i

Dry testes. — The glands from each animal were macerated and
spread out separately in a thin layer on watch glasses. These were
placed in desiccators over concentrated sulphuric acid or calcium
chloride. Drying was accomplished within eighteen hours. When
desired for use the dry substance was scraped into a mortar, and
ground up thoroughly with sand and extracted as in the previous
experiments.

VI I. The dry substance of four sets of glands was extracted in 30 c.c. H.^O
for 3 hrs.

A. Control. B. Extract: (a) 5 c.c. (unfiltered), (b) 10 c.c, (c)
5 c.c, (d) I c.c, (e) eggs in extract + equal volume ^-§ n NaCl.

Result : Within 12 hrs. no change. At the end of 24 hrs. a
very few were in initial parthenogenetic stages, 2 to 4 cell groups, in
all except (a). They could be found only after careful search and
there were as many in the control as in any of the others.
VIII. Eight sets of dried testes in 25 c.c. H2O for 4 hrs. Filtrate mixed
with an equal quantity of ^-§ n NaCl before using.

A. Control. B. Extract : 7 c.c, eggs in extract alone. C. Some
of the eggs in B were transferred to 100 c.c sea-water after i hr.,
45 mins.

Result : No segmentations or parthenogenetic forms in any.

It seemed necessary to conclude at this point that fresh water
extracts of spermatozoa do not contain substance of zymolytic
power or else that the conditions attending their use are unfavorable
to such manifestation. Enzymes which are soluble in water are also
soluble in solutions of electrolytes, so that attempts were next made
with the latter.

Salt water extract. — A common method of extracting enzymes in-
cludes treatment of the tissue with ordinary salt solution. Sea-u'ater
itself furnishes such a dilute solution, but is not so favorable to rapid
destruction of spermatozoa as fresh water or stronger salt solution.
Since spermatozoa pass through ordinary filter paper, however often
they may be subjected to filtration, it was necessary in using fresh
testes to give particular attention to killing the spermatozoa by
mechanical means. Prolonged grinding in a mortar with fine sand,
as had been done previously, followed by continuous shaking for
several hours, accomplished this.

Fresh testes. IX. Twelve sets of glands were extracted in 50 c.c. sea-water
for 4 hrs.

62 William J. Gies.

A. Controls (2). B. Extract: 20 c.c, 10 c.c, 5 c.c, i c.c, 0.25 c.c.
Result : Not a single division could be found.

The very greatest care is necessary, in this connection, in the use
of solutions of electrolytes, because of the ready osmo-parthenogenetic
response the eggs make to slightly increased concentration. There
is little reason for believing that an enzyme is present in spermatozoa
which is insoluble in dilute, but soluble in strong salt solution.
Therefore it seemed unnecessary to try the effect of more concen-
trated extractive. The tenth series shows the result of an effort to
make the best of saline extraction of fresh testes, however, in a way
somewhat different than that of the preceding.

X. Eight sets of testes in 40 c.c. § n NaCl for 2 hrs. One half was warmed
to 35-40 C. 15-20 minutes.

A. Controls (2). B. Extract (unwarmed) : (a) 5 c.c, (b) eggs
in 5 c.c. extract alone. C. Extract (warmed) : (c) 5 c.c , (d)
eggs in 5 c.c. extract alone. D. Some eggs of B and C in 100 c.c.
normal sea-water after 2 hrs.

Result : No segmentation within 6 hrs. In 12 24 hrs. a very
few 2-cell groups were found with difficulty in (a), (b), and (c) and
in one of the controls.
Dry testes. The preliminary process of drying was also resorted to in this

connection.
XI. Dry material from three animals was extracted in 5 c.c. sea-water for
2 hrs.

A. Control. B. Extract: 2 c.c. (unfilteredj, i ex., 0.25 c.c.
Result: Not a sign of segmentation.

Do the extracts possess poisonous qualities? — One condition that may
appear to be against the action of an enzyme in the extracts used in
these experiments is the possible presence of poisonous substances in
the extract. This question now required a definite answer. We had
varied the quantities of extract considerably, between all reasonable
extremes, in the belief that the most favorable amount might be
indicated, but it will be observed from the foregoing account of
results that no such relation was suggested. The eggs which had
been subjected to the extracts alone, and those placed in sea-water
with the greater proportions of extract, usually showed abnormalities
after a few hours, such as the development of enclosing membrane
or transparent periphery (thicker and not comparable to the " vitel-
line" membrane after fertilization), swelling, disintegration, discol-

Development of Mature Ova. 63

oration, agglomeration of pigment, etc., but none of these changes
were constant so far as their relation to observed conditions could
be determined. The sperm extracts contained salts and dissolved
proteids, of course, and it would be reasonable to assume that these
bodies were present in larger proportion, in some of these experi-
ments at least, than they ever are under normal conditions of
fecundation.

This important matter was definitely tested several times. The
following results of two experiments are cited to. show the facts
in the case :

A. Five sets of fresh testes were ground in the usual way and extracted for
2 hrs. in 30 c.c. fresh water. An equal quantity of ^^ n NaCl was added to
the filtrate. The eggs were placed in this mixture and samples transferred at
intervals of an hour to 100 c.c. sea-water, to which fresh spermatozoa had been
added. Results of examination at the end of 24 hours, the numerals indicat-
ing the number of hours the eggs were kept in the extract : (i) Swimming gas-
trulae. (2) Blastulse (none alive). (3) A few dead blastulae, mostly morulae.
(4) Many unsegmented, none beyond the 32-cell stage. (5) About the same as
those after the 4-hr. treatment. (6) Very few went so far as the 32-cell stage,
many were in the 4 to 8 cell groups. There were no segmentations in the eggs
kept for 24 hrs. in the extract.

B. Six sets of fresh glands were extracted in 30 c.c. sea-water, 3 hrs. Eggs
from one animal were placed in the filtered extract and also into an equal quan-
tity of sea-water (as control). At intervals eggs were withdrawn from each
supply and transferred to 100 c.c. sea-water containing perfectly fresh sperma-
tozoa. Results at the end of 36 hours from the time of the first transferral,
the numerals again indicating the number of hours the eggs were under the
direct influence of the extract or the normal sea-water: (i) Plutei in each.
(2) Advanced gastrulse in each. (3) Gastrulse in each. (4) Many gastrulae in
the control ; hardly any live ones, mostly morulae, among those treated with
the extract. (7) A large number of blastulfe were present in the control, but
no divisions beyond the 32ce]l stage could be found among the eggs which
had been in the extract ; most of the ova were unsegmented. There were no
proliferations in the eggs retained in the extract itself. In the earlier tests the
proportion of unsegmented cells was uniformly greater in the control than in
the other series, whereas the living larvae were relatively more numerous in the
latter. The extract seemed at first to stimulate, and later to inhibit karyokine-
sis. Possibly, however, the accumulation of bacteria in the bowls containing
extract was responsible for the latter effect.

It is clear, from the foregoing, that the dissolved substances of our
extracts have not prevented the eggs from sesmentino". From tVnq

64 William J. Gies.

we may safely conclude that they doubtless would not interfere with
zymolysis if such were demonstrable.

The results of all the preceding series seemed to point in the same
general direction and to indicate no mitotic action. Before accept-
ing this negative conclusion, however, we proceeded to employ various
other familiar methods for the separation of enzymes in the hope of
eventually extracting and demonstrating the presence of such a sub-
stance.

Extract of spermatozoa vrhich had been treated vrith, and preserved

in alcohol Enzymes may readily be extracted from tissues hardened

in alcohol. In fact they are frequently isolated by such preliminary
treatment, which brings about disintegration of the cellular proto-
plasm as well as coagulation of soluble proteid, and thus diminishes
the proportion of undesirable extraneous material in the final extract.
Through the kindness of Professor Loeb, I was enabled to make ex-
tracts of the spermatozoa of Strongylocentrotus purpiiratns, which had
been preserved in an excess of 95% alcohol. The testes were taken
from animals collected on the Pacific Coast about a year ago, while
Professor Loeb was engaged there in his classical researches on artifi-
cial parthenogenesis.

In these experiments, with Arbacia as well as Strongylocentrotus, the alcoholic
sperm mixture was filtered. Both the solid and fluid portions were transferred
to shallow dishes and dried in the air. The liquid soon evaporated and left an
oily residue which dissolved to a milky fluid when mixed with water.

Strongylocentrotus pnrpuratiis. XII. Three grams of the dry sperm res-
idue were thoroughly ground with sand and 30 c.c. fresh HoO. After an
hour an equal volume of '^^ n NaCl was added. Extraction in this mixture
was continued an hour.

A. Control. B. Extract: 17 c.c, 7 c.c, and eggs in 8 c.c of
extract alone. C. Some of the eggs in each of B were transferred
to 100 c.c. sea-water after 3 hrs.

Result: Not the slightest trace of segmentation.
XIII. Two grams of the finely divided dry substance were extracted in 40 c.c.
sea-water for 3 hrs.

A. Control. B. Extract: (a) 12 c.c, (b) eggs in 10 c.c. ex-
tract alone. C. Eggs from B transferred to 100 c.c. normal sea-
water after 2 hrs., 15 mins.

Result: Only a few forms in initial parthenogenesis in the con-
trol and in (a). These were found only after very careful search.
Entirely negative results in the others.

DeveloptJtent of Matttre Ova. 65

It did not seem very likely that the alcoholic filtrate would contain
a mitotic enzyme, if such a substance could not be extracted from the
portion insoluble in alcohol. Yet, since some enzymes are soluble in
diluted alcohol, the following experiments were made in order to
ascertain definitely.

XIV. Half the residue of evaporated alcoholic extract was dissolved in 40 c.c.
sea-water and filtered.

A. Control. B. Extract: (a) 15 c.c, (b) 5 c.c, (c) eggs in
20 c.c of the extract alone. C. Eggs from (c) were transferred to
100 c.c. sea-water after i hr., 30 mins.

Result : Within 6 hrs. no perceptible effect. At the end of 18
hrs. a number of irregular parthenogenetic forms and some groups of
4 and 8 cells in C. No traces of segmentation in any of the others.
XV. The result in the preceding series seemed to be due to increased con-
centration caused by the accumulated salts of the original alcoholic
extract. If this assumption were correct, dilution of the extract
should prevent the effect noticed above. Only a fourth of the
residue was next dissolved in 50 c.c. sea-water.

A. Control. B. Extract : (a) 20 c.c, (b) eggs in 20 c.c. extract
alone. C. Samples of B were transferred to 100 c.c. sea-water
after 2 hrs.

Result : Only a very few irregular shapes in the control and the
transferred eggs of (b). One 4-cell group was found among thou-
sands in the control ; none among the others even after prolonged
search.
XVI. A third experiment was made with the alcoholic residue. The solution
was made more concentrated again. The remaining portion (one
fourth) of the evaporated extract was dissolved in 15 c.c. sea-water.

A. Control. B. Extract : (a) 8 c.c, (b) eggs in 5 c.c. extract
alone. C. Samples of each of B transferred to ico c.c. sea-water
after 3 hrs.

Result : Parthenogenetic groups of small cells in the transferred
eggs of (b), but nothing of the sort in any other.

The results of the last three series emphasize the necessity of pre-
venting material change in the composition of the sea-water and
suggest how easy it might be, in cases of slightly increased concen-
tration to mistake ion parthenogenesis for enzyme proliferation.

Arbacia. Twenty-one sets of testes were treated with 500 c.c. 959^ alcohol.
After remaining in contact with the latter for two days the solid substance was
collected on a filter. *

66 William J. Gies.

X\'ll. The dry solid matter was thoroughly extracted in loo c.c. sea-water
for 12 hrs.

A. Control. B. Extract : (a) 25 c.c, (b) 15 c.c, (c) 10 c.c, (d)
5 c.c, (e) I c.c, (f) 0.5 c.c. C. Samples of B transferred to 100
c.c. sea-water after 2 hrs.

Result : A very small percentage of 2-cell groups was found
in the control, in (b) and among those of (d) which had been
transferred to normal sea-water. One 2-cell segmentation had
been found among the normal eggs immediately after they had
been taken from the ovaries.
X\'III. In 24 hours the alcoholic filtrate (500 c.c.) had evaporated to 30 c.c.
Practically all the alcohol had disappeared. The residue was made
up to 100 c.c. with sea-water and filtered.

A. Control. B. Extract: 25 c.c, 15 c.c, 10 c.c, 5 c.c, i c.c.
C. Samples of B transferred to 100 c c. sea- water after 2 hrs.

Result : An occasional 2 to 4 cell group in practically all in-
cluding the control — less than 2 per 100.

Glycerine extract Glycerine in water seems to be one of the best

of enzyme extractors. Extracts of fresh Arbacia sperm were made
by the previous general process in mixtures of equal parts of glycerine
and water. It has been assumed, of course, that the glycerine in such
extracts would exert specific deleterious effects and naturally careful
control experiments were made to ascertain its influence in the
quantities used in this series. These preliminary control tests de-
termined the influence of glycerine under three general conditions:
{a) its direct effect on the eggs, {b) its influence on normal fecunda-
tion, {c) its action on artificial parthenogenesis.

An abundant supply of equal parts of glycerine and sea-water was made
for use in all tliese experiments. Normal eggs were found to remain unseg-
mented in all proportions of this glycerine solution with sea-water, although a
few irregular parthenogenetic forms were produced by 15 c c. in 100 c.c.
normal sea-water. Quantities of this glycerine solution greater than 5 c.c. in
100 c.c. of sea-water prevented the normal segmentation by spermatozoa, but
many swimming larvje formed in the presence of 2 c.c of the glycerine solution
per 100 c.c sea-water. Even 15 c.c. of the glycerine solution in 100 c.c. of
sea-water did not, however, entirely prevent proliferation in ova which had pre-
viously been kept for 2 hrs. in 88 c c sea-water -f 1 2 c.c. -^ n KCl, yet none
of the segmentations under these conditions went beyond the 8 to 16 cell stage.
With smaller quantities, swimming larvae were obtained.

With these facts established the result of the following experiments are not
without significance.

Development of Alature Ova. 67

XIX. Seventeen sets of testes in 75 c.c. of the above glycerine solution for
48 hrs.

A. Control. B. Extract: (a) 15 c.c, (b) 5 cc, (c) 2 c.c.
C. Samples of each of B transferred to 100 c.c. sea-water after
I hr.

Result : Here and there a kidney-shaped cell was found among
those of (a) which had been transferred to normal sea-water. No
distinct segmentations.
XX. Same glycerine extract after having been shaken with the tissue 24 hrs.
longer.

A. Controls (2). B. Extract : 5 c.c, 2 c.c, 0.5 c.c, 0.25 c.c
C. Some of each of B transferred to 100 c.c. sea-water after i hr.
Result : Not the slightest suggestion of segmentation.
XXI. Twenty sets of testes were extracted in So c.c. of the glycerine solution
four days. The filtrate was poured into a litre of 95% alcohol. A
bulky, though light, white flocculent precipitate formed at once.
After 24 hrs. this precipitate was filtered off, treated with 25 c.c. of
sea-water for several hours and the filtrate used in the following
experiment :

A. Control. B. Extract: (a) 10 c.c, (b) 5 c.c, (c) 2 c.c, (d)
I c.c, (e) 0.25 c.c. C. Samples of each lot of B transferred to
normal sea-water after 2 hrs.

Result: One or two irregular parthenogenetic forms were
found in (c) and among those of (a) which had been transferred to
normal sea-water. The number of such was less than 5 per 1000.

Ether extract Substances which cause the death of the cell or

which appreciably lessen its vitality are known to favor solution of
enzyme into the surrounding medium. Small quantities of alcohol or
ether effect such results. Mathews^ has recently shown that expos.
ure of the unfertilized eggs of Arbacia to a saturated solution of ether
in sea-water for ten to fifteen minutes leads to karyokinetic division of
nearly all the eggs. In the use of ether in these experiments the
greatest care was taken, therefore, to ascertain the influence of ether
in the small quantities employed.

A solution for general use in this connection was made by mixing sea-water
and ether in the proportion of 100 c.c. of the former and 7 c.c. of the latter.
This amount seemed sufficient for any extractive usefulness ether might possess
here. Intimate solution resulted. The odor of ether from the solution was
still quite distinct at the conclusion of the experiments, though not strong at

1 Mathews : This journal, 1900, iv, p. 345.

68 William J. Gies.

any time. In three control experiments, similar to those outlined under the
head of glycerine extract, it was found that as much as 15 c.c. of this ether
solution failed to effect parthenogenesis, although after eighteen hours a few
2-cell groups and irregular forms suggesting an initial stage of mitosis were
found. As these were also present in the control, however, no importance
coukl be attached to the result. After the usual treatment with sea-water plus
2^" // KCl, swimming larvae developed when the eggs were transferred to 100 c.c.
of sea- water containing as much as 25 c.c. of the ether solution. The same
result was obtained, with as much ether solution present, when spermatozoa
were added to the eggs in 100 c.c. of sea-water.
XXII. Ten sets of fresh testes were extracted in 60 c.c. of the ether solu-
tion for 3 days.

A. Control. B. P^xtract : 25 c.c, 15 c.c, 5 c.c, i c.c, 0.25
c.c. C. Some of each lot of eggs in B transferred to 100 c.c.
normal sea- water after 2 hrs.

Result: During the first 12 hrs. no changes were mani-
fested. At the end of 24 hrs., however, all, including the control,
had a few 2 to 4 cell groups. The effect was not at all striking ;
it required careful search to find any signs of proHferation.

XXIII. The same extract, after having been 24 hrs. longer in contact with

the tissue, was again employed.

A. Control. B. Extract : 4 c.c, 2 c.c, 0.5 c.c C Eggs from
each of B placed in 100 c.c. normal sea-water after i hr., 30 mins.
Result : No sign of segmentation.

Alcohol extract — Mathews ^ has also shown that alcohol affects
Arbacia eggs much as ether does. He found that when the ova are
placed in sea-water containing 4 to 5 parts of alcohol and are left
there for from ten to fifteen minutes, they segment into several cells
when they are replaced in sea-water. In these experiments, care was
taken, therefore, to determine precisely the influence of the smaller
quantities of alcohol employed.

A general supply of 10% alcohol in sea-water was kept for the experiments.
Quantities not over 25 c.c. of this dilute alcohol, added to 100 c.c. of sea-
water, were without mitotic influence. As much as 15 c.c. in 100 c.c. of
sea-water interfered to no appreciable extent either with normal fertilization or
osmotic parthenogenesis, as swimming larvce developed within the usual period
in both cases.

XXIV. Testes from 12 animals in 60 c.c dilute alcohol solution 48 hrs.

A. Controls (2). B. Extract : (a) 25 c.c, (b) 15 c.c, (c) 5 c.c,

1 Mathews: Loc. eit., p. 346.

Development of Mahire Ova. 69

(d) 2 C.C., (e) 0.5 c.c. C. Some of each of Bin 100 c.c. normal
sea-water after i hr., 30 mins.

Result : No appreciable effect in any during the first 1 2 hrs.
At the end of 24 hrs., however, several 2, 3 and 4 cell groups were
found in both controls and also in each of those transferred to sea-
water. The eggs of (d) which had been put into sea-water had
a relatively larger proportion that showed initial division, although
the actual number was in reality small — less than 10 in 1,000.
XXV. Some of the filtrate used in the preceding series was taken to repeat
a part of the experiment just described.

A. Control. B. Extract: 2 c.c. C. Eggs from B into 100 c.c.
sea-water after i hr., 30 mins.

Result : No divisions at any time within 24 hrs.
XXVI. Seven sets of testes in 10% alcohol 4 days.

A. Control. B. Extract : Ca) 15 c.c, (b) 8 c.c, (c) 2 c.c.
C. Some of the eggs of each of B in 100 c.c. normal sea-water
after 2 hrs.

Result : Negative during the first twelve hours. At the end
of 24 hrs. there were a very few 2 and 4 cell groups in the control
and among those of (a) which had been transferred. No effect in
any of the others.

Alkaline extract. — Many enzymes showr their greatest activity in
media which are either acid or alkaline. Fluids of either reaction
are also especially efficient in transforming zymogens into enzymes.
If the latter cannot be extracted from spermatozoa, as the preceding
results may be taken to indicate, might not zymogens be detected .-'

Loeb ^ found, in his experiments on Echinoderms and Annelids
that the addition of a small quantity of acid or alkali caused the
unfertilized eggs to segment much more quickly than when they
were left in normal sea-water. NaOH seemed less effective than
KOH, but some development occurred in the presence of as little
as 2 c.c. xi NaOH in 100 c.c. sea-water. Great care had to be
exercised here, therefore. Proportionately smaller amounts were
used as a safeguard.

A saline solution was made for this series containing 8 c.c. of -^-^ NaOH for
every 100 c.c. | n NaCl. This solution was faintly though distinctly alkaline
and could hardly be considered destructive to any enzymes in the cells. In
control experiments similar to those conducted previously to ascertain the
influence of foreign substances it was found that as much as 25 c.c. of this

^ Loeb: This journal, 1901, iv, p. 438 ; also Ibid.^ 1900, iii, p. 136.

70 IVillia^n J. Gies.

solution when added to eggs in loo c.c. of sea-water caused only a few
initial segmentations and that comparatively slight influence was exerted either
on osmotic parthenogenesis or spermatic proliferation by the same quantity.
XXVII. Twenty sets of testes in loo c.c. alkaline solution 24 hrs.

A. Controls (2). B. Extract: 25 c.c, 10 c.c, 5 c.c, i c.c.
C. Some of each of B in 100 c.c. normal sea-water after i hr.
Result : Not a single division.
Extract made in fluid of alternate reaction. — XXVIII. \Vith a view of
aiding still further the transformation of any zymogen not affected by previous
extractions, twelve sets of testes were macerated in the usual way and allowed
to remain in the mortar, covered with a glass plate, for 12 hours. The normal
alkaline reaction of the fresh tissue became faintly acid to litmus during that
interval. 25 c.c of fresh water was added, the mixture neutralized and then
made faintly alkaline with {'^^ NaOH and repeatedly shaken up in this mixture
for about 6 hours. Finally it was neutralized with very dilute HCl and the
filtrate mixed with one-third its volume of 2 « NaCl to bring the concentration
of the extract close to that of ordinary sea-water.

A. Controls (2). B. Extract: (a) 20 c.c, (b) 10 c.c, (c) 1 c.c
C. Samples of Bin 100 c.c. normal sea-water after i hr., 30 mins.

Result: No effect during the first twelve hours. At the end
of 24 hrs. only an occasional 2-cell division could be found in
(c) and among those of (a) which had been transferred.

The persistently negative results of the preceding experiments,
in which the existence of neither an enzyme nor a zymogen could
be indicated, g'-adually developed the idea that possibly an enzyme
is formed from material in the ^%,%, or in the sperm, or in both, on
contact of the two living elements. If such were really the case it
would seem that extracts of the eggs which had been normally fer-
tilized might, under appropriate conditions, possess the power of
inducing segmentation in unfertilized ova.

Extracts of fertilized eggs. — The general experimental procedure by which
this matter was investigated was essentially the same in some respects as for
the preceding series. The fresh full ovaries were broken up in sea-water
in shallow dishes. Only sufficient ova were kept in each dish to form a
single layer at the bottom. The glandular tissue, with such eggs as re-
mained entangled in it, was withdrawn. A minute quantity of fresh sper-
matic fluid was thrown into 100 cc of sea- water and a few drops of this
mixture transferred to the dishes containing the eggs. Within a few hours
practically all of the eggs were developing and some spermatozoa in excess
were in active motion among them.

When the eggs were desired for extraction the fluid containing them was

Development of Mature Ova. 71

thrown into a large tunnel, the outlet of which was closed with a stopper.
The eggs quickly converged to the neck and soon settled to the bottom of
the tube in a thick layer, with a clear supernatant fluid. Practically all of
this could be eliminated by decantation, leaving a thick mass of eggs in only
a small quantity of fluid. The whole process of collection could be com-
pleted in two hours. The segmented eggs were finally thoroughly ground
with sand and appropriately extracted.

Glycerine extract. — XXIX. Eggs from 15 females, many of which had
developed to the i6-cen stage, were ground, in small quantities, with 30 c.c.
sea-water and 30 c.c. pure glycerine. They were repeatedly shaken in this
mixture. At the end of 24 hours the eggs were considerably swelled and
distorted, but were little disintegrated, in spite of the grinding. The latter
process was repeated. More of the eggs were broken up, but many were held
intact by the fertilization membrane. The extraction process was continued
36 hours longer, by which time at least half of the eggs were stiH unbroken,
though distended. A clear filtrate was obtained.

A. Controls (2). B. Extract: (a) 12 c.c, (b) 8 c.c, (c) 4 c.c,
(d) I c.c, (e) 0.25 c.c. C. Some eggs in eacli of B were trans-
ferred to 100 c.c. normal sea-water after 2 hours.

Result: No segmented cells were found in any except (d).

After 12 hours 3 or 4 irregular 2 to 4 cell groups could be found

among thousands after diligent search.^

Saline extract. — XXX. Eggs from 20 females. Development was allowed

to continue until the more advanced had reached the morula stage, when only

a very few remained unsegmented and the majority were at or beyond the

8-cell prohferation. They were ground up in 40 c.c. of fresh water, to which

40 c.c. of 1^" « NaCl was added later. Extraction was continued 36 hours.

At the end of that time many groups of cefls remained tightly held together in

the enclosing membrane ; thorough grinding had not sufficed to disintegrate

them as completely as was desired.

A. Controls (2). B. Extract : (a) 35 c.c, (b) 20 c.c, (c) 10 c.c,
(d) 5 c.c, (e) I c.c C. Some of the eggs of each of B transferred
to 100 c.c. of sea-water after 2 hrs.

Result: Negative at first. After 12 hrs. occasional irregular
forms in initial cleavage were found among thousands in one of the
controls, in (b), (c), (d), and among those of (a), (b), (c), and (e),
which had been transferred to normal sea-water — just such forms as
are sometimes found among normal unfertilized Arbacia eggs which
have been kept undisturbed in sea-water for about 24 hours.
Alcoholic extract. — XXXI. Eggs from 18 sets of ovaries, after segmenta-

^ The extracts of the fertilized eggs were no more destructive to the test-eggs
than the sperm extracts bad been. See page 63.

72 William J. Gies.

tion had proceeded in many to the blastula stage, were ground in 20 c.c. of
sea-water and extracted in this fluid plus 20 c.c. of 20% alcohol. ICxtraction
was continued for 48 hours. The alcohol favored complete disintegration, for
before 24 hours practically all of the cells were reduced to granules.

A. Controls (2). B. Extract: (a) 15 c.c, (b) 8 c.c, (c) 5 c.c,

(d) I c.c C. Some of each of B transferred to 100 c.c. normal

sea-water after 2 hrs.

Result : After 12 hrs. a small number of cells in irregular initial

segmentation were found among those of one of the controls, also in

(d) and among those of (a) which had been transferred to sea-water.

The number was less than 10 in 1,000.

Discussion of Results.

The chief feature of the results we have obtained is their negative
character. Occasionally segmentations were noted, but these were
few and rarely went beyond the 2-cell stage. Further, when the
test-eggs segmented those of the controls did also. These few
divisions could not have been due to spermatozoa, since not a single
group was surrounded with the fertilization or so-called " vitelline "
membrane, whose absence, Loeb^ has indicated, practically proves
non-spermatic influence. Thousands of eggs in the control and
extract series were carefully examined in each experiment and yet
only a trifling proportion showed initial segmentation ; excepting
very few, none of these went as far as the 8-cell stage; and no
morula or swimming larva was ever seen.

The conditions of the experiments were made as nearly normal
as possible and every precaution was taken to guard against evapo-
ration. Special ion parthenogenesis was entirely excluded, therefore.
All of the eggs were ascertained to be ripe and susceptible to seg-
mentation influences. Sufficient variety of extraction process was
employed to guard against failures in withdrawal method and the
many experiments excluded accidental sources of error. It seems
necessary to conclude, therefore, that the occasional segmentations in
initial stages that were observed were only such as have repeatedly
been seen in ripe unfertilized Arbacia eggs which have been exposed
to sea-water for from twelve to twenty-four hours.^

I have not exhausted the means commonly used for enzyme extrac-
tion. The time at my disposal for this work, and the facilities of

^ LoEB : This journal, 1901. iv, p. 454.

- LoEB : Ibid., 1899, iii. p. 136; 1900, ill, pp. 436 and 437.

Development of Mature Ova. 73

this laboratory, have not favored the trial of every known method
nor attempts to devise new ones. It may be that sperm enzyme is
as intimately connected with the structural elements of the cell, and
as resistant to extraction processes, as Fischer has found the invert-
ing ferment of Monilia cajidida to be. Buchner's experience with
zymase has not been overlooked, nor the suggestions it offers ignored.
However, unless the hypothetical sperm enzyme were very different
from most of the others, the numerous methods employed would have
succeeded in bringing it to light, if any enzyme action can be exerted
by substance in fluids surrounding the ova.

It should be recalled in this connection that Loeb^ has recently
made a series of experiments with various foreign enzymes to deter-
mine proliferative power on unfertilized Arbacia eggs, but with
negative results. He states that "the only enzyme that caused the
Qgg to segment at all was papain," but he could not be certain that
this was not due to some accidental constituent of the sample of
enzyme used. " The other enzymes were absolutely without effect."
Two years ago Mathews, in some unpublished experiments cited by
Loeb,^ tried the effect of rennin on unfertilized eggs of the sea-urchin.
The eggs were placed in sea-water solutions of rennet tablets for a
while and then transferred to normal sea-water, when segmentation
into a comparatively small number of cells resulted. The effect
closely resembled those previously described by Morgan,^ and Ma-
thews concluded that the results noted had been produced not by the
enzyme, but by the salts in the tablets increasing the concentration
of the water.

Negative results rarely justify sweeping deductions. The outcome
of these experiments, negative in detail, rather emphasizes possibili-
ties which have not yet been specially considered. It may be that
either too much extract was employed in each series for positive
results to occur or else possibly not enough was taken. Such pos-
sibility led to the wide variations of quantity and condition in these
experiments, but as no differences were noted between the effects of
the largest as contrasted with the smallest proportions of extract,
the results afford no conclusive answer in this connection.

Again, since enzymes are indiffusible, or, at most, are only very

1 LoEB : This journal, 1901, iv, p. 456.
^ LoEB : Ibid., 1900, iii, p. 437.

^ Morgan: Archiv fiir Entwickelungsmechanik der Organismen, 1899, viii,
p. 448.

74 William J. Gies.

slightly diffusible, it is possible that, in experiments of the kind con-
ducted by Loeb, Mathews, Fieri, Winkler, and myself, enzyme which
may be contained in the extract does not or cannot enter the sub-
stance of the ovum. It might be assumed that mere contact with
enzyme in such solution would not cause segmentation and that,
even if the peripheral portions of the cytoplasm should be directly
affected by such immersion, the general effect would be entirely
different if contact, or diffusion, occurred within the substance farther
toward the nucleus. Further, may not the morphological character
of the spermatozoon, specially adapted as it is for great motility and
penetration, imply that segmentation by indiffusible enzyme, con-
tained in fluid surrounding the ovum, is no more possible in artificial
than in normal fecundation. If it be ever found that enzymes, or
zymogens, are causative influences in natural fertilization, I venture
to predict, in view of the results of these experiments, that their
action will also be shown to depend on their direct delivery to points
zviihifi the ovum.

The results of this work do not warrant any additions to current
speculations on the mechanism of fertilization, but a recent sugges-
tion may seem to be connected with these results and therefore
should be considered here.

Loeb,^ referring to his experiments with Echinoderms and Anne-
lids, has expressed the view that " the spermatozoon can no longer
be considered the cause or the stimulus for the process of develop-
ment, but merely an agency which accelerates a process that is able
to start without it, only much more slowly." Accordingly it may be
assumed that " the spermatozoon carries a catalytic substance into
the ^ggl' Loeb considered that enzymes and ions may be among
these " catalytic substances."

If ions are to be reckoned among the agents of proliferation, why
it may be asked, did they not make active the sperm extracts used
in these experiments } But what is the proportion of dissociated
electrolyte in the spermatozoon and in such extracts, it may be in-
quired in return } The composition of the ash does not furnish an
accurate idea of the amount in the spermatozoon of salts pre-existent
as salts and dissociable in extracts. Arbacia spermatozoa have not
been analyzed in this connection nor the amount of dissociated Q\e.c-
trolytes in these extracts determined. We know little of the relative
proportions of the various constituents of spermatozoa and ova. As
' Loeb: This journal, 1901, iv. p. 456.

Development of Mature Ova. 75

we have no knowledge of the absolute or relative quantity of free
ions entering or acting within the ovum, we therefore know nothing
of the influence or sufficiency in this connection of the methods used
in these experiments. Further, the ions which become active in the
ovum may be originally a part of the molecules of the proteid com-
pounds of the ovum or of the sperm, or of both, until the sperm
mingles with the protoplasm of the ovum and forms new and proba-
bly simpler combinations. These experiments were neither intended
for, nor were their conditions suited to an investigation of these
particular problems. The results therefore cannot be interpreted as
having any bearing on them.

It may not be amiss to state, before concluding, that Vigier's ^
assumptions that unfertilized eggs of Arbacia develop into swimming
larvae in normal sea-water were invariably contradicted by my nu-
merous experiments. Vigier says he was unable to repeat Loeb's
results on artificial parthenogenesis. I have often used Loeb's
methods with success in order to determine the responsive character
of the eggs used in the extract series.^ Swimming larvae can be
produced and reared to the pluteus stage with ease.

Summary of Conclusions.

The positive experimental results of Fieri should be attributed to
the action of spermatozoa which had not been removed from the
extracts.

Winkler's uncertain results were doubtless the effects of osmotic
influences.

Extracts of the spermatozoa of Arbacia, which have been made by
the ordinary methods for the preparation of enzyme solutions, and
used in the proportions and under the conditions of these experi-
ments, do not possess any power of causing proliferation of the ripe
ovum.

No evidence could be furnished of the existence of a zymogen in
spermatozoa.

Extracts of fertilized eggs in the earlier stages of development
seem likewise to be devoid of any segmental activity.

The extracts did not produce the typical peripheral " vitelline "
membrane always formed immediately in Arbacia eggs, on fusion
of the male and female elements.

1 See Loeb's criticism : This journal, 1901, iv, p. 454.
^ See references in this connection on p. 57.

76 Wiiltam J. Gies.

These negative results cannot be put forward as proof that there
are no enzymes in spermatozoa which function during the normal
process of fertilization. They do not show that enzyme action is
impossible after, or at the time of union of the spermatozocin with the
ovum within the latter, although the results of Series XXIX-XXXI
might be interpreted as suggesting that enzymes are not thus
elaborated.

In conclusion I wish to thank Professor Loeb not only for the
suggestions which led me to undertake these experiments, but also
for much kindness and encouragement.

Reprinted from The Medical News, Vol. LXXIX, No. 20, Page 767 1 ^ J

November 16, 1901.

ON THE NATURE OF THE PROCESS OF FERTI-
LIZATION.!

BY WILLIAM J. GIES, M.S., PH.D.,

OF NEW YORK;
INSTRUCTOR OF PHYSIOLOGICAL CHEMISTRY IN COLUMBIA UNI-
VERSITY.

Since the time of Leeuwenhoek and his pupils
(1677) it has been known that the fluid secreted
by the male generative organs contains sperma-
tozoa. The earHer observers noted the active
movement of these innumerable minute bodies
in the fresh fluid and assumed them to be para-
sitic animalcules, "sperm animals." A century
later, about 1786, Spallanzani demonstrated that
the fertilizing power of the semen is possessed
by the spermatozoa and not by the liquid por-
tion, since the semen loses its potency when the
spermatozoa are separated from it by filtration.
Kolliker, in 1841, proved that the spermatozoa
are formed from the cells of the testis and, there-
fore, are not parasites as the earliest observers
had assumed, but, like the ova, are derived di-
rectly from the parent-body. In 1865, Schweig-
ger-Seidel and La Valette St. George showed
that the spermatozoon, like the ovum, is a pe-
culiarly-modified single cell of extraordinary
minuteness, containing a nucleus and cytoplasm,
and on the whole morphologically equivalent to
other cells. In 1875, O. Hertwig established the
fact that normal fertilization of the ovum is
brought about by immediate union with but one
spermatozoon.

Although Leeuwenhoek had assumed that the
spermatozoa must penetrate the ova in order to
effect proliferation, nearly two centuries passed
before the fusion process was actually observed.
It was first described in detail by Fol in 1879.
"In every known case an essential phenomenon
of fertilization is the union of a sperm-nucleus,
of paternal origin, with an egg-nucleus, of ma-

' The substance of this paper was given by the author at a recent
meeting of the Society of Physiological Chemists New York City.

ternal origin, to form the primary nucleus of the
embryo."

The exact nature of the process which causes
proHferation of the fertilized egg is not yet un-
derstood. During the past few years important
additions to the facts bearing on this question
have been made by Loeb, whose well known
studies of the mechanics of life phenomena have
not only added greatly to our exact knowledge
of biological events, but, also, have shown the
important influence which the modern physico-
chemical theories may have upon our understand-
ing of animal functions.

l.oeb had come to the conclusion, as a result
of numerous and varied experiments, that "some-
thing in the constitution of the sea-water pre-
vented the unfertilized eggs of marine animals
from developing parthenogenetically." It had
been known for some time that the unfertilized
ova of arthropods, echinoderms and worms seg-
ment into a few cells (2-4) when left for a com-
paratively long time in sea-water, but this was
generally considered a pathological phenomenon.
In his earlier experiments Loeb kept unfertilised
eggs of a common species of sea-urchin for two
hours in sea-water whose osmotic pressure was
slightly increased by the addition of various elec-
trol}i:es. When the eggs were returned to nor-
mal sea-water they soon began to segment, and
blastulse, gastrulse and plutei, which appeared
to be normal in every respect, rapidly developed.
In brief, the general effect in the production of
the embryo was apparently the same as that or-
dinarily caused by spermatozoa. These same
results have been obtained by Loeb with the eggs
of other animals and have been verified repeat-
edly by other observers, including the author.

In one of his first communications of the re-
sults of the work just referred to, Loeb says:
"From these experiments it follows that the un-
fertilized egg of the sea-urchin contains all the
essential elements for the production of a perfect
pluteus. The only reason that prevents the sea-
urchin from developing parthenogenetically un-
der normal conditions is the constitution of the
sea-water. The latter either lacks the presence
of a sufficient amount of the ions that are neces-
sary for the mechanics of cell division (Mg, K,
OH or others) or it contains too large a quan-
titv of ions that are unfavorable to this process^
(Ca, Na or others), or both. All the sperma-'
tozoon needs to carry into the egg for the process

of fertilization are ions to supplement the lack
of the one or counteract the effects of the other
class of ions in the sea-water or both. The
spermatozoon may, however, carry in addition a
number of enzymes or other material. The ions
and not the nucleins in the spermatozoon are
essential to the process of fertilization. . . .
I consider it possible that only the ions of the
blood prevent the parthenogenetic origin of em-
bryos in mammalians and I think it further not
impossible that a transitory change in the ions
of the blood may also allow complete partheno-
genesis in mammalians."

At a somewhat later period in his work on
marine animals, Loeb stated : "The spermato-
zoon not only starts the development of non-
parthenogenetic eggs, but it is also the bearer of
the hereditary qualities of the male. From our
experiments it becomes evident that these two
functions of the spermatozoon are not neces-
sarily bound together, for nobody would assume
for an instant that the hereditary qualities that
are carried by the spermatozoon could be im-
parted to the eg;g by a change in the inorganic
constituents of the sea- water. We have learned
to attribute the different activities of a cell to
different enzymes. We must in future consider
the possible or probable separation of the fer-
tilizing qualities of the spermatozoon from the
transmission of hereditary qualities through the
same. . . . The bulk of our protoplasm con-
sists of proteid. . . . The proteids are char-
acterized by two qualities which are of the
utmost importance in the analysis of life phe-
nomena. The proteids change their state very
easily, and readily take up or lose water. . . .
The agencies which affect these two variable
qualities of the protoplasm most powerfully are,
first of all, certain enzymes. . . . Almost
equally powerful are ions in certain concentra-
tions. . . . The third agency is tempera-
ture. In our experiments it was evidently the
second factor which affected the condition of the
colloids." The latter sentence refers, naturally,
to the colloids of the ovum.

Subsequent experiments on sea-urchins en-
abled Loeb to give a more definite answer to the
question of the nature of the process of fertiliza-
tion. He found that an increase in the osmotic
pressure of the sea-water through the addition
of cane sugar or urea can produce partheno-
genesis. "This proves conclusively," says Loeb,

"that the dei'clopment of the unfertilized egg is
produced titrough an increase in the concentra-
tion of the surrounding solution. As it is im-
material ivhether the increase in the osmotic
pressure is brought about by electrolytes or non-
conductors, there can be no doubt that the essen-
tial feature in this increase in the osmotic pres-
sure of the surrounding solution is a loss of a
certain amount of zvater on the part of the egg.
. . . . A consequence of the loss of water
on the part of the eg^ is an increase in its os-
motic pressure. The osmotic pressure inside the
egg is furnished chiefly or almost exclusively by
electrolytes. It is thus not impossible that the
ions in the egg, if their concentration is raised,
bring- about that chano^e which causes the egg
to develop. If we assume that the spermatozoon
starts the development of the egg in the same
wav as in the case of artificial parthenogenesis,
it follows that the spermatozoon must possess
more salts or a higher osmotic pressure than the
eggs. . . . But there is no reason why the
spermatozoon should not bring about the same
effects that we produce by reducing the amount
of water in the egg, in some different way. . . .
It seems as if the liquefaction of the nuclear
membrane and other constituents of the nucleus
were a prerequisite for cell division." Possibly
this liquefaction is accomplished by enzymes.

In his last paper, after many additional ex-
periments on marine fauna, Loeb stated that
"the bridge between the phenomena of natural
and artificial parthenogenesis is formed by those
animals in which physical factors decide whether
or not their eggs develop parthenogenetically.
In plant lice parthenogenesis is the rule only as
long as the temperature is high or the plant has
nlentv of water. If we lower the temperature or
let the plant dry out, sexual reproduction occurs.
The drying-out of the plant causes the tissues of
the lice to lose water. The same factor, loss of
water, makes the artificial parthenogenesis of
echinoderms and chaetoptems possible. In plant
lice the effect is of the same kind, only in the
opnosite direction."

Firrther on in the same communication, Loeb
adds: "The Sfeneral opinion concerning the role
of the spermatozoon in the process of fertiliza-
tion is that it acts as a stimulus, and that as such
it starts the development of the egg. ... If
we consider the fact that the eggs show at least
a beginning of segmentation under 'normal' con-

ditions, the act of fertilization assumes a differ-
ent aspect. The spermatozoon can no longer be
considered the cause or the stimulus for the
process of development, but merely an agency
which accelerates a process that is able to start
zvithout it, only much more slowly. Substances
that accelerate chemical or physical processes
which would occur without them are called cata-
lyzers (Ostwald). According to this definition
we may assume that the spermatozoon carries a
catalytic substance into- the tgg, which acceler-
ates the process that woiild start anyhow but
much more slowly. ... It would be wrong
to say that the K-ions are the stimulus that
causes the developmental process. They merely
act as catalyzers, accelerating a process that
ivould otherzvise proceed too slowly. The loss
of water on the part of the egg-cell must have
a similar effect, but possibly a less direct one.
It may be that the loss of water alters the chemi-
cal processes in the egg in such a way as to give
rise to the formation of a substance which acts
catalytically. . . . The introduction of the
catalytic substances which accelerate the
processes of development saves the life of the
egg. This may be made intelligible on the fol-
lowing assumption. Two kinds of processes are
going on in the mature egg after it has left the
ovary. The one leads to the formation of sub-
stances which kill the egg; the other leads tO'
the formation of substances which allow growth
and cell division and are not poisonous. We
may use as an illustration Pasteur's well-known
experiments on the behavior of yeast cells in the
presence and absence of atmospheric oxygen. In
the presence of oxygen the yeast cells multiply
on a sugar solution, while the zymase effect is
comparatively small. In the absence of oxygen
the multiplication of cells is limited or may stop,.
while the zymase effect becomes more prominent.
The products of alcoholic fermentation are com-
paratively harmless for the yeast cell, and for
this reason an increase in the fermentative ac-
tivity of the cell does not cause the death of the
yeast. I imagine that matters are similar in the
mature egg-cell after it has left the ovary, with
this difference, perhaps, that the substances
formed (by fermentation?) in the egg-cell are
more poisonous for the egg than the alcohol and
the other products of fermentation are for the
yeast. The process that causes the death of the
egg-cell and the one that causes cell division are

at least jjartly antag-onistic. They are both in-
hibited by a low temperature, so that in this case
death does not occur, althoug^h no cell division
is possible. If we succeed in finding a substance
which accelerates the process of cell division at
the normal temperature, this will at the same
time lead to a suppression or a reduction of the
antagonistic process that shortens life. In the
case of the egg of chsetopterus a trace of K-ions
acts as such a catalytic substance ; possibly a
trace of ?I-ions ; and perhaps certain substances
that are formed when the egg loses a certain
amount of water. For the echinoderm egg we
know at present only the last factor. In addition
there are the catalytic substances carried or pro-
duced by the spermatozoon (ions? enzymes?).
But there are certainly other catalytic substances,
as is proved by tumors and galls, in which the
variety of structures corresponds to an almost
equal variety of parasites. We do not need to
assume a specific parasite for each kind of tumor.
Teratomata may be explained on the basis of the
parthenogenetic tendency of the mammalian egg
in connection with some chemical change that
furnishes the catalytic substance. But it is not
impossible that even in benign tumors, such as a
teratoma, the catalytic substance may be due to
parasitic organisms.] It is very important to
realize that the introduction of catalytic sub-
stances into the egg does not prolong its life un-
less the egg has reached a critical point deter-
mined by two sets of conditions. The one is the
maturity of the egg, the other the change of con-
ditions connected with the egg leaving the ovary.
. . . . The fact that there is an age limit for
the development of carcinoma may be a similar
])henomenon. The catalytic substances which
are given off by the cancer parasite may not be
able to bring about cell division in the epithelial
cells unless the latter have reached a critical
point, which is at least partly determined by the
age of the individual."

Among the catalytic substances which Loeb
has constantly had in mind in his brilliant ob-
servations in this connection are enzymes, as has
already been indicated. With the advice and
many helpful suggestions of Professor Loeb, the
writer, working in Prof. Loeb's laboratory at
Wood's Holl, recently attempted to ascertain
whether any experimental justification can be
found for the assumption that the spermatozoon

carries substance into the ovum which effects
proHferation by zymolysis.

Fieri appears to have been the first to give
this question experimental examination. Several
years ago (1897) he reported that he had ex-
tracted soluble enzyme from the testicles of two
varieties of sea-urchin, which had the power to
bring about segmentation of ova of the same
varieties. The enzyme, which he called "ovu-
lase," was obtained, he said, by merely shaking
the testicles in distilled water or sea-water. As
he himself was not sure that all spermatozoa
were killed in the extraction process, it seems
certain that his results were due not to "ovulase,"
but to live spermatozoa.

Dubois, in 1900, arrived at the conclusion that
natural fertilization comes about through the ac-
tion of a fecundative ferment. He claims that
he was able to separate such a body, "d' une
zymase fecund ante," from the testicles of a va-
riety of sea-urchin, but, unfortunately, no ex-
periments showing its qualities or method of
preparation were detailed by him. Dubois
named the ferment ( ?) "spermase" and credited
it with the power of modifying a hypothetical
substance pre-existent in the ovum, which he
called "ovulose."

Winkler, a little more than a year ago, re-
ported the results of experiments similar to those
of Fieri. Great care was taken to destroy the
spermatozoa in the extracts and Fieri's work
was much improved. The influence of the ex-
tracts was practically negative. Sometimes with
the same extract the eggs of one individual "re-
acted," whereas the eggs of another did not.
The proliferation never went beyond the 4-cell
stage. It is well known that the unfertilized
eggs of the sea-urchin are prone to divide into
a few cells if they are allowed to remain undis-
turbed in normal sea-water for about a day —
the usual length of Winkler's experiments.
Winkler's results are hardly positive enough for
the deduction that fecundative enzyme was ob-
tained : they might, in fact, be used to show how
unwarranted were Fieri's conclusions.

Shortly after Winkler's paper appeared,
Cremer published a very brief note giving a gen-
eral statement regarding some unfinished experi-
ments by himself and Hofer. They worked with
the testicles of trout and used the Hahn-Buchner
pressure method for obtaining sperm extract.
They found that none of the expressed fluids

8

from the trout spermatozoa possessed any seg-
mental activity on mature trout ova. No de-
scription of the experiments nor methods used
in testing the extracts were given by these ob-
servers in their preUminary note.

Loeb recently made a series of experiments
with various non-spermatic enzymes to deter-
mine proliferative powder on the unfertilized eggs
of the sea-urchin, but with negative results. He
states that "the only enzyme that caused the egg
to segment at all was papain," but he could not
be certain that this was not due to some acci-
dental constituent of the sample of the enzyme
used. "The other enzymes were absolutely with-
out effect."

Two years ago Mathews, in some unpublished
experiments cited by Loeb, tried the effect of
rennin (rennet tablets) on unfertilized eggs of
the sea-urchin. Segmentation into a compara-
tively small number of cells resulted. Mathews
concluded, however, that the results noted had
been produced not by the enzyme, but by the
salts in the tablets increasing the concentration
of the w^ater.

Up to the time, then, that the author's work
was begun it seemed possible that enzyme action
might be a causative influence in normal seg-
mentation of the ovum after introduction of
spermatozoon, but no definite experimental evi-
dence had been presented to support the theory.

Regarding the writer's work a multitude of
details may be passed over and the essential facts
regarding methods of procedure, etc., stated in
the following brief account :

Because of the ease with w^hich large quanti-
ties of the spermatozoa and ova of the common
sea-urchin can be obtained, we used the sexual
organs of this marine animal, which has fur-
nished the material for many classical studies of
cell development. The normal conditions under
which fertilization and proliferation of the ova
of the sea-urchin occur can be easily maintained
in sea-water in the laboratory. Many of the
usual methods of enzyme extraction were em-
ployed on the testicles. The eggs, always normal
and mature, were kept in ordinary sea-water to
which various quantities of sperm extract were
added. Careful examination of the eggs was
made at frequent intervals -during twenty-four
hours. Concentration of the sea-water was en-
tirely prevented. The results of twenty-eight
series of three to thirteen twenty-four-hour ex-

periments were entirely negative — that is, no
proliferation resulted and every extract was de-
void of segmental power. Control experiments
were made with each series, which showed that
normal conditions prevailed and that the eggs
would have segmented had the extract possessed
proliferative power. It was also ascertained in
control experiments that the extracts were devoid
of to^ic property.

The persistently negative results of these ex-
periments, in which the existence of neither an
enzyme nor a zymogen could be indicated, grad-
ually led me to believe that possibly an enzyme
is formed from material in the egg, or in the
sperm, or in both, on contact of the two living
elements. If such were really the case it would
seem that extracts of the eggs which had been
normally fertilized might, under appropriate con-
ditions, possess the power of inducing segmen-
tation of unfertilized ova.

A large number of eggs in sea-water were ac-
cordingly treated with a drop of spermatic fluid
and allowed to develop in the normal manner to
various stages — in one experiment as far as the
blastula stage — when the fluid was separated by
decantation, the cell-groups thoroughly ground
in a mortar with sand and extracted in several
of the usual ways for the isolation of enzymes.
None of these extracts had any power of causing
fresh mature eggs to segment.

Entirely negative results rarely justify sweep-
ing deductions. Since enzymes are indiffusible,
or, at most, are only very slightly diffusible, it
is possible that in experiments of the kind con-
ducted by Loeb, Mathews, Winkler, Fieri,
Cremer and myself, enzymes which may be con-
tained in the extract does not and cannot enter
the substance of the ovum, yet it may be that di-
rect absorption of such enzyme in solution could
take place through the micropyle. It may be that
sperm enzyme, if such really exists, is as inti-
mately connected with the structural elements of
the cell, and as resistant to extraction processes,
as Fischer has found the inverting ferment of the
mould Monilia Candida to be. But even if it is
extractable, it might be assumed, with reason,
that mere contact of the ovum with enzyme in
solution would not cause segmentation and that,
even if the peripheral portions of the cytoplasm
should be directly affected by such immersion,
the general effect would be entirely different if
contact, or diffusion, occurred within the sub-

lO

stance farther toward the nucleus. Possibly the
morphological character of the spermatozoon,
specially adapted as it is for great motility and
penetration, should imply that segmentation by
indiffusible enzyme contained in fluid surround-
ing the ovum is no more possible in artificial
than it is a part of normal fecundation. If it is
ever found that spermatic enzyme, or zymogens,
are causative influences in natural fertilization, I
venture to predict, in view of the results of our
experiments, that their action will also be shown
to depend on their direct delivery to points
zvithin the ovum.

If ions are to be reckoned among the agents
of proliferation, why, it may be asked, did they
not make active the sperm extracts used in these
experiments? Unfortunately, we know nothing
at present of the proportion of dissociated elec-
trolytes in the spermatozoon and in such ex-
tracts. The composition of the ash does not fur-
nish an accurate idea of the amount in the
spermatozoon of salts pre-existent as salts and
dissociable in extracts, although the compara-
tively large quantity of ash in spermatozoa, as
found by Hammarsten and others, may suggest
proportionately large quantity of dissociable
electrolyte. We know little of the relative pro-
portion of the various constituents of sperma-
tozoa and ova, and we have no knowledge of the
absolute or relative quantity of free ions entering
or acting within the ovum. The ions which be-
come active in the ovum may be originally a part
of the molecules of the proteid compounds of the
ovum, or of the sperm, or of both until the
spermatozoon mingles with the protoplasm of
the ovum and forms new and probably simpler
combinations. The writer's experiments were
neither intended for, nor were their conditions
suited to an investigation of this particular phase
of the fertilization problem. The results cannot,
therefore, be interpreted as having any bearing
on them.

Summing up briefly, the chief experimental
results of our work are:

1. Extracts of the spermatozoa of the sea-
urchm, which have been made by the ordinary
methods for the preparation of enzyme solutions,
do not possess any power of causing prolifera-
tion of the ripe ovum.

2. No evidence could be furnished of the exist-
ence of a zymogen in spermatozoa.

3. Extracts of fertilized eggs, in the earlier

stages of development, were likewise entirely
devoid of segmental activity.

4. Enzyme seems to be excluded from the
catal5rtic substances which Loeb and others have
thought may influence the initial divisions of the
ovum after the introduction of the spermatozoon,
although it is possible that the conditions of these
and previous experiments were unfavorable to
the manifestation of activity on the part of fecun-
dative ferment. It seems more probable, how-
ever, that I.oeb's theory of the influence of sper-
matic ions in fertilization affords the true ex-
planation of the phenomena in question.

Free use in the preparation of this paper has
been made of facts and statements in the follow-
ing publications :

Wilson. The Cell in Development and Inheritance, 1898.

Loeb. Papers in the American Journal of Physiology on Arti-
ficial Parthenogenesis: iSgg, iii, p. 13s; 1900, iii, p. 434, and iv, p. 178;
1901, iv, p. 424.

Gies. Do Spermatozoa Contain Enzyme Having the Power of
Causing Development of Mature Ova? American Journal of Physi-
ology, 1901, vi, p. 53-

Reprinted from the American Journal of Physiology.

Vol. VIII. — December i, 1902. — No. III.

1

NOTES ON THE " PROTAGON " OF THE BRAlN.i
By W. W. LESEM and WILLIAM J. GIES.

SEVERAL years ago Chittenden and FrisselP made a study of
the distribution of phosphorus-containing substances in the
brain. The results obtained by them seemed to "indicate that
protagon contains but a small proportion of the total phosphorus of
the brain and that other phosphorized organic bodies, such as
lecithins, are present, preformed in the tissue, in relatively large
proportion." They concluded that " the dry solid matter of the brain
contains as much or even more lecithin than protagon." Chittenden
and Frissell also observed that, " contrary to previous statements,
protagon tends to undergo cleavage by long-continued heating at
45° C. in 85 per cent alcohol, a certain amount of an alcohol-soluble
(at 0° C.) body richer in phosphorus than protagon, being split off
while the residual protagon obtained by recrystallization at 0° C. con-
tained a somewhat diminished percentage of phosphorus.

Shortly after the publication of the brief note containing the above
deductions, Dr. Gies repeated and extended the experiments begun
by Dr. Frissell. The general conclusions of this second series of
experiments were practically the same as those previously re-
ported, but as the work was unavoidably interrupted, no further
reference was made to them. Recently, however, new experiments
on protagon have been performed by Mr. Lesem and Dr. Gies. The
results of these experiments, to which we shall refer farther on, make
it seem desirable to give here some of the related data of the earlier
experiments in which the work of Chittenden and Frissell was
repeated.

1 This work was begun by Dr. Gies under Professor Chittenden's super-
vision, in the Sheffield Laboratory of Physiological Chemistry at Yale University.
It was completed by Mr. Lesem and Dr. Gies in the Laboratory of Physiological
Chemistry at Columbia University.

2 Chittenden : Proceedings of the American Physiological Society, Science,
1897, V. (N. S.), P- 901-

183

184 ^. i^^- Leseui and IViUiam J. Gies.

I. On the General Distribution of Phosphorus-Containing
Substances in the Brain.

The brains employed in the experiments by Chittenden and
Frissell were taken from sheep. Although the brains were used
within twenty-four hours after the death of the animals, it seemed
possible that, even within that short period, bacterial changes might
have had some influence on the results.^ In repeating the first
series of experiments, this difftculty was obviated by the adoption of
the following procedure, which is the same as that used by Chittenden
and F"rissell,- except in the steps taken at the beginning to prevent
possible alterations through the influence of bacteria.

First experiment, — In this experiment glass-stoppered bottles of convenient
size, containing about 750 c.c. of 85 per cent alcohol, were accurately
weighed and removed to the slaughter house without loss of fluid. The
sheep were killed in the usual way. The greater portion of blood dis-
appeared from the brain in a minute or two, when the head was opened
with a cleaver and the entire brain quickly removed. Superficial blood
and lymph were taken off promptly with a clean dry clotli. While the
brains were still at practically the normal body temperature, they were
rapidly slashed with a scalpel and at once transferred to the bottled
alcohol. Two whole brains were deposited in each of three bottles.
Special care was taken to prevent any loss of alcohol by evaporation or by
spilling.

It would seem that this prompt treatment with alcohol prevented such
post-mortem changes as exposure for several hours to the air, a lowered
temperature, etc., might induce. We do not mean to suggest, however,
that the alcohol itself has no transforming power on the phosphorized
constituents. Such influence, if exerted, would doubtless have been no
greater, nor any different, at this point than later on.

The quantities of tissue in each bottle were 152.99. 172.19, and
148.89 gms.

Preliminary cold extracts. — The tissue remained in the original alcohol
about four hours, when the filtrate was collected and the tissue very
thoroughly macerated in a mortar. The finely divided material was next
transferred to 750 c.c. of 85 per cent alcohol, and kept under it over

> The results of the following experiments show, however, that no appreciable
changes of such character could have been effected.

2 The methods employed by Chittexoen and Frissell could not be described
in the very brief abstract of the preliminary report of their work. For that reason
we {live the methods here in some detail.

Notes on the '' Protagon'' of the Brain. 185

night, after which the filtrate was again separated. These two cold
extracts were combined.

Extracts at 4^° C. — Extraction was next made in 85 per cent alcohol (li litres
for each pair of brains) for ten hours at 45° C, and the filtrate again
collected. After standing in 2 htres of 85 per cent alcohol, at room
temperature over night, the alcohol-tissue mixture was warmed to 45° C.
and held at that temperature for twelve hours, after which the filtrate was
again obtained. The residual tissue was once more kept in 2 litres of
85 per cent alcohol over night and further extracted in the same fluid at
45° C. for fourteen hours, when the filtrate was preserved as before.
After each of these filtrations, the solid substance was washed with a little
warm alcohol (85 per cent), and the washings added to the appropriate
filtrate.

Extraction in boiling alcohol. — At this point the tissue remained in i litre of
85 per cent alcohol over night, when the mixture was boiled on a water
bath for a half hour. After filtering, the tissue was also extracted in boiling
95 per cent alcohol for the same length of time. These two hot alcoholic
extracts were combined.

Tissue residue. — The residual tissue was finally washed with cold 95 per cent
alcohol, then with absolute alcohol, and dried to constant weight at 80° C.

Ireatment of the extracts. — The extracts obtained at room temperature and
in boihng alcohol were separately evaporated in silver crucibles almost to
dryness, and the total phosphorus content determined directly. The
cold extract of our first preparation, however, was separated into protagon
and filtrate therefrom by the method referred to below.

The three extracts obtained at 45° C, in the second and third prep-
arations, were separately reduced to 0° C. with the aid of common freez-
ing mixture, and held at that point for six hours. A heavy flocculent
precipitate containing much crystalline cholesterin, protagon, etc., quickly
separated from the first of each series of three extracts. The precipitate
was considerably less in the second extract, and only a very faint turbidity
was formed in the third. Each precipitate was quickly filtered, at a
temperature slightly below 0° C, on funnels surrounded by freezing mix-
ture. The precipitates were washed once with cold 85 per cent alcohol,
and then with cold ether until free from cholesterin. The alcohol wash-
ings were added to the same filtrates. The filtrates were combined and
evaporated for the determination of phosphorus. The ether washings
were given the same treatment. The protagon products were dried at a
low temperature on the filter papers. Phosphorus was determined in the
mixture of protagon and filter papers, the latter having been free from
that element.

Phosphorus was always determined by the usual fusion method.

1 86

IV. IV. Lesoii and WilliaDi J. Gics.

Analytic results. — The following table gives our analytic results for
phosphorus in the various solids and fluids separated by the above
method :

TAHLK I.

Extracts, etc.

Phosphorus content.

1.

II.

III.

II.

III.

II.

III.

Grams.

Percentage

of total
solid matter.

Percentage

of total
phosphorus.

A. Cold extracts (2) . . .

a. Protagon

b. Filtrate from protagon

B. Extracts at45°C. (3). .

a. I'rotagons ....

b. Filtrates from prota-

gons

c. Ether washings of pro-

tagons

('. Extracts in boiling alco-
hol

D. Tissue residue ....

0.1423
0.0432

0.1348 0.1841

0.2599 i 0.2887
0.0874 0.1008
0.1370 0.1401
0.0355 0.(H78
0.0047 0.0054

0.1098 1 0.0939

1

0.35 0.43

0.68 ! 0.67
023 023
0 36 0.33
0.09 0.11
0.01 0.01
0.29 0.22

26 32

51.14
1730
27.07
6.77
0.75
21.80

32.33

50.38
1730
24.81
8.27
0.75
16.54

Total phosphorus . .

....

0.5092 0.5721

1.33 1.33

1

Weight of fresh tissue . . 1 148.89
Weight of tissue residue

Estimated solids in fresh tis-
sue (25 per cent)

Estimated weight of extracted
matter

152.99 172.19
15.25 \ 17.08

38.25 43.05
23.00 25.97

That the preliminary cold extracts contained a comparatively .small
amount of protagon seems to be indicated by the results for our first
preparation. Protagon is only slightly soluble in 85 per cent alcohol
at o' C. and is practically insoluble in ether at the same temperature.
Thus of 2 grams of protagon, 0.03 to 0.04 gram dissolved in 500
c.c. of 85 per cent alcohol at o'" C. The same quantity of ethereal
filtrate from 3.6 grams of protagon, at the same temperature, con-
tained nothing yielding a phosphorus reaction after fusion with alkali.
It is possible that the presence of the other constituents of the
alcoholic extract may increase or decrease this solubility. It is
hardly probable, however, that more than an insignificant portion

Notes on the "" Protagon'" of the Brain. 187

of the protagon remains unprecipitated on lowering to zero the
temperature of alcoholic extracts such as the above.

Second experiment. — We decided to repeat the experiment again,
but with less tissue. The results of our previous experiment had
been obtained for the whole brain. We now endeavored to ascertain
whether the above data apply equally to all portions of the brain or
whether there are wide phosphorus variations for the parts. This was
accomplished indirectly without materially altering the conditions of
the previous experiment. For the purpose indicated we took amounts
of tissue equivalent in weight to a whole brain, but made up of
different parts of two brains.

The method of treatment at the slaughter house, transportation in weighed
alcohol, extraction in 85 per cent alcohol at room temperature, at 45° C,
etc., separation of protagon. etc., were the same in this as in the first
experiment. Samples of the fresh tissue were used for determinations of
solids and phosphorus.

At the slaughter house the brains were carefully sectioned transversely into
halves just before their deposition in the alcohol. The hahes were
combined as indicated in the next table. The preliminary extracts in
cold alcohol were united with those obtained at 45° C, and the prota-
gon was removed from the mixture. Four extractions of each sample
of tissue were made at 45^ C. One litre of 85 per cent alcohol per
brain was used each time. The washing of the protagons with ether was
omitted.

Table II, on page 188, gives the essential results of this experiment.

Only insignificant differences are to be observed between the
results of the first two experiments. The analytic data are, therefore,
essentially the same for the anterior and posterior halves of the brain.
The similarity of the results of this series to those of the preceding
is especially evident from the directly comparable data given in
Table III on page 188.

The results of the first and second experiments show that the
greater portion of the phosphorus of the brain is contained in sub-
stances not precipitable as protagon. The bulk of the phosphorus
in the preliminary cold extract (Exp. i), and in the filtrates from the
protagons (Exps. i and 2), is doubtless contained in substances as
readily soluble in alcohol as lecithin. Some phosphate was also
present. Probably most of the phosphorus of the ether washings
(Exp. i) was contained in substance which was soluble in the

1 88

//'. IF. Lesnn and Williatn /. Gics.

alcohol (and in the ether), but whicli adhered to the precipitate
until it was treated with ether.

.Mil. I'. 11.

1

Extracts, etc.

Phosphorus content.

A.

Ant. half of 1.

Post, half of 2.

Grams.

B.

Ant. half of 2.

Post.half of 3.

Grams.

C.

Ant. half of 3.

Ant. half of 4.

(irams.

D.

Post, half of 4.

Post, half of 5.

Crams.

I. Ext. at room temp,
and at 45° C. .

a. Protagons (4) .

/'. Filtrates from

protagons . .

II. Extracts in boiling

alcohol . . .

III. Tissue residue . .

0.2512
0.0953
0.15.59
0.0013
0.0635

0.2152
0.0728
0.1424
0.0013

0.0432

0 244r.
0.0900

0.1546
0.0015
0 0517

0.2353
0.0869
0.1484
0.0016
0.0467

Total phosphorus :
a. Total in all parts
/'. As determined
directly . . .

0.3160

0.2597
0.2694

0.297S

0.2836 .
0.3038

Weight of fresh tissue .

S4.S6

72.07

86.44

82.23

Whether these soluble substances exist " preformed " in the brain,
as Chittenden and Frissell and others believe, or are decomposition
products resulting from the use of the reagents, as some infer, is not
made clear by these experiments. The former view seems more
probable,

TABLE III.

Exp.

Hrain.

Weight
of fresh
tissue.

Gms.

Phosphorus content.

P''°'^- ' Filtrate,
gon.

Gm. Gm.

Hot Tissue 1 -j,^,^,
extract residue, j
Gm. Gm. 1 Gm.

First

.Second

One half of III.
('.

86.10 0.0743' 0.1621-^
86 44 0 0900 0.1.546

0.0027 0 0469 ' 0 2860

0.0015 0 0517 0.2978

1

1 Including ether washintrs. - Including cold extract.

N^otes on the " Protag07t " of the Brain.

189

The results of the next experiment lead to essentially the same
conclusions as those drawn from the preceding.

Third experiment. — The methods of this experiment were, in general, the
same as those of the first and second. The following differences of
treatment are to be noted. The divisions of the brains were made longi-
tudinally instead of transversely. The alcoholic filtrates (2 ), obtained at
0° C. after separation of the protagon, were evaporated almost to dryness
on a water bath at 35 "-40° C. The residues thus resulting were thoroughly
extracted several times with a- moderate excess of cold ether. The
extracts were filtered and evaporated to dryness. The residue left after
treatment with ether was extracted with boiling 95 per cent alcohol. So
little seemed to dissolve that the alcoholic extracts were evaporated with
the ethereal. The substance remaining after the extraction with alcohol,
mostly inorganic matter, was- next treated with water. All of it dissolved
very readily. This solution was then evaporated to dryness. Phosphorus
was determined in the substance from each of these extracts and in the
protagon, with the results tabulated below:

TABLE IV.

Extracts, etc.

Phosphorus content.

A.
Same lateral

halves of

brains 1 and 2.

Grams.

B.
Opposite lat-
eral halves of
brains 1 and 3.
Grams.

C.
Opposite lat-
eral halves of
brains 4 and 5.
Grams.

■I. Protagons (2)

II. Filtrates

0.0576

0.1725
0.1531
0.0194

0.0701

0.1841
0.1603
0.0238

0.0667

0.2037
0.1750
0.0287

a. Substance soluble in alcohol
and ether

h. Residual substance soluble in
water

Total phosphorus . . .

0.2301

0.2542

•

^ 0.2704

Weight of fresh tissue

89.46

104.50

102.50

II. On the Question of the Chemical Individuality of

Protagon.

Twenty years ago Gamgee expressed himself on this subject as
follows : "There is no subject in physiological chemistry concerning
which it is more difficult to give a statement, which would be accepted

IQO IV. IV. Lescm and William /. Gics.

as correct by those who have devoted their attention to it, than the
chemistry of the complex phosphorized fats which exist in the
nervous tissue." ' The same may be said perhaps with ecjual force
to-day, in spite of the careful work done in the mean time to solve
the problems connected with the chemical constituents of the brain.

Soon after Liebreich - separated from the brain the substance he
called protagon, Thudichum ^ and others denied the existence of
such a substance. Thus, Uiaconovv,' working as did Liebreich, in
Hoppe-Seyler's laboratory, obtained results which led him to conclude
that protagon is a mixture of lecithin and ccrebrin. The later re-
searches of Gamgee and Blankenhorn,'^ iiowever, furnished data
which were generally accepted as amply confirming the original con-
clusions of Liebreich. The subsequent work of Kaumstark," Kossel
and Freytag,' and Ruppel,^ particularly, further emphasized the
growing confidence in the existence and importance of protagon
as a brain constituent. Until recently the matter seemed to be
settled in the general conviction that protagon is a chemical individ-
ual, in spite of Thudichum's claims to the contrary. As late as 1899
Hammarsten'-* indicated, as follows, the prevalent feeling toward the
non-concurrent conclusions in which Thudichum has persisted :
" Thudichum claims to have isolated from the brain a number of
phosphorus-containing substances which he divides into three main
groups : kephalins, myelins, and lecithins. Thus far, however, his
results have not been confirmed by any other investigators."

The work of Kossel and hVeytag may be regarded as an approach
to Thudichum's position with reference to the composite nature of
protagon. Kossel and Freytag discovered that protagon contains
sulphur. Variations among their several products, in spite of great
care in preparation, also led them to beliexe in the existence of
several protagons. I^\irther .than this, they found that protagons

^ GAMfJKE : A te.\t-book of tlie physiological chemistry of the animal body, 1880,
i, p. 425.

- LiEHKKiCH : Annalen der Chemie und Pharmacie. 1865. cxxxiv, p. 29.

•* Thudichum: Chemisches Centralblatt. 1875, p. 408.

■• DiACONOW : Centralblatt fiir die medicinisclien Wissenschaften, 1868, p. 97.

'" G.\MGF.E UN'I> Blankknhorn : Zeitschtift fiir physiologische Chemie. 1879,
iii, p. 260.

^ Baumstakk : Ibid, 1885. ix, p. 145.

" Kossel und Freytag : Ibid, 1893, xvii, p. 431.

* RUPPEI, : Zeitschrift fiir Biologic, 1895, xxxi, p. 86.

^ Hammarsten : Lehrbuch der physiologischen Chemie, 1899, p. 366.

Notes on the "' Protagon'" of the Brain. 191

readily yield several substances similar to or identical with some
described by Thudichum/ and which he still contends are among
the fourteen ( ! )' different bodies contained in the protagon mixture.
The subsequent work of Chittenden and Frissell also gave indications
of facts in harmony with the earliest results of Diaconow and his
view that protagon is a mixture. Lately, Worner and Thierfelder^
attacked the problem by improved methods, and obtained results
which seem to show that protagon is not an individual substance, or
else that it is a remarkable labile body, physically and chemically.

Below we give the results of our repetitions of the experiments of
Chittenden and Frissell bearing on the matter in question.

Fourth experiment. — A sample of protagon which had been prepared
by Dr. Frissell from sheep brains by the usual method — precipita-
tion from warm alcoholic extract at 0° C. and thorough washing in
ether at 0° C. — was placed at our disposal for this experiment.

We further purified the protagon by recrystallizing it once from alcohol. 25 gms.
of the product was kept in 1500 c.c. of 85 per cent alcohol at 40° C. for
twelve hours and the mixture repeatedly stirred. At the end of that
time only about half of the substance had dissolved.

First product and filtrate. — The mixture was filtered and the protagon sep-
arated from the extract by the usual cooling process, etc. The filtrate
from the protagon was evaporated to dryness.

Second prodtict and filtrate. — That portion of the original protagon which
remained undissolved was again subjected to treatment in the same
amount of alcohol. Most of the substance dissolved at the end of twelve
hours. The second portions of protagon and evaporated filtrates were
obtained as before from the filtered extract.

Third product and filtrate. — The protagon still remaining undissolved after
the second extraction with alcohol was again placed in the same amount
of warm alcohol for a similar period. Protagon was separated from the
extract and the filtrate from it evaporated to dryness as before.'^

Insoluble portion. — A fairly large proportion of the original protagon remained
insoluble under these conditions.

Alcohol-ether washings. — Each successive residual portion of protagon re-
ferred to above was washed with warm alcohol and the wasliings added to

^ Thudichum: Die chemische Konstitution des Gehirns des Menschen und
der Tiere, 1901, pp. 54-57; 328.

^ WoRNER UND Thierfelder : Zeitschrift fiir physiologische Chemie, iQoo^
XXX, p. 542.

■^ The crystalline appearance of these various protagon products was practically
the same.

ig:

IV. ir. Lcseiu and William J. dies.

the filtrates. .\ll of the samples of freshly precipitated protagon were
washeil first with a small quantity of cold 85 per cent alcohol and later
with moderate excess of cold ether. The alcoholic and ethereal wash-
ings of the freshly precipitated protagon were combined and evaporated.
Treatment of the products. — The ])ortions of protagon. and the substance in
the filtrates and washings, were carefu'ly determined quantitatively. Phos-
phorus was also estimated in each by the usual fusion method.

The following .summary gives our data in this connection : —

I'.MILK V.

Protagon, etc.

Weight in Percentage of
grams.i phosphorus.

A. Freshly precipitated protagon:

a. From first e.xtract

/'. From second e.xtract . . .
c. From third e.xtract ....

B. Insoluble protagon (residue) . .

C. Substance in filtrates from the

freshlyprecipitated protagon :

a. Of first extract

b. Of second extract

c. Of third extract

D. Alcohol-ether washings of the
freshly precipitated products

10.834
7..599
1. 729 (20.162)

2 009

0.785
0.678
0.250 (1.713)

0282

1.23
0.89
0.57

0.12

2.59
131

0.85

2.02

Total substance recovered . .
Total substance taken ....

24.17
24.34

1.16

' The weights are for substance dried in vacuo over H.2SO4 to constant weight.

Fifth experiment. — We repeated the preceding e.xperinient with two freshly
prepared samples of protagon made by us from two different quantities of
sheep brains. These samples of protagon were prepared by the usual
method and were twice recrystallized. Twelve gms. of each was used.
Two treatments were made with \\ litres of 85 percent alcohol at 45° C,
etc., as in the fourth experiment, with the results tabulated on page 193:

Among the points to be noted in Tables V and VI is the decreas-
ing percentage content of phosphorus in each successive protagon
and in the final insoluble residue. Also, the unusually high though
diminishing proportion of phosphorus in the substance of the filtrates
obtained each time protagon was separated at o C.

Notes on the " Protagon " of the Brain.

193

Our method of fractional separation was that customarily employed
in the purification of protagon. Here it was merely repeated more
frequently than usual. Instead of obtaining purer protagons in the
process, however, it appears that, with each successive precipitation,
the substance itself changed in composition and, also, that variously
composed products were liberated into the filtrates from the prota-
gons at the same time. The final residue was wax-like and quite
different from the snow-white protagon of the first extracts. We
are certain that our products were " pure " at the start.

TABLE VI.

Protagon, etc.

I.

II.

Weight

in
grams.

Percentage
of phos-
phorus.

\ Weight
in
grams.

Percentage
of phos-
phorus.

A. Freshly precipitated protagon ;!

a. From first extract . . .

b. From second extract . .

B. Insoluble protagon (residue) .

C. Substance in filtrates from the

freshly precipitated pro-
tagon :
^7. Of first extract ....

5.945
2.680(8.625)

0.655

1.613

1.21
1.01

0.91

2.22
1.30

3.659

2.009(5.668)

3.892

1.321

0.981 (2.302)

1.19

1.11

LIS

l.SO
1.45

b. Of second extract . . .

0.983 (2.596)

Total substance recovered- .
Total substance taken . . .

11.876
12150

1.26

11.862

12.150

I

1.23

1 The precipitates were washed only with col
^ See note 1 in the preceding table.

d alcohol.

The data of the last two experiments are in close agreement with
the similar facts found by Chittenden and Frissell. They are in
harmony with corresponding data recently published by Thudichum.^

These results were obtained by applying the usual purification
method. They show, we think, that protagon is either a mixture of
bodies, or else a substance decomposing quite readily under the
conditions of such experiments. If the latter conclusion appears to

^ Thudichum : Die chemische Konstitution des Gehirns des Menschen und
der Tiere, 1901, pp. 84-85.

194

//'. ir. LcscDi and William /. Gies.

be more probable than the former, it mii.st then be admitted that
thus far no standard of purity for protagon has been raised which
is not open to the objectiosi that it is based on methods involving
unavoidable decomposition.

Elementary composition of protagon. — It seemed desirable at this
point to ascertain the general elementary composition of several of
the protagon products prepared in the preceding experiments. The
summary below gives our results for four representative preparations:

TAP.LE VII.

(0

I'ercentage composition of protagons.i

4)

Fourth experiment.

Fifth experiment.

a. i

/;

I.

II.

c

To .98

1
r)6.24 66.111

66.63

66.46

6655

65.87

65.77

65.82

65.54

65.70 65.62

H 10.83

10.97 10.90

10.72

10.60

10.66

10.73

10.47

10.60

10.77

1091 10.84

N

2.09

1.95

202

2.22

2.16

2.19

1.97

1.99

1.98

2.05

2.00 2.03

P

1.23

0.89

1.25

1.26

1.26

1.21

1.25 1.23

S

..

0.77

..

0.72

0.67

0.72

02

18.97

18.99

19.67

••

.. 19 56

1 The methods of analy.sis employed were those already described by us : Hawk
and GiKS: This journal, 1901, v, p. 403.

- The amount of ash varied between 2 and 3 jier cent. It consisted very largely
of phosphate derived during the incineration process.

The results for elementary composition are in fairly clo.se accord
with those of previous observers.^ Since all of our samples were
made by practically the same method as that employed in most of
the earlier investigations, however, this harmony proves nothing
more than that the materials analyzed by all of us were of essentially
the same character. The minor variations suggest that the products
may be fairly uniform mi.xtures, but Kossel and Freytag's conclusion
that several protagons exist might also be drawn from them. In fact,
much to our surprise, these results accord as well as many analytic

^ See tiie summary lately given by Noll: Zeitscbrift fiir pbysiologiscbe
Chemie, 1S99, xxvii, p. 376.

Notes on the '' Protagon'^ of the Brain. 195

series given for what are undoubtedly individual substances. Our
data in this connection, considered by themselves, would seem to
harmonize with the older view of the integrity of protagon. In the
light of our other results, however, they illustrate the fact that uni-
formity in composition frequently hides chemical differences. In
this case general uniformity seems to give no assurance of chemical
individuality.

Application of the methods of Woruer and Thierf elder. — We have
repeated some of the recent preliminary experiments of Worner and
Thierfelder without, however, anticipating any of the steps which it
may be the intention of these investigators to take in furtherance of
their work,

Worner and Thierfelder used material from human brains. We
used purified protagon from sheep brains. The agreement between
their results and ours is, therefore, all the more significant. Our data
in this connection will be given only briefly.

We made use of freshly prepared protagon, as well as some of the
preparations already referred to. Our protagon products dissolved
almost entirely in moderate quantities of solutions of equal parts of
alcohol and chloroform, or alcohol and benzol, at 45° C. The latter
solution appeared to exert solvent action less rapidly than the other.
The crystals obtained from such fluids, after gradual evaporation at
40°-45° C, varied somewhat with changes in the composition of the
solvent and in the concentration of the solution.

The residue left behind at this point, on treatment of the protagon
with a moderate quantity of the solution, resembled that remaining
in Experiments 4 and 5 preceding. It consisted of globular forms
and amorphous substance. On cooling the filtrate from the melted
matter, a bulky precipitate of snow-white " cerebron " spheres was
deposited. The filtrate from the cerebron, on evaporation, yielded
microscopic needles. The filtrate from these crystals contained other
organic matter which, however, furnished only a slight amount of
crystalline substance on further evaporation or on longer standing.
These experiments were repeated several times with similar outcome.

Of these varous products the cerebron was the only one we
attempted to separate in any quantity for further examination. In
all the ordinary tests tried on the several preparations of purified
cerebron, we found that our products gave the reactions already
attributed to the substance by Worner and Thierfelder. All the
crystals figured for it by these investigators were observed in the

196 Jf\ Jf\ Lrscm and Williavi J. Gics.

various fluids. The typical transformation of the cerebron balls in
85 per cent alcohol at 50" C. into needles, minute plates, etc., was
also brought about several times. We were unable to make any
elementary analyses of the cerebron, but verified the statement that
on decomposition with acid a reducing substance may be detected
among its cleavage products.

In view of these results, also, it appears necessary to conclude
that protagon is not merely an unstable substance, but a mixture of
bodies.' It is not at all likely that these various products arise by
decomposition from such mild treatment. Further study of cerebron
and its related products, also of the new substance very recently
isolated by Ulpiani and Lelli,^ and called by them, " parahukleo-
protagon," may throw more light on the protagon question.

III. Summary of General Conclusions.

(i) The protagon of the brain is a mixture of substances, not a
chemical individual.

(2) The mixture called protagon does not contain the bulk of the
phosphorized organic substance of the brain.

' See very recent paper by Koch : Zeitschrift fiir physiologische Cliemie, 1902,
.\xxvi, p. 140.

- Ulpiani unij Lelli : Chemisches Centralblatt, 1902, ii, p. 292.

f:z

Reprinted from the Archives of Neurology and Psychopathology, 1899, ii, p. i.

ON THE NUCLEOPROTEID OF THE BRAIN
(CEREBRONUCLEOPROTEID).

By p. a Levene.

[From the Pathological Institute of the New York State Hospitals and the De
partment of Physiological Chemistry of Columbia University.]

Different as the activity of a nerve cell may be from that of
any other cell, there are still many features common to all, and
the main point of similarity is that the source of its specific peculiar
energy is the substance of the cell itself, that its work is being
performed at the expense of its own body. From this follows the
second point of similarity, that the nerve cell cannot work forever,
or any indefinite time without repairing its own substance, its own
body. How does it accomplish this task ? Is there in the cell a
peculiar organ for that purpose or a peculiar chemical agent that
is in charge of that function ? Cytologists have long ago observed
that when a cell is divided into two parts, so that the nucleus is
left in one of them, this last part is able to recuperate from the
loss and continue its life, while the other part has a life of very
short duration, and during the brief time it remains alive it does
not digest nor does it assimilate food, while the first part contin-
ues to do so as well as any normal cell. Thus the cytologists have
come to the conclusion that the nucleus controls the chief functions
of the cell, viz., those of repair, growth, reproduction.

Further, it is a long-established fact that the predominating
difference between the nucleus and the plasma of a cell is the
amount of chromatin substance in them. It can be justly said the
nucleus is the seat of the chromatin. Thus again biologists have
come to the conclusion that the chromatin is the most important
substance for the life of the cell and that most functions are con-
nected with some changes in that substance.

If this be true, we should naturally expect to find this sub-
stance to be, first, of a very complex nature, and second, of such
a nature that it can undergo different and manifold changes.

277

278 p. A. Levene.

So it actually is. The chromatins belong to the class of com-
pounds known as nucleoproteids, the most complex compounds
in living matter, and probably in nature. The study of these sub-
stances in different conditions of the cell, in state of rest and ac-
tivity, or better, in the state where repair predominates or dissimi-
lation prevails, is the means of finding a clue to the solution of the
problem of how the organism repairs its waste, and how we can
successfully aid the organism in the most important of its tasks,
when this power of restitution is for some reason or other dimin-
ished. We must remark, however, that our knowledge of the
composition of these substances is not quite as extensive as is
desirable, that the study of them does not date back much further
than twenty years, and that least attention has been paid to the
study of the nucleoproteids (or the proteids generally) of the
brain.

It was m\' aim to fill this gap in the study of the brain. But
before reporting my results I shall recall in a few words some of
the characteristics of the nucleocompounds and the main points
of difference between the individual compounds of this group.

The chief characteristics are that they contain phosphorus,
possess the properties of acids, and are mostly met with in com-
bination with proteids.

The points of distinction are, first, the presence or absence of
the xanthin bases in the molecule of these compounds ; the char-
acter of the bases, if present ; the amount of phosphorus and of
proteid in the molecule, and finally the character of the proteid.

Those compounds that contain a relatively higher percentage
of P and whose acidity is but little neutralized by proteids, pos-
sess a comparatively higher affinity for certain basic anilin dyes.
On account of this peculiarity the substance causing it was named
chromatin by the microscopists.

In order to understand the chemical changes accompanying
and probably responsible for the workings of the brain it is of
great interest to study the chemical changes of its chromatin in
different normal and pathological conditions of the organism.

In the nerve cell chromatin is located, in distinction from
many other cells, not only in the nucleus, but also in the cyto-
plasm (Nissl's granules), and thus naturally the question arises

NUCLEOCOMPOUNDS OF THE BrAIN. 2/9

whether the chromatin of the latter is the same substance as is
met with in the nucleus, or is it different in its nature ; in other
words, is there only one nucleoproteid in the nerve tissue or more
than one ?

Method of Obtaining the Nucleoproteid. — As far as I know,
the study of the nucleocompounds of the brain is limited to two
researches, both of them quite old, dating back to the time when
our knowledge of the nature of these substances and their classi-
fication was very unsatisfactory. Thus, Halliburton extracted the
brain tissue with H^O and precipitated from the extract with acetic
acid a proteid containing 0.3 per cent, of phosphorus. Von
Jacsch treated a few human brains with pepsin-hydrochloric acid
and from the residue extracted a nuclein — the nature of which
he did not describe with much detail. There are a few more
works dealing in a very unsatisfactory way with the general nature
of the proteids of the brain, but none of them described the
nucleocompounds.

The method that in my experience gave the most satisfactory
results was the following :

The brains from freshly killed calves were immediately placed
in alcohol-free ether and thus brought to the laboratory. After
stripping the membranes, the brains were finely divided in a chop-
ping machine and treated with large quantities of 4 per cent.
AmCl solution and on addition of chloroform left in well-stop-
pered bottles for twenty-four hours. The supernatant fluid was
then decanted, and the extraction repeated with distilled water,
two, three and even four times, until the extracts ceased yielding
an appreciable precipitate on addition of acetic acid.

. The decanted fluid was then strained through gauze and fil-
tered repeatedly till the filtrate was perfectly clear. I found
later that the filtration is greatly accelerated, and the loss of ma-
terial minimized if the strained liquid is left for several hours in
separating funnels with ether. The small particles of brain tissue
were then collected on the surface, and the liquid below was per-
fectly clear. The filtration was thereby rendered easy. The
greater part of the material I worked with, however, was ob-
tained by simple filtration without previous treatment with
ether.

28o p. A. Levene.

The perfectly clear filtrates were then treated with acetic acid,
0.5 c.c. of the acid to each 100 c.c. of the liquid, and thus a pre-
cipitate of the crude nucleoproteid was obtained.

This freshly precipitated proteid is insoluble in dilute acetic
acid, also insoluble in dilute hydrochloric acid, but is soluble in
glacial acetic acid, in weak alkalies, as one per cent, sodium car-
bonate, and 0.5 per cent, ammonium hydrate.

It is enough, however, to let the precipitate stand over night
in acidulated H.,0 to lower its solubility to a very great extent, so
that only a very small part of the precipitate will dissolve in weak
alkalies.

The usual method of purifying nucleoproteids is to redissolve
them in dilute alkalies and to reprccipitate by acids. Our proteid,
for which I would suggest the name ccrebronuclcoprotcid, could not
well be purified by this method, owing to the rapid loss of solu-
bility.

There are also some objections to repeated treatment with
alkalies in the fact that these might alter to a certain extent the
original constitution of the proteid. For these reasons I attempted
to purify the substance by repeatedly washing the precipitate first
with acidulated H.,0, then with distilled water, until the latter failed
to give the biuret reaction and was free from chlorine.

There still remained the possibility that the proteid thus puri-
fied might contain some other proteids, likewise rendered insoluble
by prolonged treatment with acidulated water. To ascertain
whether this was the case, and also to ascertain whether the usual
method of purification affects the proteids, I endeavored to redis-
solve and reprccipitate some of the substance.

In doing this I encountered great difficulty in filtering the fluid,
as the insoluble matter immediately clogs the Alter paper. Even
a constant change of the filter does not help much. In order to
overcome this difficulty I recurred again to ether. The substance
was treated with ether in a separator}' funnel and left in it a few
hours. It was then separated and filtered. The substances after
they were thus purified were treated with cold alcohol, then
boiled with 95 per cent, alcohol, then absolute alcohol and finally
with ether, until extraction was nearly complete. We found it
next to impossible even after continuous extraction during several

NUCLEOCOMPOUNDS OF THE BrAIX. 28 1

weeks to get the product in such a condition that the evaporated
alcohol or ether would leave absolutely no residue.

Of the second product there was only sufficient for an estima-
tion of the phosphorus, but a complete analysis was made of the
first product.

Preparation I.

1. 0.1675 gr. of the substance gave on combustion 0.2845 gr.

of CO,; 0 = 42.44 per cent, and 0.0987 gr. of H^O ;
H = 5.99 per cent.

2. 0.2133 gr. of the substancegave 0.3615 gr. of 00^; 0=42.28

per cent.; and o. 1 126 gr. of H,0 ; H = 5.82 per cent.

3. o. 1415 gr. digested after Kjeldahl = 0.0219 gr. of N = 15.46

per cent.
; 4. 0.458 gr. fused with NaOH and KNO3 (S-free) = 0.043 g^.
of BaSO^, S= 1.28 per cent.

5. 0.3166 gr. fused with NaOH and KNO3 = 0.0065 gr.

of Mg^P^O. ; P = 0.573 per cent.

6. o 4665 gr. fused with NaOH and KNO3 = 0.0092 gr.

Mg^P^O, ; P = 0.557 ?£!■ cent.

Preparation II.
0.4897 gr. of the substance fused with NaOH 4- KNO3 =
0.0078 gr. of Mg2P20- ; P = 0.45 per cent.

N. s. p. o.

I

2
-J

C.

42.44
42.28

H.

5-99

5.82

4

5
6

Average

42.36

5-90

15.46

1.28

0-57
0.56

15.46 1.28 0.56 34.44

Ash = 0.5 per cent.

Apparently the first method of purification affects the proteid
less than the second method, but in either case the nucleoproteid
contains very little phosphorus, probably less than any other true
nucleoproteid ; in fact it resembles in this respect the pseudo- or
para-nucleoproteids, or as Hammarsten calls them, nucleoalbu-
mins. It was of course important to ascertain to which of the

282 P. A. Levene.

two main groups of the nucleocompounds our substance belongs,
since the physiological role of the two is quite different.

For this purpose about 60 gr. of the substance was heated
in a flask with a return condenser with 2 per cent. H^SO^ for
about ten hours. It was then filtered, the greater part of the acid
neutralized by means of Ba(OH)„, filtered, the filtrate concentrated
and treated in the usual way for nuclein bases (xanthin bases).

In the xanthin fraction but a very slight precipitate of the xan-
thin silver salt was obtained — so little that the attempt to obtain a
xanthin reaction after the silver was eliminated, was without suc-
cess. The hypoxanthin fraction consisted mostly of guanin and
adenin, no hypoxanthin being found.

Thus, it was established that our substance is a true nucleo-
proteid, and that two bases take part in the formation of its mole-
cule.

Cerebroniiclci)i. — The next task was to ascertain the cause 01
the low percentage of P in the nucleoproteid. This might be due
to two different causes ; either the nuclein itself might contain
little P, or other substances might be bound to a nuclein with a
high content of P, thus giving rise to an unusually complex sub-
stance.

A considerable amount of the proteid purified by the first
method, but not extracted with alcohol and ether, was digested
with pepsin -hydrochloric acid, for a week. The digestive fluid
was then changed ever>' two days, 0.2 per cent. HCl being em-
ployed, and care being taken to have free HCl always present in
the fluid. After that, the soluble products of digestion were sep-
arated by repeated treatment with acidulated water and decantation
until the wash water gave no biuret reaction, and contained no
chlorine. The insoluble residue was then extracted with alcohol
and ether until the latter ceased extracting, which took place
after several weeks' continuous treatment. About 2.5 gr. of the
pure air-dry substance was thus obtained. A small portion of it
was then extracted with HCl water in order to ascertain whether
it contained inorganic P ; the result was negative.

The percentage of P was then estimated and 0.275 gr. of the
substance fused with NaOH and KNO3 gave o.oi40gr. of MgjPjO^ ;
P = 1.42 per cent.

NUCLEOCOMPOUNDS OF THE BrAIN. 283

In comparison with other nucleins the phosphorus is seen to
be rather low.

Cerebromideic Acid. — It is known that nucleins are com-
pounds of nucleic acid and proteids. The nuclein of the brain is
exceptionally poor in P, and we are confronted again by the two
possibilities that were met with in connection with the nucleo-
proteid itself The low percentage of P in the nuclein might be
due to the peculiar nucleic acid or to the different amounts of
proteid combined with an acid having a comparatively high con-
tent of P.

The investigation, in this direction is not completed at present,
as we found great difficulty in obtaining a sufficient quantity of
the substance. The method of obtaining the nucleic acid that
gave the most satisfactory results is the following :

The purified nucleoproteid, not extracted with alcohol and
ether, was dissolved in 2 per cent. NaOH, while being slightly
warmed on a water-bath. While still warm the fluid was neutral-
ized with acetic acid, cooled and filtered. This was found neces-
sary for the reason that that part of the proteid which was precip-
itated on neutralization (alkali albuminate) was again soluble in
an excess of acetic acid. The filtrate was rendered strongly acid
by means of acetic acid, and was then left for twenty-four hours
and filtered ; to the filtrate alcohol containing 0.3 per cent. HCl
was added until the fluid became very opalescent. After stand-
ing twenty-four to forty-eight hours, the precipitate was washed
with acidulated alcohol, then with pure alcohol and ether, dried
and weighed. The acetic solution of this proteid precipitated
albumoses and proteids from their solutions.

Seventy grams of the proteid treated with 300 c.c. of 2 per
cent. NaOH gave less than 100 mgr. of the nucleic acid. 0.0875
gr. of this substance fused with NaOH and KNO3 gave 0.0105
gr. of Mg^Pp^ ; P = 3-35 per cent.

This P estimation can be accepted for the present only as more
or less approximate to the true percentage of P in the nucleic acid.

From all these results it may be inferred that the nucleocom-
pound of the brain is a true nucleoproteid, that it differs from other
nucleoproteids by its low percentage of P, by the nature of its
xanthin bases, and by the considerably high amount of proteids
bound to its nuclein.

284 p. A. Levene.

The next aim was to iiivesti'^ate whether the residue of brain
tissue after extraction of this nucleoproteid contained another
nucleocompound different in nature from the cerebronucleoproteid.
For that purpose the residue just mentioned was extracted
during different lengths of time with dilute alkalies of different
strengths. It was found that 0.5 per cent, ammonium hydrate
will extract in twenty-four hours a considerable quantity of a pro-
teid which can be precipitated by acetic acid, and that this is a
nucleoproteid. It was also found that twenty-four hours treat-
ment of the proteid with 0.5 per cent, ammonia solution will not
split off any noticeable quantity of nucleic acid. Hence, 0.5 per
cent, of ammonia could be applied for the extraction of the residual
nucleocompounds. It remained to ascertain whether the latter
was different in nature from the cerebronucleoproteid. The esti-
mation of P in it, however, argued against such a supposition.
Thus, 0.5800 gr. of the purified substance gave on fusion 0.0105
gr. of Mg^P^O. or o. 5 per cent, of P.

We attempted also to obtain the nuclein of that residual sub-
stance, but as the quantity of the latter in our possession was
rather small, we digested the residue of forty brains with pepsin
hydrochloric acid with the same precautions as mentioned above.
After the digestion and purification was completed, the residue
was extracted with cold and boiling alcohol for several weeks,
then with ether until the myelin was nearly extracted. In order
to ascertain whether this residue contained an appreciable amount
of nucleocompound, a P estimation was made.

0-4325 gr. of the substance gave 0.0140 gr. of Mg^PjO., or
P = 0.896 per cent.

Thus, the presence of a considerable quantity of nuclein in
the brain residue was demonstrated. However, the prolonged
treatment with boiling alcohol rendered the nuclein insoluble to
such un extent that but little of it could be extracted by means of
dilute alkalies. Thirty grams of the residue was treated for five
hours with 0.25 per cent. NaOH and filtered directly into dilute
HCl solution ; a white flocculent precipitate was formed, but in a
quantity insufficient for further analysis.

No marked difference between the residue and the cerebronu-
cleoproteid could be found in the character of their xanthin bases.

NUCLEOCOMPOUNDS OF THE BrAIN. 285

Here, again, guanin was found to predominate, the other bases not
being sufficient in quantity to be identified.

These results do not bear out the supposition of the exist-
ence of more than one nucleoproteid in the nerve cell.

From these results it may also be inferred that the nature of
the chromatin of the cytoplasm does not differ from that of the
nucleus. However, this question can be fully elucidated only by
a comparative chemical study of the nerve tissue under differ-
ent physiological and pathological conditions, /. e., in conditions
when the chromatin nearly disappears from the nucleus and is lo-
cated only in the cytoplasm and vice versa.

I wish to acknowledge my indebtedness to Professor Chitten-
den for his valuable suggestions and for the privileges accorded to
me in the Laboratoiy of Physiological Chemistry of Columbia

University.*

May 20, 1899.

References.

Halliburton. Journal of Physiology, Vol. XV., 1S93.
Hammarsten. Zeitschr. f. physiol. Chem., Vol. XIX.
Von Jacsch. Pfliiger's Arch., Vol. XIII., p. 469.

*See Preface, page 7.

13

Reprinted from the Journal of Experimental Medicine, 1896, Vol. i, p. i£6.

THE MUCIN OF WHITE FIBROUS CONNECTIVE TISSUE.*

By R. H. Chittenden and William J. Gies.

(Contribution from the Sheffield Biological Laboratory of Yale University.)

All of the bodies belonging to the group of mucins and mu-
coids are possessed of considerable physiological interest, owing
especially to their peculiar compound nature and the illustration
which they afford of a possible intimate union between the proteid
group and carbohydrate radicles. That there are a number, pos-
sibly a large number, of closely related bodies belonging to the
mucins and mucoids there can be no question. Thanks to the
labors of Hammarsten f and his pupils, many of these bodies have
been subjected to careful and thorough investigation, and much
light has been thrown upon their relationships and differences.
There is still, however, much to be ascertained regarding these
bodies, and any additional facts broadening or substantiating our
present knowledge are to be welcomed as contributing toward a
more complete understanding of their genetic relationships. The
union of carbohydrate groups with proteid molecules is probably
more common than has hitherto been supposed, as witness the
peculiar gluco-nucleoproteid recently described by Hammarsten t
as a constituent of the pancreas and other glands, and the identifi-
cation by Kossel § of a peculiar carbohydrate group as a cleavage
product of certain forms of nucleic acid. Presumably in these
compound proteids of the mucin type the character of the proteid
radicle as well as of the carbohydrate radicle is subject to varia-
tion, and it is easy to conceive of differences in the nature and

* A preliminary report of this research was made by Professor Chittenden before
the American Physiological Society in Philadelphia, in December, 1895, and an ab-
stract was published in Science, January 24, l8§6 ; iii (N. S.), p. 109. No reference
was made to the fact that the report was presented for both authors.

t Pfliiger' s Archiv f. Physiol., Band xxxvi ; Zeitschr. f. physiol. Chem., Band x
and xii.

i Zeitschr. f. physiol. Chem., Band xix.

\ Du Bois-Reymond's Archiv f. Physiol., Physiol. Abtheil, 1891.

287

288 R. H. Chittenden and William J. Gies.

properties of the mucins dependent upon variations in the amount
and character of both the carbohydrate and proteid groups. The
ready formation of acidalbumin, or syntonin, albumoses and pep-
tone when mucins are decomposed by the action of superheated
water or boiling dilute acids, affords ample evidence of the pres-
ence of true proteid radicles in the bodies of this class, although
we do not know definitely the exact nature of the proteid groups
present in the original molecule. On the other hand, the simul-
taneous formation of reducing bodies whenever mucins are broken
down by the action of dilute acids, and the separation of a dextrin-
like body (the animal gum of Landwehr *) by cleavage with su-
perheated water, clearly indicate the existence of some form of car-
bohydrate matter in the mucin molecule.

Of the true mucins present in the tissues of the higher animals,
the mucin of the submaxillary gland and the corresponding body
present in or between the fibers of ordinary connective tissue are
the most important from a physiological standpoint. The former
is a product of the metabolic activity of secretory cells which are
among the most active of the secreting cells of the body, while
the latter is a product of a tissue whose activity is certainly of a
low order. That these two mucins, though closely related, are
unlike, is clearly indicated by their divergence in chemical compo-
sition as well as by their general reactions and properties.

Loebisch,t whose careful study of the mucin from tendons con-
stitutes the chief source of our knowledge regarding the chemical
composition of this body, ascribes to tendon mucin the formula
Cj^H^.^NgjSOgg, with a molecular weight of 3,936. Such a for-
mula calls for the presence of 0.81 per cent, of sulphur and this
amount was found by Loebisch in the three preparations of mucin
from ox tendons analyzed by him. In a recent examination of
mucin prepared from this same source we have obtained quite dif-
ferent results as regards the content of sulphur, and this fact has
led us to make a careful study of the composition of this form of
connective-tissue mucin. Our results in the main have afforded a
close substantiation of the conclusions arrived at by I.oebisch,

*Zeitschr. f. physiol. Chem., Band viii and ix. Also Pfliiger's Archiv. f. Physiol.,
Band xxi.^ and xl.

t Zeitschr. f. physiol. Chem., Band x, p. 40.

Tendon Mucin. 289

with the single exception of the sulphur, for which we can find no
adequate explanation. Further, some additional facts have been
found which are perhaps worthy of note.

The first sample of mucin studied was prepared from the
Achilles tendons of oxen by the following method, analogous to
the method described by Loebisch : The fresh tendons were freed
as carefully as possible from all adherent tissues, then cut into
very thin transverse sections with a razor, washed thoroughly with
distilled water, frequently renewed for twenty-four hours, in order
to remove all blood and soluble albuminous matter, and finally
pressed as dry as possible. The resultant material weighed 1,200
grammes. In order to extract the mucin, the tissue was placed
in 2.4 liters of half saturated lime water, where it was allowed to
remain for forty-eight hours with frequent agitation. At the end
of this period the pale-yellowish fluid was strained through a cloth
filter and finally filtered through paper. The clear fluid was then
treated with an excess of 0.2 per cent, hydrochloric acid — a little
more than a liter — by which a heavy flocculent precipitate re-
sulted, quickly settling to the bottom of the cylinder, leaving a
nearly clear supernatant fluid.

The residue of tendon tissue was again extracted for forty-
eight hours with 2.4 liters of half saturated lime water, and the re-
sultant solution precipitated with an excess of 0.2 per cent, hydro-
chloric acid. The precipitate so formed was nearly as heavy as
the first, thus showing that extraction of the mucin by weak lime
water is a slow and gradual process.

The precipitated mucin, separated from the acid fluid by subsi-
dence and decantation of the supernatant liquid, was washed thor-
oughly with 0.2 per cent, hydrochloric acid, by whipping up the
precipitate with the fluid and then allowing it to subside, this
operation being repeated with fresh quantities of acid until the lat-
ter failed to give any proteid reaction. In this manner it was
hoped to remove all adherent albuminous matter extracted from
the tissue by the lime water. The two portions of mucin were
then united and washed by decantation with distilled water until
the acid was entirely removed. As the fluid became less and less
acid, more time was required for the precipitate to settle, as the
latter tended to swell in the water and was more inclined to float
on the surface of the fluid.

290 R. IT. ClIlTTENDEN AND W'lLLIAM J. GlES.

The mucin was next dissolved in lialf saturated lime water, of
which a lart^e volume was required, the solution filtered through
paper, and the mucin reprecipitated by the addition of an excess
of 0.3 per cent, hydrochloric acid, a small quantity of stronger
hydrochloric acid being likewise added to induce a good floccu-
lent separation of the substance. The precipitate was again washed
by decantation with 0.2 per cent, hydrochloric acid, and lastly
with water, until the acid was entirely removed. Whenever it was
necessary for the precipitate to stand for some time with water,
the mixture was kept as cool as possible, and a little alcoholic
solution of thymol added to guard against putrefactive changes.
When the acid was wholly removed from the precipitate the water
was replaced by weak alcohol, and finally by ninety-five per cent,
alcohol, repeatedly renewed, until the substance was thoroughly
dehydrated, after which the precipitate was collected on a filter
and allowed to drain. It was then boiled with alcohol-ether (a
mixture of equal parts absolute alcohol and ether) in a suitable
flask connected with an inverted Liebig's condenser for many
days — i.e., with renewed quantities of alcohol-ether until the
latter gave no residue on evaporation. As Loebisch has shown,
this is quite an important part of the process of purification, since
a certain amount of foreign extractive matter adheres tenaciously
to the mucin, and can be removed only by long-continued extrac-
tion with the above mixture. When this process was completed
the mucin was thrown upon a filter, washed thoroughly with ether,
and finally dried over sulphuric acid. When quite dry it pre-
sented the appearance of a perfect!}' white powder, light and
fluffy. The yield amounted to twelve grammes of the dry prod-
uct, and assuming that the entire amount of mucin had been
extracted from the tendons, and disregarding the loss incidental to
purification, this quantity would imply the presence in the fresh
tendons of one per cent, of mucin.

.The composition of the product, dried at 110° C. until of con-
stant weight, was as follows : *

*The nitrogen was determined by both the absolute and the Kjeldahl method,
while carbon and hydrogen were determined by combustion in oxygen gas in an open
tube, the products of combustion passing over a layer of cupric oxide, chromate of lead,
and metallic copper.

Tendon Mucin. 291

Preparation No. i.
I. 0.2670 gramme of substance gave O.4781 gramme of CO, = 48.84 per cent.

C, and o. 1585 gramme of HjO -= 6.60 per cent. H.
II. 0.2277 gramme of substance gave 0.4082 gramme of CO, ^ 48.89 per cent. C,
and o. 1329 gramme of H2O = 6.48 per cent. H.

III. 0.1975 gramme of substance gave 0.3548 gramme of CO, = 48.99 per cent. C.

IV. 0.2363 gramme of substance gave o. 1417 gramme of H^O =^ 6.66 per cent. H.
V. 0.2426 gramme of substance gave, by the Kjeldahl method, 0.02865 gramme of

nitrogen =. 1 1. 8 1 per cent. N.
VI. 0.2754 gramme of substance gave, by the Kjeldahl method, 0.03246 gramme
of nitrogen =; 11.79 P^^" cent. N.
VII. 0.2784 gramme of substance gave, by the absolute method, 27.63 c.c. of nitro-
gen at 13.3° C, and 764.7 mm. pressure^ 11.96 per cent. N.
VIII. 0.3345 gramme of substance gave, by the absolute method, 33.3 c.c. of nitrogen
at 13.2° C, and 754.5 mm. pressure -= II. 84 per cent. N.
IX. 0.5373 gramme of substance gave, by fusion with NaOH -|- KNO3, 0.0943
gramme of BaSO^ ^ 2.41 per cent. S ; after deducting sulphur of ash ^ 2.36
per cent. S.
X. 0.4969 gramme of substance gave, by fusion with NaOH -f KNO.„ 0.0856
gramme of BaSO^ = 2.37 per cent. S ; after deducting sulphur of ash ^= 2.32
per cent. S.
XI. 0.2943 gramme of substance gave 0.0023 gramme of ash ^ 0.78 per cent. ash.
XII. Ash from 0.2943 gramme of substance gave 0.00112 gramme of BaS0^ = O.o5
cent. S.

Percentage Composition of the Ash-Free Substance.

Average,

C.

49.22

49.27 49.37

49.29

H.

6.65

6.54

6.71

6.63

N.

11.90 11.88 12.05 "-93

11.94

S.

2.36 2.32

2.34

0.

29.80

The second preparation of mucin was made in a somewhat
different manner. The fresh tendons, freed as far as pos.sible from
foreign tissue, were cut into thin transverse sections, washed with
water somewhat, then soaked for thirty-six hours in about four
Hters of ten per cent, salt solution, with, vigorous agitation from
time to time, after which the saline solution was decanted and the
tissue washed with water until the chloride was entirely removed.
The salt solution on dilution with water gave a distinct turbidity,
indicating the presence of a globulin. The application of heat
likewise produced a precipitate, as did also the addition of dilute
acetic and hydrochloric acids. It is thus evident that the salt
solution removes at the outset quite an appreciable amount of pro-

292 R. }i. Chittenoen and William J. Gies.

teid matter, with perhaps some mucin. The moist tissue, pressed
as dry' as possible, weighed i ,700 grammes. It was then extracted
with 3.4 htcrs of half saturated lime water for forty-eight hours,
two such extractions being made. From these extracts the mucin
was precipitated by the addition of 0.2 percent, hydrochloric acid,
the second extract apparently yielding as heavy a precipitate as
the first. The combined precipitates were washed repeatedly by
decantation with 0.2 percent, hydrochloric acid, lastly with water.
The mucin was next dissolved in a little o. 5 per cent, sodium car-
bonate, the solution filtered, made nearly neutral b}- the addition
of a little ten per cent, hydrochloric acid, so as to avoid undue di-
lution, and then precipitated by 0.2 per cent, hydrochloric acid.
The precipitate was again washed thoroughly with 0.2 per cent,
hydrochloric acid, and lastly with water, until the acid was entirely
removed. It was then transferred to ninety-five per cent, alcohol,
frequently renewed, and finally boiled with alcohol-ether as long
as anything could be extracted. Dried over sulphuric acid, the
product came out quite white, but not so bulky as the preceding
preparation, and weighed a little over fifteen grammes — an amount
equal to about 0.9 per cent, of the moist tissue.

As already stated, mucin is not readily extracted from tendons
by lime water ; at least four cubic centimeters of half saturated
lime water are required for every gramme of tissue in order to in-
sure a complete extraction. Thus, after the second extraction of
the above 1,700 grammes of tissue, a third extraction was made,
using again three litres of half saturated lime water. This solution,
on treatment with hydrochloric acid, gave a precipitate weighing
one to two grammes when purified, but it was noticeable that
more acid was required in order to effect a good flocculcnt separa-
tion of the mucin. Even with a fourth extraction of the tissue a
little mucin was obtained, showing as a decided turbidity when the
alkaline fluid was made distinctly acid, but it was not until four or
five days' standing that a distinct precipitate settled out even on
the addition of stronger hydrochloric acid. The amount so ob-
tained, however, was very small.

The composition of the main product obtained from the 1,700
grammes of tissue when dried at 110° C, until of constant weight,
was as follows :

Tendon Mucin. 293

Preparation No. 2.
I. 0.3194 gramme of substance gave 0.5659 gramme of COj =48.32 per cent. C,
and 0.1 815 gramme of H20 = 6.3I per cent. H.
II. 0.4197 gramme of substance gave 0.7471 gramme of €02^48.54 per cent. C,
and 0.2446 gramme of H^O = 6.47 per cent. H.

III. 0.4051 gramme of substance gave 0.7189 gramme of CO, = 48-39 per cent. C,

and 0.2353 gramme of Hfi = 6.45 per cent. H.

IV. 0.2519 gramme of substance gave, by the Kjeldahl method, 0.02965 gramme of

nitrogen = 1 1. 77 per cent. N.
V. 0.2578 gramme of substance gave, by the Kjeldahl method, 0.03026 gramme of

nitrogen = 1 1 . 74 per cent. N.
VI. 0.2954 gramme of substance gave, by the Kjeldahl method, 0.03446 gramme of
nitrogen^ II. 67 per cent. N.
VII. 0.6610 gramme of substance gave, by fusion with NaOH -|- K.NO3, 0.1131
gramme of BaSO^ = 2.35 per cent. S ; after deducting sulphur of ash ^2.32
per cent. S.
VIII. 0.5248 gramme of substance gave, by fusion with NaOH -|- KNO3, 0.0936
gramme of BaSO^^ 2.45 per cent. S ; after deducting sulphur of ash = 2.42
per cent. S.
IX. 0.6724 gramme of substance gave, by fusion with NaOH -(- KNO3, o. 1140
gramme of BaSO^ =^ 2.33 per cent. S ; after deducting sulphur of ash = 2.30
per cent. S.
X. 0.3735 gramme of substance gave 0.0025 gramme of ash = 0.67 per cent. ash.
XI. Ash from 0.3735 gramme of substance gave 0.00082 gramme of BaSO^ = o.03
per cent. S.

Percentage Composition of the Ask-free Substance.

Average.

c.

48.64 48.87

48.72

48.74

H.

N.

6.36 6.52

6.50

11.85

11.82

11.74

6.46
11.80

S.

2.32

2.42

2.30

2.3s

0.

30.65

A third specimen of mucin was prepared as follows : Fifteen
hundred grammes of ox tendons were finely divided, the tissue
extracted for twenty-four hours with three liters of ten per cent,
salt solution, and then with water until the salt was wholly re-
moved. The tissue was next extracted for sixty hours with three
liters of half saturated lime water. From this solution the mucin
could be only partially separated by the addition of 0.2 per cent.
hydrochloric acid, quite a quantity of ten per cent, acid being re-
quired to effect a flocculent precipitation of the substance. This
was purified by itself and not subjected to analysis. The tendons
were again extracted with three liters of half saturated lime water
for forty-eight hours, and from this solution the mucin was

294 ^- ^^- Chittenden and William J. Gies.

separated as a flocculent precipitate by the addition of 0.2 per cent,
hydrochloric acid. This precipitate was purified by washing with
0.2 per cent, hydrochloric acid, solution in 0.5 per cent, sodium
carbonate, reprecipitation with 0.2 per cent, hydrochloric acid,
etc., as described under the last preparation. The yield of dry
product from this second extraction of the tissue with lime water
amounted to 6.$ grammes. Dried at 110° C. until of constant
weight, this preparation gave the following results on analysis :

Preparation No. 3.

I. 0.3598 gramme of substance gave 0.6292 gramme of CO^ =^ 4769 per cent. C,
and 0.2072 gramme of HjO = 6.40 per cent. H.
II. 0.2939 gramme of substance gave 0.5150 gramme of €02 = 47.79 per cent. C,
and 0.1725 gramme H20=:6.52 per cent. H.
III. 0.3154 gramme of substance gave 0.5536 gramme of CO.^ = 47-87 per cent. C.
IV. 0.1644 gramme of substance gave 0.0944 gramme of Hj0^6.38 per cent. H.
V. 0.1965 gramme of substance gave, by the Kjeldahl method, 0.02255 gramme of
nitrogen = II. 47 per cent. N.
VI. 0.2495 gramme of substance gave^ by the Kjeldahl method, 0.02825 gramme of
nitrogen = 11.32 per cent. N.
VII. 0.2574 gramme of substance gave, by the Kjeldahl method, 0.02930 gramme of

nitrogen = 11.38 per cent. N.
VIII. 0.6046 gramme of substance gave, by fusion with NaOH + KNO3, 0.1045
gramme of BaSO.^= 2.38 per cent. S; after deducting sulphur of ash = 2.31
per cent. S.
IX. 0.5408 gramme of substance gave, by fusion with NaOH -f- KNO3, 0.0931
gramme of BaSO^ = 2.37 per cent. S ; after deducting sulphur of ash =: 2.30
per cent. S.
X. 0.3128 gramme of substance gave 0.0031 gramme of ash = 0.99 per cent. ash.
XI. Ash from 0.3128 gramme of substance gave 0.00152 gramme of BaSO^ = 0.07
per cent. S.

Percentage Composition of the Ash-free Substance.

Average.

C. 48.17 48.26 48.34 48.26

H. 6.46 6.59 6.44 6.49

N 11.59 "43 "-50 "51

S 2.31 2.30 2.31

o 31-43

A comparison of the composition of these three preparations of
mucin with each other, and with the mucin analyzed by Loebisch
and by Hammarsten, brings out certain points of interest which
merit attention :

Tendon Mucin.

295

Mucin from Tendons.

Snail Mucin.
Hammarsten.

Submaxillary-

Preparation j Preparation
I. 2.

Preparation
3.

Loebisch's
Average.

Mucin.
Hammarsten.

c

49-29 48.74
6.63 , 6.46

11.94 11.80
2.34 2.35

29.80 30.65

48.26
6.49

II. 51
2.31

31-43

48.30
6.44

11-75

0.81
32.70

50.32

6.84

13-65

1-75
27-44

48.84
6.80

12.32
0.84

31.20

H

N

S

0

Loebisch analyzed three distinct preparations of mucin from ox
tendons, in which the carbon, hydrogen, and sulphur showed prac-
tically no variation. The nitrogen, however, varied from 11.59 to
11.84 psr cent. The average content of nitrogen in his three
preparations was 11.75 P^^ cent., identical with the average of our
three preparations. It is to be noticed, however, that the carbon
of our preparations shows decided variation, and it is also to be
observed that a diminution in the percentage of carbon is attended
in each case with a diminution in nitrogen. We may suppose that
Preparation No. 3 is the purest of our products, and it is seen to
agree most closely with the results obtained by Loebisch, except
in the content of sulphur. The mucin from the submaxillary
gland, as well as the snail mucin, are both characterized by a com-
paratively high content of nitrogen, while the latter product also
shows a higher percentage of carbon.

Our results seemingly justify the assumption that white fibrous
connective tissue contains more than one mucin, or else that the
mucin obtainable from this tissue is prone to carry with it a certain
amount of some other form of proteid matter which the ordinary
methods of purification are not wholly adequate to remove. Our
experience leads us to the belief that the surest way of obtaining a
pure mucin from tendons, or at least one with a low content of
carbon and nitrogen, is first to extract the finely divided tissue with
ten per cent, salt solution, then after removal of the salt with water
to extract the tissue with half saturated lime water in the propor-
tion of two cubic centimeters for every gramme of moist tissue for
about twenty-four hours at ordinary room temperature. This ex-
tract may be rejected, as it is very liable to yield a mucin with a
higher content of nitrogen and carbon. By extracting the tissue a
second time with lime water a mucin may be obtained with a lower
content of carbon and nitrogen, as in our third preparation. It is

296 R. H. Chittenden and Wiij.iam J. Gies.

purely an assumption, howev^er, to say that this bod\- with its
lower percentage of carbon and nitrogen is pure mucin. There is
at the present time no standard of purity with regard to this body,
and it is quite as probable that fibrous connective tissue contains
two or more mucins as that there is only one mucin in the tissue,
and that any deviation from the figures obtained by Loebisch or
by us in Preparation No. 3 is due to the presence of a larger or
smaller amount of proteid impurity.

Undoubtedly, preliminary extraction of the tissue with salt so-
lution tends to remove a certain amount of proteid matter, espe-
cially globulins, which might otherwise render the product impure,
and possibly this is in part the cause of the lower content of carbon
and nitrogen in Preparation No. 2 as contrasted with Preparation
No. I. Still there is no certainty on this point, for it is to be re-
membered that precipitation of the mucin requires the addition of
considerable hydrochloric acid beyond neutralization of the alkaline
fluid, and thi5 excess of acid would naturally exert a marked
solvent action upon any albuminous matter present. That the first
lime water extract is liable to yield a mucin with a higher content
of both carbon and nitrogen the results fully indicate, and as a
direct illustration of the difference in the content of nitrogen in
mucin obtained from a first and second extract, we may instance
the following experiment: Fifteen hundred grammes of tendons
finely divided, as usual, were extracted with ten per cent, salt so-
lution for two days, then washed with water and placed in three
liters of half saturated lime water for forty-eight hours. This first
extract was then strained off, and the tissue treated a second time
with a like volume of half saturated lime water, thus giving a
second extract. From the first extract, the mucin was precipitated
by hydrochloric acid slightly above 0.2 per cent., the precipitate
washed with 0.2 per cent, hydrochloric acid, then with water, and
lastly dissolved in 0.5 per cent, sodium carbonate. From this
filtered solution a portion of the mucin was precipitated by addi-
tion of 0.2 per cent, hydrochloric acid, while a second portion
separated only on addition of a somewhat increased strength of
acid. These two fractions were washed thoroughly with 0.2 per
cent, acid, then with water, and finally boiled with alcohol-ether
until quite free from soluble matter. The yield in the first fraction

Tendon Mucin. 297

was 1.4 gramme, and in the second fraction i.o gramme. From
the second Hme water extract the mucin was precipitated with 0.2
per cent, hydrochloric acid, after which it was purified by washing
with 0.2 per cent, acid, solution in 0.5 per cent, sodium carbonate,
reprecipitation with 0.2 per cent, acid, etc. The content of nitrogen
in the three products, when dried at 110° C, was as follows, cal-
culated on the ash-free substance :

First Extract. Second Extract.

First Fraction. Second Fraction.

12.26 N. II. 91 N. II. 51 N.

It is thus seen that the first extraction with lime water furnishes
a mucin with a considerably higher percentage of nitrogen than
the second extract. It is equally noticeable that the mucin first
precipitated — as in the first fraction of the first extract — has a
higher percentage of nitrogen than the second fraction, thus indi-
cating that the higher content of nitrogen and probably of carbon
also belongs to some body more readily precipitated by acid than
the mucin with 11. 51 per cent, of nitrogen. In view of the great
care exercised in all of these preparations, and the ready solu-
bility of ordinary forms of albuminous matter in an excess of hy-
drochloric acid, especially after they have once been dissolved in
an alkaline fluid, we are very much inclined to believe in the exis-
tence of several related mucins as components of ordinary white
fibrillar connective tissue.

Such a view presents no great difificulty. Submaxillary mucin,
for example, differs from tendon mucin by only 0.5 per cent, of
carbon (48.84 per cent.) and about 0.5 percent, of nitrogen (12.32
per cent.), although it shows some other points of difference, such
as a tendency to undergo alteration by the action of lime water and
by being soluble in 0.2 per cent, hydrochloric acid. Indeed, all
of the various mucins described show minor points of difference,
although agreeing in their general reactions, and it is easy to con-
ceive of the presence of two or more closely related mucins, in
tendons, with different elementary composition.

The most remarkable thing, however, connected with the mucins
that we have separated from this form of fibrillar connective tissue
is the amount of sulphur present in the purified products. In snail
mucin, Hammarsten has shown the presence of 1.75 per cent, of

298 R. H. Chittenden and William J. Gies.

sulphur, but in the mucin from tlie submaxillary gland and in the
mucin described by Loebisch as contained in tendons, the amount
of sulphur has been placed at 0.84 to o.S i per cent. In all three
of our preparations, however, the sulphur present has amounted
to at least 2.30 per cent., and, moreover, the agreement in the
several products has been very close indeed. The greater portion
of this sulphur is closely combined, a small amount only being in
the form of the mercaptan group and responding to the reaction
with potassium hydroxide and plumbic acetate. We present these
figures with some doubt in our own minds, but, having obtained
them as the result of most careful work, we see no possible ex-
planation other than that this amount of sulphur is actually present
in the mucin molecule. The determinations of sulphur were made
after the usual method recommended by Hammarsten — viz.,
oxidizing the mucin with a mixture of ten grammes NaOH and
two grammes KNO3 in a silver crucible, etc. The sodium hy-
droxide employed was chemically pure, having been prepared from
the metal, and, furthermore, several blank tests were made to
prove the freedom of the various chemicals from sulphur. This
percentage of sulphur is greater than has ever been accredited to
a true mucin, although the mucin from the snail's membrane
(mantle-mucin), which is somewhat related to keratin, has been
found by Hammarsten to contain a fairly large amount of this
element — viz., 1.79 per cent.

With regard to the reactions of the several products that we
have studied, there is nothing special to be said. They all show
the ordinary reactions of mucin as described by Loebisch, and we
can simply substantiate what has long been published by him upon
this point.

The most characteristic feature of mucin is the peculiar cleav-
age it undergoes when heated with dilute hydrochloric acid, by
which a substance with reducing action upon alkaline copper so-
lution results. Albumose and peptone are likewise formed b)' the
action of the hot acid. We have tried several preliminary ex-
periments in this direction, the results of which may be briefly
stated: 3.25 grammes of mucin of preparation No. 2 were heated
in a boiling water-bath with one hundred cubic centimeters of two
per cent, hydrochloric acid for five hours. At the end of this

Tendon Mucin. 299

period the solution was of a deep-brown color, while suspended
through the fluid was a large amount of gelatinous matter more
or less brown in color. This was filtered off, washed with water,
in which it was wholly insoluble, until the washings gave no pro-
teid reaction. It was then tested with the following results : It
was insoluble in dilute and stronger hydrochloric acid, but readily
soluble in 0.5 per cent, sodium carbonate and in very dilute (0.5
per cent.) potassium hydroxide. From the solution in sodium
carbonate, it was reprecipitated by neutralization, and was then
readily soluble in a slight excess of 0.2 per cent, hydrochloric
acid. It gave the ordinary color reactions characteristic of pro-
teid matter. Warmed at 40° C. with an active gastric juice con-
taining 0.2 per cent, hydrochloric acid, it was wholly unaffected
even after twenty-four hours, but when warmed with an alkaline
pancreatic juice it was readily dissolved, and almost completely
converted into products soluble even on neutralization of the
fluid, thus attesting its conversion into soluble albumoses and pep-
tones. These reactions suggest that the substance in question is
a form of antialbumid.

The original acid fluid containing the soluble products formed
in the cleavage of the mucin was made neutral, by which a slight
neutralization precipitate resulted, evidently syntonin from the re-
actions tried. The neutral fluid was then concentrated to a sirup,
a strong caramel-like odor being developed during the process,
and while still warm the residue was treated with a large excess
of ninety-five per cent, alcohol, by which a thick gummy mass
was formed, hard and brittle on cooling. While warm, the alco-
holic fluid was quite clear and yellowish-red in color, but on cool-
ing, a light-yellow precipitate, very small in quantity, formed,
which was soluble in water, and gave a strong reducing action
with Fehling's solution. It was too small in quantity, however, to
study further. The gummy precipitate was washed by warming
it repeatedly with fresh quantities of alcohol. It was readily
soluble in water, gave more or less of a proteid reaction, and
showed a fairly strong reducing action with Fehling's solution.
Tested with phenylhydrazine hydrochloride, and sodium acetate,
only an amorphous precipitate resulted from which a crystalline
osazone could not be obtained. On boiling the gummy mass

300 R. H. Chittenden and William J. Gies.

with two per cent. h\-diochloric acid, however, and then extract-
ing the neutraHzed and evaporated fluid with alcohol, a very small
amount of a crystalline osazone was obtained by application of the
hydrazine test, apparently identical with that described further on.
The original alcoholic solution from the above gummy pre-
cipitate was evaporated to a small bulk on the water-bath, the
residue taken up with fifteen cubic centimeters of water, forming a
clear solution. This solution showed strong reducing action with
alkaline copper solution, and evidently contained the greater por-
tion of the reducing body formed from the cleavage of the mucin.
'to the main bulk of this solution was added one gramme of
phenylhydrazine hydrochloride and 1.5 grammes of sodium ace-
tate, after which the mixture was heated on the water-bath for an
hour and a half, the volume of the fluid being kept at fifteen to
twenty cubic centimeters. While hot the fluid was perfectly clear
and reddish in color. After standing an hour in a cool place
there was a marked separation of amorphous particles and oily
globules, but no crystals could be detected under the microscope.
After standing fifteen hours the amorphous particles were almost
wholly transformed into fine crystals. These ciystals were light
yellow in color, and were mostly arranged in rosettes or balls of
fine yellow needles, somewhat resembling lactosazone. The oily
globules were unchanged. These crystals were purified by dis-
solving them in cold alcohol, followed by the addition of water, and
heating the solution until the alcohol was practically all removed,
when the crystals again separated out as the fluid cooled. The
crystals were also insoluble in the hot concentrated fluid. In this
way the crystals were gradually freed from the oily globules
spoken of above and rendered fairly pure. Each time the crys-
tals were filtered they were also washed with a little cold water.
During the process of purification the crystals changed their ap-
pearance somewhat, tending to take on the branching form char-
acteristic of dextrosazone. This crystalline osazone, when puri-
fied as much as possible, was readily soluble in warm water, in
alcohol, ether, chloroform, and, to a certain extent, in benzol.
The amount of the purified osazone was so small that the melt-
ing point alone could be determined. This was done as usual in
a capillary tube. When the temperature reached 140° C. the

Tendon Mucin. 301

substance commenced to darken slowly, and at 160° C. it began
to melt. Further recrystallization of the osazone did not alter
this melting point. In melting point, therefore, this osazone, if
pure, differs widely from dextrosazone or lactosazone. In gen-
eral appearance and solubility, as well as in its melting point, it
appears to resemble very closely the osazone obtained by Ham-
marsten from the cleavage product of the peculiar nucleoproteid
described by him as present in the pancreas.* Whether this body
is a pentaglucose, however, we can not definitely say. We had
hoped, especially in view of the strong reducing action of the
above alcoholic solution, to obtain a fairly large amount of an
osazone, sufficient to determine its content of carbon and nitrogen,
but the yield of purified product was very small indeed.

In order to verify the above results, a second portion of mu-
cin was decomposed with dilute acid — 4.75 grammes of mucin
with two hundred and fifty cubic centimeters of 2.0 per cent, hy-
drochloric acid — the mixture being heated directly over a lamp
for about five hours. The flask was connected with an inverted
Liebig's condenser to prevent concentration, and the mixture was
kept in a state of gentle ebullition. In this case there was much
less of the antialbumid-like body so prominent in the first decom-
position, the amount being less than one fifth that found before.
The neutralization precipitate, however, was considerably larger,
and albumose and peptone were both present in abundance. The
caramel-like body precipitated by alcohol was naturally more abun-
dant than in the first case, but on analysis it was found to contain
a large percentage of nitrogen, so that its fancied resemblance to
caramel is purely superficial. By evaporation of the alcoholic
extract containing the greater portion of the reducing body a resi-
due was obtained as before, from which a crystalline osazone was
formed agreeing in all its properties' with the body previously
described. The purified osazone melted at 158° to 160° C. It
is thus evident that the mucin or mucins present in ox tendon
yield on cleavage with dilute hydrochloric acid a carbohydrate
body which forms a well-defined and crystalline osazone, although
at present we can not state definitely the exact nature of this car-
bohydrate substance.

* Zeitschr. f. physiol. Chem., Band xix, p. 19.

14

The original article appeared in the Reference Handbook of the Medical
Sciences, Revised Edition, igoi, iii, p. 223.

ANIMAL COLORING MATTERS.

By William J- Gies.

Many of the animal coloring matters are substances of consid-
erable functional consequence. Some, for example, are of special
service in respiration ; others appear to be important factors in
vision ; a large number afford protective effects ; several, also, are
attractive in their influence. A majority, however, seem to be
without any apparent physiological relations and not a few are
purely excretory products.

L Classification.

The multitude of animal pigments may be arranged conven-
iently in the following general groups :

1. Respiratory Pigments. — These coloring matters are very
important functionally. Most of them are carriers of oxygen, with
which they unite loosely, receiving it in the organs of respiration?
conveying it to the body parts, and there givdng it up to the tis-
sues. The leading ones are compound (" chromo ") proteids.
Among them are haemoglobin, haemocyanin, haemerythrin and
chlorocruorin.

2. Derivatives of Respiratory Pigments. — Some of the
best-known animal coloring matters are derivatives of haemoglobin,
and many of the colored substances in the lower animals are un-
doubtedly formed from their blood pigments. Prominent deriva-
tives of haemoglobin are bilirubin (haematoidin), stercobilin (uro-
bilin), urochrom and haematoporphyrin.

3. LiPOCHROMES. — These substances, yellow or yellowish-red
for the most part, are very numerous. They are found particu-
larly in adipose tissue, yolk of egg, butter, and in the tissues and
epidermal structures of the lower animals. In solubilities they
are much like the fats, and they show absorption bands toward

303

304 William J. Gies.

the violet end of the spectrum. Little is known of their chemical
composition. They appear to consist of only carbon, hydrogen
and oxygen. Among them are serum lutein, tetronerythrin and
the " chromophanes." •

4. Melanins. — These are brownisb -black pigments occurring
especially in epidermal structures. They consist of carbon, hy-
drogen, nitrogen and oxygen. Nearly all contain sulphur : a few,
iron. It is thought by some that they are derivatives of haemo-
globin ; by others, modified lipochromes. They have been pro-
duced outside of the body from simple proteids by prolonged
hydration (" melanoidins "), which fact suggests, of course, that
they may be so derived within the .system. Among the typical
members of the group are fuscin, phymatorhusin and sepic acid.

5. Chromogens. — These are the colorless, or less colored
precursors of actual pigments occurring in nature. The leading
ones are indoxyl compounds, which give rise to red and blue in-
digo ; melanogen ; uroroseinogen ; the chromogen of the suprarenal
medulla, related probably to the pigment of the skin in Addison's
disease ; and urobilinogen. The so-called " humous substances,"
obtained by destructive chemical methods, and such bodies as pro.
teinochromogen (tryptophan), which merely form colored combi-
nations with various reagents, are, of course, purposely excluded
here.

6. Miscellaneous Pigmexts. — This residual group includes
a very large number of protective, attractive and other coloring
matters, characteristic especially of the lower animals, studied only
spectroscopically for the most part. Among those whose chem-
ical individuality is not disputed are turacin, carminic acid, puni-
cin, chlorophyll and lepidotic acid.

II. Distribution.

Lower Animals. — Coloring matters are widely distributed
throughout the whole of the animal kingdom. In some animals
they occur only in the body fluids, in others they are also diffused
throughout various tissues. In many they occur in the form of
granules in certain cells or cellular layers. " Coloring matters
are often collected in special sacs which open and shut, producing
the 'shot' or play of color of the chameleon, dolphin, cuttlefish

Animal Coloring Matters. 305

and other animals. In many low animals the color of the pig-
ment is characteristic of genera, famiHes or even higher groups, as
among infusorians, etc." Many of the lowest types, such as in-
fusoria, sponges and hydroids, contain chloropJiyll (green) in gran-
ular form and some ciliated animalcules are colored by stcntorin
(blue). Chlorophyll is found, in several mollusks, Crustacea and
insects, and also in the so-called livers of many invertebrates {cn-
ierochlorophyll). The latter organs also contain a ferruginous
pigment, ferrin (brown) and cholechrorn or hepatocJirom (reddish
yellow), a lipochrom ; also lielicorubin (orange red). Haematopor-
phyrin (purplish red), a derivative of haemoglobin (red), occurs in
the integument of star fishes, slugs, the common earthworm and
various sponges. A number of corals and hydroids, and some
sea anemones, are colored by actinioclirom (red); also by polypery-
tJirin (red), probably identical with haematoporphyrin. Some ac-
tiniae contain a coloring matter very similar to another derivative
of haemoglobin, liaeinoclirovwgen (red), and convertible into haema-
toporphyrin. Many echinoderms contain pentacrinin (red and
purple) and the following pigments give special coloration to the
lower species from which the terms are derived : aplysiopiirpiirin
(purple), bonellein (green), echinastrin (red), astroidin (yellow),
rhizostomin (violet), ophiurin (yellowish brown), asterocyanin (blu-
ish violet) and comatiilin (red). Punicin (purple) is derived from
the colorless secretions of various mollusks on exposure to light,
and carminic acid (red) is the pigment characteristic of the cochi-
neal.

The shells of some mollusks, and also some corals, contain
" lipochromoids " and "melanoids." The brownish-black ink of
Sepia officinalis, used to color the sea water and cover the flight
of the animal, contains a melanin, sepic acid (black). The green
{chlorophari), yellow (xanthophan) and red {rhodophan) pigments,
" chromophanes," of the oil droplets in the retinal cones of birds,
reptiles and fishes, as well as the yellow substance in the yolk of
egg {o7ttochrin), are lipochromes. The Qgg of the water spider is
colored by the two lipochromes, vitellornbin (red) and vitellohdein
(yellow). Some of the characteristic coloring matters in decapod
Crustacea are lipochromes. The red crnstaceortibin is closely re-
lated to hepatochrom (cholechrom) in the livers of these animals.

3o6 William J. Gies.

The eggs of the river crab and the lobster contain the same bluish
pigment as that in the carapace of the animals. This pigment,
called cyanocrystallin, becomes red with acid and on boiling in
water. Crustaceorubin appears to be derived from it. The shells
of various birds' eggs are pigmented by haemoglobin derivatives,
among which are bilivcrdin (green); oocycmin (blue), closely related
to biliverdin ; oorliodciii (reddish brown), probably identical with
haematoporphyrin ; ooclilorin (yellow) and ooxantJiin (red).

In certain butterflies the white pigment of the wings consists
of uric acid ; the yellow pigment, of Icpidotic acid, which yields
uric acid on hydration. The red pigment of the body scales is
closely related to lepidotic acid. The wing covers of beetles con-
tain colcoptcrin (red). The showy colors in the plumage of birds
are due in part to the influence on light which the feathers them-
selves exert, causing the so-called "interference colors" ; in great
part, however, to pigments. Turacin (red) is one of the best
known of these. Among the many other feather pigments are
tiiracovcrdin (green), coonoythriyi (red), zoonibin (brown), zoofnlvin
iy tWow), picqfjdvin (yellow), tiiracobruniii (brown) And psittac of ulvin
(yellow). Nearly all of these, " lipochromoids" and " mela-
noids," seem to be very closely related to the numerous skin pig-
ments in birds, and scale and flesh pigments in fishes, such as
tetroneiythrin (red) and coriosiUfiiriii (yellow); and to lacertofiilvin
(yellow), lipoclirin (yellowish green) and others, in the skin of rep-
tiles and various amphibia. The red pigment, dicviyctylin, of
Dicitiyctylus viridcsccns, like lepidotic acid, yields uric acid on
hydration. Many invertebrates contain " histohaematins," haemo-
globin derivatives, chief of which is myohacniatin {inyoclironi) of the
red muscles ; found in the muscles of insects and mollusks, also,
whose haemolymph does not contain haemoglobin. The charac-
teristic color of the muscles of the salmon and other related fishes
seems to be due to a red lipochrom identical with tetroner}'thrin.
The nerves, particularly the ganglia, of some worms are colored
bright red by haemoglobin.

Haemoglobin is present in the circulating fluid of many species
of the invertebrate subkingdoms. It has been found in several
species of the starfish family ; in no lower invertebrate forms,
however, but in most species of all genera higher up the scale.

Animal Coloring Matters. 307

The corpuscles in the hydrolymph of sea urchins contain echino-
chrom (yellow), a " lipochromoid," with possibly respiratory func-
tion. The haemolymph of various invertebrates is colored yellow-
ish to yellowish green by Hpochromes ; violet to purplish red by
" floridins," of which haemerythrm (red) is the best known. Haem-
erythin, and also chlorocruorin (green), replace haemoglobin in the
haemolymph of worms ; haemocyanin (blue) in that of most mol-
lusks, Crustacea, and some members of the spider family. In the
haemolymph of Crustacea the lipochrom, tetronerythrin (crusta-
ceorubin, zoonerythrin), is also frequently found along with the
haemocyanin. The blood of the common house fly, and other
like species, contains haemoglobin, but that of butterflies and many
related insects is green, and contains chlorophyll derived from the
food ; although chlorophyll occurs in other parts as well. The
blood of many insects turns brown to black when it is shed, to
which process the term "melanosis" has been applied.

Higher Animals. — The various tissues and fluids of the
higher animals owe their color, very often, to mixtures of several
pigments. Colored granules are frequently derived directly from
external sources ; into the lungs (pneumonokoniosis), such as coal
dust (anthracosis), iron particles (siderosis), etc., whence they are
sometimes distributed to the liver, lymphatic glands, kidneys and
other organs. They result, also, from medicinal introduction, as
reduced silver in the alimentary tract, skin, liver, kidneys, etc.
(argyria). They enter through the skin, also (tattoo).

The following concise arrangement gives practically all the
more important pigments found in man and mammalia generally,
and will aid to reference to more extended accounts than can be
given here. The terms in italics indicate the pigments occurring
only under unusual or abnormal conditions :

Adipose tissue — lipochrom.

Bile — bilirubin, biliverdin ; also biliprasin and urobilin in
some ; bilifuscin, cholohaematin (from chromogen), hydrobiliriibin,
haemoglobin, methaemocrlobin, haematiu. Biltary calculi — bili-
rubin, biliverdin, bilicyanin, bilifuscin, bilihumin (?), biliprasin, cholet-
elin (hydrobilirubin ?). Blood — {a) corpuscles: oxyhaemo-
globin, haemoglobin ; {S) plasma : serum lutein, bilirubin (in some);
liaemoglobin and direct derivatives, haemoglobin compounds zvith

3o8 William J. Gies.

poisonous substances, hepatogenous pigments, nie/anin. Blood
CLOTS (old) — haematoiditi (bilirubin), rubigin or haemosiderin
(ferric hydroxide). Bone — lipochrom in ossein and yellow
marrow ; haemoglobin in red marrow ; Jiacviatogcnous pigments
in ossein.

Conjunctiva — /ule pigments. Connective tissues — lipo-
chrom, melanin; bile pigments. Contusion — bile pigments,
haematoidin. Corpus luteum — lutein, /laematoieiin (?) Cysts —
lipochrom : haemoglobin derivatives, including bile pigments.

Eye — (a) choroid and iris, fuscin ; (b) retina, (i) Rods —
visual purple (rhodopsin), visual yellow (xanthopsin) ; (2) Pigment
layer — fuscin, lipochrin.

Faeces — stercobilin (urobilin), indigo chromogens, urobilino-
gen, sulphide of iron ; pigments from food, such as carrotin, chlor-
ophyll, haematin ; liaemoglobin and siderous liaeniatogcnous pig-
ments, bUe and drug pigments. Freckles — haematogenous pig-
ment.

Ganglion-cells — lipochrom. Gastro-intestinal mucosa —
haemoglobin and its direct derivatives (haematochromatosis). Glands
in general—^ haemoglobin in capillaries, chromogens. haematoge-
nous pigments.

Hair — lipochrom, melanin.

Intestine — {a) conxretions : hepatogenous pigment ; (/^)
contents : essentially same as faeces, including bile pigment and
hydrobilirubin normally.

Leucocytes (phagocytic cells) — any pigment found elsewhere
in the body. Liver — ferrin, cholechrom, rubigin, non-siderous
hacmatogejious and also bile pigments. Lungs — Inhaled particles,
haemosiderin, melanin {^) Lymphatic {a) fluids — serum lutein,
haematogenous and hepatogenous pigments ; {b) glands : haemo-
globin derivatives.

Meconium — bile pigments, haemoglobiji and its derivatives.
Menstrual fluid — haemoglobin and direct derivatives. Milk
(cream, butter, cheese) — lipochrom; "blue milk," triphenyl-
rosanilin {B. cyanogenous); "red milk," pigmenthy M. prodigiosus;
"yellow milk," pigment by B. synxanthum. Mole (naevus) —
haematogenous pigment. Muscle — myochrom (diffused haemo-
globin ?), myohaematin (haemochromogen ?).

Animal Coloring Matters. 309

Pancreas — haematogenous pigment. Placenta — haemo-
globin, haematoidin, haematochlorin (biliverdin ?). Pus — lipo-
chrom, pyocyanin {B. pyocyanens), pyoxanthose, bilirubin, indigo
blue (?), haemoglobin and decomposition products.

Sebaceous secretions — lipochrom. Skin — melanin, bile
pigments (haemochromatosis), histoJiaeinatinsiJ). Spleen — haemo-
globin, riibigin, non-sideroiis liaeniatogcnotis pigment. Sputum —
blood, bile, and pns pigments ; also inhaled particles. Stomach
contents — food pigments ; blood and bile coloring viatters.
SuPRARENALS — hacmochromogen and chromogen yielding red
pigment on exposure to light. Sweat — pyocyanin, indigo blue (?),
bile pigments ; haemoglobin and derivatives (" red sweat"). Hip-
popotamus and kangaroo : reddish-brown pigment ; dwarf antelope :
blue pigment.

Tissues generally — coloration effects due to blood in capil-
laries ; bile pigments, haemoglobin and Jiaematogenons pigments.
Tumors — phymatorhusin, sarcomelanin, lipochrom, haemoglobin
and derivatives. Horse : hippomelanin.

Urine — {a^ pigments: urochrom, urobiHn, uroerythrin,
haematoporphryrin {yiXos-^^c\.x\x\),skatoxyl red, melanin, indigo (blue
and red), bile pigments, haemoglobin and direct derivatives, drug
coloring matters ; (b) chromogens : indoxyl and skatoxyl com-
pounds ; precursors of haematoporphyrin and urorosein (urorhodin,
urorubin, etc.) ; urobilinogen, hydroxybenzene derivatives {^' alkap-
tonuria''^, melanogen. Urinary calculi and sediments — uro-
erythrin, urochrom ; liaematoidin, indigo blue, bile pigments, liaemo-
globin prodiicts.

Vomit — blood, bile, food and drug pigments.

HI. Chemical and Physical Qualities.
The animal pigments have been the subject of many laborious
researches, but, owing to the great difficulties they present to the
investigator, our knowledge of the chemical characters of most of
them is very slight and uncertain. The primary obstacle in the
way of their proper chemical study is the strikingly minute
amount in which they commonly occur, and, as nearly all of them
have very great tinctorial power, their coloration effects, therefore,
are usually out of all proportion to the actual quantity in which

3IO William J. Gies.

they are present in any medium. Further, isolation of the pig-
ments by chemical means is apt to induce radical changes in them,
for many are very unstable and much confusion has resulted from
failure to recognize this important fact. Nearly all of the animal
coloring matters seem to have definite and characteristic effects
on the spectrum, and may be differentiated, to a certain extent,
by the number and position of their absorption bands. But
even the extremely delicate indications of the spectroscope have
undoubtedly led to error in some cases, since very wide spectro-
scopic differences may be brought about by very slight changes of
molecular structure or physical condition, such as often result
from ordinary chemical treatment. Consequently, there is good
reason for believing that not a few of the coloring matters which
have been dignified with special names are merely closely related
artificial derivatives (oxides, reduction products, etc.) of several
antecedent pigments or chromogens.

It would carry us far beyond the scope of this particular article
to present detailed reference to each of the pigments already men-
tioned. All of the most important are given due notice in more
extended accounts of blood, urine, faeces, bile, etc., in these vol-
umes,* so that it will be sufficient here to describe, in conclusion,
a few of the best known of those found in the lower animals.

H.\E.MOCVAXix (blue), Chlorocruokix (green). — Each of these
pigments is analogous to haemoglobin in chemical structure and
in function, the first replacing it in the haemolymph of mollusks
and related forms, the second in that of worms. Both, like
haemoglobin, unite loosely with oxygen ; oxyhaemocyanin is blue,
haemocyanin itself is colorless. Haemocyanin contains copper in
place of iron and has no special influence on the spectrum.
Chlorocruorin, on the other hand, yields haematin and shows
characteristic absorption bands.

TuR.ACiN is a red, feather pigment. It possesses a spectrum
which is almost identical with that of oxyhaemoglobin. It con-
tains seven per cent, of copper, besides carbon, hydrogen, nitro-
gen and oxygen. The quantity of turacin in the feathers of a
single bird does not exceed two or three grains. It may be ex-
tracted from the feathers with o. i per cent, alkali and precipitated

* Reference Handbook of the Medical Sciences.

Animal Coloring Matters. 3 1 1

from its solution with dilute acid. It is insoluble in water, alcohol
and ether.

Carminic Acid (Carmin). — The female cochineal {^Coccus
cacti) contains from twenty-five to fifty per cent, of this coloring
matter. The pigment is also found in the blossoms of certain
plants. Its composition is shown by its formula : Cj,Hj^O^„.
Some of its compounds produce effects on the spectrum analogous
to those of oxyhaemoglobin. Carminic acid is a glucoside ; when
it is boiled with dilute acids, and thereby hydrated, it yields an
optically inactive, non-fermentable sugar and also "carmin red"

(CuH,A) :

C,,H,p,, + 2HP = C,H,p, + C,H,,A-

Carminic acid may be extracted from the cochineal with warm
water. The pigment is soluble in alcohol and dilute acids, and
forms salts with alkalies and metallic compounds.

PuNiciN. — The colorless secretion of a glandular organ situ-
ated at the lower part of the mantle, between the gill and the rec-
tum of various species of Miirex and Pitrpiwa, assumes, on expo-
sure to light, a bluish-green color at first, then red. and lastly a
purple-violet. This coloring matter, " Tyrian purple," is the
" purple of the ancients " and for centuries was the dye of greatest
beauty and value. Punicin is the name of the pigment ; the chro-
mogen has not been isolated. Punicin is insoluble in water, alcohol
and ether ; soluble in boiling glacial acetic acid. It dissolves
readily in boiling aniline, from which it separates, on cooling, in
crystalline form.

Chlorophyll. — This important plant pigment is found in
Hydra viridis, Spongilla fliizdatihis , in the elytra of cantharides
beetles, in the blood of many insects, in the so-called livers of
many invertebrates, etc. It is insoluble in water, but dissolves in
alcohol and ether, and consists of carbon, hydrogen, nitrogen and
oxygen, and possibly iron. Chlorophyll, treated with concen-
trated acid, yields phylldcyanin. The latter, on fusion with caustic
soda, is transformed into phylloporphyrin (CjgHjj,N.,0), a close
relative of haematoporphyrin (C^gHj^N^Og), which may be produced
from haemoglobin, on treatment with acids, and is isomeric with
bilirubin (CjgHjgN203). Phylloporphyrin and haematoporphyrin

312 William J. Gies.

are probably oxide? of one and the same radicle. Tiiis kinship
corresponds to analogous physiological relations of the pigments
from which each can be deri\ed.

Tetronervthkix (Crustaceorubin, Zoonervthrvn). — The
red pigment in the warty integument around the eyes, and also in
the feathers of various birds, and in the hypoderm and haemolymph
of many invertebrates, is one of the most widely distributed of all
the pigments. It is soluble in ether, alcohol and chloroform, and
shows the absorption bands and gives the reactions of a typical
lipochrom.

Lepidotic Acid. — The \'ellow pigment in the wings and ex-
crements of butterflies {Picridi)iae). It may be extracted with hot
water or dilute alkalies, and is precipitated from such extracts on
acidification. Its solutions show a greenish fluorescence and, on
warming with dilute nitric acid, it yields uric acid. Warmed with
dilute sulphuric acid a purple product, lepidoporphj'rin, is obtained,
which shows two characteristic absorption bands. This substance
may also be derived directly from uric acid. The close relation of
lepidotic acid to xanthin and uric acid is shown by the figures for
their percentage composition :

C. I H. j N. I O.

Xanthin (dioxypurin) 39.4 2.6 36.8 21. 1

Lepidotic acid 3S.1 3.5 37.1 21.3

Uric acid (trioxypurin) 35.7 2.4 33.3 28.6

The above paper was written in the spring of 1900. Addi-
tional facts may be found in the following publications :
GriflSths. Ueber den Farbstoff von Echinus esciilentes. Chemisches

Central-Blatt, 1900, ii, p. 638.
Neumann. Das Pigment der braunen Lungeninduration. Jahres-

bericht uber Thier-Chemie, 1900, xxx, p. 882.
Rosenfeld. Ueber das Pigment der Haemochromatose des Darmes.

Ibid., p. 918.
Henze. Zur Kenntniss des Haemocyanins. Zeitschrift fiir physiolo-

gische Chemie, 190 1, xxxiii, p. 370.
Alexander. Das Labyrinth Pigment des Menschen und der hoheren

Saugethiere, etc. Centralblatt fur Physiologie, 1901, xv, p. 293.
V. Furth und Schneider. Ueber thierische Tyrosinasen und ihre

Beziehungen zur Pigmqntbildung. Beitrage ziir chemischen Physio-
logie und Pathologie, 1901, i, p. 229.

Animal Coloring Matters. 313

Jones and Auer. On the oxidation of native pigments. American

Journal of Physiology, 1901, v, p. 321.
Ducceschi. Ueber die Natur der Melanine und einiger verwandter

K5rper. Jahresbericht iiber Thier-Chemie, 1901, xxxi, p. 64.
Kuenen. Hamolyse und hamatogene Pigmentbildung. Ibid., p. 867.
Zeynek. Ueber den blauen Farbstoff aus den Flossen des Crenilabrus

pavo. Zeitschrift fiir physiologische Chemie, 1901, xxxiv, p. 148;

1902, xxxvi, p. 568.
Dubois. Ueber den inneren Mechanismus der Purpurbildung. Chemi-

sches Central-Blatt, 1902, i, p. 535. Also, Ueber die Bildung des

Purpurs bei Purpura lapillus. Ibid., 1903, i, p. 473.
Hacker and Meyer. Ueber die blaue Farbe der Vogelfedern. Cen-

tralbatt fiir Physiologie, 1902, xvi, p. 153.
Lubarsch. Ueber fetthaltige Pigmente. Ibid., p. 754.
Oppenheim. Zur Frage der Pigmentbildung aus Tyrosin. Ibid. , p. 755.
Zdarek und Zeynek. Zur Frage iiber den Eisengehalt des Sarkom-

melanins vom Menschen. Ibid., p. 757.
Zumbusch. Beitrage zur Charakterisirung des Sarkommelanins vom

Menschen. Zeitschrift fiir physiologische Chemie, 1902, xxxvi, p.

511-
Levrat and Conte. Origin of the natural coloration of the silks of

Lepidoptera. Journal of the Society of Chemical Industry, 1902,

xxi, p. 1392.
Schulz. Die physiologische Farbstoffbildung beim hoheren Tiere.

Ergebnisse der Physiologie, erster Jahrgang. I. Abteilung, p. 505.
Sieber-Schumoff. M. v. Nencki's Untersuchungen iiber den Blutfarb-

stoff und dessen Beziehungen zum Blattfarbstoff. Biochemisches

Centralblatt, 1903, i, p. 86.
Mbrner. Kleinere Mittheilungen. III. Die sogenannten gefarbten

Kalkkorper im Lederhaut der Holothurien. Ibid., p. 185.
Wychgel. Onderzoeingen over het pigment der huid, en de urine

gedurende de zwangerschap. Ibid., p. 193.
Marchlewski. Studies on natural coloring matters. Ibid., p. 215.
Gamgee and Hill. Ueber die optische Aktivitat des Hamoglobins und

des Globins. Beitrage zur chemischen Physiologie und Pathologie,

1903. iv, p. I.
Spiegler. Ueber das Haarpigment. Ibid., p. 40.

May, 1903.

Reprinted from the Archives of Neurology and Psychopathology, 1899, iij P- 557-

EMBRYOCHEMICAL STUDIES. I. SOME CHEMICAL
CHANGES IN THE DEVELOPING EGG.

By p. a. Levene.

[From the Pathological Institute of the New York State Hospitals and the Depart-
ment of Physiological Chemistry of Columbia University.]

„ L Introduction.
In his remarkable book on general physiology, Max Verworn
says : " Der I.ebensvorgang beruht in dem Stoffwechsel der
Eivveisskorper." I am not certain whether at the present state of
science we are justified in making such positive statements that
life is only a chemical process. However, it is evident to every
biologist that the workings of all mechanisms in which life mani-
fests itself to us, lead to constant wear of those mechanisms or or-
ganisms. Biologists have also observed long ago that the living
organism possesses a peculiar ability of repairing its constant
losses. In fact, there are but very lew conditions in the organism
when a substance cannot be classified among "the dead," and
when the two processes, waste and repair, are not to be noticed.
In most conditions of life we can well distinguish these two main
functions, dying and growing. And the state of any living organ-
ism, its working capacity, its " quality," so to say, depends fully
on the relation between these two functions, which Max Verworn
calls " biotonus." He further very ingeniously presents the last in
form of a fraction AjD. {A = processes of assimilation ; D =
processes of dissimilation.) Thus the different states of the bio-
tonus might be represented as

AAA

D='' Z)>^' n<'-

The significance of this is self-evident. In one case the assimi-
lation and dissimilation are in a state of equilibrium ; in the other
assimilation predominates ; in the third, dissimilation takes the
first place. It is further self-evident to any student of biology

315

3i6 P. A. Levene.

that none of these processes is a single chemical reaction, that
processes of formation, growth, as well as those of decomposition
are very complicated ; that before the body substance is trans-
formed into final decomposition products, it undergoes many
intermediate changes, and before food is assimilated and converted
into a part of the body protoplasm, it undergoes numerous trans-
formations. Thus, Verworn presents a general formula of the
" biotonus," as

This mathematical representation of the biotonus is true not
only speculatively, but is also in accord with experimental evi-
dence. It should be remarked that physiological chemistry
(organic as well as inorganic) began its work, broadly speaking,
with analytical experiments ; it began by studying the path of
transformation of that most complex substance protoplasm, into,
its final decomposition products, urea, CO.,, ammonia, etc. It
first closely followed this path in the living organism, and finally
succeeded in imitating the organism, and at the present day we
may obtain nearly all the decomposition products met with in the
organism, by mere chemical means. But if our knowledge of the
process of dissimilation has become quite extensive, we must on
the other hand own that the process of synthesis of living sub-
stance, even of proteids alone, is as dark to us to-day as it ever
has been. And yet nature offers to us conditions when the
growth of the organism is so much predominating over its wear
that it seems there ought to be little difficulty in following the
organism in its process of growing.

All the highest organisms develop from one single cell, and
in many organisms their growth takes place outside of the body
of the parent organism. In the animal kingdom the amphibia
and birds, among others, belong to the last, and they offer good
material for the study of the chemical changes in the growing
tissue or organism.

It is singular that in the development of biology, the discov-
eries of botany nearly always preceded those of the animal biolo-
gist, and this has repeated itself again in the study of the relation
of chemical changes in the growing or rather developing organism.

Embrvochemical Studies. 317

The work of E. Schulze and his school is remarkable in its re-
sults (and we refer the reader who is interested in the subject, to
the original articles), but very little has been done in this direction
by the animal physiologist.

The work we are publishing here is the beginning of a series of
articles on the chemistry of the developing egg. We think that
this general study ought to precede the special study of the de-
velopment and growth of individual tissues, as muscular, nervous,
and glandular tissues, and so on.

Of all the substances most peculiar to the living organisms are
the different nitrogenous compounds that take part in formation
of the proteid compounds and reappear on the decomposition of the
latter. These compounds may be classified in a general way into
two groups : First, those consisting only of C, H, O and N, and
second, those in which some other elements, mainly S, P and Fe
(each of them separately, or all together), join the former in the
formation of their molecule.

The first group may be again divided into substances with a
well-defined acid nature, as the monoamido acids, like leucin, and
into those of a well-defined basic nature, which are very numerous
and quite different in their composition. ;

The second group again may be divided into simple proteids,
containing only C, N, H, O and S, and combined proteids as
nucleo-compounds, mucin, etc. It is the molecule of the latter
compounds that may contain besides C, H, O and N, also P and Fe.

The aim of this work was to study the distribution of N
among the main groups just enumerated in different stages of the
development of the egg, or, to be more precise, we attempted to
estimate the quantity of N in the form of compounds not basic by
nature, like amidoacid — those in .the form of bases and finally
those in the form of proteids. Further, an attempt was made to
ascertain whether in the course of development a new formation
of the combined proteids (only the nucleo-compounds were dealt
with) was taking place or not. The amounts of ash and water
were also estimated.

The material used was the egg of the codnsh. It was exam-
ined in the following four stages : unfertilized ; 24 hours after fer-
tilization ; 1 1 days and about 20 days after fertilization.

3iS P. A. Levene.

All the material was furnished to us b\' the courtesy of the U.
S. Fish Commission, and we wish to express our indebtedness to
Doctor Bumpus and Mr. Locke, who were kind enough to supply
us with fish eggs. It was onl\' through their kind assistance that
this work could be carried out.

II. Methods.

Total nitrogen was determined, after the material was dried to
constant weight at 105° C, by Kjeldahl's method. The nitrogen
in the form of monoamido acids and related compounds was esti-
mated by the following method :

The dry substance was extracted for 24 hours with 0.2 per
cent. HCl solution. The mixture was then treated with phospho-
tungstic acid, and after standing twenty-four hours the precipitate
containing the insoluble part of the tissue and the phosphotungstic
precipitate digested by Kjeldahl's method (K.,SO, and CuSO^ used
for digestion). For estimation of the proteid nitrogen, the substance
was first extracted in a Kjeldahl digestive flask, for twenty-four
hours with boiling alcohol, then washed with ether and alcohol, and
treated with boiling water and a few drops of acetic acid for about ten
hours and with cold water for about ten hours more, and then the
N estimated by Kjeldahl's method. (All the extracts were tested
for proteids. The results were negative.)

To study the changes in the quantity of nucleo-compounds
and nucleo-bases, the eggs were extracted with cold and hot
alcohol, then dried in air, pulverized, again extracted with hot
alcohol, cold and hot ether ; again dried, first in air, then at 105° C.

To estimate the nuclein bases, the substance was heated on a
water-bath in a flask with a return condenser with 2 per cent.
H^.SOj for about ten hours. The acid was partly neutralized by
Ba (OH2), the filtrate concentrated, the silver salts of the nuclein
bases obtained and weighed as such.

Another part of the same material which was used for deterr
mination of the nuclein bases was digested with pepsin -hydro-
chloric acid for a week, and the digestive fluid changed every
second day. The residue was then washed with water, until the
latter gave a negative biuret reaction and contained no HCl. It
was then washed with alcohol, ether, dried and weighed.

Embryochemical Studies.

319

To ascertain whether the residue was really a nuclein or a sub-
stance rich in nucleins, the P was estimated ; but only in one case,
as in the other two the quantity was not sufficient for a satisfac-
tory P estimation.

We present below all the results in tabular form.

III.

Results of Analysis.*

I. HjO AND Ash Determinations.

Subst.

Dry. Subst.

A.sh,

In grms.

In grm. Per Cent.

In grm.

Per Cent.

F-0

9.7612

0.5737 5-33

0.0580

10.09

F-I

8.2201

0.4760 5.20

0. 6480

17.17

F-II

7.0600

0.5640 ■ 7.98

0.0990

17-55

F-I 1 1

8.0975

0.5315 6.31

0.1045

19.66

ii.

Distribution of Nitrogen.

Subst.

Total N

In grm.

in grm.

Per Cent.

Per Cent.

F-O

0.5405

0.059568

II. 01

10.90

0.4030

0.043800

10.80

F-I

0.3914

0.039858

10.16

9.96

0.4299

0.042048

9.77

F-II

0.2985

0.033288

II. 15

11.22

0.3225

0.036354

11.29

F-I 1 1

0.3180

0.029346

9.52

9.52

III.

N In Phosphotungstic Precipitate =

: Proteids +

Bases.

Grm. substan

ce. Grm.

Per Cent.

Per Cent.

F-0

0.3670

0.030660

8.32

8.50

0.2956

0.026280

8.88

F-I

0.1791

O.OI4OI6

7.82

7.83

0.3296

0.025842

7.84

F-II

0.2855

0.024528

8.52

8.67

0.3366

0.029784

8.85

F-I II

0.2251

0.021462

IV. Proteid Nitrogen.

9-53

9-53

F-0

0.1650

0.012264

7-43

72.9

0.2940

0.020824

7.15

F-I

0.5267

0.028470

5.40

5-33

0.5504

0.028808

5.26

F-II

0-5535

0.041610

7.52

7.27

0.6540

0.045990

703

F-III

0.2575

0.017520

6.84

6.84 ,

* F-O = unfertilized ; F-I == 24 hours after fertilization; F-[I=:II days after
fertilization ; F-III = 20 days after fertilization.

320 p. A. Levene.

V. PROrORTIONS OF ACIDS, BASES AND PrOTEIDS.

F-O F-I

Per Cent, of Per Cent, of Per Cent, of Per Cent, of

Dry Subst. Total N. Dry Subst. Total N.

N in Monoamido 10.90 — 8.60 9.96 — 7.83==

compounds ^=2.30 21.10 2.13 21.37

X in form of 8.60 — 7,29 7.83 — 5-33=

bases =1-31 12.07 2.50 25. ID

N in form of pro-

tei'Js 729 66.00 5.33 53.57

F-I I F-I I I

Per Cent, of Per Cent, of Per Cent, of Per Cent, of

Dry Subst. Total N. Dry Subst. Total N.

X in Monoamido 11.22 — 8.67^ 9.52 — 9.53

compounds ... 2.55. 22.72 = — .01 o

N in form of 8.67 — 7.27^ 9.53 — 6.84

bases 1. 40 12.48 1=2.69 28.25

X in form of pro-

teids 7.27 64.79 6.84 71-84

VI. Results ok Digestive F.xi'eriments.

Subst. in grms. Residue in grm. Per Cent.

F-I 2.0442 0.0428 2.08

F-II 1.69S0 0.0570 3.35

F-III 1-7767 0.1297 7.24

P. — Determination in the residue of F-III : 0.137 grm. of the residue = MgP20j
^ 0.014 gr. P=2.65%.

VII. DETERMIN.A.TION OF THE NUCLEO-BaSES.

Subst. in grms. Grm. bases. Per Cent.

F-0 1. 8611 0.0022 0.12

F-I 2.0227 0.0438 2.16

F-II 1-5190 0.0325 2.14

F-III 1. 2132 0.0455 3-75

IV. Gexer.al Rem.vrks.

I think it would be premature to draw any very broad con-
clusions from the little work completed at present. Such conclu-
sions should be deferred until the data have increased considerably.

The results of this work, however, tend to indicate that in the
developing egg the processes of synthesis are preceded by those
of decomposition (consult Table V.). In the first stage after ferti-
lization the proteids diminish in quantity ; basic nitrogenous sub-
stances are formed at their expense. Later the basic substances
decrease in quantity and proteids grow. Whether the molecules
oT those proteids are formed from the basic substances will be in-
vestigated in the future.

Embryochemical Studies. 321

It is also seen that the character of the proteids is changed
during the development of the egg ; the combined proteids as we
may term them, such as nucleoproteids, increase greatly in
quantity. The importance of mineral salts for the formation of
tissues can be illustrated by the increasing quantity of mineral
substances in the egg in the course of its growth.

I take occasion to acknowledge my indebtedness to Professor
Chittenden for all the kindness shown by him to me while I was
engaged in this work in the laboratory of Physiological Chemistry
at Columbia University.*

May 22, 1899.

Bibliography.

A. Tichomiroff. Chemische Studien liber die Entwicklung der Insect-

eneier. Zeitschr. f. physiol. Chemie, IX., 578.
A. Kossel. Weitere Beitrage zur Chemie des Zellkerns. Zeitschr. f.

physiol. Chemie, X., 248.

* See Preface, page 7.

B. PATHOLOGICAL AND TOXICOLOGICAL.

Reprints, Nos. 16-28.

Reprinted from the American Journal of Physiology, 1898, Vol. i, No. i, p. i.

THE INFLUENCE OF BORAX AND BORIC ACID UPON

NUTRITION, WITH SPECIAL REFERENCE TO

PROTEID METABOLISM.

By R. H. Chittenden and William J. Gies.

[From the Sheffield Laboratory of Physiological Chemistry, Yale University.]

CONTENTS.

Page.

Historical 3^5

Conduct of the Experiments 3^9

Methods of Analysis 33^

First Experiment. With Borax, 27 Days 33^

Second Experiment. With Boric Acid, 30 Days 33^

Third Experiment. With Borax and Boric Acid, 56 Days 342

Discussion of Results 334) 339) 34^

General Conclusions 35^^

In view of the wide-spread use of borax and boric acid as
food preservatives it is somewhat singular that our knowledge of
the influence of these substances upon the nutritional processes of
the body is so slight and uncertain. E. de Cyon,* M. Gruber.f
and J. Forster X have indeed studied the action of these agents
upon proteid metabolism, but with results which are utterly lack-
ing in harmony. Thus Cyon's work with borax seemingly indi-
cates that proteid metabolism is diminished under its influence,
i. e., that borax tends to protect the consumption of proteid mat-
ter in the tissues. Gruber's experiments, on the other hand, indi-
cate with equal positiveness that borax has no proteid sparing
power, but really leads to an increase in the rate of proteid metab-
olism. To add to the uncertainty, the experiments with boric
acid carried out under Forster's supervision tend to show that this

*Cyon. Sur Taction physiologique du borax. Comptes rendus, 1878, tome 87,

p. 845-

t Gruber. Ueber den Einfluss des Borax auf die Eiweisszersetzung im Organ-

smus. Zeitschr. f. Biol., 1880, Band 16, p. 198.

X Forster. Ueber die Verwendbarkeit der Borsaure zur Conservirung von Nahr-
ungsmitteln. Nach Versuchen von Dr. G. H. Schlencker aus Surakarta. Archiv. f.
Hygiene, 1884, Band 2, p. 75.

325

326 R. H. Chittenden and William J. Gies.

agent is wholly without influence upon proteitl metabolism. Ob-
viously, conclusions which arc so much at \ariance cannot be ac-
cepted without careful consideration.

Cyon's experiments were conducted simultaneously on three
full-grown dogs which were fed upon a diet almost exclusively
proteid. His observations were practically limited to determining
changes in body-weight during short periods, with an estimation
of the nitrogen of the urine. He found that during the period
when borax was included in the food, the animals gained notice-
ably in body-weight and that less nitrogen was contained in the
excreta than in the ingesta. From these very crude observations
the conclusion was drawn that borax, even to the extent of 12
grams per day, may be ingested with the food, especially when
the latter is essentially proteid in nature, without provoking the
slightest disturbance in general nutrition. Further, Cyon ap-
peared to see in his results evidence that borax, if substituted for
common salt in food, will facilitate the assimilation of the latter
and bring about a great increase in the weight of the animal.
Such deductions, however, were wholly unwarranted from the
data at hand, for not only were the periods of observation exceed-
ingly short, but, as pointed out by both Gruber * and C. Voit,t the
animals at the beginning were much emaciated and received
throughout the experiment such excessive quantities of meat that
increase of body- weight would have inevitably followed without
the presence of borax. Consequently, all that can be inferred
legitimately from Cyon's experiments is that assimilation and gen-
eral metabolism were not seriously affected b)' borax in the quan-
tities given.

In Gruber's work more scientific methods were pursued, but it
may well be questioned whether the conditions under which the
experiments were conducted were adapted for bringing out clearly
the full action of borax upon proteid metabolism. The two dogs
employed were fed simply upon meat and water, and were pre-
sumably in a condition of nitrogenous equilibrium. In the first
experiment, when the animal received daily 1,500 grams of meat
and 200 c.c. of water, the daily excretion of urea in the urine

* Gruber. Loc. cit.

t Voit. Hermann's Handbuch der Physiologie, Band 6, Theil I. p. 1C5

Borax and Boric Acid on Metabolism. 327

varied from 75.82 grams to 110.30 grams during the six days
prior to the administration of borax. Then 20 grams of borax
were introduced with the food, an amount so large that vomiting
was at once produced, leading to a loss of about 5 grams of the
borax and about 100 grams of the meat, with most of the water.
On this day, however, 108.20 grams of urea were excreted in the
urine, although the food consumed was 100 grams less than the
usual quantity. On the two following days, without borax and
with the full complement of food, the excretion of urea amounted
to 109.00 and 107.60 grams respectively. From these results
Gruber concludes that the borax increased the excretion of urea
4-6 per cent. In the second experiment, with a dog of 34 kilos
body-weight, fed on a daily ration of 1,100 grams of meat and 200
c.c. of water, the daily excretion of urea varied from 70.86 grams
to 80.60 grams for the four days of the normal period, while the
administration of 10 grams of borax was accompanied by an ex-
cretion of 82.14 grams of urea, and, on the second day following,
the introduction of 20 grams of borax was accompanied by an
excretion of 85.25 grams of urea. Further, on this latter day
the volume of urine rose to 1,310 c.c, while the largest daily ex-
cretion prior to this day was 1,040 c.c. Gruber, therefore, con-
cludes that borax does not spare proteid as Cyon asserts, but, just
as in the case of common salt, sodium sulphate, and other neutral
salts, it causes an increase in the elimination of water from the
body and induces therewith an increased proteid catabolism. It is
not to be inferred from this statement that there is simply an in-
creased washing out of urea from the tissues, for, as Voit * has
pointed out, the amounts of urea excreted on the days following
the ingestion of borax simply fall back to the neighborhood of the
average for the normal period, and do not drop below that average.
Gruber also concludes that borax has no unfavorable influence
upon the assimilation of food, since the quantity of feces, their
content of solid matter and of nitrogen are within the limits of
the normal elimination during periods when meat alone is fed.
Further, no harmful influence, even after the ingestion of the
largest dose — 20 grams — was to be observed, and the appe-
tite of the animal was found to be undiminished on the days fol-

* Voit. Loc. cit, 165.

328 R. H. ClUTTKNDKN AND WlLIJAM J. GlES.

lo\vin<j that upon which borax was given. The objection we would
make to accepting Gruber's conclusions in their entirety is that
they are based solely upon the results following the administration
of two large doses of borax, lO and 20 grams, whereas, to our
minds, longer periods with a dosage of borax continued for several
days in succession would seemingly render the conditions much
more favorable for an accurate judgment as to the character of the
influence exerted by the substance on tissue changes. Further,
since urea alone was determined in the urine, po.ssible minor
changes connected with the presence of the salt would naturally
be overlooked. Lastly, we are inclined to the view that it is ex-
tremely hazardous to draw such sweeping conclusions from one or
two short experiments of this nature, especially where, as in the
animal body, individual characteristics not infrequently give rise to
exceptional results quite foreign to those ordinarily obtainable.

In Forster's work with boric acid, Dr. Schlencker experimented
on himself, using a mixed diet and taking boric acid in daily doses
of 1-3 grams. Each experiment consisted of three periods, of
three days each, the boric acid being taken in the middle period.
The conclusions arrived at were that proteid metabolism is not in-
fluenced, the excretion of urea in the boric-acid period being mid-
way between that of the fore and after periods. It was noticed,
however, that the quantity of ethereal sulphuric acid in the urine
was considerably lessened in the boric-acid period and in the period
following, thus implying an inhibitory influence upon the putre-
factive processes of the intestine. Further, it was observed that
the amount of the feces, together with the contained nitrogen,
was greatly increased under the influence of boric acid, from which
it was inferred that this agent interferes with the assimilation of
the food and perhaps, at the same time, gives rise to an increased
secretion of mucus with a possible increase in the discharge of
epithelial cells from the intestinal mucosa. This latter, however,
is purely conjectural. Increased secretion of bile is also said to
result from the action of boric acid. On the pulse and tempera-
ture no action was observed.

It is thus quite evident that the influence of borax and boric
acid on nutrition, and especially their influence on proteid metab-
olism, is by no means wholly settled. The preceding statements

Borax and Boric Acid on Metabolism. 329

clearly emphasize the uncertainty of our present information on
the more essential features of the question before us, and we have
therefore deemed it desirable to carry out, as thoroughly as possi-
ble, a series of experiments upon the action of both borax and
boric acid on proteid metabolism and related phases of nutrition.

Conduct of the Experiments. — The experiments were conducted
wholly upon full-grown dogs ranging in weight from 8 to 12 kilos.
The animal was confined in a suitable cage partially lined with
galvanized iron and with the floor so arranged that both fluid and
solid excreta could be collected in their entirety, while the upper
portions of the cage were so constructed as to permit unrestricted
circulation of air. In view of the length of the experiments —
ranging from twenty-seven to fifty-six days each, with periods of
eight to ten days' duration — it seemed inadvisable as well as un-
necessary to empty the bladder each day with a catheter. Such
diurnal variations as might possibly occur from incomplete empty-
ing of the bladder at the end of the twenty-four hours would ob-
viously be neutralized in periods of the above length, and conse-
quently the urine was collected as naturally excreted, thus avoiding
any possible disturbance of the normal condition of the bladder,
etc. At the end of each twenty-four hours, the urine collected
was combined, and its volume, specific gravity, etc., determined,
after which the bottom of the cage, was rinsed with a little distilled
water and these washings added to the main fluid. The latter was
then made up to some convenient volume in preparation for the
daily analysis.

The feces whenever passed were collected in a weighed dish,
the mass thoroughly desiccated over a water-bath, and the dry
weight ascertained. The dried material was then pulverized and
the nitrogen-content as well as the ether-soluble matter determined
in sample portions. The nitrogen determinations were always
made in duplicate by the Kjeldahl method and rarely varied more
than 0.05 per cent. Whenever, as sometimes occurred, hair ac-
cumulated in the cage it was likewise collected and the nitrogen
determined. The ether-soluble matter was determined by extrac-
tion of the dried feces in a Soxhlet apparatus.

The animals were fed during the experiments on a mixed diet
composed of fresh lean beef, cracker dust, lard and water. The

66'

R. H. Chittenden and \Villi.\.m ). Gies.

meat was prepared as follows : fresh lean beef, freed as far as pos-
sible from all adherent fat and connective tissue, was run through
a hashing machine, after which it was enclosed in a bag of thin
cloth, placed under a heavy press, and kept there under increasing
pressure for several hours, the bloody fluid which drained off be-
ing thrown awaj'. B\' this method there results a mass of tissue
free from surplus moisture, and which, when enclosed in a bottle,
will keep perfectly fresh on ice for seven to ten days without separa-
tion of fluid. Several advantages accrue from this method. Thus,
we have a perfectly homogeneous mixture which can be drawn
from for at least a week with surety that its nitrogen-content is
constant. There is therefore no necessity for a daily determina-
tion of nitrogen in this portion of the diet, for each sample can
be analyzed when prepared and the data accepted as long as the
meat keeps fresh. Further, meat prepared in this manner at dif-
ferent times, if subjected to essentially the same pressure, varies
only slightly in its content of nitrogen. We have invariably
analyzed each lot when prepared to avoid any possibility of error,
but, as the following results show, the differences in composition
are very slight and necessitate \'ery little alteration in the propor-
tion of meat wheii changing from one lot to another. The follow-
ing results are a few of the many obtained :

1 Weight of Meat.

Absolute Content of

Nitrogen.

Percentage of Nitrogen.

I.

0.8703 gram.
0.7710 "
0.7631 "

0.03041 gram.
.02682 "
.02628 "

3^49
3.4«
3-44

2.

0.7673 "
0.9228 "
1. 0591 "

0.02716 "
.0323S "
.03723 "

3-54
3-51
352

3.

0.8478 "
1. 0014 "
0.8876 "

0.03015 "
.03591 "
.03152 "

356
3-59
3-55

4-

1.0082 "
10445 "
1.0803 "

0.03642 "
•037S3 "
.03961 "

3.6.
3.62
367

5-

1. 1977 "
0.8142 "
0.9793 '•

0.04265 "
.02902 "
■03463 "

3.5'^
3.56
3-54

The carbohydrate element in the diet, as already stated, was
supplied b\' commercial cracker dust. This was purchased in

Borax and Boric Acid on Metabolism. 331

large quantity and preserved in well-stoppered bottles. It con-
tained on an average 1.46 per cent, of nitrogen. The lard em-
ployed was entirely free from any recognizable amount of nitrogen.

The daily diet was divided into two equal portions, one half
being fed at 8 A. M. and the other half at 6 P; M. When borax
or boric acid was given, the daily dose was likewise divided and
given either with the food or directly after. The body-weight of
the animal was taken each morning just before feeding. Each
day's urine included the fluid passed from 8 A. M. of one day to
8 A. M. of the next day.

Methods of Analysis. — Nitrogen was determined wholly by
the Kjeldahl method, viz., in the daily analyses of the urine, feces
and food material. All analyses were made in duplicate, and the
figures given are based upon the averages of closely agreeing re-
sults. In analysis of^the urine 5 c.c. were used for each deter-
mination, oxidation being carried out in a long-necked Kjeldahl
flask with 10 c.c. of sulphuric acid and a crystal of cupric sulphate,
thus doing away with the necessity of adding sodium sulphide in
the distillation. The ammonia formed was distilled into quarter-
normal hydrochloric acid, the latter being titrated with quarter-
normal ammonia, using congo red as an indicator.

Sulphur and phosphorus were determined in the customary
manner by evaporating a given volume of the urine — 25 c.c. for
each determination — in a roomy silver crucible with 10 grams of
pure sodium hydroxide (made from the metal) and 2 grams of
potassium nitrate, igniting the residue until oxidation was complete
and treating the fused mass with water. For sulphur, the mixture
was acidified with hydrochloric acid, evaporated to dryness, the
residue moistened with a few drops of hydrochloric acid and dis-
solved in hot water. The filtered solution was then precipitated in
the usual manner with barium chloride, the resultant barium sul-
phate filtered, ignited and weighed, thus giving data for calculation
of the total sulphur. For phosphorus, the aqueous extract of the
oxidized urine was acidified with nitric acid, evaporated to dryness,
the residue moistened with nitric acid and dissolved in warm
water. From this solution the phosphoric acid was precipitated
in the usual manner with molybdic solution and eventually trans-
formed into ammonio-magnesium phosphate. From the weight

7,;^2 R. II. Chittenden and William J. Gies.

of magnesium pyrophosphate obtained, the total phosphorus of the
urine was calculated.

Uric acid was determined by the well-known Salkowski-Lud-
wig silver method, using 100—200 c.c. of urine.

Phosphoric acid was determined by Mercier's * modification of
Neubauer's method, /. c\, by titration of 50 c.c. of urine with a
standard solution of uranium nitrate and tincture of cochineal as
an indicator.

Total sulphuric acid was estimated by diluting 25 c.c. of urine
with 3-4 volumes of water, adding 5 c.c. of dilute hydrochloric
acid, heating to boiling and precipitating hot with barium chloride.
The barium sulphate so obtained, after standing twenty-four hours
in a warm place, was washed with hot water until free from
chlorides and lastly with hot alcohol, ignited and weighed.

Combined sulphuric acid was determined by Baumann's
method, using lOO c.c. of urine. f

Chlorine was determined in 10 c.c. of urine by Neubauer and
Salkowski's modification of Mohr's method.]: Other methods
occasionally made use of are referred to in their appropriate places.

First Experiment. With Borax. — The animal made use of in
this experiment was a short-haired mongrel bitch weighing about
1 2 kilos. She was brought into a condition approximating to
nitrogenous equilibrium only after a preliminary period of nearly
three weeks, during which time superfluous fat was lost and she
became wholly accustomed to her surroundings. The daily food,
at the time the experiment actually commenced, consisted of
250 grams of the prepared meat, 70 grams of cracker dust, 40
grams of lard and 500 c.c. of water. It contained 9.814 grams
of nitrogen. This diet, with the above content of nitrogen,
was adhered to throughout the entire experiment of twenty-seven
days, the only variation being the slight changes in the amount of
nitrogen, to be seen in the tables, incidental to the use of different
lots of meat and in the employment of gelatin capsules during the
borax period. These gelatin capsules, in which the borax was
administered, contained 14.95 per cent, of nitrogen, the four cap-

*See Neubauer und Vogel's Analyse des Harns, neunte Auflage, p. 450.
tlbid., p. 417.
+ Tbid., p. 437-

Borax and Boric Acid on Metabolism. 333

sules used each day during the borax period containing 0.12
gram of nitrogen. This amount was naturally included in the
nitrogen of the food.

The experiment extended through twenty-seven days and was
divided into three periods of nine days each : a fore or normal
period during which no borax was given, a borax period dur-
ing which 45 grams of borax (5 grams a day) were adminis-
tered, and an after period when normal conditions again pre-
vailed. During the borax period of nine days the quantity of
borax given per day amounted to nearly 0.6 per cent, of the total
food and drink ingested, while of the solid food it formed 1.3 per
cent. This dosage of borax, considering the size of the animal,
was fairly large, and with this particular dog considerable diffi-
culty was experienced in inducing the animal to take it. At first
the borax was simply mixed with the food, but its presence was
quickly detected and the food refused, although it was eventually
coaxed down, but with some difficulty. After this first day the
borax was given in capsules, as already stated, and no further
difficulty of this sort was experienced. Three times during the
borax period, however, the animal was nauseated and vomited a
portion of the food, thus showing that this quantity of borax was
sufficient to disturb the physiological equilibrium of the animal.
The vomited matter was eventually eaten, however, later in the
day, so that this occurrence did not disturb the validity of the ex-
periment. It will be remembered that in Gruber's experiment
with a much larger dog (39 kilos) 20 grams of borax likewise
caused vomiting. In his experiment, however, the entire dose of
borax was taken at one time, while in our case, 2.5 grams were
given in the morning and a like quantity at night. Hence, taking
into account the weight of the dog, it might perhaps be argued
that 0.25 gram of borax to i kilo of body-weight will produce
vomiting. This, however, is very questionable, for in the above
exeriment the dog did not vomit until the afternoon of December
5, when she had already taken 12.5 grams of borax. In other
words, the animal was without doubt suffering in part from the
cumulative action of the salt. Thus, there was a slight attack of
vomiting again on the fifth day (December 7) and a final attack
on the eighth day (December 10). During the after period of

334 l"^- ^^- ClUTTKNUKN AM) WiLLIAM J. GlES.

nine days the animal was perfectly normal, and at the close of
the period, to again test the action of the borax, 5 grams were
given at one time shortly after the morning meal. Forty-fi\'e
minutes afterwards the animal vomited, and this occurred three
times during the forenoon. We are inclined to lay particular
emphasis upon this action of the borax because it tends to show
that in this first experiment the dosage of borax throughout the
nine days' period was as large as it well could be for this particular
animal without vitiating the experiment, and that the conditions
were therefore well adapted for bringing out distinctly any possible
influence the borax might ha\e upon the metabolic phenomena of
the body.

We would also call attention to the ob\-ious advantage —
in spite of the greater labor involved — of continuing experi-
ments of this character over comparatively long periods of time.
To be sure, in some cases where the substance being tested has
a marked physiological action, a single dose may show at once
the character of the influence exerted, but too often erroneous
conclusions are arrived at through negligence of this precaution.
Where, however, the substance under examination is given for five
to ten days consecutively, with careful examination of the excreta,
the chances of detecting minor influences are greath" increased,
and at the same time the danger of being led astray by a single
exceptional result — or by other possible errors — is greatly
diminished.

The table on page 335 contains the analytical results obtained
throughout the experiment.*

Referring now to the table containing the results of the first
■experiment, it is to be noted that in the fore period of nine days
the total nitrogen ingested amounted to 88.326 grams, while in
the urine excreted during this period there were contained 87.185
grams of nitrogen, and in the feces 2.122 grams, making a total
of 89.307 grams of nitrogen ; hence the nitrogen balance for the
period of nine days is — 0.98 1 gram. The body-weight remained

* The arrangement of daily records and summaries of the metabolism experiments
has been somewhat altered in reprinting. The data have been put in more condensed
form than in the original print. \othing has been omitted. Daily averages have
been added to the summaries.

Borax and Boric Acid on Metabolism.

Table I. First Experiment.

Date.

Body.

Food.

3

pa

Urine.

! Feces.

k6

Weight.

Nitro-
gen

Vol. ^

Sp.gr.

Reaction.

Nitro-
gen.

Uric Phos-
Acid. phorus.

Sul- Total Comb'd
phur. SO3. SO3.

Dry Nitro-
Weight. gen.

kilos.

grams.

gm.

c. c.

litmus

grams.

1. Fo7-e Pei-ioJ. Ante Days.

Nov.

24

25

26

27

28

29

30
Dec.

I

2

10.9
10.9
10.9
II. o
II. o
II. o

10.8

10.9

II. o

814
814

SI4
814
814
814
814

814

814

J505 IOI8

716 IOI8

773 1017

786 1016

650 1017'

415 1017

770 1019

575 1017

439 1018

Acid.

7.945 0.058 0.468
I. 361 .049 .646

.061

.049

.047
.040
.066

11.367
12.476
10.069
6.102
1 2. ^02

.688
•763
.5S5
•325
.760

8995
6.568

.040
.038

•505
.410

491 0.962
720 1.388
671 1.343
737 I-52I:
586 1. 214
3810.765
75« 1-554

570 1. 148
405 0.804

0.05S

■ 075
.077
.084
.064
.032
.084

.062
.055

38.15 2.122

II. Borax Period. Nine Davs.

4
5
6

7
8

9
10
II

10.9

9-903

5

II. I

9-933

5

II. 2

9-933

5

II. I

9-933

5

II. I

10.016

5

II. 2

10.100

5

II. 2

10.100

■■5

"■3

10. 100

5

11-3

10.100

5

796 1021

Acid.

368 1022

Alkaline

485 1025

520 1027

686 1024

422 1024

604 1023

498, 1026

602! 1020

13 3440.054
5.909 .032

9.183

10.043

12.823

7.412

10.742

9.846
8.825

.0391

.042;

.050'
.051

•049
.031

.06^

.821 0

789

1. 631

0

•321

371

0.705

•5.S5

527

1. 103

.5681

592

1. 197

.818 '

754

1.526

•444

426 0.82s

615

596 1.228

-521

600 1. 174

456

554

1.040

097
039
057

060

042
069
060

063

35-91

2.292

24-

1.627

III. After Period. Nine Days ,

12

13
14
15
16

17
18

19
20

981
981!
981
981
981
981
981
981:
036^

[488' 1019
670 1018

Acid. 8.727 0.0421 0.441 0.59611.0241 o

691

'551
681

595
572
630

549

1017
1016
1018;
1019
1018
1017
1019

'10.632'

10 047

7.804:

10.549

10.121

9-232

9-587

9-678

•053'
-039
•032
.051
.036
.036
.056'
.0441

•589
.621
.4S2
.694
.662
-■587
• 574
-574

.716 1.247
.742 1.265
.601 0.978

•736 i.345i
.662 1. 213
.613 1.119I
.610 1.083!
.616 1. 180

055
073
083
049

073
062
069
068
069

33-25

1.995

25.45 1.629

General Summary.

Total Nitrogen.

Urine.

Feces

0

Ingested.

Excreted.) Balance.

Vol.

Nitrogen.

Uric
Acid.

Phos- j.Sul-
phorus. i phur.

Total
SO3.

Comb'd Dry Nitro-
SO3. Weight. gen.

grams.

c.c.

1

grams.

Period Tot'ils.

I.

II.

III.

88.326

89-307

— 0.981 5629 87.185

0.428 5.150 5.319

10.699

0.591

38.15

1 90.118

92 046

— 1.928 4981 88.127

.411 5.099 5.209

10.429

-555

60.59

89.884

90.001

— 0.II7 5427 86.377

-389 5-224 5-892

10.454

.601

58.70

2.122

3-919
3.624

Daily Averages.

I.

9.814

9.923

—0.109 625

9.687

0.048

ii.

10.013

10.227

— -214 553

9-792

.046

11.

9.987

10.000

— .013 603

9-597

•0+3

0.572 0.591 I.IS9 ' 0.066 4.24 0.236

.567 .579 1. 159 .062 6.73 .435

.580 : .655 1. 162 i .067 I 6.52 .403

33^ R. H. CllITTENDKN AM) WiLLlA.M J. GlES.

practically constant. The slight excess of nitrogen excreted over
the amount ingested in this period is due possibly to lack of com-
plete involution of the mammary glands ; * the deficiency, how-
ever, is too slight, considering the length of the period, to need
much consideration. For comparison, the results of the three
periods, showing the relative excretion of nitrogen, may be ar-
ranged in tabular form :

Fore Period. Rorax Period. After Period,

Nitrogen of Food . . . . 88.326 90.118 89.884

Nitrogen of Urine . . . S7.1S5]- 88.127) . 86.377)

,.,. r r- ■89.307 ;- 92.046 "." ^ 90.001

Nitrogen of Feces .... 2.122 3-919 ) 3624 j

Nitrogen Balance . . — 0.981 — 1.928 — 0.117

Ratio of Urine Nitrogen to

Food Nitrogen . . . 98.6 per cent. 97.7 per cent. 96.0 per cent.

It is thus e\'ident that in this experiment, in spite of the large
doses of borax and the length of the period, proteid metabolism is
not modified in any noticeable degree. The amount of nitrogen
eliminated through the urine in proportion to the nitrogen of the
food, during the borax period, differs from that of the fore period
only to a slight extent, and this difference is due apparently to a
diminished assimilation of the proteid food. The change in the
nitrogen balance of the borax period is plainly caused by a slight
increase in the amount of fecal nitrogen, and not to increased
metabolism, thus indicating that the borax has a tendency to
diminish somewhat the absorption of proteid food, or possibly
leads to an increased secretion of mucus. When, however, the
nitrogen of the feces of the borax period is compared with both
that of the fore and after periods the increase is seen to be so
slight that it is perhaps unwise to attach much importance to it.
Certainly the borax, though given in doses sufficiently large to
keep the animal on the verge of nausea, did not in this experi-
ment interfei"e greatly with the digestion of any of the food-stuffs,
since the feces of the bora.x period are not much greater in
amount than those of the after period, though somewhat larger
in quantity than those of the fore period.

The weight of the animal during the twenty-seven days' period
showed a tendency to rise somewhat, /. c, from 10.9 kilos to 11.5

* Marcuse. Ueber den Nahrwerth des Caseins. Pfliiger's Archiv. f. d. ges.
Physiol., 1896, Band 64, p. 2-:3.

Borax and Boric Acid on Metabolism. 337

kilos. This, however, is not to be attributed to a laying on of fat
nor to a retention of nitrogenous matter by the body, but is the
result simply of a diminished excretion of water due to the pres-
ence of the borax. The results in this connection are in direct
opposition to those obtained by Gruber with single doses of borax.
There is here no suggestion whatever of an increased excretion of
water, but on the contrary, a very marked decrease. Thus, by
reference to Table I., it will be observed that during the fore period
the total volume of urine amounted to 5629 c.c. and the body
weight remained practically constant, i. c, 10.9— i i.o kilos. Dur-
ing the borax period, however, the volume of urine fell to 4981
c.c. and the body weight gradually rose to i 1.3 kilos, while in the
after period the volume of urine rose to 5427 c.c. with a constant
body weight of 1 1.5 kilos. It is thus quite clear that borax may
decidedly check the output of water through the kidneys, and
lead, as in this case, to its retention within the body.

Very noticeable also, in this experiment, was the sudden change
in the specific gravity of the urine, as also in the reaction of the
fluid, when borax was given. Thus, in the fore period the specific
gravity of the urine stood at 1017-1018, but at the opening of the
borax period it rose at once to 1022-1027, dropping back, how-
ever, as the borax was discontinued. Similarly, the reaction of
the normal urine was acid to litmus, but on exhibition of borax,
the reaction quickly changed to alkaline. The marked rise in the
specific gravity of the urine during the borax period is not due
solely to diminished elimination of water nor to increase in the
proportion of metabolic products, but mainly to the borax itself,
which is rapidly eliminated through the urine. We have not made
any special trial to ascertain how soon the borax appears in the
urine after its administration, but we have observed that the urine
collected on the first day of the borax period gives, after acidula-
tion with hydrochloric acid, a strong reaction with turmeric paper
for boric acid. Further, that the elimination of borax in the urine
is very rapid is manifest from the fact that, at the end of the borax
period, the animal having received 45 grams of the salt, no trace of
a reaction could be obtained with turmeric paper on the second day
of the after period. In other words, elimination of the borax was
practically complete twenty-four to thirty-six hours after the last

}^S R. H. Chittenden and William J. Gif^s.

dose had been taken. These observations accord with Johnson's
statements * that borax and boric acid begin to be eHminated
through the urine a short time after their administration.

While it is clear from a study of the nitrogen excretion that
proteid metabolism, under the conditions of this experiment, is not
materially affected by borax, the other analytical results must not
be overlooked. Thus, in the borax period the excretion of phos-
phorus, sulphur, total sulphuric acid and combined sulphuric acid
is slightly below that of the fore and after periods. The differ-
ences, however, are so small that it is perhaps unwise to draw any
positive conclusions from them, other than to admit their negative
character. It can certainly be asserted with perfect safety that the
borax has failed to exert any marked influence upon the excretion
of either sulphur or phosphorus. In this connection it will be
remembered that Forster t found, on feeding boric acid to man, a
marked increase in the output of phosphoric acid. Borax, how-
ever, certainly fails to produce an}' such results, its presence in the
body (of the dog) tending on the other hand to reduce the output
of phosphorus. Further, it is evident that the slight diminution in
the excretion of combined sulphuric acid is not sufficient to indicate
any inhibitory influence upon intestinal putrefaction. Lastly, the
figures obtained in connection with uric acid are such as to indicate
a purely negati\'e action.

Second Experiment. WitJi Boric Acid. — The animal experi-
mented on was a short-haired mongrel bitch weighing 8 kilos.
Nitrogenous equilibrium was quickly established on a daily diet
composed of i6o grams of the prepared meat. 40 grams of cracker
dust, 30 grams of lard and 400 c.c. of water. This diet contained
6.144 grams of nitrogen and was practically adhered to through-
out the experiment. The latter was of thirty daj's' duration, i. e.,
three periods of ten days each. During the middle, or boric acid
period, 1-2 grams of boric acid were given daily mixed with the
food, the animal taking it without the slightest reluctance and
without any apparent effect upon the appetite. No sign of nausea

* Johnson. Ueber die Ausscheidung von I'orsaure und Borax aus dem mensch-
lichen Organismus. Jahresbericht u. Thierchemie, 1885, p. 235. See also Vigier :
Note preliminaire sur Faction physiologique du borate de soude. Comptes rend. Soc.
de BioL Paris, 1883, p. 44.

t Forster. Archiv. f. Hygiene, 1884, Band 2, p. 75.

Borax and Boric Acid ox Metabolism. 339

or vomiting was seen. With 2 grams of boric acid per day the
mixture of food and drink contained 0.31 per cent., while the dry
food contained 0.86 per cent, of boric acid. The total amount of
boric acid given during the ten days was 14.5 grams.

During the fore period often days the animal received a total
of 61.440 grams of nitrogen. The nitrogen excreted through the
urine for this period amounted to 58.119 grams, while the feces
contained 3.203 grams, thus making a total of 61.322 grams ■
of nitrogen excreted, with a nitrogen balance of + 0.118 gram.
Plainly the animal was in a condition of nitrogenous equilibrium.
The table on page 341 contains the various data obtained.

The relative excretion of nitrogen for the three periods may be
seen in the following summary :

Fore Period. Boric Acid Period. After Period.

Nitrogen of Food 61.440 62.032 61.943

Nitrogen of Urine 58.1191 ^ 59.6001 ^ „ 58.970 I ,

-NT- rtr ^61322 ^^ o '63.538 ^ ^'^y 62.923

Nitrogen of Feces 3-203) 3-93S ) 3-944 J

Nitrogen Balance -^0.118 — I- 506 — 0.980

Ratio of Urine Nitrogen to

Food Nitrogen 94.5 per cent. 96.7 per cent. 95.2 per cent.

From these figures it would appear that there is a slight ten-
dency toward stimulation of proteid metabolism. When it is re-
membered, however, that the nitrogen balance for the boric acid
period, — 1.506, is the result often days' consecutive feeding with
boric acid, it is manifest that the stimulating action is very slight,
and our results may perhaps be considered as practically in accord
with those reported by Forster, who found that in man on a mixed
diet, boric acid in moderate doses (1-3 grams) was without in-
fluence on proteid decomposition as measured by the excretion of
urea. Upon the assimilation of the proteid food there is no evi-
dence of any action, /. e., the nitrogen content of the feces during
the boric acid period is essentially the same as that of the fore and
after periods. Further, the total weight of feces for each of the
three periods is so nearly the same, it is quite evident that assimi-
lation has not been materially interfered with. In this respect our
results fail to agree with those reported by Forster, who found
that small doses of boric acid (i gram in two days) given to a
man on a mixed diet, and on a milk and &g^ diet, increased the
excretion of feces ; this increase being due, according to Forster,

340 R- H. CuiJTKNDEN AND W'lLI.IAM J. GlES.

not to ain^ decrease in the assimilation of fat nor to increase in the
volume of the secretions, but to a decreased assimilation of the pro-
teid food under the influence of the boric acid. This difference in
our results ma)' of course depend upon the difference in the char-
acter of the animal species. In our experiment, the weight of the
animal remained perfect!}- constant throughout the entire period
of thirty days.

Unlike' borax, boric acid fails to produce an\' change in the
volume of the urine. Thus, in the fore period of ten days the
total volume excreted amounted to 4,647 c.c, while in the boric
acid period of the same length the total volume was 4,665 c.c, and
in the after period 4,644 c.c. Further, there is no marked difference,
to be measured by litmus paper, in the reaction of the fluid, although,
as Table II shows, alkaline reaction is more common in the nor-
mal periods than in the boric acid period. In the latter period,
however, the specific gravity of the urine, as might be expected,
shows a higher average than in the two normal periods. This is
due, as in the case of borax, to the rapid elimination of the boric
acid through the urine. The latter shows the presence of the acid
by the turmeric test on the first day of the boric acid period, while
on the second day of the after period all trace of a reaction disap-
pears, thus showing that the acid is rapidly eliminated from the
body and is practicalh' completch' removed twent)'-four to thirt)'-
si.x hours after the last dose.

Upon the elimination of uric acid, boric acid appears to have a
slight inhibitory effect, at least under the conditions of this experi-
ment, but upon the excretion of total and combined sulphuric acid,
chlorine and phosphoric acid, no tangible effect is produced. Cer-
tainly, the results in connection with combined sulphuric acid do
not indicate any retarding effect upon the putrefactive processes
in the intestine. In this connection it will be remembered that in
Forster's experiments on man doses of boric acid, corresponding
to those used by us, apparently gave rise to a marked retardation
in the amount of ethereal sulphate excreted. As a result, Forster
arrived at the conclusion that boric acid materially reduces intes-
tinal putrefaction. Our results, however, show no action of this
kind in the dog, and we are inclined to the \'iew that both borax
and boric acid are too rapidly eliminated from the system to be

Borax and Boric Acid on Metabolism.

341

Table II. Second Experiment.

Date.

Body.

Food.

•"•d

Urine.

Feces.

1897.

Weight,
kilos.

Nitro-
gen.

^*^'Vol.' Reaction.
Spgr.

Nitro-
gen.

Uric Total
j Acid. SO3.

Comb'd ; Chlo-
SO3. i rine.

Total
P.O..

Dry Nitro-
Weight gen.

grins.

gm. : c.c. litmus.

grms.

I. Fore Period. Ten Days.

Feb.
24
2^
26
27
28

Mar.

7-9

6.144

500

1015

7-9

6.144

I4S6

1012

7-9

6.144

460

1014

8.0

6.144

I410

1015

7-9

6.144

,5«i

10T4

8.0

6.144

I325

1014

7-9

6.144

'525

1016

7-9

6.144

440

1014

8.0

6.144

370

1014

7-9

6.144

550

1015

Acid.

Acid.

6.642

0.037

0.682

0.023

0.354

0.950

5.051

■055

.533

.021

.404

0.726

5-741

.048

.638

.029

.340

0.789

4.956

.049

.560

.024

.379

0.665

7.605

.096

.830

.036

-573

1-053 I

4.067

.033

•477

.020

.210

0.506

7-613

.052

.807

.040

.404

i.ooS

5-425

.057

.581

.022

.407

0.722

4. 1 19

.026

.464

.016

.291

0.540

6.900

.051

-749

.026

.462

1.004

6. 96 0.450

11.90 0.7S0
10.50 0.657

17.30 1.316

0.911

0.946

0.550 10.20 0.710

1-073

0.5S6 9.75 :o.66o

1. 017

0.849 16.30 1. 317

0.841

0.828 11.60,0.882

0-899 5-45 I0.369

II. Boric Acid Period. Ten Days.

6
io|

II!

12

13

14
15

6.144 I I470 1016; Acid. I 5.915

6.144 I 505 1016 " I 6.390

6.183 I :38o 1014 " ! 4.479

6.223 I 525 1017 " I 7.280

16.223 1.5 400 1016 Alkaline.! 4.166

6 223 1.5 530 1017 Acid. ! 7.460

6.223 1-5,460 1017 " 6.000

6.223 2 470 1017 " 6.035

6.223 2 I480 1017; " 6.032

6.223 2 '445! 1017! " 5.843

0.040 0.678
.712
•5"
• 767
.481
.803
.664
.6S2
.648
.683

.031
.028
.041
.026
.062'
.040
.041
-035
.042

0.025
.032
.020

-034
.017

-034
.031
.031
.027
.027

0.347

-451
-259
„j6
-333
-555
538
565
488

425

III. After Period. Ten Days.

16

17

7-9
8.0

6.223
6.223

18

7-9

6.223

19

7-9

6.223^

20

8.0

6.223

21

7-9

6.223

22

23

24
25

7.9
7-9
7.9
7-9

6.041

6.188!
6.188
6.188

1432 1016 Acid.
1360 1014 Alkaline
,560 1016
485 1015
425 1013
560 1017

Acid.

Alkaline.

Acid.

490 1015 Alkaline.
:45o 1015
480 1016 "
402 1015 "

6.100 0.057 0.670 0.026 0.346 0.813 5-45 0.369

4.318 .028 .526 .020 .226 0.526 7.71 .476

7.630 .096 .874 .048 .604. 1. 106

6.284 .052 .717 .033 j .448 0.890 8.82 .655

4.412 .040 .514 .023: .366(0.674

7.947 .069 .937 .053 I .74211.205 12.25 -8x4

5.922 .044 .678 .033! .528:0.831

4.940 .036 .575 .022 .416 0.739 10.47 .798

6.827 .037 .749 .038 .296 0.979

4.599 .049 .571 .020 .260 0.686 10.90 .832

General Summary.

Total Nitrogen.

Urine. ; . Feces.

Periods.

In- 1 Ex- ^ ,
gested. creted. Balance.

Nitro- ' Uric Total Combined Chlo- Total Dry ' Nitro-
^°1 gen. Acid. SO3. iO^. rine. V^O^. Weight gen.

grms.

c c. grms.

Period Totals.

I.

61.440 61.322

-^0.118

4647 58.119

0.504

6.321

0.257

3.824 7.963

46.66

11.

62.032 63.538

—1.506

4665 59.600

.386

6.629

.278

4.317 8.500

53-30

11.

61.943' 62.923

— 0.980

4644 58.979

.508

6.8II

.316

4.232 8.449

55-60

3-203

3-938

3-944

Daily Averages.

I.

6.144

, 1
6.132:

-f-O.OI2 1

465

5.812

0.050

0 632

0.026

0.382

0.796

4.67

0.320

II.

6.203

6.354

- .iSi'

467

5.960

-039

.633

.028

-432

.850

5-33

.394

111.

6.194

6.292

— .098

404

5.898

.051

.681

.032

■423

.845

5-56

•394

342 R. H. Chittendhn and William J. Gies.

very effective in the intestine. As already stated, the elimination
of borax and boric acid through the urine commences almost im-
mediately after their ingestion, and it is very questionable, therefore,
whether, with moderate doses of these substances, enough would
remain unabsorbed at the lower end of the small intestine to exert
much influence upon the growth and development of microorgan-
isms. Certainly, the feces do not ordinarily contain any appreci-
able amount of borax or boric acid after these substances have
been administered in moderate quantities, although obviously the
length of time the feces are forming will have some influence
upon their content of soluble matter. In only one instance, to be
detailed later, where a very large dose of borax was given, could
any decided reaction for boric acid be obtained in the feces. John-
son * states that in the case of the human organism borax and
boric acid show great irregularity in their appearance in the feces,
and that he was able to detect them in the latter only in six cases
out of fourteen, although daily doses of 0.9-3.0 grams of boric
acid were given.

Lastly, it is to be noted that in our experiment with boric acid
there is no such increase in the excretion of phosphoric acid
through the urine as was observed by Forster ; our results, indeed,
fail to show any distinct influence exerted by boric acid upon the
metabolism of phosphorized matter.

TJiird Experiment. WitJi Borax and Boric Acid. — This ex-
periment was divided into seven periods of eight days each, thus
making a total of fifty-six consecutive days during which the
variations in the composition of the urine and feces were followed
as before, under the influence of both borax and boric acid. The
object in extending the experiment through this lengthy period
was to ascertain whether prolonged treatment with borax and
boric acid might not eventually result in such a disturbance of
physiological equilibrium that more positive data would be
obtained. With this end in view, a mongrel bitch of ten kilos
body-weight was brought into nitrogenous equilibrium, after which
the urine and feces were analyzed for eight consecutive days, i. e.,
the fore period. Borax was then given with the food for eight

* Johnson. Ueber die .Kusscheidung von Borsaure und Borax aus dem mensch-
lichen Organismus. Jahresbericht ii. Thierchemie, 18^5, p. 235.

Borax and I^oric Acid on Metabolism. 343

days, making the first borax period. This was followed by
another period of eight days during which neither borax nor boric
acid were administered, after which came a third period of eight
days when boric acid was fed. This, in turn, was succeeded by a
normal period of equal length, followed by eight days of borax
treatment — the second borax period — concluding with a final
after period of eight days, /. e., a total of fifty-six days. By thus
keeping the same animal under continuous observation for this
length of time it might reasonably be expected that any cumula-
tive action — assuming it to exist — would be clearly manifest.
Further, considerably larger daily doses of borax and boric acid
were administered than in the preceding experiments.

The daily diet made use of throughout the entire experiment
consisted of 160 grams of the prepared meat, 40 grams of cracker
dust, 30 grams of lard and 430 c.c. of water. Its exact con-
tent of nitrogen is shown in the table of the fore period. (Table
III.) The total amount of nitrogen ingested during the fore period
was 52.163 grams. The amount excreted during the same
period v/as 51.734 grams, thus showing a nitrogen balance for
the eight normal days of -f- 0.429 grams. The dog used in this
experiment, although short-haired, lost considerable hair daily.
This was therefore collected and at the end of each period its con-
tent oi nitrogen was determined and the amount added to the
nitrogen of the urine and feces, as seen in the accompanying
table. It is interesting to note in this connection that the loss of
hair in periods of eight days' duration may be considerable ; so
large, indeed, that an appreciable loss of nitrogen may result.
Thus, in the seven periods of this experiment the total amount of
hair shed was 61.98 grams, i. c, 8—10 grams for each period, the
total nitrogen thrown ofT in this manner amounting to 7.856
giams. These figures show that the hair shed contained 12.6
per cent, of nitrogen. Obviously, in careful experiments, this
source of loss cannot be overlooked.

In the first borax period of eight days the daily dose of borax
ranged from 2 to 5 grarns, the total amount administered being
32.5 grams. In the following boric acid period the daily dose
ranged from i to 3 grams, a total of 17 gram.s of boric acid
being given. On commencing the second borax period the daily

344

R. H. Chittenden and William ). (iiEs.

Tahlf, III. (First Part). Third Kxi'kriment.

21
22
23

24
25
26

27
28

Date.

Body. 1 Food. {

Urine.

Feces.

;s..

Drug.
Weight. Nitrogen.

Vol.
cc.

^ ufn" Nitrogen.
Sp. gr.

Uric
Acid.

Total Comb.
SO3. i SO3

Total
P.O..

Drj- Nitro-
Weight. j gen

kilos. grms.

llitmus.

grms.

lO.O
lO.O

10.0
10. 1

lO.O
lO.O
lO.O

9-9

I. Fore Period. Eight Days.

6-593

490

1015

6.770

470

1 01 3

6.770

540

1016

6.406

440

1014

6.406

640

1015

6.406

465

1012

6.406

'525

1014

6.406

|626

1015

Acid. 6.160 0.040 0.525 0.042 0.981

5.050 .032 .437 .036 0.697

" 7139 042 .641 .071 1. 117

5.231 .028 .4S9 .059 0.779

" 7.685 .060 .746 093 1.329 14.33 0.846

4.643 .031 .!J20 .0390.6381 j

" 5.641 .047 .544 I .061 0.862 I

" I 7-544 i -035 I -713 i -094 ^Zll 10.36 I 0.571

II. First Borax Period. Eight Days.

29

lO.O

6.406

30

10. 1

6.406

May

{

lO.O

6.406

2

lO.O

6.406

3

9-9

6.406

4

10. 1

6.285

5 10. 1

6.285

6

10.0

6.285

orax
2

400

1015 Alk.

4.025

0.039

0.

3

400

I022 "

6.738

.043

4

591

IOI8 "

6.542

.042

4

470

1 02 1 "

7.028

.042

4-5

520

IOI7 "

5.916

.031

5

3S0

IOI7 "

4.041

.024

5

460

1022 "

6.531

.040

5

540

1022 "

7.503

-032

401 0.049 0.597
677 .091 I. 142

704

797
565
372
597
792

,107

.0S9 I.

.126 i.o»9

.072 0.781

.040 0.409

.082 0.977

.113 1.272

2.96 0.163

20.10 0.990

20.69 1023

III. First After Period. Eight Days.

7

10.1

6.285 '

410

1015 1 Acid.

5

687

8

10.2

6.285

430

1012 "

4

3.30

9

10. 1

6.285

590

1016 "

7

671

10

10 2

6.428

390

1014

4

717

11

10.0

5.428

597

1015 "

7

425

12

lO.O

6.428

530

1013 "

5

952

n

10.1

6.428

525

1014 "

5

894

14

10. 1

6.428

490

1013 "

5

754

0.03b

.034
•044

.014

.036

.029
.029
.026

0-575

.449
.623

•563
.872
.586
.620
.568

0.0541 0.810

.040 0.458

117 1.187

.065
.106
.060
.065
•055

0.599
1.423
1.066
1.017
0.959

19.55

11.90
8.21

.627
•473

IV, BoHc Acid Period. Eight Days.

15
16

17
18

19
20
21
22

10. 1
10. 1
10.2
10.2
10. 1
10.2
10.2
10.3

6.428

6.396
6.396
6.396
6.396
6.396
6.396
6.396

Boric
Acid.

1

I

1-5
2

2-5

525
441
401
490
555
465
400
500

1015 Acid.

5677

0.039

0

1015 "

5424

•035

1014 "

4.247

•05.?

1015 "

5.909

.018

1016 "

6.934

.031

ioi6 "

6. 131

.041

1014 "

4.588

-034 .

1018 "

7.029

-059

558 0.068 1.003 2.73 0.157

627 .066 0.785

454 .038 0.502

637 .076 0.927 9.90 .529

734 .100 1.184

606 .080 0.806 9.68 .557

457 .042 0.467

689 .099 1.080 11.86 .579

V. Second After Period. Eight Days.

23

24
25
26

27
28
29
30

10.3
10.3

lO.I

10.2
10. 1
10. 1

10.2
10.3

6.396
6.396
6.410
6.410
6.410
6.410
6.410
6.410

402 1015 Acid.

5.424

0.051

0.

445 loio Alk.

3-957

.028

620 1014 Acid.

7.224

.066

521 1013 "

5-730

.051

550 1014 "

5-614

-039

470 1016 "

6.518

-033

455 1013 Alk.

4-994

.041

480 1017 .\cid.

6.977

■045

-597 0.075 0.671 3.95 0.192

.394 .031 0.289

.787 .077I 1.115

.541 .045 0.911 9.76 .542

.601 .050 0.892

.722 .066 1.103

.549 043 0.692

.769 .065 I. 113 15.04 .731

Borax and Boric Acid on Metabolism.

345

Table III (Second Part). Third Experiment.

Date.

Body.

Food.

Urine.

Feces.

May.

Weight.

Nitrogen

Drug.

Vol. ^^^'^- Nitrogen.; Uric Total Comb.
Sp. gr.' "°"- 1 : ^"'^- ^^3- SO3.

Total
P2O5.

Dry
Weight.

Nitro-
gen.

kilos. [ grms.

c.c. ! j litmus. grms.

VI. Second Borax Period. Eight Days.

31

June

I

2

3

4
5
6

7

lo.i

10. 1

10.3
10.4
10.3
10.2
10.2
10.3

6.410

6.410
6.410
6.410
6.392
6.374
6-374
6.374

Borax
10

10

5
6

7
10

530 1 1027

Alk.

420

360

1029
1026

342

S40

1020
1022

450
502

513

1025
1023
1023

7. 711
6.384

6.627

4.574
8.025

5-634
6.495
6.913

0-053
-037

.029
.029
.042

.031

.040

•034

0.836 0.070

671
761

495
828
610
629
683

.052

-099
.038

•"3

.077
.070
.076

1.474

1. 102

0.998

0.336
1. 112

0.680
1. 005
0.809

19.10

19.40

17.55

0.874

1. 019
0.844

VII. Third After Period. Eight Days

8

10.3

6.374

411

1016

Acid.

6.213

0.042

0.567

9

10.3

6-374

525

1013

"

5-834

.029

-558

10

10.4

6.374

422

lOII

a

Ar.2.d,^

.042

-447

II

10.4

6.374

500

1014

((

6.149

-037

.638

12

10.4

6.374

525

1016

a

7.560

.051

.781

13

10.3

6.409

503

1013

Alk.

5-158

-043

.518

14

10.2

6.445

652

1015

Acid.

7.917

.040

.856

15

10.2

6.445

512

1012

"

5-663

•044

.631

0-033

•043
.047

0.644

0.686

0.399
.058I 0.872

•075} 1-344
.030! 0.644
.082! I 445
-065 i 0.793

22.80

0.895

20.06 j I. 194

General Summary.

Total Nitrogen.

Urine.

Feces. ) Hair.

Period.

In- 1 Ex-
gested. 1 creted.

Balance.

Vol.

Nitro- Uric • Total \ Comb. Total
gen. ; Acid. SO3. ; SO3. P^Os.

Dry

Wght.

Nitro-
gen.

Nitro-
gen.

grms.

c. c. 1 grms.

Period Totals.

I. Normal....

52-163

51-734

+0.429

4196 49.093 0.315

4.515

0.495

7-7.38

24.69

1. 417

1.224

II. Borax ( i).

50.885

51.686

— 0.801

3761 48.324, -293

4.905

.662

7.374

43-75

2.176

1. 186

III. After

50.995

50.3341+0.661

3962 47-430 .248

4.856

.562

7.519

39-66

1.845

1-059

I^ . lioricAcid

51.200

49.026; -[-2.174

3777 45.939 .310

4.762

•569

6.754

34-17

1.822

1.265

V. After

51-252

49.130-1-2. 122

3943: 46.438 -354

4.9fo

-4S2

6.786

28.75

1.465

1.227

VI. Borax (2).

51.154

56.032-4.878

3657 52.363 .295

5.513

.505

7.516

56.05

2.737

0.932

VII. After

51.169

51.830J— 0.661

4050J 48.778, .328

4.996

•433

6.827

42.86

2.0S9

0.963

Daily Averages.

I. Normal....

II. Borax (i).

III. After

IV. Boric Acid
V. After

VI. Borax (2).

VII. After

0.520
6.361

6-374'
6.400
6.406
6.394

6.396

6.467+0.053
6.461! — o. 100

6.292
6.128
6. 141
7.004

6.479

-0.082
-0.272':
-0.265
0.610
-0.083

525

470
495
472I
493
457
506

6.137I0.039
6.041 { .037

5.9291
5.7421
5.805'
6.545

6.097

.031
-039
-044
.037

.041

0.564

0.062

0.967

3-09

0.177,

-613

-083

.922

5-47

.272

.607

.070

.940

4.96

-231

-595

.071

-844

4-27

.228

.620

.056

.848 1 3.59

.183

.689

.074

.939 7.01

.342

.624

-054

-853' 5-36

.261

>-i53
.148
.132
-158
-153
.116
.120

346

R. H. Chittenden .and William J. Gies.

dose of borax was placed at lo q,iams. This was continued
for two days, but on the third day aflcr taking the morning dose
of 5 grams the animal's ap[)etite began to fail so that it became
necessary to coax her considerably in order to have the day's
ration consumed. On this day, therefore, only 5 grams were
given, but on the following day the appetite was nearly normal
and 6 grams of borax were given. The dose was then raised
to 10 and 8 grams daily, as shown in Table III. a total of 64
grams of borax being given in this period of eight days. Through-
out the entire experiment of fiUy-six days the animal remained
perfectly well, kept a fairly constant body-weight, and showed
no symptoms of nausea or vomiting during the administration
of either borax or boric acid. The only noticeable effect was
a seeming loss of appetite on one day, as mentioned above. At
the termination of the final after period, a single do.se of 5
grams of boric acid was given. This resulted in vomiting 4-5
hours afterward.

The relative excretion of nitrogen for the seven periods is
shown in the following summary :

Foi e Period.

Nitrogen of Food 52.163

Nitrogen of Urine 49.093 )

Nitrogen of Feces I.4i7 [- 51.734

Nitrogen of Hair 1.224)

Nitrogen Balance ... + 0.429
Ratio of Urine and Ilaii
Nitrogen to Food Ni-
trogen 96.4 per cent.

4.
Boric Acid Period.

Nitrogen of Food 51.200

Nitrogen of Urine 45-939 I

Nitrogen of Feces 1.822 ^ 49.026

Nitrogen of Hair 1.265 J

Nitrogen Balance ... -|- 2.174
Ratio of Urine and Hair
Nitrogen to l-ood Ni-
trogen 92.2 per cent.

First Bora.x Period.

First After Period.

50.885

48.324)
2.176 ' 51.686

i.ise]

— 0.801

50.995
47-430 )
1.845 I 50.334

1-059 J

+ 0.661

97.2 per cent.

95.0 per cent.

5-
Second After Period.

6.
Second Borax Period.

51.252
46.438 1
1.465 ^ 49.130
1.227 j

51.154
52.363 )
2.737 I 56.032
0.932]

+ 2.122

93.0 per cent.

104. 1 per cent.

Borax and Boric Acid on Metabolism. 347

7-
Third After Period.

Nitrogen of Food 5 1 • 1 69

Nitrogen of Urine 48.778^

Nitrogen of Feces 2.089 I 51-830

Nitrogen of Hair 0.963 J

Nitrogen Balance — 0.661

Ratio of Urine and Hair
Nitrogen to Food Ni-
trogen 97-2 per cent.

In the first borax period of eight days with a total consump-
tion of 32.5 grams of borax, i. c, an average of 4 grams per
day, there is practically no change in the rate of proteid metab-
olism. There is, however, a slight rise in the amount of fecal
nitrogen similar to that noticed in the first experiment with borax,
by which the nitrogen balance is somewhat changed, but there is
plainly no effect produced on proteid metabolism. In the second
borax period, on the other hand, there is evidence for the first
time of a distinct and unquestionable influence upon proteid metab-
olism. In this period of eight days 64 grams of borax were
administered, and under its influence the excretion of nitrogen
through the urine was greatly increased. As in the other experi-
ments, the proportion of nitrogen in the feces was likewise in-
creased, implying decreased assimilation of proteid food, but the
nitrogen balance of — 4.878 is mainly due to direct stimulation
of proteid metabolism. When, however, it is considered that to
accomplish this result a daily dose of 8 grams of borax was re-
quired, and for eight consecutive days, with a dog weighing only 10
kilos, it is very plain that proteid metabolism is not readily affected
by borax.

In the boric acid period of eight days, with a total dosage of
17 grams of the acid, there is some evidence of the dimin-
ished proteid metabolism. The excretion of nitrogen through the
urine is certainly diminished ; there appears to be a sparing of pro-
teid, but it is to be noticed that in the period following, the nitro-
gen balance remains unaltered, which fact casts some doubt upon
the assumption that the result is due solely to the acid. It is of
course possible that the action of the boric acid may be continued
into the after period, but this we should hardly expect in view of
the rapid elimination of boric acid from the system. Further,

34<S R. H. Chittendkn and William J. Gies.

after the second borax: period, where the nitrogen bahmce is so
noticeably disturbed, there is a quick return to the normal, the
nitrogen balance for the final period dropping back to —0.661
gram. Consequently, while the analytical data show a retention of
nitrogen during the boric acid period, thus indicating diminished
proteid metabolism, we feel some hesitation in attributing the result
wholly to the boric acid, particularly as the earlier experiment with
boric acid gave essentially negative results.

Especially noticeable in this experiment, as in the earlier ex-
periment with borax, is the action of the latter agent in reducing
the volume of the urine. (See Table III, General Summary.)
In both borax periods the total volume of urine excreted is dis-
tinctly reduced, and the same holds true in this experiment with
the boric acid. It is quite probable that the somewhat larger
daily dose of boric acid made use of in the present experiment is
responsible for this result, although it is possible of course that the
personality of the animal may have had some influence. In the
previous experiment with boric acid, where the maximum daily
dose was 2 grams, the volume of the urine was unaltered. In
view of these facts it is perhaps proper to consider the larger
dosage of boric acid used in the present experiment as responsible
for the apparent action upon proteid metabolism likewise.

Also noticeable in this experiment is the influence of the larger
doses of borax upon the excretion of total and combined sulphuric
acid. Both of these are distinctly increased in amount during the
last borax period, in harmony with the increase in proteid metab-
olism, and there is a suggestion of the same influence in the first
borax period. Moreover, in the last borax period the excretion of
phosphoric acid is noticeably increased, while the elimination of
uric acid is slightly diminished. It is thus plainly evident, as al-
ready stated, that while moderate doses of borax, even long-con-
tinued, are without influence upon the nutritional processes of the
body, large doses may distinctly increase the rate of proteid metab-
olism, giving rise not only to an increased excretion of nitrogen,
but also of sulphuric acid and phosphoric acid.

In all of these experiments with borax there is constant evi-
dence of an increase in the weight of the feces during the borax
periods. This increase in weight is due in part to an increased

Borax and Boric Acid on Metabolism. 349

output of nitrogenous matter through this channel, but whether
the latter is caused by diminished digestion and absorption of the
proteid food or to a stimulation of the mucus or other secretions
from the gastro-intestinal tract is not so clear. It has been plainly
shown, however, in another connection * that while borax in mod-
erate quantities has no inhibitory action whatever on either gastric
or pancreatic digestion of proteids, larger proportions do retard
the proteolytic action of both digestive fluids. Further, retarda-
tion of proteolysis with borax is much more pronounced than with
boric acid ; hence it seems quite probable that the increased bulk
of feces and the higher content of nitrogen therein during the
borax periods is due mainly to slight retardation in the assimila-
tion of proteid food.

Large amounts of borax likewise interfere with the assimilation
of fatty foods ; a statement which does not appear to be true of
boric acid. In the accompanying table (Table IV) are given the
results of our analyses of the dry feces, from a study of which it
is plain that under the influence of large doses of borax — first and
second borax periods of experiment third — both the total and
percentage amounts of ether-soluble matter in the feces are greatly
increased. Boric acid, on the other hand, produces no such effect_
In the first experiment, with borax, the evidence of decreased fat
absorption is less pronounced, although both the dosage of borax
and the amount of fat fed were greater than in the first borax
period of experiment third. Quite possibly this apparent differ-
ence in action may be due to the personality of the animal. How-
ever this may be, it is plain that large doses of borax are prone to
increase somewhat the bulk of the feces, in part by diminishing
slightly the assimilation of both proteid and fatty food, and in part,
we think, through a tendency to increase the secretion of mucus.
Thus, we observed in the last experiment, during the. period when
the largest doses of borax were given, that the feces were more
sUmy than in the normal periods, and appeared to contain more
mucus than ordinarily. Further, it is to be noted that under the
influence of large doses of borax there is a tendency toward diar-
rhoea ; not very marked to be sure, but sufficient to render the
discharge of feces somewhat watery.

* Chittenden. Influence of Borax and Boric Acid, on Digestion. Dietetic and
Hygienic Gazette, 1893, vol. g, p. 25.

j:»^

R. H. CllITTENDEX AND WiLLI.Wl J. GlES.

In spite of these evidences of minor action in the intestinal
tract with large doses of borax, there is no evidence whatever of
any influence exerted upon intestinal putrefaction, either by borax

Table IV. Content of Fat and Other Ether-Soluble Matter in the Feces.

EXI'ERIMENT I.

Date. Feces

Ether-soluble Period.
Matter.

1895. Of7^W«Bit. pg^^^„,_ Q^,^3

Dec. 2

38.15

35-03

13.362

Fore

7
II

35-91
24.68

33-60
25-23

12.067
6.227

60.59

30.02

18.294

Borax

i6

20

33-25

25-45

36.51
24.36

12.140
6.198

58.70

31-24

18.338

After

Date.

1897.

Feb. 26
Mar. 2

3
5

Experiment II.

Feces. Ether-soluble
Matter.

Period.

6.96 23.70

11.90 17.88

10.50 16.95

17.30 20.82

Grams.

1.649
2.128
1.770
3.602

46.66 19.61 9.149 Fore

10
12
14
15

16
17
19
21

23
25

10.20

9-75
16.30
11.60

5-45

18.87
17.67
20.31
20.60
20.54

1.924
1-723
3-3"
2.390
1. 119

Experiment III.

Date. Feces. Ether- soluble
Matter.

1897. Dry Weight. p„ „„t.' Oram:.
jrsiuiS.

4-134
3.029

Period.

Apr. 25 14.33 28.91
28 10.36 29.09

May

29
3
6

II
13
14

24.69 29.01 7.163 Normal

2.96 29.09 0.840

20.10 36.35 7.306

20.69 37-o6 7.671

43-75 36.15 15-817 Borax

19.55

TI.90

8.21

36.18
23.50
25.89

7.091
2.797

2.II7

39.66 30.27 12.005 After

15 2.73 25.89 0.705

18 9.90 33.19 3.286

20 9.68 25.76 2.499

22 11.86 24.13 2.858

53.20 19.67 10.467

Boric
Acid

5. 45

7.71

8.82

12.25

10.47

10.90

20.54
26.63
20.28
20.72
20.01
19-31

1. 119

2.053
1.789

2.538
2.095
2.105

55.60 21.04 11.699 After j

]une

34-17

27.36

9-348

Boric

Acid

23
26

30

3.95

9.76

15.04

24.13
24.20
29-54

0.953
2.372
4-443

28.75

27.02

7.768

After

2

5
7

19.10
19.40
17-55

4501
39.06

33-94

8.596

7.579
5-940

56.05

39-46

22.115

Borax

12
15

22.80
20.06

39.27
29-99

8.954
6.028

42.86 34.96 14.982 After

or boric acid. Even with the largest doses of borax the com-
bined sulphuric acid of the urine is raised rather than lowered,
and careful examination of the urine daily with Jaffe's indoxyl test
ailed to reveal any indications pointing to an inhibitory influence

Borax and Boric Acid on Metabolism. 35

exerted by either borax or boric acid upon the production of in-
dican. If, however, one studies carefully the output of combined
sulphuric acid as shown in the various tables it will be noticed
that the highest figures are generally obtained on the day (or the
day preceding that) on which the dog defecates ; while after de-
fecation the combined sulphuric acid of the urine falls at once. In
other words, the natural obstruction of the intestine favors, as is
well known, the absorption of putrefactive products, and thus leads
to an increase of combined sulphuric acid in the urine. When, on
the other hand, defecation occurs, the combined sulphuric acid of
the urine is at once diminished in amount. Upon these natural
fluctuations of combined sulphuric acid even the largest doses of
borax and boric acid are without effect, not because these agents are
without influence upon microorganisms, but because they are too
rapidly and completely absorbed from the intestine to exert much
influence upon intestinal putrefaction. In only one instance were
we able to detect any boric acid in the feces, viz., on June 5, at a
time when the largest doses of borax were being given ; and at the
close of this period the boric acid reaction could be obtained Avith
the urine only on the first day of the after period, so rapidly was
the borax passed out of the body.

Lastly, attention may be called to the constant presence, in
appreciable amounts, of uric acid in the urine of all animals experi-
mented with, in opposition to the older statements of Liebig * and
others that kynurenic acid may entirely replace uric acid in the
urine of the dog. Our results, so far as they extend, are thus
wholly in accord with the recent observations of Solomin.f We
have, however, made no attempt to determine the amounts of
kynurenic acid present.

General Conchcsions. — Moderate doses of borax up to 5 grams
per day, even when continued for some time, are without influence
upon proteid metabolism. Neither do they exert any specific in-
fluence upon the general nutritional changes of the body. Under
no circumstances, so far as we have been able to ascertain, does
borax tend to increase body-weight or to protect the proteid
matter of the tissues.

* Liebig. Annalen d. Chem. u. Pharm. Band 86, p. 125.

I Solomin. Zur Kenntniss der Kynurensaure. Zeitschr. f. physiol. Chem., 1897,
Band 23, p. 497.

352 R. H. Chittenden' and William J. Gies.

Large doses of borax, 5-10 grams daily, have a direct stimu-
lating effect upon proteid metabolism, as claimed by Gruber ; such
doses, especially if continued, lead to an increased excretion of
nitrogen through the urine, also of sulphuric acid and phosphoric
acid.

Boric acid, on the other hand, in doses up to 3 grams per day,
is practically without influence upon proteid metabolism and upon
the general nutrition of the body.

Borax, when taken in large doses, tends to retard somewhat
the assimilation of proteid and fatty foods, increasing noticeably
the weight of the feces and their content of nitrogen and fat. With
very large doses there is a tendency toward diarrhcea and an in-
creased excretion of mucus. Boric acid, on the contrary, in
doses up to 3 grams per day, is wholly without influence in these
directions.

Borax causes a decrease in the volume of the urine, changes
the reaction of the fluid to alkaline, and raises the specific gravity,
owing to the rapid elimination of the borax through this channel.
Under no circumstances have we observed any diuretic action with
either borax or boric acid. The latter agent has little effect on
the volume of the urine.

Both borax and boric acid arc quickly eliminated from the
body through the urine, twenty-four to thirty-six hours being gen-
erally sufficient for their complete removal. Rarely are they
found in the feces.

Neither borax nor boric acid have any influence upon the putre-
factive processes of the intestine as measured by the amount of
combined sulphuric acid in the urine, or by Jaffe's indoxyl test.
Exceedingly large doses of borax are inactive in this direction, not
because the salt is without action upon microorganisms, but
because of its rapid absorption from the intestinal tract.

Borax and boric acid, when given in quantities equal to 1.5-
2.0 per cent, of the daily food are liable to produce nausea and
vomiting.

Owing to the rapid elimination of both borax and boric acid, no
marked cumulative action can result from their daily ingestion in
moderate quantities.

At no time in these experiments was there any indication of
abnormality in the urine ; albumin and sugar were never present.

17

Reprinted from American Medicine, August 23, 1902, vol. iv, p. 293.

ON THE INFLUENCE OF THE CONTENTS OF THE
LARGE INTESTINE UPON STRYCHNIN.

By William Salant, B.S., M.D.,

Fellow of the Rockefeller Institute, New York.

[A preliminaiy comrounication of an experimental study from the Rockefeller
Institute for Medical Research. Carried out under the direction of Dr. S. J. Meltzer,
New York.]

In the study of Meltzer and Salant * on the effect of submini-
mum doses of strychnin upon nephrectomized rabbits the remark-
able fact was found that notwithstanding the removal of the chief
eliminating organs, the kidneys, a good deal more than the fatal
dose can be gradually injected into these animals with hardly any
cumulative effect. As a possible explanation of this phenomenon
it occurred to us that after the removal of the kidneys perhaps
vicarious elimination into the gastrointestinal canal becomes devel-
oped. The appearance of urea in the alimentary tract in advanced
cases of nephritis seems to favor such a suggestion.

To test experimentally this hypothesis, I set out to examine
for strychnin the contents of the gastrointestinal canal of nephrec-
tomized rabbits, which gradually received a considerable dose of
strychnin. Of the methods for the separation of strychnin I first
employed those of Otto Stas and of Dragendorff, but later I fol-
lowed out scrupulously the method described by Raines. t

In four experiments in which the nephrectomized rabbits re-
ceived gradually doses of strychnin amounting respectively to 2,
3.5, 6 and 8 mgrs. the entire contents of the gastrointestinal canal
including the feces were carefully searched, but no strychnin could
be detected. To test the efficiency of the method i mgr. of strych-
nin was added respectively to blood, crushed brains, liver, etc. ;
here strychnin was easily detected.

* Meltzer and Salant. Journal of Experimental Medicine, 1902, Vol. iv.
t Haines. See Allen McLane Hamilton's System of Legal Medicine, 1894, Vol.
i, pp. 451 to 459.

353

354 William Salant.

However, before deciding that in our nephrectomi/.ed rabbits
the strychnin was not eliminated into the alimentary canal, i mgr.
of strychnin was added to the gastrointestinal contents of these
animals, and, to our surprise and disappointment, the strychnin
could not be detected. To exclude the bare possibility that in
nephrectomized animals some unknown substance is deposited in
the gastrointestinal canal which prevents the detection of strych-
nin, the contents of the gastrointestinal canals of normal rabbits
were taken for further experimentation.

In three e.Kperiments i mgr. of strychnin was added to the
entire gastrointestinal contents of one animal ; 2 mgrs. was added
to the entire gastrointestinal contents of the second animal, and i
mgr. was added to half of the contents of a third rabbit ; in none
of these e.Kperiments could strychnin be found, carefully as it was
searched for. Since there was no difficulty in detecting strychnin
in any other mi.Kture of organic substance, the failure to detect it
here could not be ascribed to imperfect methods or faulty
technic.

But now this outcome appeared to have an important practical
bearing. In numerous medicolegal cases it was claimed that
strychnin was found in the stomach of poisoned human beings,
and this finding had probably assisted more than once in deciding
the fate of an accused. The question was now, How did the
medicolegal experts succeed in separating and detecting strychnin
in the contents of the gastrointestinal canal ? Is it different with
the contents of the gastrointestinal canal in human beings? Here
another idea occurred to us. In most of these cases it was the
contents of the stomach alone in which strychnin was found. We
therefore started to examine separately the contents of some of
the sections of the alimentary tract of the normal rabbit, with the
following remarkable results :

One mgr. of strychnin added to the contents of the stomach
alone : strychnin easily detected ; i mgr. of strychnin added to the
contents of the small intestine : strychnin detected ; i mgr. of
strychnin added to the contents of cecum and colon : no reaction
of strychnin. The same uniform results were obtained in repeated
experiments. It is, then, the contents of colon and cecum alone
in which strychnin cannot be detected.

Quantitative Determination of Strychnin. 355

This failure to recover strychnin could be interpreted to mean
simply that the methods followed for its detection in organic sub-
stances are not adapted for its separation from the contents of the
cecum and colon of rabbits. However, we have seen that strychnin
could not be detected in the contents of the entire gastrointestinal
canal, which could only mean that the presence of the contents of
the large intestine prevents the detection of strychnin, even when
dissolved in another medium. This fact was now established
again directly by the following experiments :

One mgr. of strychnin was added to two thirds of the contents
of the stomach, with which one third of the contents of the large
intestine was mixed : strychnin could not be detected. One mgr.
of strychnin added to the contents of the small intestine, to which
some of the contents of the large- intestine were admixed : here,
likewise, no strychnin was found. The presence of some of the
contents of the large intestine then prevents the detection of
strychnin in any other part of the contents of the alimentary
canal.

This series of experiments leads up apparently to the very
remarkable conclusion that the contents of the large intestine of
normal rabbits contains something which, to say the least, inter-
feres with the detection of strychnin (even 2 mgs.) by the methods
at our disposal.

By experiments which are now in progress we will soon be
able to state whether and how far the contents of the large intes-
tine interfere also with the physiologic effect of strychnin.

In conclusion, I take the opportunity of acknowledging my
indebtedness to Dr. W. J. Gies, professor of physiological chemistry
at the College of Physicians and Surgeons, Columbia University,
for his generosity in according to me all the privileges of his lab-
oratory, in which the chemical work of this research was carried out.

18

[Reprinted from American Medicine, Vol. V, No. 26, pages
1027-1028, June 27, 1903.]

A FURTHER STUDY OF THE INFLUENCE OF THE
CONTENTS OF THE LARGE INTESTINE UPON
STRYCHNIN.

BY

WILLIAM SALANT, B.S., M.D.,

of New York.

Fellow of the Rockefeller Institute, New York.

(Prom the Departments of Pathology and Physiological Chemistry,
Columbia University.)

In a previous communication to this journal^ "On
the Influence of the Contents of the Large Intestine
Upon Strychnin," it was reported that small quantities
of strychnin (2 mgs.) added to the contents of the cecum
and colon of normal rabbits could not be detected when
examined according to the method recommended by
Haines.'^

In a number of control experiments, however, with
1 mg. of strychnin mixed respectively with gastric con-
tents, liver, crushed brains or urine, carried out with the
same method, strychnin was easily found. The conclu-
sion was therefore drawn that the contents of cecum
and colon of normal rabbits contain something which
interferes with the detection of strychnin by the methods
at our disposal. Beside the method referred to, other
methods, such as those of Draggendorff and Otto Stas,
were employed at first, but were abandoned in favor of
the Haines' method. I have recently made use also of
Blyth's method,^ with similar results.

A study of the physiologic efiect of a mixture of
strychnin and contents of cecum and colon of normal
rabbits was next undertaken. A quantity containing a
maximum of 2V ™S- of strychnin nitrate and injected
into a frog of about 30 gms. induced a typical tetanus
shortly after. This certainly proves that strychnin is
not destroyed by the contents of the large intestine of
normal rabbits. The failure to detect strychnin could
be due, therefore, either to its destruction by heat used
in the process of separation or to loss during the
numerous manipulations involved in the various
methods I employed. The first suggestion was put to
an experimental test. A mixture of strychnin and con-

2

tonts of lar<?e intestines of normal rabbits was lieatert on
the water-batii for four to five liours at a temperature of
75° C. to 80° C, and injected into frogs. The results
obtained, although not constant, have shown that this
temperature does not destroy strychnin in the presence
of tlie contents of the cecum and colon of normal rab-
bits. I therefore set out now to simplify the method of
obtaining strychnin. This was accomplished as follows :
After adding strychnin to the contents of the cecum
and colon of a normal rabbit the mixture was acidified
with acetic acid. To this 95 ^ alcohol was added and both
were digested on the water-bath for several hours at 75° (!.
to 80° C. Strong alcohol was now added again, and the
whole filtered, the residue washed with alcohol several
times. A second extract was made by treating the residue
with alcohol and filtering as before. The two filtrates were
united and evaporated to about 2 ounces at the same
temperature as before. This was now shaken up with
chloroform in the separatory funnel and the chloroform
drawn off. Chloroform was then added again and
the solution made alkaline with KOH. After shaking
vigorously the chloroform was drawn off. A second
chloroform extract, to insure complete removal of the
strychnin, was made, the two extracts united and
evaporated. The residue was dissolved with acetic and
water and filtered. The filtrate was made alkaline and
shaken up with chloroform, which on evaporation gave
a typical strychnin reaction with potassium bichromate
and concentrated sulfuric acid. I found, however, that
when the original mixture is digested at room tempera-
ture for 24 hours and the filtrate evaporated at ;'>0° C. to
40° C. a much purer chloroform extract is obtained. I
never failed to detect strychnin, even 2 mgs., in the
contents of cecum and colon by the method as out-
lined when the operations were carried on at a low
temperature. It is, therefore, the method that was
at fault in the failure in the early experiments to detect
strychnin in the contents of the cecum and colon of nor-
mal rabbits. Why should a simplified method give
different results? This may be explained as follows:
If a careful study be made of the various methods
I have employed, such as the Otto Stas, Draggendorff,
Haines, and Blyth, it may be seen that in all of
them the number of manipulations is quite large. The
solution is filtered many times and shaken up in the
separatory funnel a number of times successively with
several reagents before the alkaloid is readv for the final

test. As only small quantities of strychnin were experi-
mented with, the loss of even a small portion during any
of these processes would be sufJicient to prevent its detec-
tion. But the same method was successfully employed
for the separation of even smaller quantities of strych-
nin (1 mg.) from gastric contents, liver, brain, urine,
etc. This may be explained by the fact that the organic
impurities are not so numerous, and filtration much
better ; hence fewer manipulations with less loss of sub-
stance. While the acid solution of the large intestine
had to be shaken up many times with amyl alcohol,
benzin, etc., one extraction with amyl alcohol was all
that was necessary for the purification of gastric content,
etc. As amyl alcohol takes up water, it is not at all
improbable that sojne of the strychnin was lost in this
way. This would also explain why in many cases of
strychnin poisoning the alkaloid has not been found.
The large number of manipulations involved in the
methods generally employed probably interfered with
the detection of strychnin.

BIBLIOGRAPHY.

' Americnn Medicine, August 18. 1902.

2 Allen McLiane Hamilton's iSystem of Legal Medicine, Vol. i, pp.
451-59. 1895.

3 Poisons : Their Effects and Detection, third edition, p. 331.

19

LYMPH FORMATION
ASHER AND GIES.

Untersucliuiigeii iiber die Eigenschaften und die
Entstelmng der Lymplie.

D r i 1 1 0 M i 1 1 h e i 1 u n g

von

Dr. med. Leon Asher, und Dr. William J. Gies,

Privatdocent, Instructor in Physiological Chemistry

Assistent am physiolog. Institut zu Hern. Columbia University New York.

(Aus deui physiologischen Institute zu Bern.)

IV. Ueber den Einfluss von Protoplasma-Giften auf die Lymph-

bildung.

Die Anwendnng von Giften zur Erforschung der Bedin-
gungen, unter welchen die Lymphe entsteht, ist schon mehrfach
erfolgt, so z. B. durch Merunowicz, durch Camus und Gley,
durch S p i r 0 und durch Tschirwinsky. Die hierbei zu Grunde
liegende Idee wechselte, je nach den theoretischen Vorstellungen,
welche die betreffenden Beobachter iiber die Bildung der Lymphe
batten. Die Gifte wurden angewandt entweder weil sie den Blut-
druck beeinflussten oder neuerdings, weil sie gewisse Secretionen
forderten oder hemmten. Auf diesem Wege soUte also entweder
die mechanische Lymphtheorie , die Abhangigkeit der Lyraph-
bildung vom Blutdrucke, oder die Abhangigkeit vom secretorischen
VermOgen der Capillarendothelien, Heidenhaiu's secretorische
Lymphtheorie, gepriift werden. In der voraufgegangenen Mit-
theilung^) war zum ersten Male der Versuch gemacht worden,

1) L. Asher, Untersuchungen iiber die Eigenschaften und die Ent-
stehung der Lymphe. 2. Mittheilung. Zeitschr. f. Biol. 1898, N. F. Bd. 19
S. 261.

Untersuch. der Lymphe. Von Dr. med. L. Asher u. Dr. W. J. Gies. 181

Gifte zu beimtzen, um die »physiologische« mid die »physika-
lische« Componente bei den Erscheinmigen der experimentell
erzeugten Lymphbildung zu trennen. In dieser Mittheilung ge-
denken wir, den dort entwickelten Plan waiter auszuftihren.

Unter »physiologisclier« Componente verstehen wir denjenigen
Theil der Lymphbildung, welcher bedingt ist durch die specifische
Lebensthatigkeit der Zellen desjenigen Gewebes oder Organes,
aus welchem die Lymphe jeweilig stammt. Unter normalen
Verhaltnissen ist es die Thatigkeit der lebenden Zelle, sind es
die Bediirfnisse des lebendigen Protoplasmas, welche die Menge
und die chemische Zusammensetzung der gebildeten Lymphe
bestimmen. Diese Anschauung, welche alsdiecellular-physio-
logische Theorie der Lymphbildung bezeichnet werden
kann, steht im Einklange mit den Thatsachen und mit weitver-
breiteten biologischen Principien. In Bezug auf die Principien
bedarf es nur des Hinweises, dass fiir die innere Athmung und
fiir den Stoffwechsel der Nahrungsmittel die hier vorgetragene
Anschauung fast gleichlautend ziemlich unbestritten gilt. Dass
merkwiirdiger Weise fiir den unmittelbarsten Vermittler der Stoff-
wechselvorgange das cellulare Princip bisher geringere Bedeutung
besessen hat, ist offenkundig und riihrt daher, dass eine grosse
Reihe von experimentellen Erzeugungsarten von Lymphbildung
eine Erklarung zuliessen, welche mit verhaltnissmassig einfachen
mechanischen Vorstellungen auskam. Der Mechanismus der Zellen
selbst blieb hierbei ganz aus dem Spiele. Thatsache aber ist es,
wie Barb era und der Eine von uns in der ersten Mittheilung i),
sowie der Eine von uns in der zweiten zu beweisen versuchte,
dass sowohl bei der normalen wie auch bei der kiinstlich erzeugten
Lymphbildung nichts constanter Hand in Hand damit auftritt,
als Thatigkeit der Organe, weshalb die Lymphe als ein Produkt
der Arbeit der Organe bezeichnet wurde. Es mag betont werden,
dass an dieser Aussage nichts Hypothetisches ist; die Hypothese
beginnt erst bei der Erklarung des Zusammenhanges zwischen
Organarbeit und Lymphbildung. Auch bei den Vertretern mecha-
nischer Anschauungen beginnt der von uns aufgestellte Satz:

1) Diese Zeitschrift 1897, Bd. 36, N. F. Bd. 18 S. 154.

Zeitsehrift fiir Biologie Bd. XL N. F. XXU. 13

182 Untersuch. iiber die Eigenschaften u. Entstehung der Lymphe.

»DieLymplie ist ein Produkt der Arbeit derOrgane« Anerkennung
zu finden. So hat W. R6th^) sich hierzu bekannt. Erv^rkniipft
mit unserer Lebre freilich eine Reihe von physikalischen Vor-
stellungen, deneii gegeniiber mit aller Bestiramtbeit daran erinnert
werden muss, dass der Beweis fiir das factische Vorkommen
im Organismus der von ibnen (zum Tbeil im Anscblusse an
Koranyi) angenommenen »rein physikabscben « Vorgange nocb
zu erbringen ist. Wie wenig wir die Berecbtigung des Versucbes
leugnen, den Mecbanismus der Lymphbildung durcb bekannte
pbysikabscb-cbemiscbe Vorgange dem Verstandnisse naber zu
riicken, gebt wobl daraus zur Genuge bervor, dass wir selbst
eine Vorstelhmg entwickelten, welche auf den mogbcben Antbeil
der Transsudation und der Osmose binwies. Aber wir betonten
den bypotbetiscben Cbarakter dieser Vorstellung, indem wir er-
klarten: »Bei diesem Stande der Dinge kann die Vorstellung,
welcbe wir iiber die Bildung der Ernabrungsfliissigkeit baben,
nur den Wertb einer mebr oder weniger beglaubigten Hypotbese
besitzeu«. Die wicbtigsten Griinde, warum diese Einscbrankung
geboten ist, sind die folgenden: 1. Alle Versucbe werden nicbt
an der Ernabrungsfliissigkeit, sondem an der abfliessenden
Lympbe angestellt. (Dieser Unterscbied wurde scbon in unserer
ersten Mittbeilung S. 228 ausfiibrlich erortert, und wir kommen
daber bier nicbt darauf zuriick.) 2. Die Vorgange in den Gewebs-
spalten lassen sicb nicbt obne Weiteres aus den Erfabrungen ab-
leiten, welcbe durcb osmotiscbe Experimente an Membranen ge-
wonnen werden; denn jene Vorgange werden durcb das lebendige
Protoplasm a beeinflusst, dessen pbysikaliscb-cbemiscbe Eigen-
scbaften recht wenig bekannt sind. 3. Da die einzelnen Organe
in Bezug auf ibren Cbemismus specifiscb verscbieden sind,
diirften deren Zellen in entsprecbend verscbiedener Weise an
der L}Tnpbbildung mitwirken; die bisberigen mecbaniscben Hypo-
thesen beriicksicbtigen diese Unterscbiede nicbt, sondem sprechen
nur von iiberall gleicben Kraften. 4. Die angenommenen und

1) W. R6th, Ueber die Permeabilitat der Capillarwand und deren Be-
deutuDg fiir den Austausch zwischen Blut und Gewebsfliissigkeit. Arcbiv f.
Anat. u. Physiol. Phys. Abth. 1899, S. 416.

Von Dr. med. L. Asher u. Dr. W. J. Gies. 183

theoretisch durchaus moglichen osmotischen Krafte lassen sich
gar nicht selten bei reiuen physiologischen Versuchen — z. B.
bei den schonen Resorptionsversuchen Cohnheim's, den wich-
tigen, von den Mechanisten noch nicht hinreicbend gewtirdigten
Erfabrungen Hamburger's an der Halslymphe des Pferdes
oder den interessanten Versuchen von Cushny und Wallace
iiber Abftihrmittel — direkt ausschliessen. Da wo scheinbar
osmotische Krafte zur Erklarung glatt ausreichen, wie bei den
Versuchen Roth's an der Peritonealhohle, handelt es sich oft
um die Untersuchung eines Vorgangs, welcher gar nicht zu den
normalen Functionen des betreffenden Korpertheils gehort. Man
konnte also sagen, dass in Bezug auf einen solchen Vorgang
die betrefTenden Zellen ohne Leben seien.

Im Gegensatze zu dem hypothetischen Charakter der bisher
vorgetragenen mechanischen Anschauungsweisen lasst sich die
Arbeit der Organe als Faktor bei der Lymphbildung thatsachlich
nachweisen. So stehen beispielsweise in Bezug auf die theore-
tisch ausserst wichtigen Lymphagoga als einzige gesicherte ex-
perimentelle Erkenntnisse da : erstens Heiden bain's Entdeckung
eben ihrer eigenartigen lymphagogen Wirkung, zweitens unser
Nachweis, dass dieselben die Leberthatigkeit intensiv steigern,
weshalb wir vorschlagen, dieselben als Leber gift e zu bezeichnen.
(In der vierten augenblicklich im Drucke befindlichen Mittheilung
wird diese Frage im Anschlusse an die Untersuchung liber die phy-
siologische Arbeit der Leber eingehend behandelt werden.) Lehren,
wie die Starling'sche von der Veranderung der Permeabilitat
der Capillarwand, oder Koranyi's von dem durch Eiweisszerfall
erhohten osmotischen Druck, oder unsere eigene von der Aende-
rung der osmotischen Beziehungen zwischen Blut und Lymphe
durch Dissimilationsprodukte der Zellen, sind entweder tiberhaupt
nicht experimentell nachgewiesen oder gewinnen erst dadurch
einen festen Ausgangspunkt der Prtifung, dass der physiologische
Factor der Arbeit der Organe gesichert ist.

Wahrend in der Arbeit der Organe wesentlich die »physio-

logische Componente« bei der Lymphbildung beruht, besteht

daneben eine »physikalische Componente«, welche von der

13*

184 Untersuch. iiber die Eigenschaften u. Entstehung der Lymphe.

specifisclicn Zellmechaiiik unabhangig is(. Die »physikalische
Componente« tritt am besten bei gewissen kiinstlichen Steigerungen
der Lymplibildung zu Tago und die Annahme liegt nabe, dass
eben dnrch die Kiinstlicbkeit derVersucbseingriffe diese Erscbei-
nung begiinstigt wird. Unter der »physikaliscben Coniponente«
bei der Lymphbilduiig versteben wir alles das, was sicb einwands-
frei und ausschliesslich auf die pbysikaliscben Factoren der Fil-
tration, der Diffusion und Osmose zuriickfiibren lasst. Die An-
erkennung einer Erscbeinung als rein pbysikabscb verursacbt,
ist vor AUem abhangig von der ErfuHung der Bedingung, dass
die specifische Zelltbatigkeit nacbweisbar bei der Mitwirkung
ausgeschlossen ist — wir balten dies methodiscb fur eine un-
erlassHcbe Voraussetzung. Der Gang unserer Erkenntnisse in
der Biologie ist zumeist der gewesen, dass eine Zeit lang die
beobachtbaren Erscheinungen sich auf verhaltnissmassig einfacbe,
mechanische Weise erklaren liessen, bei weiterer Analyse aber
immer wieder, wie Heidenbain eindringlich betont hat, die
»Vorgange der lebenden Zelle« als mitwirkend erkannt wurden.
Das ist auch der augenblickliche Stand der Lympbfrage.

Gemass den entwickelten Anschauungen haben wir in dieser
Mittheilung Giftwirkungen versucht, um die physiologiscbe von
der pbysikaliscben Componente zu trennen. Die Anwendung von
Giften ist insofern ein Notbbebelf, als die Giftwirkung meist sehr
vielgestaltig, und die Art und Weise, wie sie die lebendige Zelle
beeinflusst, ziemlich dunkel ist. Immerbin gibt es einige
wenige Gifte mit gewissen so bervorstecbenden Merkmalen,
dass sie methodiscb brauchbar erscheinen. Als solche boten
sich fiir unsere Zwecke in erster Linie das Chin in und das
Arsen dar.

Das Chinin gilt als ein ganz allgemeines Protoplasma-
Gift; es sollte daher dazu dienen, zu priifen, wie sich die Lympli-
bildung verhalten wiirde, wenn bekannte, lymphvermebrende Ein-
griffe statt batten, wahrend die specifischen Zellen gleichzeitig
dem Einflusse eines allgemeinen Protoplasmagiftes unterworfen
waren. Andererseits darf das Arsen auf Grund der Untersuchungen

Von Dr. med. L. Asher u. Dr. W. J. Gies. 185

von Bohm^) und besonders auch voii Magnus 2) als ein typi-
sclies Gefassgift bezeichnet werden; es sollte daher dazu
dienen, zu untersuchen, welche Bedeutung einer bekanntermaassen
vorbandenen, nicht etwa bloss hypothetisch angenommenen, er-
hobten Permeabilitat der Gefasswande fiir die Lympbbildung bei-
zumessen sei. Dass die etwas scbematische Trennung der beiden
Gifte als Protoplasma- und Gefassgift eine streng durchftibrbare
sei, liegt uns fern zu bebaupten: es kommt nur darauf an, dass im
Symptomenbild der Vergiftung quantitativ die Unterscbiede der
beiden Wirkungsarten so bervorstecbende seien, dass etwaige Ab-
weicbungen von bekanntenVorgangenbei der Lympbbildung obne
Weiteres auf Protoplasma- oder Gefassvergiftung bezogen werden

konnen.

Metb odiscbes.

Die Praparation des Brustlympbganges geschab in der Art
und Weise, wie sie in den friiberen Mittbeilungen gescbildert
wurde. Mit der Form der Cantile baben wir wiederum gewecbselt,
ein Ereigniss, was wobl raancbem Untersucber des Lympbstromes
als Notbwendigkeit sicb aufgedrangt bat. Wir bedienten uns
dieses Mai der Heidenbain'scben Form der Lympbcaniile,
mit Weglassung der zweiten Biegung. Wir baben dieselbe nicbt
durcb Nabte befestigt, sondern die Cantile wurde wabrend der
ganzen Beobacbtungszeit von uns mit der Hand gebalten. Ob-
wobl dies, namentlicb wabrend langdauernder Versucbe, etwas
unbequem ist, verlobnt es sicb docb, der kleinen Miibe sicb zu
unterzieben ; denn das Halten mit der Hand erwies sicb dessb alb
so vortbeilbaft, weil man den kleinsten Verlagerungen der Cantile,
welcbe sicb aucb bei tiefer Narkose nicbt vermeiden lassen, mit
der nacbgiebigen Hand sofort Recbnung tragen kann; bingegen
ist man bei dem scbweren Gewicbte der Cantile durcb das blosse
Annaben an die Haut oder die Muskeln nicbt vor unliebsamen
Zerrungen oder Compressionen des Lympbganges gescbtitzt. Die
Bestimmung des Trockengebaltes der Lymphe gescbah auf be-

1) Bohm u. Unterberger, Beitrage zurKenntnissd. physiol.Wirkung
der arsenigen Saure. Archiv f. exp. Pathol, u. Pharmak. 1874, Bd. 2 S. 89.

2) Magnus, Ueber die Entstehung der Hautod erne bei experimenteller
hydramischer Plethora. Archiv f. exp. Pathol, u. Pharm. 1899, Bd. 42 S. 250.

186 Untersuch. iiber die Eigenschaffen u. Entstelmng der Lyniphe.

kannte Weise ; es wurde, wenn moglich, jede aiifgefangene Lymph-
portion auf ihre Concentration gepriift, weil, wie schon fruher
ausgefiihrt wurde, dem Trockengelialte der Lymphe in zahlreichen
Fallen ein grosserer Werth zur Beurtheilung der Ereignisse im
Quellgebiete der Lymphe zukommt als der blossen Austluss-
menge. Zur Zuckerbestimmung wurden Blut und Lymphe nach
Drechsel's Methode vorbehandelt. Zuniiohst wurde eine ab-
gemessene Portion in die zehnfache Menge 95proc. Alkohols lang-
sam zugelassen; nach 24 Stunden wurde mit der Saugpumpe vom
Niederschlage abfiltrirt und der gut ausgewaschene Niederschlag
nochmals im Morser mit Alkohol verrieben und filtrirt. Die
vereinigten Filtrate wurden abgedampft und der Riickstand mit
etwa 200 ccm heissen Wassers aufgenommen; hierzu wurden
etwa 2 g reinen Paraffins und 6 — 7 Troi)fen Phosphorsaure zu-
gesetzt. Bei starkem Kochen ballen sich Verunreinigungen und
Fett zusammen und nach dem Erkalten kann die klare Fliissig-
keit von der festen Paraffindecke abfiltrirt werden. Der Paraffin-
kuehen wird noch drei Mai unter Zusatz von einem Tropfen
Phosphorsaure mit Wasser aufgekocht. Die vereinigten sauren
Fliissigkeiten wurden mitNaoCOs neutrahsirt und auf ein passendes
Volum eingeengt. Der Traubenzucker wurde nach Kiihne's
Methode mit ammoniakahscher Ku])fersulfatlosung bestimmt.^)
Wir fanden es vortheilhaft, die auf Zucker zu priifende Losung
ganz gleichmassig und allmahlich zufiiessen zu lassen, bis der
Moment kam, wo die blaue Farbung entschieden abzublassen
beginnt, dann nichts mehr zuzugeben und zwei Minuten lebhaft
weiter zu kochen; das vOUige Verschwinden der blauen Farbe
nach zwei Minuten Kochen giebt die scharfe Endreaction. Bei
den ersten Titrationen lasst man leicht zu viel LOsung zufiiessen,
man erhalt aber bald constante Minimalwerthe.

Lymphbildung unter der Einwirkung von Chinin.

Alle Eingrilie, welche ktinstlich eine Besclileunigung des
Lymphstroms, eine vermehrte und qualitativ veranderte Ljonph-
bildung hervorrufen, sind unserer Auffassung nach auf das

1) 0. Cohnheim, Ueber die Diinndarmresorption. Zeitschr. f. Biol.
Bd. 36 N. F. Bd. 18 S. 134.

Von Dr. med. L. Asher u. Dr. W. J. Gies. 187

Innigste verkntipft mit veranderten Thatigkeitszustanden der Ge-
webe. Von solchen bekannten und sowohl von anderer Seite
als auch von uns mehrfach discutirten Eingriflten unterzogen wir
zunachst die Lymphbildung nach Injection von Traubenzucker
einer Untersuchung auf ihr Verhalten unter der neuen Versuchs-
bedingung, dass gleichzeitig der Organismus einer starken Chinin-
vergiftung ausgesetzt war. Wir wissen, dass die Injection von
krystalloiden Substanzen zu einer regen Thatigkeit der verschie-
densten driisigen Organe Veranlassung gibt; leider liegen noch
keine Untersuchungen iiber etwaige Differenzen je nach der an-
gewandten Substanz vor, aber nach Allem, was wir tiber den
Stoffwechsel wissen, miissen sich unzweifelhaft die Verhaltnisse
anders gestalten, je nachdem beispielsweise Zucker, Kochsalz
oder Harnstoff injicirt wird. Es ist von alien Seiten zugestanden
worden, dass gerade die Erscheinungen nach Injection von
krystalloiden Substanzen zmn guten Theile sich erklaren lassen
ohne Zuhilfenahme specifischer Zellthatigkeit. Da der Eingriff als
solcher, vor allem in der bisher beliebten Methode, weit abweicht
von physiologischen Zustanden, ist es nicht verwunderlich, dass
die Antheilnahme der physiologischen Zellthatigkeit nicht ohne
Weiteres entschleiert werden kann. In der letzten Mittheilung
hat der Eine von uns eine Erscheinung beschrieben, welche als
>;physiologische Componente« bezeichnet wurde: es war das die
Thatsache, dass nach Injection verhaltnissmassigkleiner Mengen
von krystalloiden Substanzen eine vermehrte Stoffabfuhr aus den
Geweben durch die Lymphe stattfand. Wir haben zunachst
gepriift, ob diese »physiologische Componente« irgendwie durch
Chininvergiftung beeinflusst wtirde.

(Siehe Tabelle auf S. 188.)

Der Versuch ergab, dass eine wesentliche Veranderung in
den Erscheinungen, trotz einer hohen Chinindosis, nicht zu er-
kennen war. Es trat sowohl nach intravenoser Injection einer
nicht allzu grossen Menge Traubenzuckers eine merkliche, wenn
auch nicht sehr grosse Lymphbeschleunigung ein, als auch er-
folgte die charakteristische Vermehrung des Procentgehaltes der
Lymphe an festen Substanzen. Diese vermehrte Stoffabfuhr durch

183 Untersnch. iiber die Eigenschaften n. Entstelinn<: der Lyinphe.

Tabelle I.

Vers. 1. Hnnd 7 — >> kg. 24 Std. ol)no Xnhriiiis ; Morphium-Aethernarkose.

Zoit

I.ympli-
menge
in CCIU

Lymph-

menge

pro Min.

in com

I'rocent-

gehalt

an festen

Substnnzen

IJemcrkungon

10 h 8

— lOh

44'

4,3

0,12 5,27

10. 44
11 > 21

— 11 >

— 11 »

20'
57'

6,6
6,0

0,18
0,17

6,02
5,53

10 h 15 — >')0'. :;0 ccni Kochsalz-
losung enthalteiid 10 g Tnuiben-
zucker-|-0,') g Chinin mur. in die
Vena feinoralis ; k e i n e anfiing-
licheX'erlnngsamung; tropft selir
gut ab; (Jerinnung viel weniger
als friJher.

12. 5'
12 45 '

— 12>

— 1 >

41'
21'

5,4
3,0

0,15
0,08

6,00
6,41

*

12 h 5'. 0,5 g Chinin mur. in die
Vena femoralis.

1 > 27'
1 . 37'

1 > 47'

— 1 >

— 1 >

37'
47'
57'

1,0
1,2

2,8

0,10
0,12
0,28

> 6,31

1 h 27 '. 10 g Traubenzuck. in 30 com
Kocbsalzlosung in die Vena fe-
morali.s; keine anfangliche Ver-
langj-ainung.

1. 57'

— 2,

3'

2,1

0,35

.

2. 3'

— 2^

13'

2,2

0,22

'

2. 13'

— 2.

23'

2,3

0,23

. 6,20

2» 23'

— 2.

33'

2,1

0,21 '

2> 33'

— 2»

39'

0,5

0,08

die Lymphe wurde auch nicht verringert, als in einer spateren
Periode des Versuches durch eine abermalige Zuckerinjection
eine enieute Lymphbeschleuniguiig hervorgerufen wurde. Aus
dieser letzieren Thatsache gebt hervor, dass die Concentrirung
der Lymphe in spateren Stadien solcher Versuche nicht etwa
darauf beruhe, dass der Lymphe nicht iiiehr geniigende Wasser-
mengen zur Verfiigung stehen. Nachdem wir so erkannt batten,
dass dem Chinin nicht das Vermogen innewohne, die Vorgange
im Lymphsystem nach Injection von kleinen Mengen von Trauben-
zucker erkennbar zu beeinflussen, schritten wir zur Untersuchung
der Lymplibildung unter dem gleiclizeitigen Einflusse einer
intravenosen Lijection von gross en Mengen Traubenzuckers und
einer starken Chinin vergiftung. Es kam hierbei darauf an, folgende
Momente zu berucksichtigen : die Vermehrung der Lymphmenge,
die Verhaltnisse der Concentration der Lymphe an festen Sub-
stanzen, die Zuckerausscheidung aus dem Rlute und das Ver-
halten der Zuckerconcentration in der Lymphe. Die beiden letzten

Von Dr. med. L. Asher u. Dr. W. J. Gies. 189

Punkte beanspruchen desshalb besonderes Interesse, well Heiden-

hain bekaiintlich an ihnen einige auffallende Thatsachen ent-

deckte, in denen er Merkmale eines activen, secretorischen Ein-

greifens der Capillarendothelien sah.

Diese Annahme ist mit gewichtigen Griinden von Cohnstein

und Starling bekampft worden, und auch wir kounten uns,

wenn auch aus ganz anderen Griinden wie die genannten Forscher,

vorlaufig der secretorischen Hypothese nicht anschliessen. Die

Ergebnisse der besprochenen Versuche sind in Tabelle II nieder-

gelegt.

(Siehe Tabelle auf S. 190.)

Diese Versuche lehren zunachst, dass trotz der Chinin-
vergiftung nach Zuckerinjection eine erhebliche Beschleunigung
des Lymphausfiusses eintritt; vielleicht ist dieselbe nicht ganz
so gross wie sie ohne Chinin gewesen ware, wenigstens, wenn
man als Maassstab die von Heidenhain in seiner grossen
Arbeit mitgetheihen Zahlen wahlt. Dort find en sich unter zwolf
Versuchen Beschleunigungsquotienten, welche vom 4,8fachen bis
zuni 3X,5fachen gehen. Doch wollen wir auf diesen Unterschied
kein grosses Gewicht legen; zunachst kommen viele individuelle
Schwankungen der Reaction auf Traubenzuckerinjection vor, wie
sich am besten daraus ergibt, dass zwischen der pro 1 kg Korper-
gewichtinjicirtenZuckermenge und demBeschleunigungsquotienten
gar keine Proportion ahtat nachweisbar ist; ferner haben wir bei
einer so schweren Chininvergiftung, dass bald nach der Zucker-
injection der Tod eintrat, eine ganz ungemeine Beschleunigung
des Lymphflusses sich entwickeln sehen. Auf dieses wichtige
Experiment kommen wir weiter unten in einem anderen Zu-
sammenhange zuriick. Auch die Art und Weise, wie sich der
Procentgehalt der Lymphe an festen Substanzen, namentlich aber
wie sich die Ausscheidung des Zuckers aus dem Blute und die
Anhaufung desselben in der Lymphe gestaltet, weicht nicht von
den Befunden an unvergifteten Thieren ab. Ganz wie bei den
letztgenannten verlasst der Zucker ausserordentlich rasch die
Blutbahn und tritt in die Lymphe tiber, wo er sich so anhauft,
dass lange Zeit die Zuckerconcentration hoher ist, nicht allein

190 Untersuch. tibcr die Eigenschaften u. Entstehung der Lymphe.

TabeUe U.

Versuch 2. Hund 20 kg. Morpliiuin Aethornarkose.

Zeit

Lymi>h-
menge
in com

Lymph- Zucker-
menge gelialt

pro dor

10 Min. ' Lymphe i ^ , .
in ccm in i'roc. 5'U'>''ia'iz

Procent-

gehftlt an

festen

liciiicrkungon

9h 20'— 9h 34'
9 > 45 ' — 9 » 58 '

9, 58' — 10- 12'

10. 12' — II . 00'

11 . 00' — 11> 20'
11 . 20 ' — 11 . 40 '

11 > 40 ' — 11 59 '

11. 59' — 12» 17'

11,0
24,0

29,0
32,0

9,8
12,0

10,1
10.2

7,8
16,2

20,7
6,7

4,9
6,0

5,3

5,6

0,451
0,464

I 0,364

4,90

4,91
3,38

5,54
5,49

9 h 45 ' — 48'. 40gTrauhcn-
zufker+0,5gChiniii mur.
in 80 coin Koehsalzlosung
in dieV. feinoralis; kcine
aiiilingl. Vorlangsaraung.

10 h 2'. 0,5gChinin niur. in
die V. femoralis.

10 h 2'2'. 2S ccm Blut iius
der Art. femoralis , ent-
haltend 0,357 "/o Zuckcr.

Uh 25'. 54 ccm Blut aus
der Art. femoralis; ent-
lialtend 0,2080/0 Zucker.

12 h 17'. 50 ccm Blut aus
der Art. femoralis; ent-
haltend 0,128O/o Zucker.

llh 4'
11 > 15 '

11 . 25

11 » 31 '
11 » 41'

Versuch 3. Hand 20kg. Morphium-Aethernarkose.

llh 14
11 > 25

11 31

11 . 41
11 > 51'

11 ^ 51 ' — 12 . 13

12. 13'

— 12. 50

12. 50'

— 1. 10

1 . 10 '

— 1. 25

1 . 25'

1 > 40

5,3

5,3

6,30

8,2

8,2

0,843

9,6

16,0

5,46

10,0

10,0

0,870

9,0

9,0

3,86

12,2

5,6

0,748

10,0

2.7

0,376

5,6

2,3

5,27

7,0

4,7

0,518

5,0

3,3

0,780

11 h 15'— 17'. 40gZueker+
1 g Chin. mur. in 80 ccm
Koehsalzlosung in die V.
femoralis ; k e i n e anfiing-
liche Vcrlangsamung.

11 h 28'. 35 ccm Blut aus der
Art. femoralis, enthaltend
0,538 o/o Zucker.

llh 45'. 33 ccm Blut aus der

Art. femoralis, enthaltend

0,288"/o Zucker.
12 h. 30 ccm Blut aus d. Art.

femor., enthaltend 0,247°/o

Zucker.

1 h 10'— 12'. 20 g Trauben-
zucker i. 100 ccm Koehsalz-
losung in die V. femoralis.

als die gleichzeitige, sondern sogar als diejenige, die dreiviertel
Stunden friiher im Blute nachweisbar war. Aus dem dritten Experi-
raente geht sehr deutlich hervor, dass selbst zwei Stunden uach
der Einfiihrung des Giftes auf eine erneute, gar nicht sehr grosse

Yon Dr. med. L. Asher u. Dr. W. J. Gies. 191

Zuckerinjection bin Lymphbeschleunigung und Zuckeraustritt aus
dem Blute in die Lymphe in charakteristischer Weise sich geltend
macht. Das Gesammtergebniss unserer Versucbe iiber combinirte
Wirkung von Chinin- und Zuckerinjection auf die Vorgange am
Lymphstrome wiirde sicb also dahin aussprecben lassen, dass
Cbinin dieselben nicbt deutlicb erkennbar zu beeinflussen vermag.
Wenn die Voraussetzung ricbtig ware, dass Cbinin als allgemeines
Protoplasmagift die specifiscben Zellfunctionen tief scbadigen
miisse, so mtisste man zu dem Scblusse gelangen, dass weder
die Bildung einer vermebrten und anfangbcb weniger, spater
mebr concentrirten Lympbe, nocb die ungebeuer rascbe Aus-
scbeidung des Zuckers aus dem Blute, nocb scbliesslicb das
ganzlicb unparallele Verbalten der Zuckerconcentration im Blute
und in der Lympbe irgend etwas mit aktiver Zelltbatigkeit zu
scbaffen baben. Man wird denjenigen, welcbe die gescbilderten
Vorgange in bekannter, ausscbliesslicb mecbaniscber Weise zu
erklaren gewillt sind, zugeben mtissen, dass die soweit mitgetbeilten
Versucbsergebnisse einen zwingenden Grund nicbt entbalten,
diesen Standpunkt zu verlassen, im Gegentbeil eber eine Be-
statigung desselben zu geben scbeinen.

Eine nabere Discussion iiber die Wirkungen des Cbinins auf
den Organismus lebrt, dass die Verhaltnisse nicbt gar so einfacb
liegen. Leider ist mancbes, was iiber die Cbininwirkungen als
bekannt vorliegt, nicbt eindeutig oder nicbt binreicbend experi-
mentell beglaubigt. Zunacbst geben alle Beobacbter an, dass
toxiscbe Dosen den Blutdruck erbeblicb mindern; nacb der
mecbaniscben Lympbtbeorie soil die Lympbvermebrung nacb
Injection von Krystalloiden auf Capillardruckerbobung beruhen:
bier liegt also scbon eine Scbwierigkeit vor. Ferner scbeint aus
einer grossen Reibe von Beobacbtungen bervorzugeben, ^j dass
toxiscbe Dosen auf die Blutgefasse stark erweiternd wirken;
unter diesen Umstanden wird die Annahme nabe gelegt, dass die
Capillarzellen selbst in ibrer Function leiden konnten. Da nun

1) Die Literatur hieruber findet sich in vorziiglicher Weise zusammen-
gestellt in Wood, Therapeutics; its principles and practice. 9. Ed. Phila-
delphia 1894.

192 Untersuch. iilier die Eigenschaften u. Entstehung dor T.ynij)he.

die Zuckerausscheidung aus dein Blute trolz Chiiiiiivergiftung
ungestort verlauft, wiirden unsere Vor.siiche eiiie weitere Stiitze
fur die Ableuguuug secretori.scher Functionen der Capillar-
endotlielien darbieten. Am wichtigsten orscheint uns aber, dass
sowohl die Unter.suchungeii von Strassburg wie auch die von
Chittenden ergaben, dass selbst grosse Dosen Chinins keine
nierkliche Stornng der Kohlensaurebildung verursachten. (Etwas
abweichend davon sind die Angabt^n von Boeck und Bauer.)
Daraus geht hervor, dass durchaus nicht alle Stoffwechsel-
vorgange unter der Giftwirkung des Chinins zu leiden haben ;
unzweifelhaft hat aber die intravenose Injection von Trauben-
zucker niit jenen Processen, welche zur COa-Bildung fuhren, enge
Beziehungen. Diese Erwagungen fuhren zu dem nahehegen-
den Schlusse, dass moghcher Weise die Erscheinungen am
Lymphstronie nacli Injection von Kry stall oiden nur desshalb
nicht durch Chininvergiftung nierklich geandert werden, weil
das Chinin denjenigen physiologischen Processen gegeniiber, wel-
che durch intravenose Zuckerinjectionen angeregt werden, niacht-
los ist. Wir niiissen daher die Frage nach der physiologischen
Componente bei der Lymphbildung in Folge von intravenoser
Zuckerinjection als eine durch Chininversuche ungeloste be-
zeichnen.

Unsere nachste Aufgabe war, die Wirkung eines der Heiden-
hain'schen Lymphagoga unter gleichzeitiger Anwendnng der
Chininvergiftung zu priifen. Wir hatten in unseren friiheren
Mittheilungen den Nachweis zu erbringen gesucht, dass die Ver-
mehrung und gewaltige Veranderung in der Lymphbildung durch
dieselben eine Theilerscheinung der intensiven Leberthatigkeit sei,
welche durch jene Mittel ausgelost wiirde. Da wir auf dieser
Erkenntniss fussten, erschien die Anwendung des Chinins im
Hinblick auf die ziemlich sichergestellte Thatsache (namentlich
durch die Untersuchungen von Prior), dass durch Chinin die
Harnstoffbildung sehr bedeutend herabgedriickt wird, geradezu
geboten. Denn die letztere Thatsache weist ja auf eine tiefe
Schadiguug desjenigen Organes hin, dessen Thatigkeitsgrad be-
sonders maassgebend fiir die Art und den Unifang der Lymph-

Von Dr. med. L. Asher u. Dr. "W. J. Gies. 193

bildung ist, wie wir wiederholt nachgewiesen haben. Wir wandten
fiir unsere Versuche als Lymphagogum (oder Lebergift) Extract
von Blutegelkopfen an. Blutegelkopfextract hat vor manchen
anderen Mitteln gleicher Wirkungsart den grossen Vortheil
voraus, dass es dem Herzen und den Gefassen gegeniiber in den-
jenigen Dosen, die zur Anregung der Lymphbildung erforderlich
sind, unschadlich ist. Beim Pepton liegen die Verhaltnisse viel
verwickelter, da dasselbe nicht allein das Herz, sondern auch, wie
aus den Untersuchungen von Thompson i) hervorgeht, sehr aus-
gepragte Wirkungen auf die Gefasse besitzt. Worauf es aber wesent-
lich ankommt, das ist Pepton und Blutegelkopfextract gemein-
sam: denn das Letztere regt in gleicher Weise, wie Barbera und
der Eine von uns und auch Gley fiir Pepton nachwiesen, nach
Gley's in der Festschrift der Societe de biologie (1900) nieder-
gelegten Beobachtungen stark die Leberthatigkeit an. Wir
wandten fiir unsere Versuche ein Blutegelinfus an, gestiitzt auf
die Erfahrungen von Eguet^), der in Sahli's Klinik nach-
gewiesen hat, dass dieses Praparat am wirksamsten und von
der grossten Constanz war. Ausser dem jeder Zeit frisch be-
reiteten lufus bedienten wir uns noch eines von Haussmann
(St. Gallon) hergestellten Glycerinextractes , von dessen Wirk-
samkeit auf die Hemmung der Blutgerinnung wir uns durch
einen eigenen Versuch iiberzeugten. Ein Cubikcentimeter dieses
Extractes entspricht zwei Blutegelkopfen. Aus den Ergebnissen
von Versuch 4 ist mit ziemlicher Deutlichkeit zu erkennen, dass
die charakteristische Wirksamkeit des Blutegelinfuses auf die
Lymphbildung durch die Chininvergiftung ganz wesentlich modi-

ficirt wird.

(Siehe Tabelle auf S. 194.)

Es wird zwar, wie Tabelle III lehrt, die Lymphmenge nach
der Injection von Blutegelinfus recht erheblich gesteigert, aber
das, was so charakteristisch fiir die Wirkung eines solchen

1) W. H. Thompson, The physiological effects of ^peptone* when
injected into the circulation. Journ. of. Physiol. 1899, Vol. 24 p. 874.

2) Eg net, Ueber den Einfluss des Blutegelinfuses auf die Thromben-
bildung. Inaug.-Dissert. Bern 1894.

194 Untersucli. iiher die Eigenschaften u. Entstehuntr <ler Lymphe.

Tahellc III.

Versuch 4. Hund 10 kg 1^ eg Morplumii, spater Aether.

Zeit

Lymph-
menge
in PCin

Lymph-
mcnge

pro Min.
in ccm

Procent-

gehnlt

nn festen

Substnnzen

Bemerkungen

9h

48

— 9h

58'

2,5

0,25

7,34

9.

58'

— 10.

11'

3,0

0,23

6,96

9h .^8' — lOh 8'. O.egChinin mur.
in SO ccm Kochsalzlosiinn in die
V. fcmoralis.

10

11'

— 10.

21'

1,8

0,18

6,93

10 >

21

— 10»

33'

7,3

0,61

6,99

10h22'. 30 ccm Rlutesclinfiis in
die V. femoral. (10 Rlutc.irclknpre
in 50 ccm Salzltisung infnndirt.)
10 h 27' Ausfln.ss beschlcunigt.

10 >

33

- 10 .

42'

10,0

1,11

6,64

Lymphe gerinnt viel weniger. 10 h
37'— 40' 6 ccm Infus in d.V.fem.

10 .

42

— 10.

53'

G,6

0,60

6,34

10 h 47'— o2' der Rest des Infuses
in die Vene.

10

53

— 11

5'

11,0

0,92

5,41

10 h 64'— 11 h 5'. 360 ccm 0,85 proc.
Kochsalzlosung in die V. fenior.
10 h 59' deutl. Beschleunigung ;
vorhcr Verlangsamung.

11 .

5

— 11 .

14'

19,0

2,11

4,00

11 .

14

— 11

23'

10,0

1,11

4,32

11 »

23

— 11 .

32'

5,8

0,64

4,71

Mittels ist: die bedeuteude Steigerung de.s Procentgehaltes der
Lymphe, bleibt vollstandig aus. Auf Grand aller bisherigen
Beobachtungen ware bei einem so imgemein hohen Beschleuni-
gungsqiiotienten der Lymphe wie 4,5 im Gegentheil eine ent-
sprechende grosse Vermehrung der festen Substanzen in derselben
zu erwarten gewesen. In dem vorliegenden Versuche nimmt
die Concentration nnansgesetzt ab. Gerade die.ser Contrast
zwischen Menge and Concentration erscheint besonders werthvoll,
weil er darauf hinweist, dass zwar dem Infus als solchem Wirk-
samkeit innewohnt, aber dessen Wirksamkeit diirch das Ein-
greifen eines anderen Momentes in die durch dasselbe sonst aus-
gelosten Vorgange gestort worden ist. Dieses andere Moment
ist die Chininvergiftung. Die Chininvergiftung hat die Aus-
losung einer Leberthatigkeit von solcher Intensitat durch das
Blutegelinfus verhindert, dass dadurch nicht allein ein vermehrter
Fliissigkeitsiibertritt, sondern auch eine gesteigerte Stoffzufuhr
in die Lymphe veranlasst wiirde. In der Thatsache, dass Chiiiin
die charakteristische Wirkung der Lymphogoga erster Klasse

Von Dr. med. L. Asher u. Dr. W. J. Gies. 195

(Lebergifte) unterdrtickt, liegt ein neuer Beweis dafur vor, dass
der Erfolg derselben gekniipft ist an das Stattfinden einer er-
hohten Leberthatigkeit, Wir haben im vorliegenden Versuche
durch Injection einer grossen Menge von Kochsalzlosung zum
Scblusse untersucht, ob die Permeabilitatsverhaltnisse der Gefass-
wande irgendwie gelitten batten : das aus dem Grunde, weil man
geneigt gewesen ist, die Wirkung der Lymphagoga auf blosse
passive Veranderung der Permeabilitat der Gefasswande zuriick-
znfiibren. Der prompte Erfolg der Kochsalzinjection erwies, dass
die Permeabilitat der Gefasswande von der Norm nicht abwich;
es ist somit der Einwand nicht zulassig, dass die Cbininvergiftung
durch Storung der Permeabilitat der Gefasswande hinderlich
gewesen sei. Andererseits ist die Schadigmig der specifischen
Leberfunctionen durch Chinin experimentell bewiesen; erstens
durch den schon erwahnten, von Prior^) gelieferten Nachweis,
dass gerade derjenige Stoffwechsel, an welchem die Leber einen
so hervorragenden Antheil nimmt, unter Chininzufuhr stark dar-
niederliegt, zweitens durch den neuerdings von Cavazzani^)
erbrachten Beweis, dass Chinin die Glykogen bildende Func-
tion der Leber hemmt. Wir theilen in der folgenden Tabelle
noch zwei weitere Versuche mit, wo nach der Chininvergiftuug
Blutegelinfus ohne jede Wirkung auf den Lymphstrom war.
(Siehe Tabelle IV auf S. 196.)

Im 5. Versuch, in welchem offenbar durch das Chinin ein
hoher Grad der Prostration erzielt war, hatte Blutegelinfus iiber-
haupt keinen nachweisbaren Einfiuss auf die Lymphbildung. In
Versuch 6 bentitzten wir als Injectionsweg fiir die anzuwendenden
Mittel die V. lienalis; iiber die Methodik wird in der vierten
Mittheilung berichtet werden. Auf diese Weise wurde sowohl
das Chinin wie auch das Blutegelinfus direct der Leber zugeleitet
und konnte so moglichst verdiinnt in demjenigen Organe ihre
Wirkungen entfalten, welches bei dem vorliegenden Probleme

1) Prior, Ueber den Einfluss des Chinins auf den Stoffwechsel des
gesunden Organismus. Pfliiger's Archiv 1886, Bd. 34 S. 237.

2) Cavazzani, Influence de la quinine sur la glycogenese et sur la
tbermogenese du foie. Arch. ital. de Biol. 1899, T. 32 p. 350.

196 Untersucli. iiber die Eigenschaften u. Entstehung der Lym])he.

Tabelle IT.

Versuch 5. Hund 6,25 kg. 6 eg Morphium, hernach Aether.

Zeit

I.yniph-
menge
in ccin

Lymi>li-

inoiige

pro Min.

ill com

I'rooeiit-

gchalt

an fcsteii

SulistanziMi

Bcmci'kuiigeii

10 h 30'

— lOh

5r, '

^,-

0,31

9,1^7

l.yiiiplie von Aiifant; an blutig; 10 li
33'— 53' O.T g Chin. mur. in 40 ccm
Kochsalzlosiing in d. V. femoral.;
viel (ierinnnng in der Lymphe.

10 > 53'

-11 .

8'

2,4

0,16

9,30

11> 8'

— 11

43'

1."-^

9,43

11 h 9'— 28'. 20 cf-ni Blutegeliiifus
(2o ccm = 7 Blntcgplkfiyife); fort-
wahrendeGeriniiung; 11 h 43' — .55'
nene Caniile in den Brustlymph-
gang eingebunden.

11 > 55'

-12.

7'

6,2

0,52

8,40

11 h 58' 5 ccm Blutegclinfns.

12 » 7

-12»

18'

3,0

0,27

8,53

12 h 19'— 22' 8 ccm Glycerinlilut-
egelextract in "0 ccm Kochsalz-
losung in die V. fern.

12 » 18

-12>

40'

6,2

0,28

8,65

12 » 40

-12.

53'

4,2

0,40

8,49

12 h 49'— 53' 8 ccm Glycerinbhitegcl-
extract in 30 ccm Koclisalzlcisung
in die V. fern

12 . 53

— 1 .

15'

6,5

0,33

8,46

1 li 1' liccm (;iycerinl>lutegelextract
ill 10 ccm Kochsalzlosung in die
V. fein.

1 . 15

- 1 »

30'

7,0

0,47

7,95

Wahrend des ganzen Versuches tiefe
Prostration des Thieres.

Versuch 6. Hand 12kg. Morphium; dann Cura're.

3h 47'— 4h
4 » 2'— 4 >
4» 12'— 4 .

4> 27

43 ' — 5
2'— 5

2 '

3,3

0,22

12'

3,6

0,36

27'

2,8

0,11

1

43'

4,4

0,29

2 '

5,1

0,27

12'

5,0

0,50

i

4,65
5,20
5,13

5,38

5,69
5,69

3 h 52'— 4 h 8' 1 g Chinin mur. in
die Vena lienalis.

4h 12'— 20' Bhitegelinfusausl2Blut-
egullcopfen in die V. lienalis. 4 h
15' Speichelflus.s; einige Beweg.

4h 30' — 39' C ccm Glycerinblntegel-
extract in die V. fern

4 h 47' Speichel flies.st a. d. >funde.

Curare wirljung vertieft sich wtihrend
des Versuches.

iiberwiegend in Frage kam. Das Curare, welches wir anwandten,
um vollkommene Bewegungslosigkeit zu erhalten, hat seinen
bekaimten Einfluss auf den Ljaiiphstrom ausgeiibt. Aus den
Uiitersuchungen Paschutin's^) ist bekannt, dass nach dem
Eintritte der Curarevergiftung die Geschwindigkeit der Absonde-
rung wachst, sowie der Gehalt an festen Substanzen, namentUch

1) Paschutin, Ueber die Absonderung der Lyuiphe iin Arme des
Hundes. Ludwig's Arbeiten 1873, S. 197.

Von Dr. med. L. Asher u. Dr. W. J. Gies. 197

an Eiweiss, erheblich zunimmt. Hand in Sand mit der sich ver-
tiefenden Curarevergiftung geht eine Concentrirung der Lymphe
einher; der Hauptsprung erfolgt von der ersten zur zweiten
Lymphportion, also vor jeder Beeinfiussung durch Blutegelinfus.
Das Infus selbst hat keine sich wesentHch bemerkbar machende
Wirkung auf den Lymphstrom gehabt, und wir glauben nach
AUem, was ausgefiihrt worden ist, dem Zusammenhange der Dinge
am meisten durch die Annahme gerecht zu werden, dass auch
hier die Chininvergiftung durch Hemmung der Thatigkeit der
Leber eine Begleiterscheinung dieser Thatigkeit, namhch die
vermehrte und veranderte Bildung der Lymphe, unterdriickt
habe.

Es erhebt sich die Frage, lehren die mitgetheilten Versuche
etwas Tiber die Betheihgung der Gefasswandzellen an der Lymph-
bildung? Leider sehen wir uns, wie bisher stets in dieser Frage,
vor der Nothwendigkeit des Verzichtes auf unbedingt einwands-
freie oder tiberzeugende Auskunft. Chinin stort die Erschei-
nungen nach Zuckerinjection nicht, wohl aber diejenigen nach
Injection von Blutegehnfus. Die Anhanger von Heidenhain's
Anschauungen, denen zu Folge in beiden sich die active Thatig-
keit der Capillarendothehen offenbart, miissen hierdurch in einige
Verlegenheit gerathen, sich zu entscheiden, aus welchem Grunde
sie fiir den einen Fall eine Gefasswandschadigung annehmen
wollen, fiir den anderen aber nicht. Wenn man hingegen an-
nehmen will, dass mit jeder Organthatigkeit normaler Weise ein
besonderes Verhalten der Gefasswande auf das Innigste verbunden
ist — eine Moglichkeit, auf welche wir wiederholt schon
hinwiesen, — wiirde man schliessen konnen, dass in den zuletzt
betrachteten Fallen das Chinin mit den Processen in den speci-
fischen Leberzellen zugleich auch die dazugehorigen in den
Gefasswandzellen betroffen habe. Aus biologischen Griinden
wollen wir diese Auffassung nicht vollstandig ablehnen, betonen
aber, dass andererseits unsere Versuche Denjenigen nicht Liige
strafen, welcher eine active Betheihgung der Gefasswande
leugnet.

Zeitschiift fiir Biologie Bd. XL N. F. XXII. . 14

198 Untersuch. iiher die Eigenschaften u. Entstehung der Lymphe.

Lymphbildung ynter der Einwirkung von Arsen.

Die angestellten Betrachtungen iiber die etwaige Rolle der
Gefasswande bei der Lymphbildung leiten zu den Versuchen mit
einem typiscben Gefassgifte iiber. Magnus hat in seiner oben
citirtenArbeit die von Schmiedeberg aufgestellte Ansicht, dass
Arsenik in eigenartiger Weise die Wandungen der Capillaren ver-
giftet, so dass ausser der Erweiterung eine tiefgreifende Storung des
Stoffaustausches zwischen ihnen und den Geweben besteht, ex-
perimentell gut gestiitzt, indem er direct die Steigerung der
Durchlassigkeit der Capillaren der Haut nachwies. Dass aber
auch namentlieh die Capillaren des Darmes betroffen werden,
geht aus den Untersuchungen von Bohm und Unterberger,
sowie von Pistorius (nahere Literaturangaben finden sich in
Magnus' oben citirter Arbeit) hervor. Bei der Bedeutung, welehe
von vielen neueren Forschern der blossen Aenderung der Per-
meabilitat der Gefasswande zugemessen wird, ist es sehr werthvoll,
ein Mittel zu besitzen, welches nachweisbar diese Aenderung ver-
ursacht; es ist nun zu erwarten, dass durch das Experiment sich
erkennen lasst, welehe Beziehungen zwischen vermehrter Permea-
bilitat der Capillaren und Lymphbildung bestehen. Auch fiir
Heidenhain's Vorstellungen von der secretorischen Function
der Capillarendothelien bietet sich in dem Arsenik, kraft seiner
geschilderten Eigenschaften, ein willkommener Priifstein dar.

Wir benutzten zur Injection in die Vena femoralis Losung
eines Praparates reinen arseniksauren Natriums in Kochsalzlosung ;
1 ccm derselben entsprach 0,01g Natrium arsenicosum. In Tab. V
(S. 199) sind Versuchsdaten niedergelegt, welehe iiber mehrere
der hier interessirenden Punkte Aufschluss geben. Arsenik ver-
mehrt, wie mit aller Deutlichkeit aus dem Versuche hervorgeht,
den Ausfluss der Lymphe aus dem Brustgang. Auf der Hohe
der Arsenikbeschleunigung betragt der Beschleunigungsquotient
nicht weniger als 3,5. . Hiermit ist der Nachweis geliefert, dass
Arsenik ein lymphtreibendes Gift ist. Da sich keine mechanischen
Verhaltnisse, welehe etwa nur die Austreibung einer durchaus
nicht vermehrt gebildeten Lymphe begiinstigen wiirden, aus-
gcbildet haben, muss es sich um die vermehrte Bilduug von

Von Dr. med. L. Asher u. Dr. W. J. Gies.

199

Tabelle Y.

Versuch 7. Hand 17 kg. Morphiumnarkose.

Zeit

Lymph-
menge
in ccm

Lymph-
meuge
pro Min.
in ccm

Procent-
gehalt an

festen
Substanz.

Zucker

in
Procent

Bemerkungen

10 h 00

— lOh

10'

2,0

0,20

7,02

10 » 10'

— 10.

23'

3,2

0,25

6,98

10 h 11'— 12' 10 ccm Arsen-
losung und um 10 h 121^2
bis I6V2' ^0 ccm Arsen-
losung in die V. fern. =
0,03 g Natr. arsenicosum.

10. 23'
10. 33'

— 10.

— 10.

33'
53'

2,8
6,2

0,28
0,31

7,08
7,17

10 h 36'— 38' lOccmArsen-
losung = 0,01 g Natr. ars.

10. 53'
11 . 10 '

— 11 .

— 11 .

10'

20'

5,6
2,7

0,38
0,27

7,18
}7,28

llh 71/2'— 8Vs' 10 ccm Arsen-
losung = 0,01 g Natr. ars.

11 > 20'

— 11 .

30'

3,4

0,34

11 h 20'— 21'/2' 10 ccm Arsen-
losung, 11 h 251/2'— 26V2'
10 ccm Aisenlos. = 0,02 g
Natr. ars.

11 . 30 '
11 . 40 '

— 11.

— 11.

40'
50'

4,2
6,4

0,42
0,64

}7,31

llhSO'- 38Va' SOccmAxsen-
losung = 0,03 g Natr. ars.

11 . 50 '

— 12.

5'

10,4

0,70

7,38

12. 5'

12. 12'

12. 17'

— 12.

— 12.

-12.

12'

17'
22'

10,5

17,0
11,0

1,50

3,48
2,20

6,97

} 1,411

12 h 5 ' — 8 ' 30 g Trauben-
zucker + 0,01 g Natr. ars.
in die V. fem.

12. 22'
12 1 27'

— 12.
-12.

27'
32'

7,0
5,0

1,40
1,00

> 5,73

1,095

Herzschlag nicht wie ge-
wohnlich beiZuckerinjec-
tion verstarkt.

12 » 32 '

— 12.

37'

4,0

0,80

12. 37'

— 12.

57'

9,8

0,49

0,959

Lymphe handeln. Was die Aenderung der mechanischen Ver-
haltnisse durch das Gift anbetriiit, so liegen sie alle eher nach
der Richtung der Hemmung fiir das Wegschaffen der Lymphe.
Unzweifelhaft liegt der Blutdruck tief darnieder und sind eine
Reihe motorischer Elemente, welche gleichfalls den Lymphausfluss
fordern konnten, in einem lahmungsartigen Zustande. Unser
Versuch Hefert, wenn man von der durch Magnus gesicherten
Erkenntniss der erhohten Durchlassigkeit der Capillarwande aus-
geht, einen neuen Nachweis dieser Thatsache fiir das grosse
Gebiet der Eingeweidelymphe . Als weitere Stiitzen fiir die Ansicht,
dass die vermehrte Lymphbildung durch Arsenikvergiftung auf

14*

200 Untersuch. iiber die Eigenschaften ii. Entstehung der Lymphe.

der erhohten Permeabilitat der Gefassvvande berulien miisse,
konnen die bekannten, sehr heftigen Vergiftungserscheinungen
ail der Schleimhaut des Verdauungskanals angefiihrt werden,
welclie von jeher auf eine vermehrte Exsudation aus den Ge-
fassen bezogen wurden. Da die Veranderung der Durchlassigkeit
der Gefasse vornehmlicb die Eingeweidegefasse betrifft, steht der
Durchtritt einer wesentlich concentrirteren Fliissigkeit als sonst
zu erwarten; das ist in der Tliat der Fall.

An und fiir sich wiirde im Verlaufe eines lilnger dauernden
Versuches die Concentration der Lymphe unausgesetzt sich
mindern ; in dem vorliegenden Versuche niramt die Concentration
von 7,02% bis zu 7,38% zu. Diese Zunahme ist nicht erheblich,
aber immerhin mit Riicksicht auf die eben genannte, nicht zu
vernachlassigende Thatsache eine ins Gewicht fallende. Ueber-
blicken wir die Voraussetzungen und die Erfolge des Versuches
bis hierher, so haben wir fast alle Momente beisammen, welche
bei der Einwirkung der Heidenhain 'schen Lymphagoga (der
Lebergifte) auf den Lymphstrom zur Beobachtung gelangen und
welche von Seiten Starling's und seiner Anhanger zur Er-
klarung derselben angefiihrt werden. Nach Injection von Krebs-
muskelextract , Blutegelextract , Pepton etc. wird die Lyniph-
bildung vermehrt, die Lymphe concentrirter ; beim Pepton ist
zudem noch eine Beeinflussung der Gefassweite und der sog.
»Vasomobilitat« constatirt worden, welche die gr5sste Aehnlich-
keit mit der Arsenikwirkung auf die Gefasse besitzt. Und doch
besteht ein frappanter Unterschied, welcher auch in dem nachst-
folgenden Versuche zur Geltung kommt.

(Siehe Tabelle S. 201.)

Auch dieser Versuch zeigt wiederum die Vermehrung des
Lyniphstromes und die Erhohung der Concentration. Eine
weitere Aehnlichkeit mit den Erfolgen der Injection von Leber-
giften besteht ferner noch in den Concentrationsverhaltnissen
des Blutes; wie bei der letztgenannten steigert sich auch wahrend
der Arsenikvergiftung der Gehalt des Gesammtblutes an festen
Bestandtheilen, woraus abermals folgt, dass Arsenik einen ver-
mehrten Austritt von Plasma aus den Blutgefassen veranlasst.

Von Dr. med. L. Asher u. Dr. W. J. Gies.

201

Tal)eUe VI.

Versuch 8. Hund 12,5 kg. 16 eg Morphium; sehr tiefe Narkose.

Zeit

Lymph-

menge
in ccm

Lymph-
menge
pro Jlin.
in ccm

Proeent-

gehalt

an festen

Substanzen

Bemerkungen

9h 40'

— lOh

11'

5,4

0,25

6,40

10 h 4 ' 2, 1.558 g Blut aus der Art.
fem. mit 17,02% fester Substanz.

10 » 11

— 10.

33'

8,5

0,39

lOhll' — 18' 0,03 g Natr. ars. in
die V. fem.

10 » 33

— 10»

55'

9,2

0,42

6,59

0,03 g Natr. ars. in die V. fem.

10. 55

— 11»

11'

8,7

0,44

6,05

10 h 59' — 11 li 4' 0,03 g Natr. ars.
in die V. fem.

11 » 11

— 111

39'

7,2

0,36

6,48

11 h 22 ' 2,2795 g Blut aus der Art.
fem. mit 18,29 «/o fester Substanz.

11 » 39

— 12^.

1'

10,3

0,47

6,54

12. 1

— 12:.

15'

7,3

0,52

7,03

12hl5' Tod des Hundes ; nacbdem
Tode Lymphfluss sehr langsam ;
fast ganz stockend v. 12 h 55' an.

12 > 15

— 1 .

15'

7,8

0,13

Aber nicht minder tritt der Unterschied der Arsenikwirkung auf
den Lymphstrom gegeniiber derjenigen der Heidenhain'schen
Substanzen zu Tage. Wie seltsam contrastiren beim Arsenik
auf der einen Seite die tiefgreifenden Schadigungen der Gefass-
und Darmschleimhautzellen und die eventuellen profusen Exsuda-
tionen, auf der anderen Seite die verhaltnissmassig geringfiigige
Beschleunigung und die sich in engen Grenzen haltende Con-
centrirung der Lymphe mit der gewaltigen Vermehrung der
Lymphmenge und deren sehr starker Anreicherung an festen
Substanzen durch die unvergleichlich unschuldigeren Lebergifte.
Was den Contrast noch verscharft, ist, dass das Arsenik tiberall
im Korper als ein Capillargift sich erweist, ein Lymphagogum
aber nur auf dem beschrankten Gebiete der Leber und des
Darmes (was iibrigens bis jetzt nur fiir das Pepton erwiesen ist).
Hierzu kommt ferner noch die Thatsache, dass Arsenik eine
Steigerung des Zerfalls der Gewebszellen und so bedeutsame
Stoffwechselveranderungen wie Fetttransporte nach besonderen
Stellen des Korpers veranlasst; den Anschauungen zu Folge,
welche wir bei friiherer Gelegenheit entwickelt haben, mtissen
solche Vorgange zur Bildung einer stoffreicheren Lymphe bei-
tragen. Dieses Moment muss also mit der Erhohung der Per-
meabiUtat der Gefasswande concurriren, wenn es sich um die

202 Untereuch. iiber die Eigenschaften n. Entstehung der Lymphe.

ursacliliche Erklarung der Lymphbildung unter dem Einflusse
von Arsenik handelt,

Wir glauben, durch die Darlegung der Unterschiede zwischen
den Wirkungen des Arseniks einerseits, wie sie aus den zwei
besprochenen und einem dritten sofort mitzutheilenden Versuche
sich ergeben baben, andererseits denjenigen der Lymphagoga
1. Classe, neue Belege dafiir erbracht zu baben, dass die Hypo-
tbese, nacb welcber die Wirkung der letztgenannten Substanzen
ausschliesslich auf Rechnung erbohter Permeabilitat der Unter-
leibsgefasse zu setzen sei, unhaltbar ist. Die Ueberlegenbeit der
Lebergifte als lympherzeugende Mittel gegeniiber dem deletaren
Protoplasma- resp. Gefassgifte Arsen berubt auf dem Hinzutreten
eines physiologischen Momentes, dem von uns nachgewiesenen
gesteigerten Tbatigkeitszustande der grossten Unterleibsdrtise.

Ein actives Eingreifen der Capillarendothelien in Heiden-
hain's Sinne wiirde gleicbfalls die Ueberlegenbeit der Lympha-
goga vor dem Arsen erklaren. Die Beobaehtungen, welche wir
im weiteren Verlaufe des 7. Versuches (Tabelle V) gesammelt
baben, gibt uns auf neue Veranlassung, vorlaufig von dem activen
Eingreifen der Capillarendotbelien wegen Mangels an bestimmten
Beweisen fur dasselbe abzusehen. Denn als auf der Hobe der
Arsenikvergiftung eine intravenoseTraubenzuckerinjection gemacht
wurde, traten die gewobnten Folgen am Lymphstrome auf. Zu-
nachst einmal die sebr starke Bescbleunigung des Lymphflusses.
Das Gelingen dieser ausserordentlichen Bescbleunigung — der
Bescbleunigungsquotient erreichte den hohen Wertb 17,4 — be-
seitigt den etwaigen Einwand, dass die Schwere der Arsenik-
vergiftung verbindert babe, dass die Folgen der erhohten Permea-
bilitat der Gefasswande sich geltend machten. Worauf es aber
im Augenblicke noch mehr ankommt, ist die Tbatsache, dass die
Zuckerausscheidung aus dem Blute mit so grosser Geschwindig-
keit vor sich geht, dass schon in dem Zeitraume 4 — 14 Minuteu
nach der vollendeten Zuckerinjection die Zuckerconcentration
der Lymphe den sehr hohen Werth 1,411% erreicht hat. Man
wird schwerlich annehmen konnen, dass ein so heftiges Capillar-
gift wie das Arsen die Zuckerausscheidung ungestort belassen

Von Dr. med. L. Asher u. Dr. W. J. Gies. 203

hatte, wenn diese wirklich, wie Heidenhain andeutete, auf
einer secretorischen Leistung der Capillarendothelien beruhte.
Wir haben somit das interessante bisherige Ergebniss, dass so-
wohl Chinin wie auch Arsen auf die Entfernung des Zuckers
aus dem Blute ohne Einfluss ist und erblicken darin experi-
mentelle Sttitzen fiir die Annahme, dass den Capillarendothelien
nicht das Vermogen zukommt, Zucker aus den Gefassen aus-
zuscheiden. Es mag freilich noch einmal daran erinnert werden,
dass den Chinin versuchen, fiir sich allein betrachtet, keine er-
hebliche Beweiskraft aus friiher erorterten Griinden beigemessen
werden kann.

Die Permeabilitatsverhaltnisse bei der Arsenikvergiftung haben
wir noch auf eine andere Weise in dem Versuche, iiber welchen
Tab.VII (S. 205) Auskunft gibt, der Priifung unterzogen. Was die
reine Arsenikwirkung auf den Lymphstrom anbelangt, so lehrt
dieser Versuch, wie die friiheren, die erhebliche Steigerung der
Lymphbildung und der Concentration unter dem Einflusse des
Giftes. Die Beschleunigung des Lymphstromes ist eher etw^as
grosser als in den beiden anderen Versuchen; der Concentrations-
zuwachs ist zwar sehr ausgepragt, wiederum aber nicht gleicher
Grossenordnung als wie bei den Lymphagogis, trotz der durch
die Lymphvermehrung erwiesenen erhohten Permeabilitat. Als
weiteres Prtifungsmittel der schon durch die Verhaltnisse des
Lymphstromes erwiesenen erhohten Durchlassigkeit der Gefasse
wandten wir ein zuerst von 0 r 1 o w , dann von Cohnstein
naher untersuchtes Verfahren an. Orlow^) hatte mit dem Blut-
plasma isotonische Fltissigkeiten in die Peritonealhohle gebracht
und gefunden, dass dieselben daraus resorbirt wurden, ohne dass
eine merkliche Aenderung des Lymphstroms aus dem Brust-
lymphgange eintrat. Cohnstein ^j hatte nach Infusion von
2 1 Kochsalzlosung in die Bauchhohle nur bei Massage des

1) W. N. Orlow, Einige Versuche uber die Resorption in der Bauch-
hohle. Pfliiger's Archiv 1894, Bd. 59 S. 170.

2) W. Cohnstein, Ueber Resorption aus der Peritonealhohle. Central-
blatt f. Physiologic 1895, Bd. 9 No. 13 S. 401.

204 Untersuch. ttber die Eigenschaften u. Entstehung der Lymphe.

Tabelle VII.

Versuch 9. Ilniul 12 kg. 16 eg Morphiumnarkose.

Zeit

Lymph-
menge
in ccm

Lymph-
menge
pro Min.
in ccm

Procont-

gehalt

an festen

Substanzcn

Bemerkungen

9h 37

— 9h

47'

2,7

0,27

4,37

9. 47

— 10.

2 '

4,9

0,33

4,38

9h 47'— 52Vj' 0,03 g Natr. ars. in
die V. fern.

10. 2
10 > 17

— 10.

— 10.

17'
27'

6,9

7,8

0,46
0,78

4,51
4,55

10 h 3' 2'— 8' 0,03 g Natr. ars. in die
V. fem.

10 . 27
10 » 37'

-10.
— 10.

37'
44'

10,8
9,7

1,08
1,39

4,59
5,30

10 h 28'— 31' 0,03 g Natr. ars in die
V. fem.

10. 44'

11 » 0'

— 11 .

— 11 .

0'
10'

19,4
9,4

1,21
0,94

4,73
4,54

10 h 48'— 52' Peritonealhohle wird
croffnet, um in die Oeffnung cine
Pipette einzufuhren; 53'— 59'
20ccm ciner 0,85 proc. Kochsalz-
losung in die Peritonealhohle.

11 » 10'

— 11 .

30'

23,0

1,15

4,80

11 > 30'

— 11 »

55'

27,5

1,80

4,79

11 . 55'

— 12.

10'

15,0

1,00

4,«4

Thier starb um 1 h; bei der Section
flnden sich in der Bauchhohle
90 ccm Fliissigkeit.

Leibes und Hochbinden der Hinterbeine Ansteigen der Lymph-
menge, und bei Infusion der gleichen Menge nach 1^/2 Stunden
Dauer des Versuches eine Abnahine der Concentration von 5,73
auf 5,42% beobachtet. Nach unseren Erfahrungen wiirde sich
auch ohne den Versuchseingriff in Bezug auf die Concentration
so ziemhch das Gleiche ereignen, Wir ftihrten nur 200 ccm
isotonischer Kochsalzlosung in die Bauchhohle ein, von welcher
im hochsten Falle 110 ccm resorbirt wurden. Es hat nun, wie
die Versuchsergebnisse lehren, die Aufnahme dieser geringen
Fliissigkeitsmenge in das Blut gentigt, um die durch die Arsen-
vergiftung herbeigefiihrte Steigerung der Concentration der Lymphe
von der erreichten Hohe herabzudriicken und langere Zeit auf
einem niedrigeren Werthe festzuhalten. Es geht daraus hervor,
wie wenig leistungsfahig die blosse Erhohung der PermeabiHtat
der Gefasswande in Bezug auf die Concentrirung der Lymphe
ist, obwohl in dem vorhegenden Versuche die Arsenvergiftung
fortfuhr, sich zu vertiefen. Die gute Durchlassigkeit der Ge-
fasse wird ferner im vorliegenden Versuche durch die verbal tniss-
massig rasche Resorption der isotonischen Losung erwiesen.

Von Dr. med. L. Asher u. Dr. W. J. Gies. 205

Uebrigens lehren zahlreiche Erfahrungen der Pathologie, dass
schon ziemlich gewaltsame Eingriffe an den Gefassen und Geweben
stattfinden mtissen, um die Durchlassigkeit der Gefasse so weit
zu erhohen, dass sehr eiweissreicbe entziindliche Transsudate
entstehen.

Einen Augenblick miissen wir noch bei der Discussion der
Bedeutung erhohter Permeabilitat der Gefasse verweilen, aus
Anlass einiger anderen Beobachtungen, welche zu der gleichen
Auffassung fiihren wie die bisher entwickelte. Heidenhain's
Lymphagoga soUen nach Starling ihre merkwiirdige Wirkung
vermehrter Durchlassigkeit der Lebercapillaren verdanken, eine
Hypothese, welche angesichts der vielen Vorgange, die im
lebenden Organismus sich als gekntipft an den Einfluss der
Lymphagoga erwiesen haben, der schwachste Punkt der mecha-
nischen Lymphtheorie ist. (Wir sehen im Augenblicke von den
in unserer ersten und zweiten Mittheilung niedergelegten Beob-
achtungen tiber Anregung der Leberthatigkeit ganz ab.) Nun
hatte Heidenhain seiner Zeit schon einen interessanten Ver-
such mitgetheilt, welcher beweisen sollte, dass die Wirkung der
Lymphagoga ein Lebensvorgang sei ; er hat namlich gezeigt, dass
nach zeitweiliger Verschliessung der Aorta die charakteristische
Wirkung der Lymphagoga vollig ausbleibt. Daraus zog er den
Schluss, dass durch Schadigung einer physiologischen Function
die lymphtreibende Wirkung jener Substanzen unterdriickt worden
sei und zwar glaubte er, gemass seinen ofters erorterten An-
schauungen, dass die Erregbarkeit der activ secretdrischen Capillar-
zellen ftir jene Gifte durch die Anamie aufgehoben worden sei.
Dieser, nach vielen Analogien, wenigstens was die Schadigung
irgend eines physiologischen Vorganges anbetrifft, durchaus be-
rechtigten Vorstellung setzte S t a r 1 i n g i) die Muthmaassung ent-
gegen, dass durch die lange Anamie Verhaltnisse geschaflten
worden seien, dass die Folgen der vermehrten Durchlassigkeit
der Gefasse sich nicht ausbilden konnten. Eine Reihe von Be-
obachtungen nun, welche der Eine von uns gemeinsam mit

1) E. H. Starling, On the mode of action of lymphagogues. Journ.
of Physiol. 1894, Vol. XVII p. 30.

206 Untersuch. iiber die Eigenschaften u. Entstehang der Lymphe.

Dr. J. P. Arnold aus Philadelphia gelegentlich einer anderen,
demnachst zu verofEentlichenden Untersuchung gemacht hat,
lehren im Gegentheil, dass die zeitweilige Verschliessung der
Aorta der Ausbildung erhohter Pemieabilitat der Gefasse ausser-
ordentlich forderlich ist. Diese Thatsache ergab sich aus fol-
genden Erfahrungen : Nach Verschliessung der Aorta am Aorten-
bogen und WiedererOffnung derselben geniigte sehr oft eine geringe
Menge von intravenos injicirter Kochsalzlosung, welche sonst
spurlos am Organismus voriibergeht, um Transsudationen in den
verschiedenen serosen Hohlen zu veranlassen. Es ist dies ein
sicherer Beweis fiir die erhohte Dnrchlassigkeit der Gefasse,
Magnus hat in seiner citirten Arbeit die ungemein erhohte
Durchlassigkeit der todten Gefasse exi)erimentell schlagend er-
wiesen. Ware also wirklich die wesentliche Ursache der Wir-
kung der Lymphagoga in der vermehrten Permeabilitat zu suchen,
so miisste sich dies gerade nach zeitweiliger Verschliessung der
Aorta offenbaren. Thatsachlich beweist also der negative Aus-
fall von Heidenhain's oben beschriebenen Experimenten, dass
die Wirkung seiner Lymphagoga nicht zureichend durch die An-
nahme erhohter Permeabilitat der Gefasse erklart werden kann.
Ueberblicken wir nochmals die Ergebnisse der Arsenversuche,
so lehren sie jedenfalls, dass Arsen einen grossen Einfluss auf
die Lymphbildung hat, dass seine Wirksamkeit aber trotz erweis-
licher, stark erhohter Durchlassigkeit der Gefasse weit zuriick-
steht hinter derjenigen so viel harmloserer Mittel wie Krebs-
muskel- oder Blutegelkopfextract. Es hat sich auf diese Weise
durch die Anwendung des Arsens den friiheren positiven Be-
weisen ftir die i>physiologische Componente* der zuletzt genannten
Mittel ein neuer Beweis zugesellt. Audererseits ergibt sich aus
der Art und Weise, wie wahrend einer tiefen Arsenvergiftung
dem Organismus kunstlich zugefiihrtes Wasser und Zucker aus
dem Blute in die Lymphe iibertritt, kein Anhaltspunkt fiir die
Auffassung, dass eine active, secretorische Thatigkeit der Capillar-
endothelien regelnd hierbei eingriffe. Es ist vielmehr wahrschein-
lich gemacht worden, dass diese Erscheinungen zur sphysika-
lischen Componente« bei der Lymphbildung gehoren; aber auch

Von Dr. med. L. Asher u. Dr. W. J. Gies. 207

nicht mehr wie wahrscheinlich , denn welche Gewahr besitzen
wir daftir, dass das Arsen alle physiologischen Vorgange, welche
in Betracht kommen konnten, beseitigt habe?

Einiges iJber Lymphbildung nach dem Tode.

Mit unserem Hauptthema, dem Einflusse von Protoplasma-
giften auf die Lymphbildung, steht die Untersuchung der Lymph-
bildung nach dem Tode scheinbar in einem nur losen Zusammen-
hange. Thatsachlich war auch der Zufall, dass gelegentlich eines
nicht gewollten Vergiftungstodes ganz tiberraschende und fiir die
Theorie der Lymphbildung bedeutungs voile Erscheinungen zu
Tage traten, die nachste Veranlassung fiir ein Eingehen nach
dieser Richtung bin. Aber doch besteht auch ein mehr innerer
Zusammenhang ; denn der Tod des Organismus ist der machtigste
Zerstorer des lebenden Protoplasmas. Da diese Zerstorung aber
eine ganz allmahliche ist, das Erloschen der einzelnen Functionen
fiir die verschiedenen lebenden Theile zeitlich ein ganz getrenntes
sein kann, konnte auch daran gedacht werden, dass die Unter-
suchung der Lymphbildung nach dem Tode als eine Methode
der Analyse sich brauchbar zeigen wiirde.

In Tab. VIII (S. 209) ist einVersuch mitgetheilt, in welchem das
Versuchsthier in Folge der schweren Chininvergiftung starb. In
der 8. bis 4. Minute vor dem Tode waren dem 9^/2 kg schweren
Thiere 25 g Traubenzucker intravenos beigebracht worden, also
pro Kilo 2,6 g. Trotz der Schwere der Vergiftung, welche nach
Allem, was wir wissen, ein tiefes Darniederliegen der Kreislaufs-
verhaltnisse bedingen musste, hob sich so fort, d. h. innerhalb
der vier Minuten Injectionsdauer und den zwei darauf folgenden
Minuten die ausfliessende Lymphmenge um das 4^/2fache. Dies
mag hervorgehoben werden, weil von Seiten der Anhanger der
Filtrationstheorie Gewicht darauf gelegt wird, dass die erste Folge
der intravenosen Krystalloidinjection eine anfangliche Verringe-
rung des Lymphflusses sein musse.^) Das Nichteintreten

1) W. Cohnstein, Ueber die Einwirkung intravenoser Kochsalz-
infusionen auf die Zusammensetzung von Blut und Lymphe. Pfltiger's Arch.
1895, Bd. 59 S. 508.

208 Untcrsucli. iiber die Eigenschaften u. Entstehung der Lyraphe.

Versuch 10.

Tabcllo VIII.

Hund 9,5 kg. Morphiumnarkose.

Zeit

Lymph-
inenge
in ccm

Lymph-
menge
pro Min.
in ccm

Procent-
gehalt an

festen
Substanz.

Zucker

in
Procent

Remerkungen

nil 7'

— l)h

20'

5.2

0,4

5,62

9 > 20'

— 9»

39'

7,6

0,4

0,192

9 h 20-31' 1 g Chinin raur.
in die V. fern.

9» 40'

— 9 >

46'

11,0

1,83

1,095

9 h 40 — 41 ' 25 K Traubon-
zncker + 0,3 g Chinin mnr.
in 80 ccm. StiV/Aosung in
•lie V. fem. 9 h ir>' 35 ccm
Kliit aus d. .\rt. fem. mit
0,7070/0 Zucker.

9 * 46 '

— 9»

50'

15,0

3,75

1,646

Tod des Hundes 9 h 48'.

9» 50'

— 9»

53'

9,5

3,17

1,875

9 » 53 '

— 9»

59'

10,5

1,75

1,920

9» 59'

— 10.

9'

10,5

1,05

2,031

10 » 9'

— 10.

24 '

11,0

0,73

2,138

10 » 24'

-10.

44'

12,0

0,60

2,165

10. 44'

-10.

54'

6,0

0,60

5,77

10 » 54'

— 11.

54'

26,5

0,44

5,80

2,237

11 . 54'

— 12>

54'

15,0

0,25

5,90

1,825

dieser Verringerung , welche wir iibrigens niemals beobachten
konnten, liegt in diesem Falle mit aller erwiinschten Deutlichkeit
zu Tage. In den nachsten vier Minuten, innerhalb welchen das
Thier stirbt, wachst die Beschleunigung bis iiber das 9 f ache.
Wie aus unseren friiheren Chinin versuchen, geht auch aus diesem,
vielleicht mit noch grosserer Scharfe, hervor, dass Chinin gegen-
iiber der Lymphbeschleunigung durch Zuckerinjection machtlos
ist. Dass dieses Versagen des Chinins aber der Filtrationstheorie
zu gute kommt, erscheint uns wenig annehmbar angesichts des
vorliegenden ^^e^suchszustande. Auch hinsichtlich der Frage
der Zuckerausscheidung ist dieser Versuch lehrreich; denn der
Zucker A^erlasst mit der gewohnten erstaunhchen Raschheit die
Blutbahn: schon in den ersten 6 Minuten wachst die Zucker-
concentration der Lymphe auf 1,095%, M'ahrend in derselben
Zeit die Zuckerconcentration des Blutes auf 0,707% offenbar
wieder gefallen ist. Der Anstieg erreicht in den nachsten vier
Minuten den Werth von 1,646 "/q. Wiederum ist, wie in den

Von Dr. med. L. Asher u. Dr. W. J. Gies. 209

friiheren Chininversuchen, jene merkwiirdige Erscheinungsreihe,
welche nach H e i d e n h a i n in dem Secretionsvermogen der Capillar-
endothelien wurzelte, unversehrt geblieben. Wir verweisen auf
unsere am. Schlusse der Chininversuche vorgetragenen Erorte-
rungen iiber die Frage, woher es kommen moge, dass Chinin
spurlos an jener »physiologischen Componente« vorubergehen
konne, vorausgesetzt, dass eine solche in diesen Processen vorliegt.

Weit interessanter ist aber das Verhalten des Lymphstroms
nach dem Tode. Drei Stunden lang nach dem Tode
fliesst aus dem Brustlymphgang, ohne jede ktinst-
liche Mithilfe, ein ergiebiger Lymphstrom. Wohl als
erster Eindruck drangt sich die Ueberzeuguug auf, dass die Lehre
von der unmittelbaren oder gar zwingenden Abhangigkeit der
Lymphbildung vom Blutdruck, die neuere Filtrationstheorie,
diesem Experimente gegenliber ganz und gar versagt,

Vor der weiteren Discussion des eben Gesagten eriibrigt es
noch, kurz die Verhaltnisse der Zuckerconcentration in der post-
mortalen Lymphe zu erledigen. Zwei Stunden lang steigt die
Zuckerconcentration der Lymphe an und erreicht ganz ungewohn-
Hch hohe Werthe. Zwei Griinde, glauben wir, liegen in den
Versuchsbedingungen hiefiir zur Erklarung vor: erstens der Weg-
fall der Zuckerausscheidung durch die Niere (bei Ausschaltung
der Nierenfunction durch Unterbindung der Nierenarterien beob-
achtete Heidenhain das gleiche Verlialten), zweitens das ver-
muthhche Erloschen einer Reihe von physiologischen Zellfunc-
tionen, welche sonst zur rascheren Beseitigung des Zuckers aus
der Lymphe beitragen wtirden. Da sich der Umfang, welche
diese beiden Momente gewinnen, gar nicht bemessen lasst, darf
nicht allzuviel Gewicht auf die Thatsache gelegt werden, dass
lange Zeit aus dem zuckerarmeren Blute Zucker in die zucker-
reichere Lymphe hiniibergeschafft wird. Immerhin ist das post-
mortale Auftreten dieser Erscheinung sehr bemerkenswerth und
kann gemeinsam mit den mannigfachen friiher mitgetheilten Er-
fahrungen gegen die Annahme von dem secretorischen Vermogen
der Capillarendothelien verwerthet werden : fiir sich allein beweist
aus naheliegenden Grtinden diese Erscheinung niclits dagegen.

210 Untersuch. iiber die Eigenschaften u. Entstehung der Lvmphe.

Dass es sicli bei diesem Versuche um Zucker allein handle und nicht
etwa um andere postmortal gebildete reducirende Substanzen, haben wir da-
durch zu beweisen versucht, dass wir eiweissfrei gemachte Lymi)he vergilhren
lieesen und nach der Vergahrung keine Reduction niehr constatiren konnten ;
ausserdem stellten wir Phenylosazon dar.

Die nahere Betrachtung des vorliegendeii Versuches lehrt,
dass die Beschleunigung, wenn auch abnehmend, eine Stunde
lang nach dem Tode anhalt, und auch wahrend der ganzen
zweiten Stunde betragt die Menge pro Minute immer noch ein
klein wenig mehr als zu Anfang des Versuches vor der Chinin-
vergiftung. Selbst in der dritten Stunde ist der Lymphfluss kein
schlechter. Es erhebt sich die Frage, wie erklart sich die Bil-
dung der Lymphe und woher kommen die Triebkrafte zum Aus-
stossen derselben im vorhegenden Falle? Dass die todthche
Chininvergiftung nichts damit zu thun habe, lehrt Versuch 11
in Tabelle IX. Sofort mit dem Tode stockt der Lymphstrom

Tabelle IX.

Versuch 11. Hund 12 kg. Morphiumnarkose.

Zeit

Lymph-
menge
in fcm

Lymph-
menge
pro Min.
iu ccm

Procent- .
gehalt der

festen
Substanzen

Bemerkungen

9h 21

— 9h

36'

2,4

0,16

4,87

9»

36

— 9>

53'

3,8

0,22

5,24

9 h .3G'— 51' 1 g Chinin mur
Salzlosung in d. V. fem.;
leichte Convulsionen.

in 80 ccm
am Ende

9>

5-S

— 10.

5 '

2,25

0,19

6,26

Lymphe wird wahrend des Versuches

blutiger; 10 h 4' Tod.

10.

5

— 10.

13'

6,8

6,15

Kein Ausfluss ausser durch Pumpen.

und lasst sich nur, wie das schon lange bekannt ist, durch
Pumpen kiinstlich im Gauge erhalten. Hingegen wurde in dem
oben beschriebenen Versuche 8 (Tabelle VI) nach dem Vergif-
tungstode durch Arsen eine Stunde lang vollstandiges Ausfliessen
der Lymphe beobachtet, also in einem Falle, wo ein lymph-
treibendes Agens angewandt worden war. Aber jener Lymph-
fluss verlangsamte sich, ganz anders wie in diesem Versuche,
momentan ganz erheblich mit dem Tode und blieb an der Grenze
des Versiechens. Das lymphtreibende Mittel in unserem Falle
ist die vorausgegangene intravenose Traubenzuckerinjection und

Von Br. med. L. Asher u. Dr. W. J. Gies. 211

hierin liegt die grosse theoretische Bedeutung des Experimentes.
Die Filtrationstheorie, deren plausibelste Seite — wenn auch
durchaus nicht einwandsfrei — die mechanische Deutung der
Lymphbeschleunigung nach intravenoser Krystalloidinjection war,
lehrt, dass dnrch die Salzinfusion der osmotische Druck des
Blutes iiber die Norm steigt, in Folge dessen das Blut aus den
Lymphspalten Wasser anzieht und nun durch den abnormen
Fltissigkeitszuwachs der intracapillare Druck steigt; entsprechend
den Filtrationsgesetzen filtrirt dann eine grossere Menge verhaltniss-
massig wasserreichen Blutplasmas. Beim todten Thiere kann
von einer derartigen Erhohung des Capillardrucks keine Rede
sein; selbst wenn man den arteriellen Blutdruck mit Starling
nicht als maassgebend fur die Hohe des Capillarblutdrucks an-
sieht, wird man nicht annehmen diirfen, dass bei stillstehendem
Herzen und arteriellem Nulldruck nach dem Tode langere Zeit
ein Capillardruck bestehen kann, der fahig zu vermehrter Fil-
tration sei. Wir behaupten, dass aus diesem Experimente folgt,
dass die Lymphbeschleunigung nach Krystalloidinjection nicht
ihre Ursache in gesteigertem Capillardrucke habe.
Nach der Widerlegung der Filtrationshypothese tritt die ursprting-
liche Heidenhain'sche Erklarung in ihre Rechte wieder ein:
»die injicirten Substanzen treten durch Diffusion schnell aus
dem Blute in die Lymphraume und wirken hier wasseranziehend
auf das Gewebswasser der Zellen, Fasern u. s. f. ; das diesen ent-
zogene Wasser fliesst zum Theile durch die Lymphkanale ab.«
Wenn diese Annahme richtig ist, so muss die Lymphbeschleu-
nigung abhangen von der Zuckermenge, welche Gelegenheit hat,
vor dem Tode in die Gewebsspalten tiberzutreten. Der Versuch
bestatigt, dass diese Bedingung von dem grossten Einflusse ist.
In Vers. 12 (Tab. X, S. 213) war das Thier schon eine Minute nach
der voUendeten Traubenzuckerinjection gestorben; es kommt
zwar zur sofortigen Beschleunigung und diese halt zehn Minuten
nach dem Tode an, dann aber mindert sich der Ausfluss und
hort drei Viertelstunden nach dem Tode ganz auf. Dem ersten
Versuche hingegen vollkommen gleich verhalt sich der letzte hier
mitzutheilende in Tabelle XI (S. 213).

212 Untersuch. iiber die Eigenschaften u. Entstehung der Lymphe.

V e r 8 u c h 12.

Tabollo X.

Hiind 7 kg. Morphiumnarkose.

._ -

I.ynil)h-
mengc

Lyinph-

Procont-

Zeit

nienKe
l)ro Min.

gehalt der
festen

Beinerkuugeu

in ccm

in ('('in

Siibstanzen

Ih

0

—

2h

0'

6,8

0,11

4,66

2 h 10' - i:! 21 g Traul)eu7.iifkur In
die V. jugularis, sofortige Be-
schleunigung. 2 h 13' Chloroform
in das Herz. 2h 14' Tod constatirt.

2.

14

—

2 >

24'

10,0

1,0

4,6n

2 >

24

—

2 .

40'

3,0

0,19

1

2.

40

—

3 .

0'

1,0

0,5

1

Tabelle XI.

Versuch 13. Hund 12 kg. Morphiumnarkose.

11 h 15'

12 > 9 '

12 > 15!'
12 » 25'
12 . 30 '
12 » 35'
12 . 40 '

11 h 55'

12 > 15i'

12
12
12
12
12

25'
30'
35'
40'

45'

3,1

0,078

5,51

1

1,8

0,28

5,73

4,6

0,49

4,71

1,8

0,36

\

2,8
1,4

0,56
0,28

> 4,17.

1,2

0,24

12 h 7'— 9' 30 g Traubenzucker iu
die V. jug. 12 h lr>' Chloroform
in die V. jugiil. 12 h 15'/2' Tod.

Wfthrcnd der ganzen Zeit starker
Speichelfluss u. starkes Secerniren
der Augendriisen. 1 h 5' noch
lobhnftes Ausfliessen yon Speichel
und Lymphe, wean auch lang-
samer als vorher.

Hier verlaiift alles so, als ob das Thier noch lebte. Das
Maximum der Beschleunigung, das 7,2fache gegeniiber dem
Lymphflusse vor dem Versuchseingriffe, tritt 21 bis 26 Minuten
nach Vollendung der Traubenzuckerinjection ein, zu einer Zeit,
wo das Thier schoii iiber eine Yiertelstunde todt ist. Langer als
drei Viertelstuiiden halt die sehr ausgepragte Beschleunigmig
des Lymphstromes an. Auch die Concentrationsverhaltnisse der
Lymphe entsprechen den bekannten Erfahrungen bei den nam-
lichen Versuchen am lebendeu Thiere. Die Erklarung fiir den
geschilderten Gang der Ereignisse liegt in den Versuchsbedin-
gungen deutlich zu Tage. Hier war nach vollendeter Zucker-
injection dem Zucker 6^/0 Minuten Zeit geboten, um sich in den
Gewebsspalten anzuhaufen; in diesem ersten Zeitraume findet ja
bekanntlich die grosste Abnahme der Zuckerconcentration des
Blutes statt. Die dargelegten Versuche beweisen, wenn wir sie
zusammenfassend betrachten, dass die vermehrte Lymphbildung

Von Dr. med. L. Asher u. Dr. W. J. Gies. 213

nach Injection von Krystalloiden nicht eine Function des ge-
steigerten Blutdruckes ist, wohl aber nach Heidenhain in ein-
fach physikalischer Weise durch die Anziehung der krystalloiden
Substanzen zu dem Gewebswasser erklart werden kann.

Die Triebkraft zum Ausstossen der vermehrt gebildeten Lymphe
kann in unseren Versuchen auch nicht in dem Blutdrucke gesucht
werden. Es kann durch die grundlegenden Arbeiten Ludwig's
und seiner Schiiler als gesichert betrachtet werden, dass unter
physiologischen Verhaltnissen der Blutdruck eine wesentliche
Rolle bei der Mechanik des Lymph stromes spielt. Dass aber
noch andere Momente mitwirken, lehren die vorliegenden Ver-
suche. Dass die blosse Mehrbildung von Lymphe nicht noth-
wendiger Weise eine vermehrte Abfuhr derselben bedingt, beweisen
zahlreiche Beobachtungen ; Oedeme konnten nicht so hartnackig
bestehen, wenn mit der Bildung die Wegschaffung der Lymphe
Hand in Hand ginge. Es liegt die Annahme nahe, dass in den
vorliegenden Versuchen der osmotische Druck des Zuckers, wie
er die Ursache der vermehrten Lymphbildung ist, auch diejenige
des postmortalen Fliessens ist. Aber neben dieser Annahme
sind noch andere Moglichkeiten denkbar, die aber hier nicht
weiter discutirt werden mogen.

Nur ein letzter wichtiger Punkt bedarf im Anschlusse an
die mitgetheilten Beobachtungen der naheren Berticksichtigung.
Im letzten Versuche war der postmortale Lymphstrom von einer
lebhaften postmortalen Driisensecretion begleitet. Die Speichel-
secretion nach dem Tode ohne jeden Blutstrom ist, neben
Ludwig's klassischem Speicheldruckversuch, die Fundamental-
thatsache, auf welche sich die allgemein anerkannte Lehre
stiitzt, dass die Speichelsecretion kein Filtrationsprocess sei. Der
vollkommene Parallelismus der beiden Vorgange im letzten Ver-
suche weist darauf hin, dass Driisensecretion und Lymphbildung
Processe gleicher Grossenordnung sind und nicht etwa
der letztere ein einfacher Filtrationsvorgang ; er macht es auch
wahrscheinlich , dass die »physiologische Componente« bei der
Lymphbildung zum guten Theile in der Thatigkeit der speci-
fischen Zellen und nicht der Capillarendothelien gegeben sei.

Zeitschrift fiir Biologie Bd. XL N. F. XXn. 15

214 Untersuch. (Iber die Eigonschaften u. Entstehung der Lymphe.

Wenn Lymphbildung iind Driisensecretion einigermaassen analoge
Processe sind, so wird dadurcb verstandlich, warum wir so wenig
liber die Triebkrafte des Lymphflusses wisseii; diejenigen der
Secretion sind ja gleichfalls noch nicht entwickelt.

Wir fassen die Ergebnisse dieser Untersucbung in Folgen-
dem zusammen :

1. Cbinin hat auf diejenigen Vorgange, welche nach intra-
venoser Zuckerinjection am Lyrapbstrorae in Bezug auf
Menge und Concentrationsverbaltnisse der festen Sub-
stanzen, sowie besonders des Zuckers zur Beobachtung
kommen, keinen erkennbaren Einfluss.

2. Die Unwirksamkeit des Chinins in dieser Beziehung
gestattet nicht mit Bestimmtheit, eine »physiologische
Componente« bei dieser iVrt der Lymphbildung auszu-
schhessen, da diejenigen Stoffwechselvorgange, welche
im Organismus zur COo-Bildung fiihren, nicht nachweisbar
gestort werden.

3. Da bei tiefer Chininvergiftung die Gefasse in Mitleiden-
schaft gezogen werden sollen, sprechen die unveranderten
Ausscheidungsverhaltnisse des Zuckers in die Lymphe
nicht zu Gunsten eines Secretionsvermogens der Capillar-
endothehen.

4. Die Wirkung der »Lebergifte« oder von Heidenhain's
»Lymphagoga erster Art« werden durch tiefe Chinin-
vergiftung unterdriiekt oder gehemmt. Hiermit ist ein
neuer Beweis dafiir gegeben , dass diese Mittel eine
»ph3^siologische Componente«, bestehend in erhohter
Leberthatigkeit als Ursache der Lymphbildung, besitzen.
Damit steht die anderweit bekannte Thatsache im Ein-
klange, dass Chinin diejenigen Processe, welche zur
Harnstoff- und zur Glykogenbildung fiihren, hemmt. Die
Wirkung der Lebergifte kann nicht ausschliesslich auf
vermehrter Durchlassigkeit der Lebercapillaren beruhen.
Die zum Mindesten nicht verminderte Durchlassigkeit
der Gefasse bei der Chininvergiftung lasst sich experi-
mentell nachweisen.

Von Dr. med. L. Asher u. Dr. W. J. Gies. 215

5. Arsen, ein »typisches Capillargift«, bewirkt den Ausfluss
einer vermehrten und hoher concentrirten Lymphe. Ob-
wohl aber die Schadigung der Eingeweidecapillaren viel
grossere sind als diejenigen weit schwacherer Mittel, wie
Krebsmuskel- und Blutegelkopfextract, ist der Umfang
der Lymphbildung durch Arsen viel geringer als bei den
letztgenannten. Hieraus folgt wiederum, dass blosse er-
hohte Permeabilitat der Gefasswande die Wirkungsweise
der Lymphagoga nicht ausreichend erklart.

6. Die Zuckerausscheidung aus dem Blute in die Lymphe
nach intravenoser Tranbenzuckerinjection verhalt sich
wie beim iinvergifteten Thiere, wesshalb eine active Be-
theiligung der Capillarendothelien hierbei unwahrschein-
lich gemacht wird.

7. Da sich auch bei tiefer Arsenvergiftung durch geeignete
Eingriffe wesentlich beschleunigter Lymphstrom erzielen
lasst, konnen Begleiterscheinungen der tiefen Arsen-
vergiftung nicht der Grund sein, warum trotz erhohter
Permeabilitat der Gefasswande nicht so machtvolle Wir-
kungen am Lymphstrome auftreten, wie durch die Leber-
gifte (Lymphagoga).

8. Zeitweilige Aortenverschliessung sowie Tod der Capillaren
fuhren zu experimentell nachweisbarer , ungemein ver-
mehrter Durchlassigkeit der Gefasse ; Heidenhain's
Nachweis, dass Aortenverschliessung die Lymphagoga
unwirksam macht, beweist gleichfalls, dass diese Sub-
stanzen nicht bloss durch Erhohung der Gefassdurch-
lassigkeit wirken konnen.

9. Lange Zeit nach dem Tode dauert ein beschleunigter
Lymphstrom in Folge von intravenoser Zuckerinjection
an; die Beschleunigung kann ihren Maximalwerth erst
eine Viertelstunde nach dem Tode erhalten. Bedingung
ftir das Eintreten eines langer andauernden postmortalen
Lymphstromes ist, dass zwischen der Vollendung der
Zuckerinjection und dem Tode vier bis sieben Minuten
vergehen. Diese Thatsachen beweisen, dass die Lymph-

15*

216 Untersuch. der Lymphe. V(jn I)r. nied. L. Ashcr n. Dr. W. J. Gies.

bildung nicht eine Leistung des Blutdruckes ist, hin-
gegen wird Heidenhain's Erklarung der Lymphbesehleu-
nigung durch intravenOse Krystalloidinjection aus der
Auziehung der Sake zu dem Gewebswasser den That-
sachen gerecht.
10. Der vollkonimeiie Parallelismus der postmortalen Speichel-
secretion and dor postmortalen Lymphbildung beweist
nicht allein die Unabhangigkeit beider Vorgange vom
Blutdrucke, sondern weist auch darauf bin, dass beiden
physiologische Processe ahnlicher Art zu Grunde liegen.

Die Mittel zu dieser Untersuchung sind von der hohen
Koniglichen Akademie der Wissenschaften zu Berlin bewilligt
worden.

[Reprinted from the Philadelphia Medical Journal, Vol. VII, No. 12, Page 566,
March 23, 1901.]

THE TOXICOLOGY OF TELLURIUM COMPOUNDS, WITH
SOME NOTES ON THE THERAPEUTIC VALUE OF
TELLURATES.

By WILLIAM J. GIES, M.S., Ph.D.,

of New York.
Instructor of Physiological Chemistry, Columbia University.

A. Action on Plants and Microorganisms. — The earliest
as well as most important researches on the biological
influence of tellurium compounds were conducted on
domestic animals and on man. It was not until 1885
that the results of a study of their action on plants was
announced. Knop, in that year, after an investigation
of the influence of various substances on growing plants
(maize) by the water-culture method, reported that
telluric acid'^ to the amount of 0.05 to 0.1 gm. per litre
of nutrient fluid, (0.005-0.01%), exerted no observable
influence on their development, although analysis of
the plants showed that tellurium had been absorbed.

Bokorny, a few years later, working with tellurous
oxide and potassium tellurite, found that aqueous solu-
tions containing only a trace of the very insoluble oxide
had no effect whatever on algae and infusoria, even after
5 days' treatment, and that 0.02% solutions of potassium
tellurite (containing, also, 0.1% of dipotassium phos-

1 Tellurium was discovered in 1782 by Miillervon Reiehenstein, and identified
and named (from iellus, the earth) by Klaproth in 1798. The metal is silver-
white, of markedly crystalline structure, with strong metallic lustre. Its atomic
weight is still uucertain, but closely approximates l'Z8. Tellurium is very nearly
related chemically to sulphur and selenium. Its chemical qualities have made
it a difiacult problem from the time of its discovery, and at first it was called aurum
paradoxum and metallum problematicum. It is one of the rarer elements and
occurs in nature mostly as telluride in combination with bismuth, lead, mercury,
silver, and gold. The following formulae show the composition and relation-
ships of the tellurium compounds referred to in this paper :

Tellurous oxide TeOj.

Telluric oxide TeOs.

Tellurous acid HaTeOa.

Telluric acid HaTeO^.

Sodium tellurite Na^TeOa.

Sodium tellurate Na2Te04.

Hydrogen telluride H^Te.

Methyl telluride (CH3)2Te.

Ethyl telluride (CaHsJgTe.

Tellurium tartrate Te (CiHgOe)*.

phate) were likewise without toxic influence, although
the algae had been kept in the fluid for a week. Under
the microscope the cells were seen to be perfectly nor-
mal in all outward aspects. Even with a 0 1 % solution
of potassium tellurite (containing a trace of potassium
hydroxide), only one form of spirogyra seemed to be
affected. All of the rest vegetated normally, even at the
end of a week of treatment. Continuing his experi-
ments, Bokorny, in the following year, reported that
when various algae, such as Spirogyra communis, S.
nitida, conferveae, diatomaceae, etc., and also infusoria,
were placed in 0.1% solution of telluric acid and kept
there in diffused light for some time, little, if any, in-
fluence was exerted. At the end of 48 hours the Algae
remained perfectly normal, and the infusoria swam
about in very lively fashion. Even after 14 days some
of the algae were still growing, in spite of the fact that
the faintly acid solution contained no mineral or other
nutrient material. Potassium tellurate (slightly alka-
line in reaction), in like quantity, was just as in'-
nocuous.*^

Scheurien, very recently, wishing to grow hdcillus an-
thracis in pure cultures, and in the absence of atmos-
pheric oxygen, sought a medium which, containing
loosely bound oxygen in oxyhemoglobin-like combina-
tion, would be almost as favorable to their growth as
blood itself. Having previously found that selenious
acid on warming with organic substances is reduced
and red selenium deposited, he experimented with so-
dium selenite and also with sodium tellurite, which on
similar treatment yields grayish black metallic tellu-
rium. He found that not only B. anthrads but also
all of the growing bacteria he worked with were col-
ored by reduced metal in the presence of sodium salts
of these acids. The bacteria themselves were colored,
not the nutrient medium. They were grown on 10 cc.
of a meat infusion peptone agar with 1 to 3 loopfuls
of a 2% solution of the salts.

These results led directly to the detailed work con-
ducted by Klett, who studied the growth of numerous
species of bacteria and some moulds under the influ-
ence of selenium and tellurium compounds, and found
that the development of various forms, such as staphyl-

* Further reference to effects on lower animals is made farther on in the re-
views of Hofraeister's and Czapek and Weil's work.

ococcus pyogenes aureus and B. mesentericus vulgatus, as
well as the various moulds, was not materially hin-
dered by slight quantities of sodium tellurite, although
several others, such as B. fluorescens liquefaciens, were
strongly retarded in growth by only traces of the tellu-
rite, which seems to be more inhibitory than the
selenite. Yet a few, such as the bacillus of malignant
edema and of symptomatic anthrax, which are
markedly arrested in growth by selenite, not only re-
duce tellurite, but appear to continue their develop-
ment in the presence of a larger proportion of the latter
salt. Most of the experiments were made on 10 to 12
cc. of nutrient medium (gelatin, agar-agar), containing
1 to 3 loopfuls of 2% solution of the tellurite. Increasing
amounts of tellurite wrought more destructive effects,
of course. The colonies in all cases, as in Scheurlen's
experiments, were colored grayish black by metallic
tellurium, the intensity of the coloration having been
proportional to growth. Grayish particles were de-
posited within the bacteria. Since the colonies only
were pigmented by the metal and the surrounding me-
dium was left entirely colorless, Klett concluded that
the reduction took place in the protoplasm of the
bacterial cell and not outside the cell by secondary ac-
tion of metabolic products. For this reason, then, he
considers tellurites, with selenites, the most satisfactory
reagents for detecting and determining accurately re-
ducing action on the part of bacteria. It was observed,
further, that the oxygen set free from tellurite during
the reduction could not be utilized by aerobic bacteria
in anaerobic environment, nor was the presence of
tellurite favorable to the growth of anaerobic forms,
Klett found, also, that tellurite, in the quantities used,
did not decrease the virulence of such forms as B.
anthracis. Sodium tellurite was the only tellurium
compound tried in this connection. Sodium selenate
in slight quantity was found to have little or no effect
on the growth of bacteria and was not reduced. Klett
appears to have concluded, from analogy, that tellurates,
also, would not be reduced by them.'

B. Effect on Cold-Blooded and on Domestic Animals. —
Chr. Gmelin appears to have been the first to give

3 The author is greatly indebted to Dr. P. H. Hiss for the references to the
work of Scheurlen and Klett, and for suggestions in connection with this reTlew
of their results.

special attention to the action of tellurium compounds
in the animal body. Early in the last century he
experimented with tellurous acid on a dog and a rabbit.
The former he gave 3 grains (0.2 gram) in a single •
dose; the latter, 14 grains (0.9 gram) in the course of
three days. The dog lost its sprightliness at first and
also its appetite, but in a few days recovered both.
The rabbit's appetite remained normal throughout the
experiment, but on the fourth day it died. On post-
mortem examination of the poisoned animals Gmelin
noted that a peculiar garlicky odor proceeded from the
abdominal cavity ; that the mucous membrane of the
stomach and intestines was much sv/ollen and covered
with a thick layer of tough mucus ; and that from the
pylorus to the rectum the walls of the intestines were
very black. The liver was covered with minute red
spots, the blood-serum colored violet, the gallbladder
widely distended and the heart full of coagulum.

A more extended series of experiments was next
carried out by Hansen, who, working in Wohler's labor-
atory, found that 0.3 gm. of potassium tellurite,
introduced directly into the stomach of a medium-sized
dog, was followed almost immediately by an unpleas-
ant, garlicky odor in the breath, similar to that which
Gmelin had noted on opening the bodies of the pois-
oned animals and which Wohler and his pupils had
attributed to ethyl telluride. Twenty minutes after
dosage repeated vomiting ensued. The symptoms
noted by Gmelin (languor and loss of appetite) were
also observed and recovery was not long delayed. The
same dose twice on the following days, morning and
afternoon, induced identical results, while the odor in
the breath became stronger each day and persisted long
after the conclusion of the experiment. The vomit
and feces were slimy and black with tellurium granules.

In a second experiment on a dog of average size, 0.5
gm. of tellurous a.cid per os on two succeeding days caused
no toxic symptoms, although the odor of the breath
became more and more marked, and the feces were
blackened by metallic tellurium. On the third day,
0.7 gm. of acid potassium tellurite induced vomiting of
grayish-black slimy, material in addition to the previous
results, and the odor of the breath rapidly grew stronger.
On the fourth day another dose of 0.7 gm. of the tellurite
caused vomiting, and considerable thick mucus ran

from the mouth. On the seventh day 0.5 gm. of the
same potassium salt, in solution, was injected into the
jugular vein. Convulsions resulted at oDce and death
followed in four minutes. The body cavity gave off
the characteristic odor and the alimentary tract as well
as the kidneys and all other glands, except the spleen
and parotids, were colored bluish-black. The liver was
not covered with the inflammatory spots, nor was the
blood-serum colored violet, as Gmelin had previously
found. The lungs, brain and spinal cord retained
their normal appearance. The pigmentation of the
glands, etc., was caused by deposition of microscopic
granules which were shown to consist of tellurium.
The peritoneal cavity contained a small quantity of
serous fluid, but neither hyperemia nor inflammation
was observed. The wall of the urinary bladder was
bluish in color and the urine, acid in reaction, con-
tained the odoriferous compound. The right side of
the heart and the vena cavae were swollen with blood.
In the crystalline lens of each eye, as reported by
Hansen's friend. Dr. Schrader, there was a deposit of
chalky granules of varying size. They were least in
quantity in the center. The cataract was greatest in the
left eye. The humours of the eye gave off the odor of
garlic. Tellurium was separated from the urine, liver,
stomach and intestines. Two additional experiments
on dogs gave results that were identical with the above
in practically all particulars. The blood-serum was
normal in color in each case.

Hansen concluded his paper with the opinion that
the pigmentation of the contents of the gastrointestinal
tract was due to deposition of tellurium by a process
of reduction and that direct absorption of the metal
through the intestinal wall was indicated by the bluish-
black color of the mucous membrane. He suggested,
further, that the violet color of the blood -serum, noted
by Gmelin, was due to the presence of absorbed metal
in suspension, and that it was not observed in his
own experiments because there had been time in
each for the tellurium to be deposited in the tissues.

Kletzinsky,* also, in experiments on animals noted
that administered tellurium was eliminated, in part, in
the urine. Rabuteau, 15 years after Hansen's results had

* Kletzinsky : Ueber die Ausscheidung der Metalle in den Secreten, Wiener:
med. Wochenschr., 1858, viii, 355.

heen recorded, found tellurium to be exceedingly
poisonous and considered it very similar in its action
to selenium, although stronger. This deduction was
based on the results of only one ex[)eriment, however,
with sodium tellurite. Following an intravenous injec-
tion of O.OS gram of that substance in a dog, vomiting
■ensued within 2 hours, after which profound dyspnea
eet in, with anesthesia, opisthotonus, and finally death
from asphyxia in 4 hours. Postmortem examination
12 hours after death showed marked congestion and
ecchymosis of the whole of the intestinal canal ; also of
the liver, spleen, lungs and especially the kidneys.
The latter were almost black as a consequence and the
tubuli were studded with fat globules. In the heart
the right side was filled with blood, the left side on the
contrary was empty.

The contents of the right side of the heart, and also
of the larger bloodvessels, held a multitude of small
prismatic crystals of unknown chemical composition —
0.002 to 0.004 mm. in width and from 5 to 10 times as
long — which, in the opinion of Rabuteau. presented a
mechanical obstacle to the movement of the blood and
thus eventually caused the death of the animal in
asphyxia. These crystals were apparently identical
with those Rabuteau reported he had found under simi-
lar conditions after intravenous injections of sodium
selenite and administrations of the same per os. They
were not produced, he says, by selenates — only by
selenites and tellurites. Rabuteau states, further, that
they were more numerous than the corpuscles. He says
nothing about their color, but his sketch of them suggests
that they may have been hemoglobin or some deriva-
tive of it. Radziejewski' seems to entertain this oi)inion.

It should be remarked, in passing, that Chabrie and
Lapicqufc® were unable to find these crystals in the blood
of animals poisoned with sodium selenite and, also, that
Czapek and Weil, whose work with tellurium is sum-
marized farther on, obtained the same negative result,
both with selenites and tellurites, after intravenous in-
jections. Rabuteau's observations in this connection
have never been confirmed. Consequently, his theory
that death after injection of tellurites results from a

» Radziejewski : In abstractor Rabuteau's paper. Cent. f. d. med. Wits., 1869,
vii,446.

» Cbahrie et Lapicque : Sur Taction phjsiologique de I'acide selenieux,
Com/jl. rend , 1890. tx, 152.

6

" mechanical poisoning," which produces asphyxia,
cannot be accepted. Rabuteau makes no reference
whatever to the work of Hansen, or any of his prede-
cessors, and says nothing definite about odor in the
expired air of the dog to which he had given tellurite.''

Czapek and Weil, in perhaps a more thorough
research than any of the preceding, learned that, in its
toxicological influence, tellurium behaves very much as
does its close chemical relative, selenium, although the
symptoms it induces appear later and are, for the most
part, weaker — just the reverse, in the latter respect, of
Rabuteau's deduction. Sodium tellurite, in quantities
of 0.002 gm., under the skin, caused the death of frogs
within 48 hours ; 0.01 gm. of sodium tellurate was
required to produce the same result. In cold-blooded
animals these quantities of tellurium gradually brought
about paralysis of the central nervous system and death.
The heart was arrested in diastole, apparently because
of paralysis of the so-called excito-motor ganglia.
Atropin did not restore the beats, and the heart-tissue
itself remained susceptible to mechanical and electrical
stimulation. The garlic odor was detected about the
animal in most of these cases. Muscular fibrillations
were almost always observed in frogs into which tel-
lurium had been injected, but neither clonic nor tetanic
convulsions followed its introduction in the quantities
employed.

In warm-blooded animals these same observers found
that 0.02 gm. of sodium tellurite, and 0.05 gm. of
sodium tellurate, per kilo of body-weight, gave very
toxic effects.^ Dogs very soon became restless. Vomit-
ing quickly ensued, followed by diarrhea, weakening
of the reflexes, somnolence, unconsciousness, general
paralysis, stoppage of respiration, and death after con-
vulsions. Within five minutes of the time of adminis-
tration of the poison, the garlic odor in the expired air
was intense. There was no muscular fibrillation as in
the case of frogs, and, with the exception of the spasm
just before death, no clonic or tetanic convulsions. In
all cases a lowering of blood-pressure followed the in-

f The odor caused by selenates Babuteau mistakenly ascribed to hydrogen
selenide instead of methyl selenide. Hofmeister, whose experiments are referred
to on a subsequent page, assumes that the usual odor was recognized by Rabuteau,
and, from analogy apparently, that it was referred in error to hydrogen telluride.

8 We are left to infer the manner of introduction of tellurium in these e.^peri-
ments. It seems to have been both, by way of the mouth and under the skin.

jection of tellurium salts. This was due, not to central
influences, but to direct peripheral action on the blood-
vessels, resulting in impaired tonic contraction, for the
vasomotor center remained sensitive to stimulation and
the vagi were able to carry impulses. The abdominal
capillaries, particularly, were very greatly distended.

The blood from animals poisoned with tellurium was
dark-colored and had a distinct garlic odor. Spectro-
scopically it was normal and the corpuscles showed no
change. Czapek and Weil could not confirm Rabuteau's
observation in this connection. Postmortem exami-
nation showed profound changes in the intestinal
mucous membrane, in which edema, congestion, and
extravasations were especially prominent. Desqua-
mation of the villi was also observed in most cases.
Destructive changes were the rule in the tubules of
the kidneys. The urine was bloody now and then,
and frequently tellurium could be detected in it.
Nearly all of the body parts, in the cold as well as
warm-blooded animals experimented on, were colored
grayish by metallic tellurium, but no deposit of the
metal in granules was observed, on microscopic exam-
ination, in any of the tissues. It seemed to be in solu-
tion. The muscles of the poisoned animals retained
their susceptibility to stimulation.

Tellurium was found to differ from selenium, in toxi-
city, mainly quantitatively.® Czanek and Weil concluded
that the diflference between the two lies in the dififerent
modes of elimination. Tellurium salts are less toxic, they
think, because the tellurium is quickly transformed by
reduction to the metallic state and so is rendered
comparatively passive at once. The results of their
experiments indicate that in its toxic action tellurium
behaves much as do selenium, arsenic and antimony.'"

Although the garlic odor in the breath and about the
organs of animals to which tellurium salts had been
administered was thought at first to be due to ethyl
telluride, its resemblance to methyl telluride, when
that substance was first made, satisfied Wohler and his
pupils that it resulted from a formation of that organic

» Also In having anidrotic action. See footnote further on, where additional
results of Czapek and Weil's work are given.

•" It is interesting to note, in this connection, that tellurium is believed by
some cheiuists to be in reality a mixture of elements, containing an antimony
arsenic-like body. Brauner calls one of the presumed constituents of the tellu-
rium complex, attstriacum, which appears to be the drvitellurium, predicted by
MendelfeefT.

8

compound. This conclusion was generally accepted for
some time. Hofmeister, in some very exact experi-
ments, finally determined in a chemical way that the
methyl synthesis, assumed by previous investigators,
really does take place when tellurium is administered
and that the garlic odor arising as a consequence is
caused by methyl telluride.^^

In experiments on warm and cold-blooded animals
he confirmed the observations of previous workers that
the various body parts take on the same odor, and
showed that it is strongest, or in other words the
methyl synthesis is relatively greatest, in the testes and
the lungs, and pronounced in the blood, liver and
kidneys. He found that when the organs of an animal
into which sodium tellurite had been injected intraven-
ously, are put in a warm place (at 36° C), the smell of
methyl telluride is intensified about those having that
odor to begin with and is gradually made distinct in
others. Under the same conditions, blood loses it,
however. Time and intensity vary, of course. These
facts show that the cells of the glands are able to
absorb tellurium and that they also have the power, at
the body temperature, of forming methyl telluride from
it. This substance is formed also by minced fresh
organs from dogs and rabbits when they are treated
with the same substance at the body temperature.
Hofmeister proved that this synthesis, with production
of the characteristic odor, takes place, also, in frogs,
fishes, crabs, and even in earthworms, when small
quantities of tellurite are given them." The tellurium
was deposited in the animals experimented on in large
part in metallic form in many parts of the body, the
reduction, judging from the discoloration, varying con-
siderably.

In the body of a dog weighing 850 gms., into which
0.04 gm. of sodium tellurite had been injected intra-
venously, and which after bleeding to death had been

11 Sodium tellarate, 0.03—0.06 gram, was injected subcutaneously into dogs
and cats. As soon as the garlic odor became evident in the expired air the
latter was passed through saturated solution of iodine in potassium iodide for 20
to 48 hours The solution decomposed the methyl telluride, but retained each
group and from it methyl was separated in the form of methyl sulphide by treat-
ment with sodium sulphide. Tellurium after evaporation of the solution and
treatment with nitric and hydrochloric acids, was precipitated in metallic flakes
with sodium sulphite.

12 Of the other influences of tellurium salts on these animals, Hoiineister says
nothing oxcept that injection of sodium tellurite into the soft parts of crabs is
followed by paralysis and death.

kept at normal temperature for four hours, practically
all parts were pigmented by tellurium except cartilage,
bone and the white matter of the nervous system.
When dosage was not too great, however, it was found
that in the lungs and testes the tellurium, instead of
having been deposited was transformed wholly into
methyl telluride, which accounts for the fact that these
organs are rarely colored by the bluish-black metallic
deposits usually found in practically all of the glands.
The long-continued elimination of methyl telluride in
the breath, Hofmeister shows, is due to gradual syn-
thetic transformation of the tellurium which had been
deposited in the tissues in metallic form soon after its
introduction. He suggests that the reduced tellurium
is slowly transformed into the soluble sodium tellurate
by the action of the alkaline tissue fluids before it
reaches the lungs, and that it is there changed to the
methyl compound. In this way he explains the persis-
tence of the odor in the breath.

Hofmeister was unable to determine the specific
source of the methyl for this synthesis, but, as the
liberation of methyl groups, and also their incorpora-
tion in other substances like cholin and creatin, seem
to be intermediate processes in general metabolism,
he concluded, from his experiments, that the tellurium
unites with methyl groups set free in some manner in
the cells. He showed that this conversion of tellurium
to methyl telluride, and the process of reduction of
tellurium compounds, may take place quite inde-
pendently of each other, for when fresh normal glands
after maceration are warmed a few minutes, at 50 to
55° C, and then treated with sodium tellurite, their
power to reduce is undiminished, although no methyl
telluride is formed by them. The synthetic process is
entirely prevented, also, after treatment of the tissues
with solutions of various chemicals — even physiolog-
ical salt solution.

Beyer, following the general suggestions of Ludwig,
demonstrated, in some transfusion experiments on per-
fectly fresh kidneys with oxygen free and arterial
blood containing sodium tellurate, that the methyl
synthesis does not take place in the absence of oxygen,
although reduction to the metallic state occurs in the
cells quite independently of the character of the trans-
fused blood. He sought also, by histological methods,

10

to determine just where in the tissues the reduction of
tellurium from its salts occurs. He injected small
quantities of sodium tellurate, dissolved in physiolog-
ical salt solution, into the jugular veins of dogs and
rabbits, and found that granular metallic tellurium
was deposited only in form elements ; in nerve and
glandular cells, leukocytes and striated muscle espe-
cially. Endothelium, unstriated muscle, nerve and con-
nective tissue fibers, on the other hand, were found to
have no affinity for tellurium. The deposit of metallic
element in the cells did not appear to cause their
degeneration. Destruction occurred only occasionally.
The cells, for the most part, seemed to have the power
of gradually removing the foreign material without loss
of normal function, and even when quite full of the
deposit behaved toward all the various staining re-
agents exactly as normal cells do. Even three weeks
after injection of tellurate, while the breath still smelled
strongly of methyl telluride, Beyer found metallic
tellurium in the glandular cells. Its transformation
must, therefore, have been gradual, as Hofmeister has
shown was the case in other connections.

Increasing amounts of sodium tellurate injected into
the blood of rabbits induced clonic convulsions, respir-
atory paralysis and death. The blood became laky.
Lakiness was not produced by tellurate in rabbit's blood
outside the body, which fact suggests that a tellurium
transformation product caused it in Beyer's experi-
ments. Intravenous injections, in dogs, of quantities of
sodium tellurate ranging from 0.025 to 0.04 gm. per kilo
of body -weight were quickly followed by death in some
cases ; at other times, by vomiting and loss of appetite,
with recovery in several days. These quantities also
brought about general paralysis ; sometimes only of the
hind legs and masseters, but usually also of the inter-
costals, making respiration very labored. Fatty degen-
eration of the hepatic cells and destructive changes in
the uriniferous tubules also resulted. The lymphatic
vessels of the liver were found to be much enlarged
and other structural changes were observed. In one
case lymph from the thoracic duct had a grayish color,
due to suspended tellurium.

The urine under these same conditions was turbid,
greenish brown to a dark green in color, and gave off
the odor of methyl telluride. It contained metallic

11

tellurium, crystals of urocanic acid and triple phos-
phate ; also, blood-corpuscles, albumin and bile pig-
ment. The latter appeared in the blood-serum also.
From the urine of a dog, collected during the first 24
hours after intravenous injection of 0.75 gm. of sodium
tellurate (0.27 gm. Te), Beyer separated 0.062 gm. of
metallic tellurium. From the urine of the second day,
0.081 gm. There was only a trace in that of the third.
None in the fourth. More than one-half of the tellu-
rium administered was, therefore, eliminated through
the kidneys.

Until recently, a brief and imperfect experiment by
Beyer, on the excretion of urea after intravenous injec-
tion of sodium tellurate, had been the only one to sug-
gest the metabolic influence of tellurium. Beyer found
that the normal amount of urea eliminated in the urine
of a healthy dog, during three preliminary days, was
9.45, 10.41 and 7.62% respectively, an average of 9.16%.
After injection of 0.75 gm. of sodium tellurate into the
jugular vein, the urea in the urine on five successive
days was 1.79, 6.06, 8.50, 7.98, 9.00%, an average of
6.67%. This marked falling-off in the amount of urea
was due mainly to the refusal of the dog to eat on the
first and second days of the tellurium period, and as
Beyer does not give any analytic data regarding the
food, it is impossible to attach any special importance
to his results in this connection.

The author, very ably assisted by Mr. L. D. Mead,
recently completed a series of experiments on dogs in
which an attempt was made to ascertain, among other
things, the effects of continued dosage of tellurium
compounds. It was found that nontoxic doses of tellu-
rium (in quantities several times as great as therapeutic
doses and in the forms of oxide, tellurite, tartrate and
tellurate) did not materially afi'ect metabolism in dogs
brought to a state of nitrogenons equilibrium, even
when dosage was continued for a week. These sub-
stances appeared to stimulate proteid catabolism only
slightly. They increased somewhat the weight of dry
matter in the feces and diminished, in small degree, the
absorption of fat. The urine was unaffected in volume,
specific gravity, and reaction, but became dark brown
in color during the dosage periods.

Excesive doses retarded gastric digestion; induced
violent vomiting, loss of appetite and somnolence. They

12

caused, besides, inflammation and disintegration of the
mucous membrane of the gastrointestinal tract and,
also, intestinal hemorrhage. Introduced under the
skin, tellurium (tartrate) caused restlessness, tremor,
weakening of the reflexes, somnolence, diarrhea, paral-
ysis, unconsciousness, stoppage of respiration and death,
in convulsions from asphyxia. At the point of injection
much of the tellurium was deposited in metallic form,
but it was also distributed in large quantity to most of
the organs and tissues.

It was found, also, that tellurium compounds, even
in small proportion, markedly arrested the secretion of
acid in the stomach — the direct cause, probably, of the
indigestion brought about, not only in dogs but, as
will be pointed out later, by tellurium compounds in
man, also. Intestinal putrefaction was not influenced
in any degree. The action of trypsin and pepsin out-
side the body was not very perceptibly diminished by
quantities of tellurium compounds under 0.6 % . Zymol-
ysis was almost unaflFected in the presence of as much
as 1.25% of some of the salts. Ptyalin was more easily
afi"ected, even by the faintly alkaline tellurate. Trypsin
appeared to be least sensitive to destructive influence,
acting rapidly in the presence of even 2.5% of tellurite.

Tellurium was eliminated in metallic form in the.
feces; as methyl telluride in the breath, urine, feces,
and epidermal secretions ; in a soluble form, in small
quantity, in the urine and in the bile. The urine was
colored brown to yellowish green after heavy dosage
with tellurium compounds, but return to normal colora-
tion was rapid after administration had been discon-
tinued. Albumin and bile pigment, besides tellurium,
were the abnormal constituents of the urine found after
subcutaneous injections. Toxic quantities given by the
mouth caused the appearance of coagulable proteid,but
no bile pigment, in the urine.

0. Influence on Man. (a) General. — Berzelius," who
led the way for so long in chemical studies of tellurium,
found, from personal experience, that hydrogen tellu-
ride is irritant in its action and more poisonous in effect
than the corresponding compound of sulphur. Ber-
zelius and Kolreuter" have reported that the oxides of

18 Th. Husemann und A. Husemann : Handbuch der Toxikologie, 1862, 773.
"L. Gmelin: Handbook of Chemistry (Watts), 1856, iv, 898,399, 402, 403.
AlsoJbid., 1856, x, 309, and Berzelius : Trait§ de Chimie, 1846, ii, 225, 230.

13

tellurium, as well as a number of salts of telluric and
tellurous acids, have a very unpleasant metallic taste
resembling that of compounds of antimony and that
some have a nauseating action and are strongly emetic.

Wohler, at the time of his discovery of ethyl tellu-
ride,'* referred to the disagreeable odor of that substance,
and stated that it is very poisonous. At that time, and
subsequently, while engaged in his chemical researches
on ethyl telluride, Wohler observed that his sweat and
breath took on an odor closely resembling that of the
substance he was working with.'* One night, while
perspiring very freely, the garlic odor in his sweat be-
came so great that he himself could hardly bear it. It
persisted in his breath for weeks. These facts led
Wohler to suggest the physiological researches made in
his laboratory by his pupil, Hansen.

The latter was the first to experiment systematically
on man with tellurium compounds. For 7 successive
days he himself took neutral potassium tellurite an
hour before dinner. On the first 4 days 0.04 gm., on
the 2 following days 0.05 gm., and on the last day 0.08
gm. — a total of 0.34 gm. During the first two days very
unusual sleepiness was the main symptom. Later it
disappeared. At the beginning there was increased
appetite, but later the appetite was reduced. After
dosage on the last day there was a sense of oppression
in the cardiac region, also nausea and abundant saliva-
tion. The tongue was heavily coated with a white
deposit, and there was complete loss of appetite. The
gastric symptoms did not disappear completely until
after a lapse of 2 weeks, and the alliaceous odor of
the breath continued 7 weeks.

The characteristic odor of the breath was noticed
within a few minutes after the first dose had been taken,
and soon became so strong and so obnoxious to others
that his own seclusion was necessary for their comfort.
At that time the odor was attributed to a volatile com-
pound of tellurium identical with or similar to ethyl-
telluride. Hansen was unable to separate any tellurium
from the urine ; not even from that passed during the
first 24 hours after the last dosage. Experiments on
his friend, von Roder, who took 0.04 gm. of acid potas-
sium tellurite before dinner one day, and nearly 0.05

"Wohler: Telluraethyl, Ann. d. Chem. u. Phann., 1840, xxxv, 112.
"Qjrup-Besanez: Lehrbuch der physiol. Chemie, 1878, 552.

14

gm. at the same time the next, presented essentially the
same results. Hansen refers to Wohler's previous
experience and says that during these later experi-
ments in the latter's laboratory Wohler observed the
same phenomena, with regard to himself, a second time.

Heeren," also working under Wohler's direction, on
the chemical nature of various compounds of ethyl
and methyl tellurides, noted that the garlic odor of the
breath was especially strong in his own experience
when methyl telluride or any of its derivatives was
under examination. He states that even when these
products are merely touched with the fingers their
characteristic odor is carried to all parts of the body
and in a few days the breath also acquires it, the odor
quickly becoming so obnoxious that, as he puts it, " one
must avoid all social life for months, so as not to annoy
others."

Sir J. Simpson records a case^^ in which a student
inadvertently swallowed a dose of tellurium, which was
followed by the evolution of such a persistent odor that
for the remainder of the session he had to sit apart
from his fellow students.

Prof. Victor Lenher, who for several years has been
engaged in chemical studies of tellurium, greatly favored
the author with a statement of his toxicological experi-
ences for use in this connection After inhalations of
the volatile tellurous oxide, which he formed repeatedly
in preparing metallic tellurium by the fusion method,
Prof. Lenher's breath and the excretions from his skin
took on the usual garlic odor. Metallic taste was noted
and nausea also frequently experienced. The odor of
the breath in one case persisted for about a year.
General depression followed continuous inhalation of
the oxide, and in one instance a prolonged period of
somnolence resulted, an experience similar to Hansen's
after ingestion of tellurite. Severe constipation was also
a marked symptom following tellurium inhalation. At
no time could Prof. Lenher detect any tellurium in
his urine, not even during the periods of his worst
experiences.

The author has found in his own experience that
when the methyl telluride which had been exhaled by

17 Heeren : Ueber Tellurathyl und Tellurmethyl-Verbindungen, Cfiem. Cen-
tram., 1861, vi, 916 (N. F.)
i« Quoted from Blyth : Poisons, their EflEects and Detection, 1885, 559.

15

the dogs he experimented with was taken into his own
lungs, an alliaceous odor of the breath and excretions
from the skin soon became noticeable and continued
persistently. Also, that such inhalation was accom-
panied frequently by short periods of drowsiness and
nausea.

(/;) The cause of ''bismuth breath^^ and the minimal
quantity of tellurium that ivill produce it. As early as 1875
tellurium had been suspected in commercial prepara-
tions of bismuth." The evidence on this point at that
time was not of an analytical character, but was based
upon the observation that people to whom certain bis-
muth preparations had been administered suffered from
fetid breath. The presence of tellurium in bismuth
preparations has since been repeatedly shown,^" and
their medicinal use implies frequent incidental action
of this tellurium impurity.

Reisert, in 1884, after an investigation of the cause of
the so-called bismuth breath, ascertained that it was
due, as had been supposed by some, to the minute tel-
lurium impurities often found in the commercial bis-
muth compounds used in medicine, and not to arsenic
or bismuth itself, as had been assumed by others." He
not only demonstrated, in some experiments on himself
and friends, that the "bismuth breath" did not follow
dosage with chemically pure bismuth sesquioxide, or
arsenious oxide, but also determined the minimal
amount of tellurium which would produce the alli-
aceous odor in the breath. He found that as little as
0.000,000,5 gm. of tellurous oxide, given in solution to
men, was followed by the smell of garlic in 75 minutes,
and that it continued for about 30 hours ; 0.000,000,3
gm., given to three different individuals, failed to pro-
duce a detectable quantity of the odor. In one experi-
ment, three doses of 0.005 gm. each were taken on the
same day at intervals of 3 hours. "In 15 minutes after
the first dose the breath had a strong garlic-like odor,
and in an hour a metallic taste was observed. An hour

» Bly th : A Manual of Practical Chemistry, 1879, 428.

^ Lately again : Druggists' Circular and Chemicnl Gazelle, 1894, xxxviii, 256,
referring to observations of Janzon in Phnrm. Zeilschr.

21 The author is greatly indebted to Prof. John Marshall for calling his atten-
tion to Reisert's work. It seems that subsequent foreign investiKatnrs of the
behavior of tellurium in the animal body were unaware of Reisert's results. It is
probab e, however, that Kunkel refers to these results when he says, " The odor
(of methyl telluride) has Ijeen detected in the fects of man over two months, and
in the breath more than a half year, after the last dose of tellurium." Hnniibuc/t
der Tojnkoleyie, 1899, 365.

16

after the second dose the urine and sweat had the garlic-
like odor, which was also observed in the feces, 4 days
later. The metallic taste was observed for 72 hours ;
and the garlic-like odor in the urine for 382 hours, in
the sweat for 452 hours, in the feces for 79 days, and
in the breath it was still present, though very faintly,
after 237 days."

Reisert passed his breath through a tall column of
distilled water for several hours, in the hope of catching
the odoriferous compound which seemed to be elimi-
nated from the lungs in appreciable quantity, but analy-
sis of this water afterwards gave negative results. He
assumed, therefore, that the quantity of substance
responsible for the odor was too small to be detected by
known chemical means and suggested that the " physio-
logical test " is much more delicate than any purely
chemical one for this purpose.^^ Reisert concluded his
paper with the remark that idiosyncrasy did not seem
to have any influence in his experiments, since the
breath of every one to whom the tellurous oxide had
been administered, in quantities not less than 0.000,-
000,5 gm., was affected with the alliaceous odor.

(c) Antihydrotic Action and Therapeutic Use. — Neusser
was the first to show that tellurium compounds are of
therapeutic value. In about fifty clinical experiments,
on as many consumptives, he observed that the night-
sweats were very perceptibly reduced after administra-
tions of potassium tellurate in daily doses of 0.02 to 0.06
gm.^' In a majority of cases 0.02 gm. was sufficient,
although cumulative dosage was necessary at times to
effect continued results. He noted, also, that these
amounts did not cause any particularly toxic symptoms,
although mild dyspepsia (eructations, coated tongue,

22 Reference has already been made to Hofmeister's method for separating
tellurium eliminated in the form of methyl telluride in the expired air. This
was not applied, of course, until after Reisert's work had been reported. Reisert
knew, however, that Wohler and his pupils attributed this odor to methyl tellu-
ride, but he failed to use adequate means for the retention and chemical detec-
tion of such a volatile compound.

23 In order to test the anidrotic action of tellurium, Czapek and Weil, whose
work has already been reviewed, made careful experiments in this connection
on kittens with results that entirely confirmed Neusser' s original observation.
Moderate nontoxic doses (presumably of tellurates) were given and before any
of the usual sickening influences had manifested themselves the moisture on
the soles of the hind paws became less and less, until they were quite dry, when
even the strongest electrical stimulation of the peripheral end of the divided
sciatic nerve was insufficient to call forth secretion ; after the tellurium had
reached its fullest efiect, pilocarpine, however, was able to induce seeretion.
These investigators were unable to determine any pathological changes in the
structure of the sweat glands and concluded that the interference with secre-
tion was a direct peripheral action of the tellurium and not one upon the central
nervous system.

17

loss of appetite) was produced now and then by the use
of the largest dose. In some cases there appeared to
be stimulation of appetite at first and, in quite a num-
ber of instances, Neusser received the impression that
slight narcotic action had been manifested. The breath
of each individual experimented on always quickly
assumed the characteristic alliaceous odor even with the
smallest quantities of the tellurate. This was the only
undesirable feature that occurred regularly. Neusser
stated that the odor was not noticed by the patients
themselves except in a few cases. Sulphurous and
camphoraceous odors in eructations were sometimes
complained of. His experiments were conducted on
patients in advanced stages of phthisis, but with none
of these was any favorable influence of the tellurium
observed on the disease itself.

Pohorecki, following Neusser's lead, confirmed, in a
large number of clinical experiments, the latter's re-
sults in practically all particulars. lie reported that
increased appetite and better general nutrition resulted
from dosage with 0.01 — 0.02 gm. of potassium tellurate
in the eariier stages of phthisis. Anidrotic action was
manifested in fifteen minutes to an hour, and continued
five to seven hours. The garlic odor of the breath could
be detected fifteen minutes after administration and
continued four to eight weeks. Even in people who
were perfectly well it was observed that potassium
tellurate greatly hindered the secretion of sweat.

Combemaleand Dubiquet found that sodium tellurate
m daily doses of 0.02 to 0.05 gm. had a pronounced
antidiaphoretic action and was more eflfective in this
respect than even camphoric acid. Anidrosis was
obtained not only with patients suffering from phthisis,
but also in other cases in which sweating is often pro-
fuse (rheumatism, dyspepsia, etc.). Administration of
sodium tellurate was followed by diminished perspira-
tion in 18 of 20 cases. In 6 of the 18 it was arrested
completely. 0.02 gm. was found to be the minimal
dose which would induce anidrosis ; 0.05 gm. the most
effective quantity. Repeated dosage with this amount
for a few days brought about the result, if it was not
manifested immediately after the first administration.
These observers, unlike Neusser in his experiences with
the potassium salt, did not find that any gastrointestinal
disturbances were set up and report the alliaceous odor

18

of the breath in but a few instances as the only objec-
tionable feature following its administration in the
doses indicated and for reasonable lengths of time.^*
Combemale and Dubiquet consider sodium tellurate
the very best anidrotic agent and prefer it as a result of
their experiments to camphoric acid, white agaric,
atropin, phosphate of lime, etc. Combemale favors the
view that excessive sweating, in such disorders as
phthisis, is due to the action of ptomaines elaborated
by the specific germs of the disease and he supposes
that sodium tellurate exerts an antihydrotic influence
by rendering these soluble septic products innocuous.
He presents nothing, however, in direct evidence to sub-
stantiate this deduction. His theory would not explain
the reduced sweating in perfectly well people, which
Pohorecki observed after administration of potassium
tellurate.

Mr. Mead and the author have shown, as has already
been pointed out, that tellurates, in quantities not ex-
cessive and yet much greater than the therapeutic
doses in man, exerted no particularly deleterious effects
on the nutritional processes in dogs, even when dosage
was continued for a week, although proteid catabolism
seemed to be slightly stimulated after a time, and
secretion of acid in the stomach retarded. The
alliaceous odor imparted to the breath appears, there-
fore, to be the chief objectionable feature constantly fol-
lowing the use of therapeutic amounts of tellurates.

BIBLIOGRAPHY.

Chr. Gmelin. 1824. Versnche iiber die Wirkungen des Baryts, Strontians,
u. s w., auf den ibierischen Organismus. Tubingen, 43.

Hansen. 1863. Versnche iiber die Wirkung des Tellurs auf den lebenden
Organismus. Ann. d. Chem. u. Pharm., Ixxxvi, 208.

Kabuteau. 1869. Eeeherches sur les proprietes et sur I'glimination des
composes oxyggnes du selenium et du tellure. Gaz. hebd. de Med. et de Chir.,
xvi, 194; 24).

Reisert. 1884. Thie so-called bismuth breath. American Journal of Pharm.,
Ivi, 177.

Knup. 1885. Ueber die Aufuahme verschiedener Substanzen durch die
Pflanze, welchenicht zu den Nahrstoffen gehoien. Botan. CentralbL, xxii, 35.

Neusser. 1890. Ueber tellursaures Kalium als Mittel gegen die Nacht-
schweisse der Phthisiker. IVien. klin. Wochenschr., iii, 437.

Pohorecki. 1891. Ueber den Einfliiss des Kali telluricum aui die Schweisse
der Phthisiker. Jahresber. ii. d. ges. Med., xxvi, (i), 398.

Combemale et Dubiquet. 1891. Le tellurate de soude comme medicament
antisudoral. Sem. medic, xi, Annexes, 24.

24 The reported absence of the garlic odor in the breath in a large majority of
these cases is in direct disagreement with the results of Reisert's quantitative
experiments and the observations of all previous and subsequent investigators,
except Rabuteau, each of whom has found that it invariably follows the intro-
duction ol very small quantities of tellurium compounds both in man and
lower animals.

19

Combemale. 1891. Recherclies cliDiqii&i sur deux agpnts antisudoraux ;
I'acidecanjphorique et le lelliiraie Je sonde. Bull. gen. de. Therap., cxx, 14.

Czapek und Weil. 1893. Uel>er die Wirkung de» Seleiis und Tellurs auf den
thierischen Organismus. Archiv. }. ezp. Path. u. Pharm., xxxii, 4;-!8.

Bokorny, (a). 1893. Ueber die phy.siologische Wirkung der toUuriaen Saure.
Chem. Zeitung, xvii, (ii), 1598: (6). 1894. Toxikologische Notizeii uber einige
Verbindungen des Tellur, Woifranij u. s. w.. Ibid., xviii, (ii), 1739.

Hofuielster. 1894. Ueber Methyhningim Thierkorper. Archiv. f. ezp. Path,
u. Pharm., xxxiii, 198

Beyer. 1895. Durch welchen Bestandtheil der lebendigen Zellen wird die
Tellu'rsaure reducirt? Archiv/. Anal. u. Physiol., Physiol. Abth'l'g. 225.

Scheiirlen. i900. Die Verweudung der selenigen und tellurigeu .Saure in
der Kakteriologie. Zeilschr./. Hyg. u. Inf.-krank., xxxiii, l'<5.

Klett. 1900. Zur Kenntniss der reducireuden Eigenschaften der Bakterien.
Zeilschr./. Hyg. u. Inf.-krank, xxxiii, 137.

Mead and Uifei. 1901. Physiological and toxicological effects of tellurium
compounds, with a special study ol their influence on nutrition. Amer. Jour.
0/ Physiol., T, 104.

20

21

TEIvLURIUM TOXICOI<OG12;
MEAD & GIES.

Reprinted from the American Journal of Physiology.
Vol. V. — IVIakcii i, 1901. — No. II.

PHYSIOLOGICAL AND TOXICOLOGICAL I'FFI^CTS OF
TELLURIUM COMPOUNDS, WITH A SPECIAL STUDY
OF THEIR INFLUENCE ON NUTRITION.^

Bv L. D. MEAD and WILLIAM J. GIES.

[From the Lahoratorv of Physiological Che?nist?y of Columbia University, at the College
of Physicians and Surgeons, A'e^a Vori'.]

CONTENTS. Page

I. Influence on metabolism 105

a. Conduct of the experiments 106

b. First experiment. With tellurous oxide 10*^

c. Second experiment. With tellurous oxide 116

d. Third experiment. With sodium tellurite and telluiium tartrate . . 121

e. Fourth experiment. With sodium tellurate 126

f. Review 130

II. Influence on digestion and on the gastrointestinal tract 133

a. Experiments on normal dog 133

b. Experiments on dog with gastric fistula 137

c. Influence on zymolysis 139

d. Effect on absorption and on the fceces 140

III. Effects and distribution after subcutaneous injection 141

IV. Elimination of tellurium 143

V. Personal experiences 14.5

VI. Summary of conclusions 147

VII. r.ibliography 149

ABOUT two years ago Professor Victor Lenhcr was engaged at
this University with extended studies of the properties of
tclkiriuni and its compotinds.- The ill effects which Professor Lcnher
experienced from involuntary inhalations of volatile products formed
in preparing tellurium impressed him with the desirability of a
systematic study of its physiological effects. lie generously offered
to furnish Dr. Gies with pure telliu'ium preparations for such an
investigation. We wish to thank Professor Lenher for the suggestion

^ A preliminary account of some of the experiments referred to in this paper
was given in abstract in the Proceedings of the American Physiological Society.
This journal, 1900, iii, p. xx.

- See Journal of the American Chemical Society, 1899, xxi, p. 347; 1900, xxii,
pp. 28, 136. .

Effects of TelluriM7n Compounds. 105

which led to these experiments, and for the costly material without
which they would not have been possible. VVe are also greatly
indebted for valuable facts communicated by Professor Lenher from
his large chemical experience.

I. Influence on Metabolism.

With the exception of a brief and very imperfect experiment
by Beyer (13),^ on the excretion of urea after intravenous injec-
tion of sodium tellurate, no special study has ever been made of
the influence of compounds of tellurium on the nutritional pro-
cesses in the body.^ Neusser (6) was the first to note that potassium
tellurate induces anidrosis. In about fifty clinical experirrents, on
as many consumptives, he observed that the night-sweats were very
perceptibly reduced after administrations of potassium tellurate in
daily doses of 0.02-0.06 gm. Subsequent investigators, principally
Pohorecki (7), Combemale and Dubiquet (8) and Czapek and Weil
(10) confirmed this observation of the physiological action of tellu-
rates, and Combemale (9) even expressed the conviction that sodium
tellurate is one of the very best antisudorific agents. Consequently,
both potassium and sodium tellurates have been employed for the
purpose of arresting sweating, particularly the colliquative sweats
of phthisis.^ Further, tellurium is repeatedly found, in small quan-

1 The numerals in parentheses correspond with those preceding the references
in chronological arrangement at the end of this paper.

^ Tellurium was discovered in 1782 by Miiller von Reichenstein and identified
and named {telhts, the earth) by Klaproth in 1798. The metal is silver-white, of
markedly crystalline structure, and possesses a strong metallic lustre. Its atomic
weight is still uncertain, but closely approximates 128. (See note, page 148.)
Tellurium is very nearly related chemically to sulphur and selenium. Its chemical
qualities have offered difficulties from the time of its discovery, so that at first it
was called aur-usn pa7'adoxiini and metallum problematiacin. It is one of the
rarer elements and occurs in nature mostly as telluride in combination with bis-
muth, lead, mercury, silver, and gold. The following formulae show the com-
position and relationships of the tellurium compounds referred to in this paper -.

Tellurous oxide, Te02. Sodium tellurate, Na.2Te04.

Telluric oxide, TeOs. Hydrogen telluride, HoTe.

Tellurous acid, IlaTeOg. Tellurium tartrate, Te(C4HgO^)4.

Telluric acid, H2Te04. Methyl telluride (CHglgTe.

Sodium tellurite, NaaTeOg. Ethyl telluride (CoHs)2Te.

^ Cerna : Notes on the newer remedies, 2d ed., 1895, pp. 164 and 185. See
also. New York medical journal, 1891, liii, p. 370, on camphoric acid and tellurate
of sodium as anidrotics, referring to the recommendations in La province medi-

io6 L. D. Mead and II'. J. Gics.

tit)-, in commercial bismuth preparations/ and their medicinal use
implies frecjucnt incidental action of this tellurium impurity. In
view of these facts, we ha\'c attempted first of all in our experi-
ments to determine the influence of small non-toxic quantities of
tellurium on metabolism, as measured especially by fluctuations in
the excretion of nitrogen.

Conduct of the Experiments.

Animals and Environment. — The experiments were performed on
full-grown dogs weighing from lO to 1 6 kilos. The general methods
were those outlined in the report of some previous investigations
made by Dr. Gics under Professor Chittenden's supervision.- The
animals were confined in a suitable cage, well adapted for the col-
lection and separation of fluid and solid excreta. The cage was
open at the top so as to permit of free circulation of air, and was
kept in a comfortable room with a constant temperature.

Character of Diet. Feeding. — The animals received regularly a
mixed diet of hashed lean meat, cracker dust, lard, and water.
Former experience proved this to be a very acceptable, digestible
and nutritious mixture. The hashed meat was prepared by a
method similar, in general, to that previously described b}- Dr.
Gies.^ The hash was preserved frozen with results which were sat-
isfactory throughout all the experiments. Commercial cracker dust,
containing only 1.5 i per cent of nitrogen, afi'orded the carboh}-drate
element of the diet. This was kept entirely dry in large quantity
in well stoppered bottles. The lard employed was perfectly fresh.
Ordinary river water was used. Neither lard nor water contained
appreciable quantities of nitrogen.

The daily mixed diet was given regularly in two equal portions, in
the morning at nine and in the evening at six o'clock. The water
was stirred with the other ingredients, until the whole mixture had the
consistency of very thick soup. This mixture, while not very appe-
tizing in appearance, possessed an agreeable odor and was always
lapped eagerly by all the animals in the normal periods. The food

cale. Tellurates have not, however, come into general employment, becau.se of the
obnoxious odor imparted to the breath after their administration. See page 130.

^ See Ekin and Bkowxen : American journal of pharmacy, 1S76, xlviii, p. 133
(Abstr.). Blvth : Poisons, their effects and detection, 1885, p. 559. Janzox :
Druggists' circular and chemical gazette, 1894, xxxviii, p. 256 (Abstr.).

2 Chittendex and GlES : This journal, 1898, i, p. 4. ^ ihid., p. 5.

Effects of TelluriMm Compounds. 107

was presented in a common glass crystallization dish, a receptacle
especially suited for the licking up of last traces.

Dosage, "Weighing, etc. — The daily doses of tellurium were also
divided equally. Each half was enclosed in a capsule made of a
small portion of the weighed hash. This was always quickly swal-
lowed, in eager anticipation of the rest of the meal, which followed
immediately, so that the tellurium entered the stomach almost simul-
taneously with the main portion of the food.

In the records of the experiments each period of twenty-four
hours ended at 9 o'clock in the morning, when the first food of
the new day was given. The animal was weighed just before that
hour. The daily analytic data are for the twenty-four hours end-
ing at 9 A. M. The figures representing weight are therefore for
the weight at the end of each experimental day.

Collection of Excreta. — It was found in the experiments already
alluded to ^ that diurnal variations in the elimination of urine were
practically neutralized at the end of a week or ten days. Conse-
quently, in these experiments, in which the periods were of from
seven to ten days' duration, it was unnecessary to remove any urine
with a catheter. We collected the urine as it was excreted naturally
and thus avoided the disturbances which may arise from cathe-
terization. At the end of each day the interior of the cage was
thoroughly sprayed, and rubbed with a stiff test-tube brush. After
the physical qualities of the combined 24 hours' urine had been
noted, the cage washings were used in making up the daily volume
of urine to a litre, in preparation for analysis. Powdered thymol
was added in order to prevent bacterial changes. This was at
times particularly desirable, for not all the analyses could be begun
on the day of collection.^

No special indigestible substance was introduced with the food
to mark off the faeces. As the elimination of solid excreta from the
dog is quite regular imder normal conditions, and also when equi-
Hbrium is maintained, it seemed best to refer the excrementitious
matter from the intestines to the period of their collection. While
this course permits of error, only unimportant influences on char-
acter and elimination would be hidden under these conditions. The

^ Chittenden and Gies : This journal, 1898, i, p. 4.

2 Some of this urine remained in the laboratory for ahnost two years, without
undergoing any change in nitrogen content. A very thin scum formed during tliat
time and the urine became a little darker in color.

io8 L. D. Mead and IV. /. Gics.

inaccuracies of deduction resulting from this i^roccdurc certainly
could not have been material in our work, since the figures for the
nitrogen in the f?eces of whole periods, to be given farther on,
are essential!}- the same for each period in a group.' The fitces
were thorough!)' tlesiccated over the water bath on a weighed dish
immediately after collection, then weighed, thoroughly ground, pre-
served in dry, well-stoppered bottles, and anal\'zed at convenient
intervals.

An appreciable quantity of hair falls from most dogs during such
an experiment. This was collected daily, combined for each period,
and the nitrogen content determined. It will be observed, in the
tables giving analytic data, that the nitrogen thrown off in this way
is so considerable that it must be taken into account in equilibrium
experiments. From long-haired dogs the loss of hair is especially
marke ' The nitrogen eliminated in this way is not the same for
each p(_ 1, as our results will show.^

Analytic methods. — Nitrogen of the food and excreta was deter-
mined by the Kjeldahl process, in all except the last experiment.
Oxidation was accomplished with sulphuric acid aided by copper
sulphate.'^ In the urine of the last experiment nitrogen was esti-
mated by the hypobromite method with Marshall's apparatus.*
Urea was calculated from the nitrogen thus obtained (i c.c. N
= 0.00282 gm. urea). Total sulphur and phosphorus were deter-
mined by the usual fusion methods ; "' phosphoric acid by Mercier's
modification of Neubauer's method ; ^ total and combined sulphuric
acid gravimetrically by customary methods, the former with Salkow-
ski's precaution,' the latter by Baumann's process ; *^ uric acid by
Ludwig's well-known silver method ; ^ fat (ether-soluble matter) in
the faeces by extraction with anhydrous ether in the Soxhlet apparatus
in the usual manner. The total solids in the urine were calculated
from the volume and the specific gravity (" Christison's formula")
with the aid of Haeser's coefficient.^*^ Indoxyl was estimated quali-

1 See tables giving quantitative elimination of faeces, composition, etc., under
similar conditions : Chittenden and Gies, Ioc. cit., p. 37.
- See also, Ibid.., pp. 24 and 33.

8 Marcuse: Archiv fur die gesammte Physiologic, 1896, Ixiv, p 232.
* Marshall: Zeitschrift fiir physiologische Cliemie, 1S87, xi, p. 179.
s Given in detail by Chittenden and Gies : Ioc. cit., page 7.

« Neubauer and Vo(;el : Analyse des Harns, zehnte Auflage, 1898, p. 731.
^ Ibid., p. 721. 8 //,i,f^ p, 72^

9 Ibid. p. 820. 10 /i>i,f,^ p. 703.

Effects of Telluritim Compounds. 109

tatively with the Jafife-Stokvis test.^ The specific gravity of the urine
was ascertained with the ordinary urinometer. The reaction to Htmus
was taken. When the urine was amphoteric, the stronger reaction
was recorded. The quantities of which analyses were made were
those customarily employed.

Tellurium was determined quantitatively in the following manner:
Solid excreta, after fine division in a mortar, and also concentrated
urine, were treated with strong hydrochloric acid and potassium
chlorate over the water bath until completely disintegrated and
almost perfectly dissolved. After that had been accomplished the
fluid was kept on the bath until it Avas entirely freed of chlorine
gas. It was then concentrated to 400-500 c.c. and filtered. The
clear acid filtrate was next saturated, while warm, with sulphur
dioxide gas and allowed to stand for 24 hours. The bluish black
metallic tellurium which had separated in this process was then fil-
tered on a weighed paper, washed with dilute acid, dried at 110° C
to constant weight, and determined gravimetrically.^

First Experiment; with Tellurous Oxide.

The animal used in this experiment was a long-haired bitch weigh-
ing approximately 1 5 kilos. A preliminary period of six days sufficed
to bring her into nitrogenous equilibrium. The dail}^ diet throughout
the experiment was 250 gms. of prepared meat (9.099 gms. N), 50
gms. of cracker dust (0.755 gm. N), 40 gms. of lard, and 700 c.c. of
water, containing a total of 9.854 gms. of nitrogen. The experiment
continued twenty-four days, and was divided into three periods : a
fore period of seven days during which normal conditions prevailed ;
a longer period of ten days during which doses of tellurous oxide,
averaging nearly o.i gm., were given twice daily; and an after period,
equal in length to the first, during which no tellurium was adminis-
tered. During the tellurous oxide period of ten days a total of 1.6
gm. of the oxide was retained after ingestion, or 0.16 gm. per day.
The smallest dose was 0.05 gm. in half of the food for the day; the
largest was 0.5 gm. in the same quantity of food. ^

^ Neubauer und Vogel: Analyse des Harns, zehnte Auflage, 1898, p. 166.

2 This method, Professor Lenher assures us, gives accurate quantitative results.
The methods employed by Hansen, Kletzinsky and Hofmeister were much
the same. Kletzinsky: Wiener medicinische Wochenschrift, 1858, viii, p. 355.

^ The daily dose of tellurate, in therapeutic use, recommended by Neusser,
PoHORECKi and Combemale and Dubiquet, varies from o.oi to 0.06 gm.

iio L. D. Mead and W. J. (lics.

On the first day two doses of 0.25 gm. were given. A few min-
utes after the first dose was administered, the characteristic aUiaceous
odor became quite noticeable in the expired air and it increased
steadily during the rest of the day. On the following morning the odor
in the room was of sickening intensity. No special change except
languor and sleepiness had been noticed in the animal itself up to
this point. Tlie dose in the morning meal (second day) was raised to
0.5 gm. But this was clearly a mistake, for, although the food with
its contained tellurium o.xide was eaten eagerly and quickly, the
whole meal was \'omitccl in less than ten minutes afterward.^ The
vomit was collected quantitatively. The evening portion of food con-
tained only 0.25 gm. The dog ate it very slowly, but before swallow-
ing all of it, vomited violently what had just been eaten. This
vomited material was also gathered quantitatively and added to that
collected in the morning. The uneaten portion of the evening meal
was mi.xed with the vomit of the day, and the whole thoroughly desic-
cated on the water bath for determination of its nitrogen content,
which was found to be practically equivalent to that of the day's
food.^ The dog was sick throughout the second da\'. The
urine, 220 c.c, was coffee colored.'^ It contained no granular tel-
lurium, although some was held in solution, l^ile pigment, albumin,
and sugar were also absent.

At this stage of the experiment it was obvious that the animal had
been thrown completely out of physiological equilibrium.'* The
quickest way to restore the equilibrium seemed to be to feed the dog
an e.xtra amount of food equivalent to the previous day's meal.

1 In a few preliminary experiments on two other dogs of about the same size
it was found tiiat 0.75 to i.o gm. of the oxide administered in the same manner
caused vomiting, but that 0.5 gm. did not. We had hoped, therefore, that this
dose would be safely ingested, at least once, so that we should be able to deter-
mine very definitely what metabolic influence tellurium might exert under condi-
tions approximately toxic; and yet not toxic enough to vitiate tlie experiment.
It will be seen that this was practically accomplished, in the case of this par-
ticular dog.

" It contained 10335 gm- of nitrogen. The food contained 9.S54 gm. The
difference (0.4S1 gm.) was doubtless due to the nitrogen of the mucus, etc., thrown
from the stomach.

3 Somewhat darker than No. 8 in \'ogel's well known scale of urine tints. See
Tyson: A guide to the practical examination of urine, 1896, 9th ed., frontispiece.

* The dosage period was lengthened to ten days on account of this occurrence.
See Chittexdkx and Gies, loc. di., page 9, for an account of similar experiences,
with favorable outcome.

Effects of Tellurium Compounds. 1 1 1

This amount was given in two equal portions on the third day, with
the gratifying results shown in the tables for this experiment. Al-
though cumulative action of the tellurium had been manifested, the
dog's appetite did not seem to be at all impaired at this time. The
food on the third day contained 0.25 gm. of the oxide. During the
remaining seven days of the oxide period, the dosage was kept as
high as was deemed expedient. On the evening of the fourth day
the animal was again nauseated, although the food with its dose of
0.125 gm. of tellurous oxide was finally eaten and none thereafter
vomited. For the rest of the period the daily amount — o. i gm. — gave
no special trouble. The dog was very stupid on the third and fourth
days of the dosage period, and manifested a constant tendency to
sleep. On the fifth day it was more lively and toward the end of the
period was entirely normal. At the close of the experiment 0.5 gm.
of the oxide in the usual quantity of food induced vomiting within an
hour.

The color of the urine throughout the tellurous oxide period was
considerably darker than normal, but this difference was less and less
perceptible after the day of the greatest dosage. Only now and then
could the odor of methyl telluride be detected. Indican was present
in samples of each period. Bile pigment, sugar, coagulable proteid
and abnormal sedimentary material were absent. Tellurium in small
quantity could be detected in the urine during the first half of the
period. The faeces were not greatly changed ; they were somewhat
more bulky, contained more mucus, and were bluish-black instead of
brown, as in the fore period, and late in the after period. Occasionally
the odor of methyl telluride in the fresh faeces was recognized, though
usually it was lost in that of the normal fsecal aromatic compounds.
The alliaceous odor in the dog's breath was most marked at about the
middle of the experiment, when it began to diminish, although, so
long as the animal remained under observation — for almost five
weeks after the last dosage — it was very marked. The shed hair
gave off distinctly the odor of the methyl compound, yet we were
unable to separate any tellurium from it.

The accompanying tables, pp. 112 and 113, give the various
analytical results and other data of the first experiment.^

1 The first three metabolism experiments were performed before Mr. Mead
had been invited to assist in this research, and during the year when the routine
labor connected with the equipment of the Department of Physiological Chemis-
try and its organization for regular work was most exacting. Hence it was im-

1 1

L. D. Mead and IV. J. Gics.

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114

L. D. Mead and IV. J. Gics.

The tables show at a glance that during this experiment telhniiun
had no material influence on the weight of the animal, that the
volume and reaction and specific gravit}' of the urine were not particu-
larly altered, that the quantities of phosphorus, sulphur and uric acid
excreted were uniformly the same, and that the nitrogen elimination
was but little affected. The following summary gives the quantitative
and the percentage distribution of nitrogen for each period :

Total nitrogen.

Fore period.
Grams.

Tellurous o.xide
l^eriod.
Grams.

After period.
Grams.

Nitrogen of food
Nitrogen of urine
Nitrogen of fasces
Nitrogen of hair

68.978
65.359]
2.374 68.787
1.054 J

98.0591
94.752 1
5.154 J- 101.138
1.232]

68.978

63.194 ]
3.291 K'7.669
1.184]

Nitrogen balance

+ 0.191

- 3.079

+ 1.309

Ratio to nitrogen ingested.

Per cent.

Per cent.

Per cent.

Nitrogen of urine
Nitrogen of faeces
Nitrogen of hair

94.8
3.4
1.5

96.6
5.3
1.3

91.6
4.8
1.7

Nitrogen balance

+ 0.3

-3.2

+ 1.9

1 Quantity remaining after subtraction of the nitrogen of the vomit. See footnote, p. 110.

possible to make daily detailed analyses of each 24 hours' urine, and Dr. GlES
had to be content, in some cases, with results obtained from the urine of several
days combined. The totals and daily averages were, of course, in no wise affected
by this. Thus, throughout the three periods of the first experiment, the data for
phosphorus, sulphur, and uric acid are for urine passed during several days. The
figures are recorded on the last day of each separate combination. The dry weight
of the fa;ces is recorded on the days of elimination. The 0.75 gm. of tellurous
oxide given on Dec. 9 is not included in the total for the period (1.6 gm.), because
practically all of it was ejected in the vomit. See pages no and 11 1 for references
to the latter and the variations in quantity of food on Dec. 9 and 10. The average
daily weight of hair shed was 1.24 gm. in the fore period (0.150 gm. N). 0.99 gm.
in the tellurous oxide period (0.123 gm. N), and 1.35 gm. in the after period
(0.169 gm. X).

Effects of Telluriu7n Compounds. 115

These results show that in spite of the relatively large doses of tel-
lurous oxide (quantities greater than therapeutic doses for man), given
repeatedly during a period of ten days, the animal remained approxi-
mately in nutritive equilibrium. They also show that the immediate
ingestion of food equal to that vomited, sufficed to restore promptly
the balance that had been disturbed on the second day of the oxide
period. It should, of course, be remembered, in considering the
effect of quantity in this connection, that tellurous oxide is a com-
paratively insoluble substance — insoluble in water and dilute acids,
soluble in dilute alkahne fluids ; also that its reduction to the metallic
state quickly follows ingestion and that its absorption is therefore
comparatively slow and very incomplete. The odor of methyl tellu-
ride in the breath proved that some tellurium had been absorbed,
but much of the tellurium was eliminated in the faeces in metallic
form, a fact which will be referred to again.

The slightly increased elimination of nitrogen during the second
period cannot be attributed solely to the influence of tellurium,
because of the lack of food on the second day, and the excessive
amount of food on the third day of that period. It is very well
known that unusual amounts of ingested proteid stimulate nitrogenous
catabolism and cause immediate increase in the output of urea ; also,
that when no food is eaten proteid catabolism, although diminished,
still continues. In this experiment we could not well avoid a com-
bination of both circumstances. The animal had been brought into
nitrogenous equilibrium. On the second day of the tellurium period,
however, when no food was retained, proteid catabolism continued at
the expense of the body proteid. On the third day much of this lost
proteid was made up from that ingested, but undoubtedly a good
proportion of the nitrogen of the double quantity of food on this day
was quickly passed into the urine. Nitrogenous equilibrium was
probably very soon restored, but the small balance of 3 gms. in favor
of excreted nitrogen was doubtless largely due to enforced irregular-
ity in the feeding on the second and third days of the period.

The faeces, also, it will be seen, were not greatly altered chemi-
cally, although they were considerably increased in quantity. The
percentage of nitrogen rose somewhat during the second period,
but this increase was probably due to the greater quantity of mucus
eliminated, to which we have already drawn attention, and was
not a result of impaired digestion. There seems to have been a
slight interference with the absorption of fat, since the quantity of

ii6

L. D. Mead and IF. / Gics.

ether-soluble matter is somewhat increased in the second and third
periods. This fact seems to harmonize with the cause assumed for
increase in the faecal nitrogen, for since tellurium is deposited
in the mucous membrane of the stomach and intestines, and thereby
increases the number of cells and the quantity of mucus thrown into
the canal, it can be safely argued, that it may in some measure inter-
fere with absorption. However, this increase in the quantity of
ether-soluble matter, in the feeces, like the increase of nitrogen, is so
slight that little importance can be attached to it.

We ha\'e already called attention to the bluish-black appearance of
the faeces after administration of tellurous oxide. The color is due
to metallic tellurium present in fairly large proportion. Since only
traces of tellurium were present in the urine early in the oxide
period, and none could be separated from a little more than lO gms.

Periods.

Grams.

Pur cent.

Faeces.

Ether-sol. matter.

Nitrogen.

Ether-sol. matter.

Nitrogen.

P'ore

Tell, oxide
After

46.67
81.24
64 33

12.626

27.308
20.110

2.374
.S.1,S4
3.291

27.1
33.6
30.1

6.3

,vl

of hair shed during the same time, it seems very probable that the
comparatively small quantity of tellurium which succeeded in getting
through the walls of the intestine was finally converted into methyl
telluride and that it was all being gradually eliminated in that form
through the lungs. The largest proportion left the body in the
faeces. ^

Second Experiment; wrni Tellurous Oxide.

Although the analytic results of the first experiment indicated that
there had been but slight stimulation of catabolism, we felt it desirable
to make a second trial with tellurous oxide. In this second experi-
ment we sought to avoid the vomiting which in the first had tem-
porarily upset the equilibrium, while at the same time we aimed to
keep the dose as large as possible in order to determine the maxi-
mum influences. We used a dog weighing approximately 10.5 kilos.
Equilibrium was established in eight days. The diet consisted of

' See analytic results, Exp. i, page 135.

Effects of Tellurium Compounds. 117

175 gms. of prepared meat (6. 121 gms. N), 40 gms. of cracker dust
(0.604 gn^- N), 30 gms. of lard, and 450 c.c. of water; it contained a
total of 6.725 gms. of nitrogen. The experiment lasted three weeks
and was divided into three periods of equal length. Throughout the
second week tellurous oxide was given as before, in two equal doses
averaging 0.21 gm. per diem; and each day there was retained 0.05
gm. more than in the previous experiment. The largest single dose
was 0.15 gm., the smallest 0.05 gm.

On the fifth day, when a total of 0.3 gm. was given, the dog ate
with reluctance and it was only after considerable coaxing and pet-
ting that all was swallowed. Loss of appetite had also been shown,
during the previous day, when an equal amount of the oxide had
been administered. We assumed, therefore, that increased dosage
on the following day would cause vomiting, so the daily quantity
given with the food was reduced. It was evident, however, that for
that particular time we had administered the maximum quantity that
could be borne without toxic manifestation. Loss of appetite was
evident to the end of the period in spite of reduced dosage, but
appetite quickly returned when the oxide was discontinued. Within
an hour after the first dose had been swallowed the garlic odor of the
breath, noticed in the previous experiment, was again recognized. It
remained in evidence throughout the experiment and for some days
thereafter. The languor and sleepiness prominent in the first experi-
ment were not especially noticeable in this. There was no sickness;
loss of appetite was the only approach to it.

The urine was not quite as dark in color as before. Albumin, bile
pigment, sugar, and abnormal sediment were absent from the urine
in all cases. None of the samples of urine gave off sufficient methyl
telluride to be detected by the sense of smell. The faeces were
little altered, although they acquired the characteristic bluish-black
appearance during the oxide period, due, as previously stated, to
metallic tellurium. They contained no unusual quantity of mucus ;
only once was the garlic odor perceived. In this experiment also,
the cast-off hair had the usual garlic odor, but we were unable to
detect any appreciable quantity of tellurium in the hair shed during
the oxide period.

The tables given herewith (pages 118 and 1 19) present the data of
this experiment.^ They show conclusively, we think, that tellurous

^ Indoxyl was determined with uniform quantities of urine and reagents so as
to make colorimetric observations directly comparable. The dry weight of the

ii8

L. D. iMcad and IV. J. Gies.

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Effects of Telhirhmi Compounds.

119

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I 20

L. D. Mead a7id IV. J. Gics.

oxide in ciuantitics as large as coukl well be retained had little meta-
bolic influence that could be measured chemically. Body weight
was constant; volume, reaction, and specific gravity of the urine
showed little variation, total phosphoric and sulphuric acids were
unchanged in quantitative elimination ; and nitrogenous excretion was
only slightl)- in excess of ingestion in each of the three periods.
The distribution of nitrogen in the excreta is stated in the following
summary :

Total nitrogen.

lore period.
Grams.

'rdhirous o.xide
period.
Grams.

After ])eriod.
Grams.

Nitrogen of food
Nitrogen of urine
Nitrogen of faeces
Nitrogen of hair

47.075

44.9081

1
1..S39 1-47.341

0.894 J

47.075

45.2171

1
1647 \-\'i!-)~il

1
1.108 J

47.075

45.437 1
1
1.832 H8-215

1
0.946 J

Nitrogen balance

- 0.266

- 0.897

-1.140

Ratio to nitrogen ingested.

Per cent.

Per cent.

Per cent.

Nitrogen of urine
Nitrogen of faeces
Nitrogen of hair

95.4
3.3
1.9

96.1
3.5
2.4

96.5
3.9
2.0

Nitrogen balance

-0.6

-20

-2,4

These results are in accord with those of the pre\ious experiment.
The unimportant excess of excreted nitrogen in each period can
hardly be given much significance from any standpoint, as each
amount is within the ordinary limits of error in work of this kind.

It is worthy of note that no particular influence on normal putrefac-
tive changes in the intestine was manifested, for indoxyl could be
detected in ever}^ day's urine. The normal fluctuations were quite
noticeable. The indoxyl reactions were obtained most distinctly on or

faeces is recorded on the day of elimination. The nitrogen of the faeces was
determined in the combined excreta of each period. The average daily weight
of shed hair was: fore period, 1.02 gm. (0.128 gm. N) ; tellurous oxide period,
1.30 gm. (0.158 gm. N); after period, i.iogm. (0.135 N).

Effects of Tellurium Compounds. 121

about the days of defecation, indications that the formation of indigo
bodies was greatest when the matter in the intestines was largest in
amount. The faeces collected throughout this experiment showed
even less variability than was noticed in the previous experiment.
Not only were the quantities eliminated in each period approximately
equal, but nitrogen content, also, was practically the same. It may
be assumed, therefore, that there was little interference with absorp-
tion in this experiment.

This animal seemed to bear the tellurium dosage especially well-
At the end of the equilibrium experiment 0.75 gm. of the oxide was
given with the usual morning meal. It did not cause vomiting,
although a few hours thereafter the odor of methyl telluride in the
expired air was almost unbearable, and it remained strong for several
weeks. Even languor and sleepiness were not particularly noticeable.

Third Experiment; with Sodium Tellurite and
Tellurium Tartrate.

In several preliminary experiments both the tellurite of sodium
and the tartrate of tellurium seemed to be more distinctly toxic than
tellurous oxide, facts which are doubtless dependent on the greater
solubility of the former compounds.^ The dog weighed 9.8 kilos.
Equilibrium was established in four days. The daily food was com-
posed of 160 gms. prepared meat (5.856 gms. N), 40 gms. cracker
dust (0.604 gm. N), 30 gms. lard, and 400 c.c water. The total
nitrogen was 6.460 gms. The experiment was carried through four
periods, each a week in length. Throughout the second period
sodium tellurite was given in meat capsules with the food as before ;
in the fourth, tellurium tartrate. The third or intermediate period
gave the animal time to recover from any influence of the tellurite,
and, serving as an " after" as well as a " fore" period, enabled us to
note any possible cumulative effect of the dosage.

The largest dose of the tellurite was 0.15 gm. with half the daily
quota of food, the smallest 0.05 gm. The greatest amount of tel-
lurium tartrate given with any one meal was 0.025 gm., the smallest
0.0125 gm. On the evening of the sixth day of the sodium tellurite
period, the dog ate the usual portion of food only after much
persuasion. Loss of appetite was very marked. On the next day,

1 It should not be forgotten, however, that tellurites are transformed into the
hydrated oxide by the acid of the gastric juice. The oxide likewise becomes
tellurite in the alkaline liquids of the intestines.

12 2

L. D. Mead and ]V. J Gics.

assuming that the limit of dosage had been reached, and wishing to
prevent vomiting, the dose was decreased to the smallest quantity of
the period. No trouble was experienced with the tellurium tartrate.
\Vc were, however, afraid to increase the dose over 0.05 gm., as o. i
gm. had caused vomiting in another dog. Possibly for this one the
dose might have been raised somewhat.

Within half an hour after the ingestion of the first dose of tellurite,
the garlic odor of the breath was very noticeable. It continued
throughout the whole experiment. On the day the tellurium tartrate
was first administered, nothing resulted save an unmistakable in-
crease in the odor. With the exception of the loss of appetite on the
sixth day of the tellurite period, and the garlic odor of the breath,
there was nothing at any time to indicate that the dog was not
normal. The urine showed little variation in color and nothing
abnormal could be detected in it. Even the faeces were only a little
blackened by metallic tellurium ; in all other outward appearances they
were perfectly normal. No methyl tclhnide could be detected at
an}' period in the solid excreta even directly after passage.

The accompanying tables, pages 124 'and 125, giving detailed
analytic data^ for this experiment, point to the same general conclu-
sions that were drawn from the first and second experiments. These
non-toxic doses induced very little alteration in the course of meta-
bolic events. The weight of the animal fluctuated very little; the
volume, specific gravity, and reaction of the urine were practically
constant throughout; and the quantity of sulphuric acid excreted was
the same in each period. The nitrogen showed little deviation from
the normal, although slight stimulation, after dosage, was again indi-
cated. On page 123 are the figures for the distribution of nitrogen in
the various excreta, which emphasize the conclusions already drawn.

In this experiment we determined quantitatively the amount of
combined sulphuric acid in order to measure more definitely than
was the case in the previous experiment the effect of tellurium on
intestinal putrefaction. It will be noticed that the normal fluctuations

' Nitrogen was determined, every two or three days, in combined urines. (See
note, bottom of page iii). Total SOg of the urine, and the nitrogen and ether-
soluble matter of the faeces, were determined in the excreta for the whole period.
Combined SO3 was determined in the urine passed on the days of elimination and
also in the combined urines of each period. Dry weight of fsces is recorded on
days of defecation. The average daily amount of cast-off hair varied between
0.77 gm. and 0.89 gm. ; the content of nitrogen between 0.099 o'^- ^""^ o.i [5 gm-
The dosage appeared to have no influence in this connection.

Effects of Tellttrium Compounds.

123

are here again emphasized and that the combined sulphuric acid was
greatest in quantity in the urine on or about the days of defecation.
In only one case was the amount of combined sulphuric acid in the
urine of the day of defecation less than the average daily output of

Total Nitrogen.

Periods. 1

Normal.
Grams.

Sod. tellurite.
Grams.

Intermediate.
Grams.

Tell, tartrate.
Grams.

Nitrogen of food
Nitrogen of urine
Nitrogen of faeces
Nitrogen of hair

45.220

41.8781

3.126 1-45.757

1
0.753 J

45.220

41.4521

3.896}- 46.042

1
0.694 J

45.220
40.432 ]

3.781 J-4+-934

1
0.721 J

45.220
41.300 1
3.812 1-45.916
0.804 i

Nitrogen balance

- 0.537

- 0.822

4- 0.286

- 0.696

Ratio to nitrogen
ingested.

Per cent.

Per cent.

Per cent.

Per cent.

Nitrogen of urine
Nitrogen of faeces
Nitrogen of hair

92.6
6.9

1.7

91.7
8.6
1.5

89.4
8.4
1.6

91.3
8.4
1.8

Nitrogen balance

-1.2

-18

+ 0.6

-1.5

the same for the whole period. The ratios of combined to total
sulphuric acid are here summarized ; from these it is evident that
tellurium, in the quantities and forms administered, had no material
influence on intestinal putrefaction.

Periods.

Grams.

Ratio.

Per cent of Total.

Combined SO3.

Total SO3.

Combined to
Total.

Combined SO3.

Normal

Sodium tellurite
Intermediate
Tellurium tartrate

0.361
0.411
0.461
0.427

4.461
4.398
4.537
4.316

1 : 12.4
1 : 10.7
1 : 9.8

1 : 10.1

8.1

9.3

10.1

9.9

124

L. D. iMcad and JF. J. Gics.

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Effects of Tellurium Compounds.

125

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I 26

L. D. Mead and W. J. Gics.

The increased quantit\' of ether-soluble matter in the faeces, re-
corded in the table of the first experiment, is repeated in this experi-
ment after the administration of the tellurium compounds. The
ratio of the fat and nitrogen to the whole quantity of the faeces for
each period is shown in the summary :

Periods.

Grams.

1

I'er cent.

Faeces.

Ether-sol. matter.

Nitrogen.

Ether-sol. matter.

Nitrogen.

Normal
Sod. tellurite
Intermediate
Tell, tartrate

55.15
63.67
59.82
62.33

16.201
22..581
19.S7S
IS. 792

3.126
3.896
3.781
3.812

29.4
35.4
33.2

30.1

5.7
6.3
6.3
6.1

There is seen to be a rise in the quantity of both ether-soluble and
nitrogenous matter during the dosage periods ; this, though very
slight, indicates some interference with absorption, and probably an
increase in the quantity of mucus and epithelial cells. The action
here may be relatively more marked because the soluble substances
would naturally have more decided local action than the insoluble
oxide. However, these differences are entirely too slight for more
than reasonable guesses.

At the close of the experiment o.i gm. of tellurium tartrate given
with the usual morning meal caused vomiting in little less than an
hour. Two days thereafter 0.5 gm. of the tellurite produced the
same effect in three hours. The odor of methyl telhu-ide in the
breath was especially strong at the time of vomiting.

Fourth Exi'Krimext; with Sodium Tellurate.^

With the results of the first three experiments before us it ap-
peared altogether unlikely that non-toxic amounts of tellurates
would have a more decided action than that already observed. It

1 The preparation of tellurates in a pure condition is a most difficult problem.
After working several months, with the assistance of the late Dr. Herman A. Loos,
Professor Lenher succeeded in making for us 9.5 gms. of almost cliemically pure
sodium compound. This preparation was recrystallized at least twenty times.
Its only impurity was a very small proportion of sodium tellurite. It is probable
that commercial tellurates are no purer than this preparation and that their effects,
when given as drugs, are modified by the small quantities of tellurite which they
contain.

Effects of Tellurium Compounds. 127

seemed desirable, however, to determine experimentally the influ-
ence of sodium tellurate on metabolism, because of the therapeutic
employment of this particular compound. The dog used in this
concluding experiment weighed 15.5 kilos. ^ The diet consisted of
275 gms. of prepared meat (9.675 gms. N), 50 gms. of cracker dust
(0.755 g™- N)) 30 gms. of lard and 600 c.c. of water; it contained in
all 10.430 gms. of nitrogen. This diet was given for eight days, until
the weight of the animal remained constant, when the experiment
was begun. It was carried through three periods ; the first and third
were each a week in length ; the second, eight days. During the
second the tellurate was given daily with the food in the accustomed
way. The largest dose of the tellurate, i gm., was given on the last
day of the second period with the morning meal. With the first
food of the tellurate period 0.5 gm. was given, and none for the rest
of the day. The amount regularly administered was 0.25 gm. with
each portion of food.

During the night of the third day the dog vomited a little greenish
mucus. As this indicated cumulative action no tellurate was given
on the fourth day. The vomited mucus was mixed with the food
given the next morning. There were no manifestations of illness other
than vomiting, and no toxic symptoms were exhibited even after the
administration of the unusual dose during the morning of the last day
of the tellurate period. Sleepiness, however, was very marked at
the end of the second and at the beginning of the third periods.
Within a very short time after the first ingestion of tellurate the
alliaceous odor of the breath was very marked. It seemed to in-
crease steadily, and was, of course, strongest after the administration
of the largest dose ; for more than two months thereafter it was
still very perceptible.

The urine manifested the customary coloration changes — became
more coff"ee colored with tellurium dosage — but no abnormal
constituents could be detected in it, except occasionally a garlic
odor.^ Its reaction was acid throughout and indoxyl could be

^ Six months previous to this experiment agastric fistula had been made in this
dog for experimentation in other connection. At this time the cannula had not been
opened for a little more than a month. The fistula was kept closed throughout
each of the three periods. The dog remained in perfectly healthy condition to
the end of the experiment.

2 By an unfortunate oversight we failed to look for tellurium in the urine.
After the largest dosage it is probable that the urine did contain the substance.
See results in tliis connection on pages in and 135.

L. D. Mead and W. J. Gics.

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Effects of Tellurium Compounds.

129

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detected in each sample. The fieces contained a httlc more than
the normal amount of mucus, during the second and part of the third
period, and the bluish-black color of deposited tellurium which had
been noticed before was again observed ; otherwise there was noth-
ing unusual to be noted.

In this experiment nitrogenous metabolism was measured by the
output of nitrogen in the urine only. The nitrogen was determined
by the hypobromitc method. Urea was calculated from the nitrogen.
The accompanying tables summarize the data of this experiment
(pages 128 and 129).

Here again the results are essentially a repetition. Body weight
as well as the volume, reaction and specific gravity of the urine
were unaffected. The total solids of the daily urine were practi-
cally the same in each period, but nitrogen (urea) was increased
enough to indicate, as in the case of all of our previous experi-
ments, that metabolism had been slightly stimulated. With the
exception of the vomiting on the third day and the continuous
elimination of methyl telluride in the expired air, there were no
visible toxic effects of the tellurate. The dog was particularly
sleepy for a short time, as already mentioned, but did not suffer
from loss of appetite, a symptom observed in each of the preced-
ing experiments. In fact, the tellurate seemed to be especially
devoid of toxicity, for even 1.5 gm. given on an empty stomach
with a small piece of meat at the close of the experiment, caused
vomiting only after seven hours. The quantitative eliminations
of the faeces, it will be seen from the tables, were so constant that
it may safely be said that no particular effect was produced on intes-
tinal absorption, except, perhaps, a slightly diminished assimilation
of fat.

Review.

In reviewing the results of these metabolism experiments it should
be mentioned that the occasional vomiting was quite in accord with
the original observations of Hansen (2) and the experience of subse-
quent workers. The alliaceous odor of the breath after the introduc-
tion of tellurium has been observed by all investigators except
Rabuteau (3) and Combemale and Dubiquet. Reisert (4), inquiring
into the cause of the so-called bismuth breath, found that when
men took only 0.000,000,5 gi^i- o^ tellurous oxide, in solution, the
odor of garlic could be noticed in the breath 75 minutes after-

Effects of Tellurium Compounds. 131

ward, and that it continued for about 30 hours. Before Wohler
and Dean's ^ and Heeren's ^ observations were made this odor had
been attributed to ethyl telluride by Wohler and his pupils.^ Heeren
assumed that the volatile substance exhaled was in reality the tellur-
ide of methyl. Hofmeister (12) has lately proved by chemical
means that synthesis of methyl telluride occurs in almost all parts of
the body after the introduction of tellurium in any form, and Beyer
has found that the process does not take place in the absence of
oxygen. Hofmeister has also shown that methyl telluride is formed
in worms and Crustacea, as well as in dogs and rabbits, and Hof-
meister and Czapek and Weil observed similar production after
administration of tellurium to frogs. Neither Knop (5) nor Bokorny
(11), who have found that small quantities of tellurium compounds
exert little or no destructive action on plants, observed this synthesis
on the part of vegetable cells.

The very evident languor, sleepiness, and loss of appetite in
some of these experiments, first noted by Gmelin (i), were re-
ported by Hansen among the results of experiments on himself,
and were observed also by Neusser. The color and odor of the
urine and fasces, the increase of mucus, and the presence of tel-
lurium metal, in the latter, confirm previous observations by Hansen,
Beyer and Reisert. The latter found that the garlic odor, after
ingestion of 0.015 g^^i. of tellurous oxide, could be perceived in
the urine 382 hours; in the sweat, 452 hours; in the faeces, 79 days.
In the breath it was still present at the end of 237 days.^

Tellurium appears to have had no influence at all on intestinal
putrefaction. This result, however, harmonizes with the very recent
observations of Scheurlen (14) and Klett (15), who found that the
development of various forms of bacteria, for example, Staphylococcus

1 Wohler und Dean : Annalen der Chemie und Pharmacie, 1855, xciii, p. 233.

2 Heeren : Chemisches Centralblatt, n. F., 1861, vi, p. 916.

^ Wohler: Annalen der Chemie und Pharmacie, 1840, xxxv, p. ill; Ibid.,
1852, Ixxxiv, p. 69. Also, Mallet: Ibid., 1851, Ixxix, p. 223. Also, Wohler:
Journal fiir praktische Chemie, 1840, xx, p. 371.

^ We are greatly indebted to Professor John Marshall for calling our attention
to Reisert's work. It seems that subsequent foreign investigators of the behavior
of tellurium in the animal body were unaware of Reisert's results. It is probable,
however, that Kunkel refers to these results when he says, " The odor (of methyl
telluride) has been detected in the fasces of man over two months and in the
breath more than a half-year, after the last dose of tellurium." Handbuch der
Toxicologie, erste Halfte, 1899, p. 365.

I ;2

L. D. JMcad and ]\\ J. Gics.

pxogcncs aureus and B. mcscntcricus vuli^atus, was not materially
hindered by small proportions of tellurite. Klett observed that the
virulence of such bacteria as B. anthracis was not perceptibly de-
creased by the action of small quantities of the same salt.' In all
of our experiments much of the ingested tellurium was quickly
transformed to the passive metallic state. As a consequence, the
proportion of active tellurium in the intestinal contents must ha\c
been very slight.

Attention has alrcad\' been called to the fact that Beyer's brief
and imperfect experiment on the excretion of urea after intravenous
injection of sodium tcllurate was the onl\- previous attempt to deter-

Tellurium
compound

used.

Nitrogen

ingested daily.

Grams.

Nitrogen

excreted daily.

Grams.

Total balance of nitrogen

for each period.

Grams.

Fore.

Dos-
age.

After.

Fore.

Dos-
age.

After.

Fore.

Dosage.

After.

1. TeOo

2. TeO.,

3. NagTeOs
Te(C4n50,)4

4. Na./re04

9.854
6.725
6.460
6.460
10.430

10.839
6.725
6.460
6.460

10.430

1
9.854 '

6.725

6.460

10.430

9.827
6.763
6.537
6.419
9.070

11.147
6.853
6.577
6.559

10.850

9667
6.888
6.419

10.200

+ 0.191
-0.266
- 0.537
+ 0.286
+ 9.510

- 3.079

- 0.897

- 0.S22

- 0.696

- 3.380

+ 1.309
- 1.140
+ 0.286

+ 1.590

The figures for excreted nitrogen in Experiment 4 represent only that eliminated
in the urine, so that the corresponding figures under "total balance" represent
differences Ijetween food and urine nitrogen.

mine the metabolic influence of tellurium. He found that the normal
amount of urea eliminated in the urine of a healthy dog during three
preliminary days was 9.45, 10.41 and 7.62 per cent respectively, an
average of 9.16 per cent. After injection of 0.75 gm. of sodium
tellurate (0.27 gm. tellurium) into the jugular vein the urea in the
urine on five successive days was 1.79, 6.06, 8.50, 7.98, 9.00 per cent,
an average of 6.67 per cent. This falling-ofif in the amount of urea
was due, undoubtedly, to the refusal of the dog to eat on the first
and second days of the tellurium period, and as Beyer does not give
any analytic data regarding the food, it is impossible to attach any

1 Our attention was first called to the work of Scheurlen and Klett by Dr. P.
H. Hiss, to whom we are also indebted for valued suggestions.

Effects of Tellurium Compou7ids. 133

special importance to his results in this connection. After the injec-
tion of tellurium, albumin and bile pigment were eliminated in the
urine for several days. On the first day after injection of tellurate,
0.062 gm. of metallic tellurium was eliminated, on the second 0.08 1
gm., on the third a trace. More than half the amount injected,
therefore, was eliminated through the kidneys.

Our own results with respect to nitrogenous catabolism are shown
in the above general summary, page 132.

II. Influence on Digestion and on the Gastro-Intestinal
Tract in General.

In our metabolism experiments we noted that vomiting occurred
in the first and fourth experiments, soon after ingestion of 0.5 gm.
of tellurous oxide and several hours after 0.25 gm. of sodium tel-
lurate had been administered. At times there was loss of appetite
and in practically all of the experiments the elimination of mucus
in the faeces was increased. We saw, also, that tellurium compounds
were reduced in the gastro-intestinal tract, that absorption of fat was
diminished and that methyl telluride mingled with the faecal gases.
We have attempted to determine by additional experiments some
of the other special influences of compounds of tellurium in the
alimentary tract.

Experiments on the Normal Dog.^

The following abbreviated reviews present the essential points
observed in this connection, together with other facts of interest :

I. "With tellurous oxide. 1809. Jan. 7 . — Dog weighed 14 kilos.
Had received no food during previous 24 hours. Was given a total of 3.5 gms.
of Te02, with 280 gms. fresh meat, in equal portions — 0.5 gm. TeOs in pieces
of meat weighing 40 gms. •^- at 1.30, 3.30, 5-oo, 8.15, 9.15, 10.15, ^n*^ io-4S p-m.
Drank 200 c.c. water with first dose. Odor of methyl telluride in room very
strong at 2.30 p. M. At 9.00 animal very sleepy and odor sickening. Continued
so throughout experiment. At midnight had neither vomited nor passed
urine. Jan. 8. — Considerable vomit found in morning ; full of undigested
pieces of meat, with heavy white and greenish black mucus. Contained much
undissolved TcOq. Was acid to litmus ; no free acid. Dog very languid.

1 The dogs of these experiments were kept in the cage used in the metabolism
work. Its arrangement favored separation of solid matter in the vomit from fluid,
as well as the separation of fseces from urine. Tests for free acid were made with
Giinzburg's reagent and tropEeolin 00.

1 34 L. D. Mead and IV. J. Gies.

12.30 p. M., first food offered — 20 gms. meat with 0.5 gm. Te02 — eagerly
eaten; water refused. 1.45, all vomited, with much greenish black mucus in
strings and lumps. Acid to litmus ; no free acid. Contained undissolved
TeOo. 11-30 P.M. (no food or water during interval), vomited again.
Mostly clear fluid with much mucus. Acid to litmus ; no free acid. Jan. 0.
— ID A. M., drank 500 c.c. water; no food given. Ten minutes later 100 c.c.
vomited: neutral to litmus. 10.30, 175 c.c. urine eliminated. The urine
yellowish green, like diluted bile, though no bile pigments were present.
No coagulable proteid. 150 gms. meat at 6 p.m. Jan. 10. — ^150 gms.
meat, 200 c.c. water at 9 a. m. 30 gms. meat with 0.5 gm. TeO.^ at 5.15 p. m.
At 8.00, fffices — bluish-black, streaks of blood, iliuch mucus. Urine also, 80 c.c,
not as dark in color as on 9th. No coagulable proteid. 8.30, 30 gms. meat
with 0.5 gm. TeOo. Jan. 11. — No vomiting since last doses. 11 A. m., re-
fused food and water — none for 26 hrs. Nose very warm and dry. Refused
food repeatedly all day. Persisted in sleeping. Fever high at midnight.
Dog not easily roused from stupor. Jan. 12. — 9 a.m., 100 gms. meat, in
several pieces, eaten; vomited in 10 minutes. Solid portion eaten; again
quickly thrown up. This occurred three times in half hour. Fluid each time
acid to litmus ; no free acid. Greenish mucus very abundant. 2 p. m.,
vomited again ; acid to litmus ; no free acid. Jan. 13. — Ate small quantities
of meat and drank water, with increasing appetite throughout day. 300 c.c.
urine in morning ; not particularly dark. Jan. 14.. — Recovering rapidly.
Odor of methyl telluride undiminished. Jan. 15. — 10 a. m., 50 gms. meat in
one piece with i.o gm. Te02 and 200 c.c. H2O. 12 m., 60 gms. piece with
i.o gm. TeOa. 4 p. m., 30 gms. piece with i.o gm. TeO.,. Up to midnight
no action except increased methyl telluride and stupor. Jan. 10. — Vomit
found at 9 a. m. — 30 and 40 gms. pieces meat unchanged, with contained
Te02 i" place, (ireenish fluid, full of greenish and bluish shreds of mucus.
Strongly acid to litmus ; no free acid. Urine normal in appearance, 250 c.c.
at 9.30. At 12.30 p. M., vomited again. 60 gms. piece meat tlirown up,
undiminished in size ; putrid. Strings of blue mucus half foot in length.
Some TeO^ undissolved. Vomit acid to litmus, none free. 5.00, bloody
f?eces; bluish-black in places. Jan. 17. — 9 a. m., unusually lively. 30 gms.
meat, 1.0 gm. TeOo, 200 c.c. water. 3 p.m., fluid vomit; green and blue
mucus ; acid to litmus, none free. 5.00, tried to vomit, without success.
5.30, vomited 30 gms. piece meat given at 9 a. m. TeOo powder in blue
mucus. Acid to litmus; none free. Midnight, 115 c.c. very dark urine.
Contained coagulable proteid ; no bile pigment.

Jan. IS. — Post-mortem (chloroform, 9 a.m.). Methyl telluride from ab-
dominal cavity and separate organs. Blood, liver, lungs, brain, spleen, normal
in outward appearance. Gall bladder greatly distended. Alimentary tract
lined throughout with greenish and bluish-black layer of metallic tellurium in
granules. Small intestines much inflamed. Contents of stomach acid to

Effects of Tellurium Compounds. 135

litmus ; no free acid. Pepsin present. Intestinal contents bluisli-black ; much
mucus. Kidneys very dark, cortical layer black. Urine in bladder very dark ;
no tellurium in suspension. Walls of bladder normal in appearance.

Analytic results. Qualitative analysis of various parts by method outlined
on page 109 gave following results for tellurium : positive, liver, blood, stomach,
intestines, muscle from back, urine, contents of stomach and of large and small
intestines, bile, faeces ; negative, lungs, spleen, pancreas, brain, heart. The
amount in the faeces was surprisingly large, 75 gms. of the desiccated material
yielding 0.977 gm. of tellurium — 1.3 per cent of dry substance.-"^

2. "With tellurous oxide. 1899. Mar. 13. — Bitch weighed 16 kilos.
9.30 A.M., i.o gm. Te02, 125 gms. meat, 300 c.c. H2O. Ten minutes later
nearly all vomited ; all solid portion licked up at once. At i i.oo, large quantity
thrown up again ; all eaten quickly. This repeated at 3.00, 6.30, 8.45 and
1 1. 1 5 p. M. Vomit less and less each time ; propordon of bluish-black mucus
correspondingly greater. Samples of each vomit acid to Htmus ; no free acid.
No haemoglobin in any, but bile pigment in some. Increasing number of
bacteria. Each gave good precipitate with AgNOs and HNO3 after removal
of albuminate and proteose. Kelling's and Uffelmaim's ^ tests for lactic acid
gave negative results. Urine had usual coffee color. Odor of telluride of
methyl very strong soon after first dosage.

3. With sodium tellurite. 1899. Apr. 7. — Bitch weighing 6.2 kilos.
9 A.M., full meal meat, bread, water. 3.30 and 4.30 p.m., 15 gms. meat
enclosing o.i gm. NagTeOa. 5.30 and 6.30, same quantity meat with 0.25
gm. NaaTeOa. At 4.00, methyl telluride very noticeable about cage ; more and
more intense throughout day. 6.35, vomit — fluid and mucus. Acid to litmus,
no free acid. 7.35, more vomit — three pieces of meat given during afternoon
thrown up litde altered, with parts of fourth. Blue mucous strings. Fluid
acid to litmus; none free. 9-30, refused food. Sleepiness pronounced.
10.00, 150 c.c. urine, somewhat darker, otherwise normal.

4. With tellurium tartrate. 1899. Apr. 8. — Same dog used Apr. 7th,
8.30 A.M., urine normal in appearance. 9-30, 100 c.c. water, 15 gms.
meat in piece enclosing 0.3 gm. Te(QH506)4. 10.45, same quantity water
and meat with 0.43 gm. Te(C4H506)4- Methyl telluride stronger an hour
after first dosage. Vomit at 11.15, 11.30, 11.40, 11.55 a.m., and 12.10 and
12.35 P'^^- Unchanged pieces of meat came up at 11. 15 and 11.30 a.m.
Much fluid and mucus thereafter. Each vomit acid to litmus, with no free
acid. Dog very ill during afternoon ; recovered rapidly during evening. At
first refused food. 10 p.m., ate largely and eagerly; food retained. 11-30,
125 c.c. normal urine.

5. With sodium tellurate. 1900. Apr. 16. — Dog weighed 7.3 kilos.
Good meal night before. 12.30 p.m., 0.5 gm. Na2Te04 with 100 gms. meat,

1 See quantitative results on page 143.

2 Simon: A manual of clinical diagnosis, 1897, pp. I56-I57„

1^6 L. D. Mead and \V. J. Gics.

two pieces. Methyl telluride very strong within an hour. 5 p. m., 0.5 gni.
Na.>Te04 in 100 gms. meat, tliree pieces. Sleepiness vcr\' marked, odor un-
usually strong at 7 i>. m. No other marked symptoms. .Ipr. 17 . — 9 a. m.,
odor of methyl telluride in room almost unbearable.' No food given. Very
sleepy. 10.00, vomited — two pieces meat each weighing nearly 30 gms.,
with considerable quantity grayish-black mucus. Vomit acid to litmus ; none
free.

Post-inortcvi (Chloroform, 11 a.m.). Only i)athological conditions noted
were inflammation of intestines ; bluish-black lining of gastro-intestinal tract
due to granular tellurium in epithelium ; and methyl telluride from abdominal
cavity and organs. No tellurium could be separated from the lungs.

Many of the results in the abo\e experiments confirm observations
made in our metabolism experiments and in those of previous in-
vestigators, especially Hansen, Rabuteau and Beyer; but particularly
strikinij is the fact that there was never any free acid in any of the
mixtures thrown from the stomach. It is quite e\ident from these
experiments that irritation of the gastric mucous membrane is usually
very marked, although it required at times a surprisingly large quan-
tity of tellurium compound to cause irritation. The intestines were
also much inflamed by tellurium. The mucous cells appeared to be
greatly stimulated, judging from the large quantities of mucus
secreted. Slight intestinal hemorrhage was also produced, as was
occasionally shown by the bloody fsces. The results of each of these
experiments seem to combine to prove that tellurium exerts an inhibi-
tory action on the secretion of acid in the stomach. Certainly not
enough acid is found to furnish free acid, even when only a small
amount of proteid is present there to combine with it. This must be
one of the causes of the indigestion repeatedly observed throughout
these experiments. It does not seem probable that mere transforma-
tion of the small quantity of tellurium compounds administered could
account for the disappearance of free acid. We have not recorded,
above, the individual results regarding the presence of proteol\-tic
enzyme. Pepsin was contained in active quantity in each particular

^ A dog of 15 kilos weight which liad been perfectly healthy during the six
months he was in our charge was chained near the animal on which the experi-
ment was being performed. During the night he vomited twice. This seemed
to be due entirely to inhaled methyl telluride. The windows and doors of the
room had been closed for the night, so that the telluride accumulated. See
personal reference on page 147.

Effects of Tellurium Compounds, 137

vomit. When an equal amount of 0.2 per cent hydrochloric acid
was added, giving distinct blue reaction with congo red, fibrin in
relatively large quantity was quickly digested in all samples.

ExPERIiMENTS ON A DOG WITH GASTRIC FiSTULA.

In order to test the above conclusion regarding interference with
secretion of hydrochloric acid, we conducted on a dog with gastric
fistula some experiments designed to give even more direct evidence
in this connection. The dog weighed 15.5 kilos. The cannula was
put in place, toward the pyloric end, on the 9th of November, 1899,
five weeks before the experiments were begun. Entire recovery
speedily resulted and the dog seemed to digest normally.^

1, Preliminary control experiments. — /. 1S99. Dec. 15. — 12.15 p.m.,
free acid in stomach contents. 12.45, ^5^ g'^s. of meat given in four pieces
of equal size. 5.50, free acid in contents. Time from feeding to first ap-
pearance of free acid, 5 hr. 55 min.'^

II. 1899. Dec. 17. — 9. 50 a.m., free acid in stomach contents. 10.00,
50 gms. of meat given in one piece. 12.00, free acid detected. Time re-
quired for appearance of free acid, 2 hr. o min.

2. Experiments •with tellurium compounds. — I. With tellurous oxide.
— 1899. Dec. 18. — 9 a. m., free acid in contents of stomach. 9.15, fed
150 gms. of meat in four pieces of equal size, each containing o. i gm. TeOj.
2 p.m., some undissolved TeOo in contents. 3-oo, vomited small amount of
thick mucus. Stomach contents scanty. Was given 25 c.c. HoO. 4.20,
drank 150 c.c. H^O. 7-oo, stomach contents faintly alkaline to litmus. 9.30,
still no free acid. Contents neutral to litmus. 9.30, time since ingestion of
food, with no free acid, 12 hr. 15 min. At 9.30 p. m., 50 gms. of meat given
in one piece with 100 c.c. water. 10.30, free acid. The fresh meat seemed
to act as a special stimulant, and in the absence of the oxide, which we assume
had been mostly removed, was able to call forth abundant secretion of acid.

II. With sodium tellurite. — 1899. Dec. 19. — 10.30 A. M., no free acid
in stomach. Given 50 gms. of meat in single piece with o. i gm. NaoTeOs.
1 2.45 p. M., trace of free acid. Interval to appearance of free acid, 2 hr. 15 min.

1 Methods. On the day preceding each experiment the dog was well fed and
received all the water it desired. On the day of the experiment only the meat
mentioned in the above summaries was fed; no water was given except when
specially recorded. About 10-15 c.c. of fluid were taken from the stomach at
intervals of from 15 minutes to an hour. Acidity to litmus, congo red, Giinzburg's
reagent and tropaeolin 00 was determined qualitatively in each sample withdrawn.

2 See Chittenden, jVTendel, and Jackson: This journal, 1898, i, p. 194.
The time until free acid appears is here lengthened, probably because no fluid
was ingested. Note, however, the result of our last control experiment.

I vs

L. D. Mead and W. J. dies.

III. "With tellurium tartrate. — 18VU. Dec. 20. — lo A. M., no free acid
in contents. 10.15, 150 gms. of meat in four pieces, equal in size, with
total of 0.3 gm. Te(C4H506)4. 10.15 ^•'^^•■> still no free acid. 10.30, interval
of no free acid, 12 hours. At 10.30, 50 gms. of meat given in one piece.
12.15 A.M., no free acid. Experiment discontinued. These results might in-
dicate that tellurium tartrate has even more decided inhibitory action than the
oxide.

JV. With sodium tellurate. — 1000. May 25. — 10 a.m., no free acid
in contents. 10.15, 5° g"''^- °^ nieat in single piece with 0.3 gm. of NaoTeO^.
2.15 i\ M., first appearance of free acid. First appearance of free acid at the
end of 4 hours.

V. With sodium tellurite. (Direct continuation of Exp. IV.) — 2.45 P. M.,
abundance of free acid. 3.00, 100 gms. meat in two ])ieces, with 0.3 gm.
NaoTeO:;. 10.15, first trace of free acid. First trace of free acid after an
interval of 7 hr. 15 min.

Note. — The odor of methyl telluride in the exhalations always became more
pronounced an hour or two after the ingestion of the meat containing the tel-
lurium compounds. Frequently bile pigment was detected, with Gmelin's test,
in the stomach contents after tellurium dosage, but not at any other time. All
of the various samples tested contained pepsin which, after the addition of an
equal quantity of 0.2 per cent HCl, showed vigorous digestive action on fibrin
shreds. Contents almost always acid to litmus.

3. Final control experiment. — 1000. Jime 1. — 11.15 A.M., no free
acid in contents. 11.30, 50 gms. of meat fed; one piece. 11-45, fr*-'^' a<^id.
Same at 12.00, 1 2.30 and i p. M. Time from feeding till free acid was detected,
15 min.

No.

Meat.
gms.

Time of
feeding.

First trace
free acid.

Time
interval.

Conditions.

Average
interval.

KH)

.=iO

10.00 A.M.

12.00 m.

2 hr. 0 min.

Prelim, control )
Final control )

1 hr. 7 min.

3

50

11.30 a.m.

11.45 a.m.

0 hr. 15 min.

1(1)

150

12.45 P.M.

5.50 P.M.

5 hr. 55 min.

Prelim, control

5 hr. 55 min.

2(11)

50

10.30 a.m.

12.45 P.M.

2 hr. 15 min.

0.1 gm. NaaTeOg )
0.3gm.Na2TeO4)

3 hr. 7 min.

2(IV)

50

10.15 a.m.

2.15 P.M.

4 hr. 0 min.

2(V)

100

2.45 P.M.

10.15 P.M.

7 hr. 15 min.

0.3 gm. Xa,,Te03

7 hr. 15 min.

2(1)

150

9.15 a.m.

*

12 hr. 15 min.t

0.4 gm. TeOa

12 hr. 7 min.§

2(111)

150

10.15 A.M.

*

12 hr. 0 min.t

0 3 gm.

Te(C4H506)4.

* No free acid when experiment was discontinued. t .\t least. § Minimum.

Effects of TellMvium Compounds. 139

Direct comparison of the results, in the preliminary and final
" control " experiments with those in which the meat fed contained
tellurium, clearly brings out the fact that free acid invariably appeared
in shorter time when no tellurium was given. The above summary
of these experiments, page 138, in which the results for equal portions
of meat are grouped together, shows this, and our data indicate, we
think, that the secretion of hydrochloric acid in the gastric juice is
markedly inhibited by tellurium compounds.

Influence on Zymolysis.

All evidence in our experiments up to this point, bearing on diges-
tive conditions, appeared to favor the view that tellurium compounds,
in the quantities given, have no special inhibitory action on pepsin
proteolysis in the presence of free hydrochloric acid. The secretion
of pepsin did not seem to be materially affected. When it is re-
called, however, that traces of pepsin manifest great proteolytic power
under favorable conditions, it cannot be safely inferred, from any re-
sults we have presented, that its secretion was not interfered with.
In the case of the acid, however, its more definite quantitative rela-
tionship to proteolysis in the stomach makes deduction regarding
its formation in these experiments much more reliable.

With a view of ascertaining roughly the action of percentages of
tellurium compounds, equal to and somewhat higher than those in
the stomach throughout the previous experiments, we conducted a
few test tube experiments with " pepsin — HCl " and fibrin, and then,
incidentally, also determined the effects of similar quantities on
ptyalin and trypsin under appropriate artificial conditions.^ We give
our results briefly in summary :

I. Pepsin — HCl, 0.2%.

I. With sodium tellurite. (Alkaline in reaction to litmus. In quantities
above 0.6% , is transformed in great part into hydrated Te02, which

1 Methods. I. "Pepsin — HCl" was prepared by dissolving 0.5 gm. of pepsin
scales (P. D. & Co., 1-2000) in a litre of 0.2 per cent HCl. II. Neutral solution
of trypsin was made by Kiihne's method. (Given in Studies from the Yale
Laboratory of Physiological Chemistry, vol. i, p. loi.) III. Neutralized, filtered
saliva was used in the amylolytic experiments. IV. Proteolysis was determined
by the disintegration and disappearance of purified fibrin in shreds ; amylolysis on
starch paste, 0.5 per cent, with iodine and Fehling's solutions as indicators. The
volumes of the digestive mixtures were 15-20 c.c. Time: usually 30 minutes to
an hour, at 40° C. In all cases control experiments were made to determine the
activity of the enzyme solutions.

140 L. D. Mead ami IV. J. Gies.

is precipitated. Reaction of mixture also becomes alkaline).
Digestive action quickly obtained with amounts not over 0.625%.
In presence of this quantity some acid is uncombined.
II. With tellurium tartrate (acid). Rapid digestion with as much as
1.25%.!
III. With sodium tcllurate (containing trace of tellurite; slightly alkaline).
Digestion with 1.25%.

2. Trypsin (neutral).

I. With sodium tellurite. Rapid digestion in presence of 2.50%. ^

II. With tellurium tartrate. Some digestion in presence of 0.85%.

III. With sodium tcllurate. Rapid digestion in presence of 2.50%.'

3. Ptyalin (neutral).

I. With sodium tellurite. No digestion with quantities above 0.027o-
II. With tellurium tartrate. No digestion with quantities above 0.02%.
III. With sodium tellurate. No digestion with quantities above 0.35%.

It seems quite evident, from these results, that pepsin and tr)-psin
arc not destroyed by quantities of telkirium compounds under' 0.6 per
cent and are active with as much as 1.25 per cent and 2.5 per cent,
respectively, of some compounds. Ptyalin appears to be the most
sensitive to destructive infUicncc, tr\'psin least so. The reactions of
the compounds appear to influence greatly these results, the tellurate
(only very faintly alkaline from admixed tellurite) having the least
destructive action. It may be reasonably concluded, then, that inter-
ference with digestion in the dog, after dosage with comparatively
small amounts, has resulted more from disordered secretion than from
direct influence on zymolysis itself.

Effect ox Aijsorption .\nd on the F.eces.

From the experimental data here presented we can draw hardly
more than very general deductions regarding influence on absorption.
The chief evidence of disturbed absorptive function is given in the
figures for ether-soluble matter in the faeces of the first and third
metabolism experiments, indicating decreased fat assimilation. Dur-
ing the dosage periods the cells of the villi take up metallic tellurium
and their absorbing capacity may therefore be much diminished.
The variations in nitrogen content of the faeces shown in the tables
of the first three metabolism experiments arc too slight to warrant the
conclusion that food proteid had accumulated in the intestines.
Besides, it has been very evident, in almost all our experiments that
the secretion of mucus was considerably increased in the presence of
tellurium, and the larger quantity of nitrogen in the fjeces after dosage

^ Effects of larger quantities were not determined.

Effects of TellMrium Compounds. 141

may have been due entirely to that cause. It is perhaps unwise, how-
ever, in the absence of direct experimental evidence, to lay any
stress on these points, since the digestive and absorptive changes in
the intestines are far too complex, and are influenced by too many
interdependent relations, for us to ascribe the increase of ether-soluble
matter and nitrogen of the fseces to any one general disturbance, or
to consider it a result of any specific abnormality.

Since secretion of acid in the stomach is interfered with, it may be
reasonably supposed that secretory inhibition results in the intestines
also and that perhaps digestion of fat was retarded for that reason.
Certain it is, at all events, that loss of appetite, gastric indigestion,
irritant action resulting in vomiting and disturbed secretion of gastric
juice, result from sufficient dosage of tellurium ; that the mucous
cells in the membrane lining the gastro-intestinal tract throw out an
abnormal quantity of their product; that excessive doses of tellurium
may cause intestinal hemorrhage ; that the cells of the mucous mem-
brane reduce tellurium compounds to the metallic state ; and that the
faeces, somewhat more bulky in the dosage periods, carry off, in the
form of the metal, much of the ingested tellurium. Intestinal putre-
faction does not seem to be especially influenced, and methyl telluride
is formed somewhere in the tract and eliminated in part, at least, per
rectum.

III. Effects and Distribution after Subcutaneous

Injection.

No effort has previously been made to determine quantitatively the
distribution of tellurium, although its presence in almost all parts of
the body, after intravenous injections, has been shown quite satisfac-
torily by histological methods. We give here the toxicological data
of one experiment in which tellurium tartrate was injected under the
skin, together with the results of some analyses of the glands and
tissues.

I. Injection experiment. "With tellurium tartrate. 1899. April 9.
Bitch weighed 6.2 kilos (same animal had previously been used ; in ex-
periments 3 and 4, page 135). 10 a.m., full meal given. 3.30 p.m., 0.25 gm.
Te(C4H506)4 (5 CO. of 5 per cent sol.) injected on side, posteriorly. Marked
local irritant action. 3.50, very restless. 4.00, tremor in limbs. 4. 10, garlic
odor very strong. 4.20, tongue and jaws moving continually, as if to get rid
of ill-tasting matter. 4-30, 0.2 gm. Te(C4H506)4 injected, near same place
(4 c.c. of 5 per cent sol.). 4-50, breathing more labored. 5.10, muscles

142 L. D. Mead ami \V. J. Gics.

twitching all over body. 5.30, i.o gm. Te(C4H50o)4 injected, opposite side
(5 c.c. of 20 per cent sol.). 6.00, very unsteady. 6.20, movements of
tongue and jaws less frequent. 8.30, stupor; aroused with tlifficulty. 8.45,
90 c.c. urine — coffee colored, containing coagulable proteid and bile pigment ;
no sugar. (Urine, night before, normal.) 9.1 5, defecated — very watery. 12.00,
midnight, hardly able to stand. Refused food. Senses dulled. Nose cold
and moist. April 10, 8.30 a.m., nose dry and warm. Unable to rise.
8 p. M., remained in any unnatural position, however uncomfortable. 9.00,
arose with difficulty to defecate — diarrhoea. Food refused. 10.15, profound
stupor. April 11. 9 A. M., odor of telluridc remarkably strong. Temperature
very much lowered — extremities cold. 10.30, convulsive movements. Un-
able to rise. 12.15 p. m., breathing slow and deep for several hours. Faeces —
watery and bluish-black (color doubtless due to tellurium from ingested com-
pounds in previous experiment). 3-15, no control of movements. 5-10,
brownish red vomit, with much mucus. Acid to litmus, none free ; contained
pepsin. 8.00, unable to move, even with mechanical stimulation. 8.30,
reddish black urine, 110 c.c, containing coagulable proteid. 9-15, coma.
9.45, convulsions. 9.50, breathing intermittent. 9-55, convulsions ; death.

Post-mortem. 10.15 p.m., garlic odor from abdominal cavity. Blood very
black. Not laky. No crystalline forms found in blood, such as Rabuteau de-
scril^ed. Kiilncys very black in cortical layer. Heavy deposit of metallic tel-
lurium about points of injection, and some pus. Intestines very much inHamed.
Gastro-intestinal tract lined with metallic tellurium (from previously ingested
compounds). Stomach contents deep red, alkaline ; contained pepsin. Liver
congested. No other lesions observed. Parts removed for analysis.

These results tend to show that subcutaneous injections of tellu-
rium salts are followed essentially by the general effects noted
after intravenous injections, especially by Rabuteau and Czapek
and Weil, except that with subcutaneous injections the effects are
much more gradual. Particularly noticeable in this experiment were
general depression, weakening of the reflexes, increasing stupor,
paralysis, coma, and conx'ulsions preceding death from asphyxia.

2. Distribution of tellurium. — We determined quantitatively^ the
amounts of tcHurium distributed to the various organs of the dog into
which the tellurium tartrate had been injected,- with results agreeing

1 By the metliod outlined on page 109.

'^ This same animal had previously ingested 0.7 gm. Na.3Te03 and 0.73 gm.
Te(C4H506)4, in experiments 3 and 4, page 135. Most of this was vomited, how-
ever, and much that remained in the tract finally passed out with the faeces, or
was held in the intestinal mucous membrane-. The total quantity of Te(C4H50e)4
injected under the skin was 1.45 gm. — containing approximately 0.31 gm. Te.

Effects of TelluriMTn Compounds. 143

in the main with the quahtative conclusions drawn by previous ob-
servers. The figures given below show relative distribution, and they
indicate that tellurium is readily soluble in the tissue fluids and, as
Beyer has demonstrated histologically, may be carried to and depos-
ited in almost all parts of the system :

Te in nigs.

Muscle and skin about points of injection 1 (300 gms.) 38

Liver 12

Kidneys 9

Blood, clots from heart and large vessels (150 gms.) 8

Bile, 11 c.c 7

Stomach 5

Urine, 110 c.c. (April 11) 4

Brain . . • 4

Bladder 2

Stomach contents 2

Muscle, from shoulders and fore legs (150 gms.) trace

Lungs, pancreas, spleen trace

We see from the above results that the liver and kidneys contained
a fairly large proportion of tellurium, and it is obvious that these
organs have much to do with its separation from the blood and sub-
sequent elimination. The comparatively large quantities in the urine
and bile show this conclusively. In spite of the strong odor of the
breath, the lungs contain at any one moment only traces of tellurium.

IV. Elimination of Tellurium.

Tellurium compounds appear to be quickly reduced after they
enter the body. In all our feeding experiments the faeces con-
tained much of the bluish-black metal, the walls of the gastro-intesti-
nal tract were lined with reduced tellurium and even the material in
the vomit — pieces of meat as well as mucus — showed reducing
action by holding tellurium in metallic form. Consequently a great
part of ingested tellurium is eliminated in metallic form with the
intestinal excrementitious matter. When dosage was excessive, or
when tellurium was introduced under the skin, appreciable quantities
were eliminated in solution in the urine.^ When the quantities car-
ried into the stomach were small, only traces of tellurium appeared in
the urine -^ frequently none could be detected. After subcutaneous

, ^ Discoloration (bluish black) extended far beyond the limits of the excised
tissue, so that much more tellurium was deposited near by.

2 Identical results vi^ere obtained by Hansen, Czapek and Weil and Beyer.
Also by Kletzinsky : Wiener medicinische Wochenschrift, 1858, viii, p. 355.

144 L. D. Mead and IT. J. Gics.

injection wc have found tellurium in the urine and in the bile — proof
of the elimination of that substance from the body by both the liver
and the kidneys. The glandular and tissue cells appear to reduce
the bulk of soluble tellurium compounds coming in contact with
them and to retain the metal, although, as Hofmeister and ]^c>cr
have shown, they form methyl telluride also — probably from the
metal.

This reduction takes place very readily, in contact with any
protoplasmic substance. We ourselves have observed it when tellu-
rium compounds were brought in contact with fresh meat. Scheurlen
and also Klett have lately shown that bacteria reduce tellurite to
tellurium and that the bacterial cell is colored by the metal under
such conditions, thus furnishing a very satisfactory indicator of
reducing power on the part of these organisms. Hansen first
referred to this process in explanation of the pigmentation of the
glands and the contents of the gastro-intestinal tract. Hofmeister
noted that the methyl synthesis and the process of reduction arc
entirel}' independent of each other, and that the latter may take place
all over the bod)^ Beyer, working by histological methods, observed
that granular tellurium was deposited only in form-elements — in
nerve and glandular cells, leucocytes and striated muscle particularly.
Endothelium, unstriated muscle, nerve and connective tissue fibres,
on the other hand, were found to have no affinity for tellurium.

The continuous evolution of methyl telluride in the breath (noted
by practically all observers under all circumstances, and a symptom
in all our experiments), implies transformation of deposited metal
into soluble and diffusible form and subsequent transference to the
lungs. This elimination, as we have seen, invariably continues so
long after the last dosage of tellurium that gradual transformation
of deposited metal seems to be a necessary deduction.^ Tellurium in
the form of methyl telluride is thrown from the body, not only by the
lungs, but also with the epidermal excretions, in the faces and intes-
tinal gases, and may, as Neusser has pointed out, give special odor to
eructations.

1 Hofmeister has, in fact, proved this. He injected pulverized, chemically pure
metallic tellurium, suspended in 0.7% NaCl solution, into the jugular veins of rab-
bits. At first there were no special symptoms. After 2-3 days, however, the odor
of methyl telluride appeared in the expired air and continued to develop. In this
way, also, much of the metal deposited under the skin in our subcutaneous injec-
tion experiment must have been slowjv transformed (page 142).

Effects of Tellurium Compounds. 145

V. Personal Experiences.

There are no cases of fatal tellurium poisoning on record, so far as
we have been able to ascertain, although comparatively small quanti-
ties have been destructive of life in the lower animals. Compara-
tively few facts have been collected regarding the action of tellurium
in the human system. Sir J. Simpson records a case ^ in which a
student inadvertently swallowed a dose of tellurium, which was fol-
lowed by the evolution of such a persistent odor that for the remain-
der of the session he had to sit apart from his fellow-students.

Berzelius ^ found hydrogen telluride more irritant in its action and
more poisonous in effect than the corresponding compound of sul-
phur. Both he and Kolreuter ^ have reported that the oxides of
tellurium, as well as a number of salts of telluric and tellurous acids,
have a very unpleasant metallic taste resembling that of compounds
of antimony,* and that some have a nauseating action and are
strongly emetic. Wohler, at the time of his discovery of ethyl tellur-
ide,^ stated that it is very poisonous. At that time and subsequently,
while engaged in his chemical researches on ethyl telluride, Wohler
observed that his sweat and breath took on an odor closely resem-
bling that of the substance he was working with.^ One night while
perspiring very freely, the garlic odor in his sweat became so great
that he himself could hardly bear it. It persisted in his breath for
weeks. During seven successive days Hansen took a total of 0.34
gm. of potassium tellurite. Unusual sleepiness, oppression in the
cardiac region, nausea and abundant salivation were the chief symp-
toms observed. At the end of the dosage period there was complete
loss of appetite. The gastric symptoms did not disappear completely
until after a lapse of two weeks. The characteristic odor of the
breath continued seven weeks. Hansen was unable to separate any
tellurium from his urine. An experiment on his friend Von Roder
presented essentially the same results. Heeren" states that when

^ Quoted from Blyth : Poisons, their effects and detection, 1885, p. 559.

2 Th. Husemann undA. Husemann : Handbucli der Toxikologie, 1862, p. 773.

^ L. Gmelin : Handbook of Chemistry (Watts), 1850, iv, pp. 398, 399, 402,
403. Also, Ibid., 1856, X, p. 309, and Berzelius : Traite de chimie, 1846, ii, pp.
225, 230.

■* See foot-note, page 148.

^ Wohler: Annalen der Chemie und Pharmacie, 1840, xxxv, p. 112.

^ Quoted from Hansen's paper. Also referred to by Gorup-Besanez : Lehr-
buch der physiologische Chemie, 1878, p. 552.

■^ Heeren : Chemisches Centralblatt, n. F., 1861, vi, p. 916.

146 L. D. Mead and W. J. Gics.

compounds of ethyl and nictlnl tclliirides are merely touched with
the fingers their characteristic odor is carried to all parts of the
body, the breath acquiring it, also, in a few days. In addition to the
facts, already referred to in the experience of Reisert,^ metallic taste,
after ingestion of 0.015 of tellurous oxide, was observed in an hour
and persisted for three da)'s. We have already alluded to the clinical
observations of Neusser, Pohorccki and Combemale and Dubiquet."^

We are highly favored in being permitted to present the following
statement from Professor Victor Lcnhcr in this connection.

Professor Lenher sa)"s, " My work with tellurium was largely from
a metallurgical standpoint. I frequenth' had occasion to make large
quantities of tellurium. The oxide is volatilized at high tempera-
tures. In the process of fusion of the metal some of it escaped into
the air and a considerable quantity was involuntarily inhaled into the
lungs. Inhalation of the volatile tellurous oxide was accompanied
by a distinctly metallic taste, and the breath and secretions from the
skin quickl)' took on the characteristic garlic odor. In my own
personal experience this disagreeable odor remained for months. In
one case it persisted for about a year. When particularly large
quantities of the o.xide were inhaled, great depression and weakness
followed. One day, after having fused metallic tellurium in the open
air for several hours, I was so overcome by the influence of the
volatile oxide that I lay on my bed to sleep for a little while, intend-
ing to arise shortly and resume my work ; but I slept soundly for
eighteen hours without awakening once during that time. Severe
constipation followed the inhalation of the oxide and even purga-
tives, such as compound cathartic pills and Rochelle salt, failed to
move the bowels for several days at a time and occasionally for a
week. The inhaled oxide did not diminish intestinal putrefaction.
The faecal odors were stronger than normally and, besides, distinctly
garlic. As the tellurium disappeared from the system a return to
normal conditions was experienced and the odor of the expired air
steadily diminished. A few days after my worst experience I
analyzed a large quantity of the urine, but could not detect any
tellurium in it. The faeces were not closely examined, but they were
not blackened by metallic tellurium. After inhalation of fumes of
the oxide I have frequently felt nauseated, although I have never
vomited."

We ourselves have had no particularly toxic experiences, although
1 Pages 130 and 131. 2 Pages 105 and 131.

Effects of Tellurium, Compounds. 147

the following facts observed by Dr. Gies may not be without some
interest : At the close of the first metabolism experiment (see foot-
note, page III) Dr. Gies had occasion to make a journey of some
length. He was very much surprised to learn that a pronounced
alliaceous odor was observed not only in his breath but also in the
excretions from the skin. This information was offered independently
by several friends. It seems probable, therefore, that some of the
tellurium, in the methyl compound breathed out by the dog, was
inhaled by him and retained in his system and then was gradually
eliminated in the same form. Dr. Gies is certain that he did not at
any time come in personal contact with the oxide, but while stooping
over the dog to hold the dish containing the weighed food — from
five to ten minutes at a time twice a day for over two weeks —
he breathed the eliminated telluride in relatively large quantities.
These brief intervals of special inhalation were usually followed by
drowsiness, and sometimes by nausea. Each symptom was, however,
of short duration.

VI. Summary of Conclusions.

Non-toxic doses of tellurium (in the forms of oxide, tellurite, tar-
trate and tellurate) did not materially affect metabolism in dogs
brought to a state of nitrogenous equilibrium even when dosage was
continued for a week. These substances appeared to stimulate pro-
teid catabolism only slightly. They increased somewhat the weight
of dry matter in the faeces and diminished, in small degree, the absorp-
tion of fat. The urine was unaffected in volume, specific gravity and
reaction, but became dark brown in color during the dosage periods.

Large doses retarded gastric digestion, induced violent vomiting,
loss of appetite and somnolence. They caused, besides, inflammation
and disintegration of the mucous membrane of the gastro-intestinal
tract and also intestinal hemorrhage.

Introduced under the skin, tellurium (tartrate) caused restlessness,
tremor, weakening of the reflexes, somnolence, diarrhoea, paralysis,
unconsciousness, stoppage of respiration and death, in convulsions,
from asphyxia. At the point of injection much of the tellurium was
deposited in metallic form, but it was also distributed in large quan-
tity to most of the organs and tissues.

Methyl telluride invariably appeared in the breath a few minutes
after introduction into the system of even very small quantities of
tellurium. It persisted for months after the last dosage. The odor

148 L. D. Mead and W. J. Gics.

of this substance was also detected in the faeces and urine, about the
viscera and in the epidermal excretions.

Secretion of mucus in the stomach and intestines was greatly stimu-
lated by tellurium. Regurgitation of bile into the stomach was a
frequent result. Tellurium compounds, even in small proportion,
markedly arrested the secretion of acid in the gastric juice.

In the gastro-intestinal tract tellurium compounds were quickly
reduced and the metal deposited in great part in, and on, the mucous
membrane. Intestinal putrefaction did not appear to be influenced
in any degree. The intestinal contents were pigmented by reduced
tellurium and much of the ingested substance was eliminated in me-
tallic form in the f^ces.

The action of tr\'psin and pepsin outside the body was not very
perceptibly diminished by quantities of tellurium compounds under
0.6 per cent. Zymolysis was almost unaffected in the presence of
as much as 1.25 per cent of some of the salts. Ptyalin was more
easily affected, even by the faintly alkaline tellurate. Trypsin ap-
peared to be least sensitive to destructive influence, acting rapidly in
the presence of even 2.5 per cent of tellurite.

Tellurium was eliminated in metallic form in the faeces; as methyl
telluride in the breath, urine, fneces and epidermal e.xc^retions ; in a
soluble form, in small quantity, in the urine and in the bile.

The urine was colored brown to yellowish green after heavy
dosage with tellurium compounds, but return to normal coloration was
rapid after administration had been discontinued. Albumin and bile
pigment, besides tellurium, were the abnormal constituents of the
urine, found after subcutaneous injections. Toxic quantities given by
the mouth caused the appearance of coagulable protcid but neither
bile pigment nor sugar in the urine.

In man tellurous oxide taken into the lungs in fairly large quan-
tity caused nausea, metallic taste, somnolence, depression and consti-
pation. Meth\'l telluride was excreted in the breath, through the
skin and with the fseces. Inhalations of methyl telluride induced
sleepiness and nausea and the breath and the excretions from the
skin under these circumstances acquired, and retained for a long
time, the odor of that substance.

In many respects the action of tellurium in the body is like that of
selenium, arsenic and antimony.^

^ CzAPEK and Weil have come to the same conclusion. It is interesting to
note, in this connection, that tellurium is believed by some chemists to be in

Effects of Telkivium Compounds. 149

VII. Bibliography.

1. Chr. Gmelin.

1824. Versuche iiber die Wirkungen des Baryts, Strontians, u. s. w., auf den
thierischen Organismus. Tubingen, p. 43.

2. Hansen.

1853. Annalen der Chemie und Pharmacia, Ixxxvi, p. 208.

3. Rabuteau.

1869. Gazette hebdomadaire de medecine et de chirurgie, xvi, pp. 194, 241.

4. Reisert.

1884. American journal of pharmacy, Ivi, p. 177.

5. Knop.

1885. Botanisches Centralblatt, xxii, p. 35.

6. Neusser.

1890. Wiener klinische Wochenschrift, iii, p. 437.

7. POHORECKI.

1891. Jahresbericht iiber die gesammten Medicin, xxvi, I, p. 398.

8. COMBEMALE ET DUBIQUET.

1 891. Semaine medicale, xi, Annexes, p. 24.

9. COMBEMALE.

1 891. Bulletin general de therapeutique, cxx, p. 14.

10. CzAPEK UNO Weil.

1893. Archiv fiir experimentelle Pathologic und Pharmakologie, xxxii, p. 438.

11. BOKORNY.

(a) 1893. Chemiker Zeitung, xvii, ii, p. 1598.
ib) 1894. Ibid., xviii, ii. p. 1739.

12. Hofmeister.

1894. Archiv fiir experimentelle Pathologic und Pharmakologie, xxxiii, p. 198.

13. Beyer.

1895. Archiv fiir Physiologie, p. 225.

14. Scheurlen.

1900. Zeitschrift fiir Hygiene und Infectionskrankheiten, xxxiii, p. 135.

15. Klett.

1900. Zeitschrift fiir Hygiene und Infectionskrankheiten, xxxiii, p. 137.

References in which only casual mention of effects of tellurium appear are
given in the footnotes throughout this paper, pages 105, 131, 143, 145, and 146.

reality a mixture of elements, containing an antimony, arsenic-like body. Brauner
calls one of the presumed constituents of the tellurium complex, atistriacum, which
may be the dwitelluruim predicted by Mendeleeff. See Brauner: Journal of
the Chemical Society (London), Trans., 1889, Iv, p. 382, and Grunwald : Ibid.,
Abstracts, 1890, Iviii, p. 434; also. Dictionary of applied chemistry, Thorpe,
1893, iii, under "Tellurium." (See footnote, p. 105.)

Reprinted from THE THERAPEUTIC MONTHLY.
Vol. II, No, 4. APRIL, 1902. Pages 144-145.

CHEMICAL CHANGES IN THE BODY IN WHICH THE
METHYL GB,OUP MAY BE INVOLVED.

By WILLIAM J. GIES, M. S., Ph. D.,
of New York.

Thewriter has read, with much interest and profit,
the very valuable resume of leading facts in our
knowledge of the "changes of substances in the or-
ganism," written by Dr. J. W. Wainwright, and re-
cently published in this journal (this volume, p. 92).
The paper referred to presents a timely and systema-
tic review of many important facts which could be
brought together so admirably only by one
thoroughly versed in the subject.

For the sake of imparting further historical accur-
acy to Dr. Wainwright's very acceptable review,
which no doubt will serve as a guide to many in the
future, the writer would call attention to several
slight inaccuracies in the discussion of synthetic
changes in which the methyl group is involved.

Dr. Wainwright states (at the bottom of page 96)
that we "know three cases in which the methyl
group has paired in the body. One is the appear-
ance of methyl tellura^e' after feeding with telluric
acid (Hofmeister). The substance was recognized
by the odor, but not by analysis, none being made. The
glandular organs, and especially the testicles, are able
to form a large amount of methyl tellura/t\ Selenic
acid similarly yields methyl selenafc."

The author is not prepared to say that methyl
tellurc/f and methyl seleno/t' may not be formed after
administrations of the acids — nobody knows, he
thinks — but it should be pointed out that no one
has yet demonstrated their synthesis in the organ-
ism. Compounds aiialogotis to methyl sulphide, tellur-
ide and also selenu/c of methyl, however, are formed
in abundance after the entrance of tellurium and
selenium compounds into the body and, quite con-
trary to Dr. Wainwright's statement, it has been
shown very definitely, in a chemical way, that such
syntheses of these volatile, alliaceous bodies can and
do occur.

In his early experiments on dogs with tellurous
acid, Gmelin, (1824), on post mortem examination,
detected a peculiar garlicky odor on opening the ab-
dominal cavity. Hansen (1853) observed this same
odor in the breath of dogs to which potassium tellur-
ite and tellurous acid had been given. The odor was
assumed, by Wohler and his pupils at that time, to
be due to ethyl telluride. Various investigators,
chiefly Rabuteau (i86q), Reisert (1884), Neusser
(1890), Czapek and Weil (1893) and Hofmeister
( 1894), have since confirmed the fact that this pecu-
liar odor may be detected in the breath of animals
and man after the administration of tellurium and
selenium in various forms (including the metallic),
the so-called ''bismuth breath" being due to tellur-
ium impurities is l)ismuth products used medicallx .

Reisert's investigation of the cause of "bismuth
breath," following the therapeutic use of various
commercial preparations of bismuth, showed that as
little as o. 000,000,5 gram of tellurous oxide, given
in solution to men, was followed by the smell of gar-
lic from the expired air in 75 minutes and that it
continued for about 30 hours.

The odor has also been found to proceed from

— 3 —

the secretions of the skin, from the urine and
feces and from the blood of poisoned animals ; also,
from the minced fresh glands, etc., after treatment
outside of the body with tellurium or selenium com-
pounds.

Experiments with tellurium compounds already
] blished by the author, and additional ones in pro-
gress with selenium, confirm all of these facts.

On the discovery of methyl telluride the resem-
blance of its odor to the garlicky odor of the breath,
etc., after administration of tellurium compounds
convinced Wohler that methyl not ethyl telluride, as
he had previously assumed, was formed in the body
and eliminated from it under such circumstances.
This view was at once generally accepted. Hofmei-
ster eventually proved, in a chemical way, the fact
of methyl telluride synthesis and also showed satis-
factorily the formation of the corresponding methyl
selenide on administration of selenium compounds.
Hofmeister's method of detection of methyl tel-
luride was as follows: Sodium tellurate, 0.03-0.06
gram, was injected subcutaneously into dogs and
cats. As soon as the garlic odor became evident in
the expired air, the latter was passed through satura-
ted solution of iodine in potassium iodide for 20 to
48 hours. The solution decomposed the methyl tel-
luride, retaining each radicle. From it, methyl was
separated in the form of methyl sulphide by treat-
ment with sodium sulphide. Tellurium, after
evaporation of the solution and treatment with nitric
and hydrochloric acids, was precipitated in metallic
form with sodium sulphite.

The literature on this subject was reviewed some-
time ago by the author: See, "The toxicology of
tellurium compounds, with some notes on the thera-
peutic value of tellurates," Philadelphia Medical Jour-
nal, igoi, Vol. ii, p. 566.

[Reprinted from The Medical News, Vol. LXXX, No. ^C-^-t
5, Page 20I, February, 1902.]

NOTE ON THE GLYCOSURIA FOLLOWING EXPERI-
MENTAL INJECTIONS OF ADRENALIN.*

BY C. A. HERTER_, M.D.^

PROFESSOR OF PATHOLOGICAL CHEMISTRY, UNIVERSITY AND BELLE"
VUE MEDICAL SCHOOL,

AND

A. N. RICHARDS^ PH.D.,

OF NEW YORK;
RESEARCH SCHOLAR OF THE ROCKEFELLER INSTITUTE.

It is the object of this communication to call
attention to the fact that when a considerable
quantity of adrenalin (Takamine) is injected into
the peritoneal cavity of a normal dog there fol-
lows a rapid and usually considerable excretion
of dextrose with the urine. This fact was noted in
the course of a research upon the functions of the
pancreas, which is being carried on by the writers
under the auspices of the Rockefeller Institute for
Medical Research.

Apparently the first observations on the influ-
ence of the suprarenal gland upon carbohydrate
metabolism were those of Blum^ who found that
the urine of animals subcutaneously injected with
considerable quantities of an extract of fresh su-
prarenal gland regularly contained sugar, even
when the diet of the animals (chiefly dogs) had
been free from carbohydrates. The highest per-
centage of dextrose observed was 3.8 per cent. ;
usually the concentration of sugar in the urine
was much lower.

The discovery of Blum has recently been con-
firmed by G. Luelzer^ and, in this country, by
Croftan.^ The latter writer reaches the conclu-
sion that the glycosuria observed by him in rab-
bits and dogs depends on the action of a diastatic

* The observations referred to in this paper were made partly in
the laboratory of Dr. Herter, partly in the Laboratory of Physiolog-
ical Chemistry, College of Physicians and Surgeons, Columbia Uni-
versity.

^ Ueber Nebennierendiabetes, Deutsches Archiv fiirklinische Med-
icin, Bd. 71, Heft. 2 and 3, p. 146, igoi.

-Zur Frage des Nebennierendiabetes, Berliner klinische Wochen-
schrift. No. 48. s. 1209, igoi.

3 Concerning Sugar Forming Ferment in Suprarenal Extract. A
Preliminary Report on Suprarenal Glycosuria, American Medicine,
Tan. 18, igo2.

ferment in the suprarenal gland which converts
the glycogen of the liver into sugar.

The observations made by us on the action of
adrenalin in producing glycosuria are strongly op-
posed to the idea that the suprarenal glycosuria
is connected with a diastatic ferment contained in
this organ. Further reference will be made to
this point.

Two very recent publications* relating to the
chemistry and pharmacology of adrenalin fail to
make any reference to the ability of this prepara-
tion to induce glycosuria and it seems fair to infer
that this action of adrenalin has not heretofore
been noted. The omission is the more noticeable
as one of these papers is by Takamine (to whose
ingenuity we owe adrenalin) w'hom one may pre-
sume to be fully familiar with the investigations
that have been made on the properties of the ex-
tract.

The following are brief extracts from the proto-
cols of our experiments :

Experiment I. — A small terrier (about i6 lbs.)
received 6 c.c. of a i-i,ooo adrenalin solution (Ta-
kamine) in the peritoneal cavity. During the fol-
lowing two hours there were repeated vomiting
and great excitement which gave way to pros-
tration and unsteady gait. Urine previous to in-
jection contained no reducing substance. Urine
passed six hours after injection contained 6 per
cent, of sugar by fermentation (6.11 per cent,
by Fehling) and yielded a typical glucozazone.
No more urine was secreted. The animal died
about twelve hours after the injection, after hav-
ing had bloody diarrhea.

Autopsy. — Peritoneal cavity contained about 30
c.c. of bloody fluid. Entire gastro-enteric tract,
from lower end of esophagus to anus, intensely
congested. Mucosa deep purple color, surface
denuded of epithelium. Congestion most intense
in descending colon and rectum. Pancreas un-
evenly hemorrhagic and congested throughout.
Liver and spleen normal. Kidneys slightly con-
gested. Suprarenals appear slightly congested at
junction of cortex and medulla. Tissues preserved
in Ohlmacher's fluid. Sections through descend-
ing colon show mucosa to be entirely denuded of
epithelium in places. Submucosa is seat of nu-
merous and extensive hemorrhages. Pancreas
shows considerable congestion, most noticeable in

^E. M. Houghton, The Pharmacology of the Suprarenal Gland
and a Method of Assaying Its Products. Jour. Amer. Med- Assoc,
Jan. 18. 1902, and J. Takamine. The Blood-Pressure Raising Princi-
ple of the Suprarenal Gland, Jour. Amer. Med. Assoc , Jan. :8, 1902.

the capillaries close to the islands of Langerhans.
Many lobules are the seat of hemorrhage and near
such hemorrhages the secreting cells are in all
stages of disintegration. Numerous focal ne-
croses involving nearly entire lobules. In some
parts the acini are well preserved. Between the
necrotic areas and the well preserved acini are
zones of acini showing various grades of cell de-
generation. In places the cells composing the
islands of Langerhans show only slight degenera-
tive alterations of the cell-bodies, the nuclei
retaining their normal structure and staining
properties. In very many places these cells are
markedly altered, the protoplasm of the cell-bodies
showing a high degree of granular degenerative
change. The cell nuclei have in many instances
lost their chromatin, and are barely distinguish-
able. Many of these nuclei are very pale and
some show no coloration by hematoxylin. It is
noticeable that in many places the acini about
these much damaged cells are comparatively well
preserved. The acini that are not necrotic are
widely separated and the connective tissue is
looser than normal, suggesting the presence of
edema. The kidney shows only the signs of acute
congestion. The suprarenal structures are un-
altered.

Experiment 11. — Small dog (about 20 lbs.).
Animal bled; 30 c.c. for sugar determination.
Urine collected at this time reduces Fehling's
very slightly. Soon after bleeding received 6 c.c.
adrenalin in peritoneal cavity (1-1,000 solution).
Urine passed forty-five minutes later contained
an abundance of reducing substance (Fehling's) ;
by fermentation 4.20 per cent, glucose. Vomited
several times within a few hours of injection;
later had bloody diarrhea. Urine passed during
night after injection reduced Fehling's strongly.
Urine in bladder twenty-four hours after injection
contained 0.4 per cent, glucose. Animal bled for
sugar determination, then bled to death.

Autopsy. — Congestion of mucous membrane of
stomach, lower jejunum and ileum in patches, in-
tense congestion of greater part of rectum, irregu-
lar congestion of colon. Pancreas irregular and
finely mottled with pink. Appears normal on sec-
tion. Kidneys and spleen look normal. Supra-
renals look congested in cortex. Blood drawn be-
fore injection contained 0.163 per cent, sugar.
Blood drawn after injection (when urine con-
tained 0.4 per cent.) contained 0.174 per cent, glu-
cose.

Experiment III. — Large mongrel terrier (about
40 lbs.) received 10 c.c. adrenalin solution (i-
1,000) intraperitoneally. Urine drawn within five
minutes after injection contained 0.25 per cent,
glucose. Urine collected next day did not reduce
Fehling's. Animal vomited after injection, but
showed no prostration and had no diarrhea. Re-
covered.

Experiment IV. — Large dog (about 30 lbs.),
bled for sugar determination. Urine collected
after bleeding free from sugar. Injected intra-
peritoneally 18 c.c. adrenalin solution (1-1,000)
which had been boiled for five minutes. In half
an hour animal became violently excited and re-
mained so for more than one hour, howling con-
tinually. Vomited several times. Refused food
six hours after injection, but drank water. Urine
collected four hours after injection contained 3
per cent, sugar by fermentation and reduced Feh-
ling's strongly. Recovered.

Experiment V. — Large dog (about 50 lbs.) ;
urine free from sugar. Received 8 c.c. adrenalin
solution (1-1,000) in peritoneum. After remain-
ing quiet about half an hour after injection, vom-
ited and became violently excited for more than
one hour. Excitement then gradually w'ore away.
L^rine collected four hours after injection con-
tained 9.17 per cent, of glucose and 1.84 per cent,
nitrogen. The ratio of nitrogen to dextrose
was thus 4.98. Six hours after injection, animal
refused meat, but drank water. Day following
injection animal appeared in good condition and
urine was free from glucose. Recovered.

Experiment VI. — Small dog (about 18 lbs.) ;
urine free from sugar. Received 8 c.c. adrenalin
solution (1-1,000) in peritoneal cavity. One hour
later urine contained 6.9 per cent, glucose. Eigh-
teen hours after injection urine contained 2.0
per cent, glucose. Forty hours after injection no
reducing substance was present in the urine
passed at this time. Recover}'.

Experiment VII. — Small dog (14 lbs.) re-
ceived 5 c.c. adrenalin solution (i-i.ooo) intra-
peritoneally. Soon vomited. No excitement but
some prostration and drowsiness. Urine before
injection 0.46 per cent, glucose TPavy's method) :
after injection (twenty-one hours later) urine
contained 5.26 per cent, glucose.

Experiment VIII . — Large dog (about 40 lbs.).
On Januars' 14th given i gm. phloridzin under
skin. Next day urine, previously free from sugar,
contained large quantity of glucose. Januar}'

15th received i gm. phloridzin under skin. Jan-
uary 1 6th urine continued to show large quantity
of glucose. January i8th tirine contained about
I per cent, glucose. Animal fasted from January
14th to i8th. On evening of January i8th re-
ceived a moderate allowance of lean meat. Ani-
mal then fasted until evening of January 23d. On
January 226., urine being entirely free from re-
ducing substance, the dog was given 10 c.c. adre-
nalin solution (1-1,000) intraperitoneally. Urine
collected four hours after injection failed to re-
duce Fehling's solution in the slightest degree.
Urine passed about twenty hours later reduced
Fehling's solution slightly (o.i per cent, glucose).
Soon after last collection animal received i gm.
phloridzin. Next urine passed contained an
abundance of glucose (1.28 per cent, glucose).

Experiment IX. — Dog (24 lbs.) received 7.5
c.c. adrenalin solution (1-1,000) under skin.
Urine before injection 0.31 per cent, reducing
substance (Pavy) ; twelve hours after injection
0.47 per cent, reducing substance. After this col-
lection animal received good meal. Eight hours
later 10 c.c. adrenalin (1-1,000) in peritoneal cav-
ity. Four hours later urine contained 5 per cent,
glucose.

Experiment X. — Dog (30 lbs.) received 10
c.c.^ adrenalin solution (1-1,000) under skin.
Urine before injection 0.48 p er cent, reducing
substance (Pavy) . First collection after injection
0.66 per cent., second collection 0.52 per cent, re-
ducing substance.

Experiment XI. — Dog (50 lbs.) received
I5_c.c. adrenalin solution ( 1-1,000) . Urine before
injection contained 0.33 per cent, reducing sub-
stance (Pavy) ; second collection, 0.57 per cent.;
third collection, 0.42 per cent.; fourth collection,
0.46 per cent. ; fifth collection, 0.44 per cent, re-
ducing substance. Second, third, fourth and fifth
collections were made after injection.

The observations here recorded show that the
intraperitoneal injection of adrenalin solution (i-
1,000) in doses varying from 6 to 10 c.c. was in
each instance followed by the appearance of glu-
cose in the urine. ^ In one instance the percentage
reached 9.17 per cent.; in another it was as low

^ Since the foregoing was written an observation has been made
regarding the local application of adrenalin solution to the pancreas
Dog, previously used in Experiment V., after his recovery and the
disappearance of glucose from the urine, was etherized and the ab-
dommal cavity opened. Pancreas exposed and i c.c. of adrenalin
solution (i-iooo) plus ic.c.of water applied to its surface by meansof •
a soft brush. After a shght immediate blanching of the gland, con-
gestion followed simultaneously with the appearance of glucose in
the urine, ten minutes after application of the adrenalin solution.

as 0.25 per cent. In one instance the sugar ap-
peared in the urine in less than five minutes. Two
of the dogs died. All vomited and showed more
or less violent excitement after the injection.
Those which recovered showed some degree of
prostration twenty-four hours after the injec-
tion. With two exceptions the dogs used had
heen on a diet of lean meat previous to the injec-
tions.

Experiment VIII. appears of special interest,
because an effort was made greatly to reduce the
store of carbohydrate material by means of in-
jections of phloridzin together with deprivation
of food. It is noticeable that urine collected four
hours after the injection did not contain any su-
gar, although glucose subsequently appeared in
very small amount.

Another feature of interest is the fact that an
abundant excretion of glucose followed the injec-
tion of an adrenalin solution which had been
boiled for five minutes. We should expect any
diastatic ferment contained in the extract to be
destroyed by this treatment. Indeed the method
described by Takamine*' for the preparation of
adrenalin renders it most unlikely that any dias-
tatic ferment would resist the injurious action of
the heat employed in the course of the process for
the purpose of getting rid of albuminoid sub-
stances. To accomplish this involves an exposure
to 90' to 95° C. for a period of one hour.

It may also be pointed out that adrenalin can
be added to a solution of glycogen and kept in the
incubator for twenty-four hours without any con-
version of glycogen into sugar. These different
considerations show that there is no reason for
attributing the glycosuria from adrenalin to the
presence of a diastatic ferment. There is also no
satisfactory ground for referring to a diastatic
ferment in the suprarenal body the glycosuria
caused by any extract of this gland.

Attention is directed to the observation that the
dogs which received adrenalin subcutaneously
showed only a slight increase in the reducing sub-
stance of the urine. With doses of equal size
these results contrast sharply with the glycosuria
following intraperitoneal injections.

The alterations noted in the intestine and pan-
creas in Experiment I. are remarkable. They in-
dicate that the suprarenal extract is capable of in-
ducing changes of a highly destructive character
in these parts. How these alterations are brought

*Loc. cit.

7

about we shall not undertake to discuss here, as it
is our intention to speak of these lesions more
fully m another connection.

While we do not at present care to express an
opinion as to the relations between the glycosuria
caused by adrenalin and the lesions observed in
the pancreas, it may be stated that we have already
made observations which suggest that this glyco-
suria is in reality of pancreatic origin. It is not,
however, maintained that advanced pancreatic le-
sions like those to which we have referred are es-
sential to the glycosuria in question.

Among the topics which we reserve for future
discussion in connection with adrenalin glycosuria
or diabetes are the glycogen content of the liver
and muscles and the sugar content of the blood.

We feel justified in emphasizing the following
facts :

1. Adrenalin given intraperitoneally is capable
of inducing a marked glycosuria, in which the
percentage of sugar may reach 9.17 per cent., and
the ratio of nitrogen and dextrose 4.98.

2. Adrenalin glycosuria is not dependent on
the presence of a diastatic ferment stored or
farmed by the suprarenal gland.

3. Adrenalin injections are sometimes followed
by destructive lesions of the gastro-enteric tract
and pancreas.

4. After a fatal dose of adrenalin the cells com-
posing the islands of Langerhans were found to
be the seat of granular degeneration, very pro-
nounced in some places. The nuclei of many of
these cells showed extensive loss of chromatin
substance. In some parts of the pancreas the
cells of the islands of Langerhans were much
more injured than the surrounding cells of the
secreting acini.

6. With equal doses of adrenalin the intra-
peritoneal injections proved much more efficient
in the production of glycosuria than injections un-
der the skin.

"j^

Reprinted from the American Journal of Physiology.

Vol. IX. — March 2, 1903. — No. I.

STUDIES ON THE INFLUENCE OF ARTIFICIAL

RESPIRATION UPON STRYCHNINE SPASMS

AND RESPIRATORY MOVEMENTS.

By WILLIAM J. GIES and S. J. MELTZER.

[Fro/n the Laboratory of Physiological Chemistry of Columbia University, at the College of
Physicians and Surgeons, New Yorh.l

I. Historical.

PREVIOUS to the discovery of the effect of artificial respiration
upon strychnine convulsions, the observation was made by
Meissner and Richter (i) that artificial respiration in curarized
animals will prevent the outbreak of strychnine convulsions even
after the paralyzing influence of curare has worn off. These authors
did not ascribe this favorable result to the effect of artificial respi-
ration, but assumed that, during the period of rest enforced by
curare, the strychnine was partly eliminated from the body and
partly neutralized within the body.

Leube (2), however, came to a different conclusion. Under the
direction of I. Rosenthal, Leube studied the alleged immunity of the
chicken to strychnine. He found that if artificial respiration be in-
stituted during a strychnine tetanus, the tetanus will soon give way.
If the dose of strychnine be too large, or the artificial respiration
last only a short time, the convulsions may return.

Uspensky (3), also working under the direction of Rosenthal, a year
later studied the influence of artificial respiration upon the spasms
brought on by other poisons. He found that the convulsions which.

I

2 William J. Gies and S. J. Mcltzcr.

followed poisoning with brucin, thebain or caffein may be inhibited
by artificial respiration, but that artificial respiration has no influence
upon convulsions following poisoning with nicotin or picrotoxin. The
poisons of the latter group, though capable of producing spasms, do
not increase reflex irritability, while those poisons, the convulsions of
which are affected by artificial respiration, have the common charac-
teristic that they do increase reflex irritability. It appears evident,
therefore, that artificial respiration inhibits only such spasms as are
of reflex origin.

From the results of the preceding experiments, Rosenthal (4) con-
cluded that artificial respiration exerts its influence upon the spasms
by means of the increased oxygenation of the blood. He compared
this influence with the effects which artificial respiration exerts upon
the mechanism of respiration itself, in the production of apnoea. In
both cases the oxygen reduces the irritability of the central organs ;
in respiration it is the natural irritability of the respiratory centre in
the medulla oblongata, whereas in strychnine poisoning it is the
exaggerated irritability of the spinal cord.^

Shortly after the experiments by Rosenthal and his pupils had
been published, Schiff (5) obtained essentially the same results.
Schiff observed, also, that after prolonged artificial respiration a few
animals survived very large doses of strychnine.

During the succeeding interval a few publications have dealt with
the facts and views presented by Rosenthal and his pupils. There
is one assertion on record which is in contradiction to previous state-
ments of fact. This was made by Rossbach and Jochelsohn (7) in
a brief preliminary communication, which was never supplemented
by a full publication of their experiments. They claim that artificial
respiration has no soothing influence whatsoever upon strychnine
spasms. These observers make additional statements in this con-
nection which are not in conformity with the general experience,
but which need not be discussed here. All other investigators
confirm, unreservedly, the fact that artificial respiration exerts an in-

^ According to this view the favorable results in the experiments of Meissner
and RiCHTER were due solely to the artificial respiration. We should like to call
attention, however, to the experiments of Ch. Richet (6) in this connection.
RiCHET found that after poisoning with large doses of strychnine, the life of the
animal is greatly prolonged, if, in addition to the artificial respiration, curare is
also administered. Richet makes no reference to the work of either Meissner
and RicHTER, or to that of Rosenthal and his pupils.

Artificial Respiration and Strychnine Spasms. 3

hibitory influence upon the strychnine convulsions. There is, how-
ever, a divergence of opinion regarding the nature of this inhibitory
influence.

Ebner (8) and Buchheim (9) stated that they were able to induce the
same soothing effect by simple movements of the body and extremi-
ties of the animal, and denied, therefore, that oxygenation has any-
thing to do with the favorable action. They believe that the muscular
movements are the favorable factors in the relaxation of the spasms.
L. Pauschinger (10), however, working under Rosenthal's direction,
could easily dismiss this contention by showing that the authors sim-
ply employed the now well-known Schultze's method of instituting
artificial respiration without opening the trachea.

Brown-Sequard (11), after confirming the fact that the convulsions
may be relieved by artificial respiration, denied that the favorable
effect is due, as Rosenthal believed, to a greater charging of the blood
with oxygen. He was of the opinion that artificial respiration causes
a mechanical stimulation of the nerves of the lungs, thorax, and dia-
phragm, and thus affects an inhibition of the reflex centres.

The statements of Brown-Sequard were contradicted by Filehne
(12). Later we shall have occasion to return to the works of both
these observers.

Rosenthal's view was supported by Ananoff (13), who, in a brief
communication, reported that animals breathing pure oxygen show
a greater resistance to the effects of strychnine.

From 1878 to 1900 there is no publication to be found bearing
on this subject. In the last-named year this question was studied
by Osterwald (14) in the Pharmacological Institute of Gottingen.
Osterwald, like Ananoff, put animals under glass bell jars through
which a stream of oxygen was conducted. Experiments with mice
did not yield striking results, but a few positive results with guinea-
pigs led Osterwald to the unreserved support of the opinion that the
favorable influence of artificial respiration is due to the greater intro-
duction of oxygen into the blood.

Similar experiments were made last year by Von Czyhlarz (15)
with guinea-pigs as well as with rabbits. His experimental results
may be more appropriately discussed farther on.

The present status of this subject is, then, as follows: It is now
a well-established fact that artificial respiration may prevent the out-
break of convulsions due to strychnine poisoning, or may inhibit

4 Willi a 111 J. Gics a fid S. J. Meltzcr.

them if already present, provided the dose of strychnine be not too
large. The striking feature of its action is the perfect relaxation of
the convulsed muscles, the absence of any muscular rigidity or any
kind of tremor.^ Artificial resi:)iration here, ap[)arently, inhibits the
artificial increase of reflex-irritability. It is now the consensus of
opinion that this inhibition is produced by increased introduction of
oxygen into the blood, and that the mechanical effect of the expan-
sion of the lungs, suggested by Brown-Scquard, has no share in the
result.

This position, did not appear to us to be entirely satisfactory, for
the following reasons : the soothing influence of artificial respiration
upon the increased reflex-irritability due to strychnine is apparently
identical with its soothing influence upon respiration itself, /. c, with
the production of apnoea.- We have already stated above that Ros-
enthal, who may be said to be the discoverer of these phenomena,
looked upon both as processes of identical character — the inhibition
of the normal reflex-irritability in one and inhibition of the increased
reflex-irritability in the other. As regards the causation of apnoea it
now seems to be a settled conviction that this state can be brought
about by hyperoxygenation as well as by the mechanical distention
of the lungs. Why, then, should it be different with the inhibitory
effect of artificial respiration upon the strychnine spasms?

Furthermore, the evidence upon which the present prevailing
view is based does not appear to us to be entirely conclusive. The
chief points in the evidence are: i. Filehne's work in disproving
Brown-Sequard's claims of the disappearance of the effect of arti-
ficial respiration after section of the cord or the vagi ; 2. Ananoff's,

^ Some writers, when speaking of the favorable effect of artificial respiration,
mean simply that it prolongs life. Life can be prolonged by artificial respiration,
however, even if the administered strychnine dose is very large: but then the
tonic and clonic convulsions continue even during the most energetic artificial
respiration.

- The inhibitory effect of artificial respiration upon the complex mechanism
of respiration may show itself in several ways: i. The animal stops its normal
abdominal and thoracic respiratory movements. 2. The concomitant respiratory
movements of mouth and nose stop during artificial respiration. 3. All respiratory
movements remain quiet for some time immediately after discontinuation of the
artificial respiration. Usually only the last form of inhibition is termed apnoea.
It is obvious, however, that the arrest of the respiratory movements during arti-
ficial respiration also belongs to the inhibition phenomena, and ought to be
included in the term apnoea.

Artificial Respiration and Strychnine Spasms. 5

Osterwald's, and Von Czyhlarz's experiments in producing the same
favorable effect by simple normal inhalation of pure oxygen.

Considering the last line of evidence first, we have to exclude at
the outset the testimony of Ananoff. In his short communication
Ananoff speaks only of artificial respiration prolonging life, and does
not mention the absence of spasms during the process which, as
remarked above, is the essential point.

Osterwald's successful experiments were made on guinea-pigs
(animals which are very resistant to strychnine), and were few in
number. These strictures appear the more important when we read
them in the light of the results reported by Von Czyhlarz. This
last-named observer made nine experiments with guinea-pigs. In
each experiment one animal inhaled pure oxygen and the other (con-
trol) inhaled air. In four of these experiments both animals had only
marked hyperassthesia. In one both animals had tetanus and survived.
In the remaining four experiments the oxygen animals had marked
hyperaesthesia, whereas the control animals had non-fatal convulsions.
In the majority of the experiments, therefore, there were hardly any
differences between the oxygen-breathing animals and the control
animals, while the differences observed in the minority of the exper-
iments were only of a minor character.

Von Czyhlarz's experiments on rabbits are still more instructive.
Here he records eight experiments. In three experiments both
animals had non-fatal tetani. In two both had fatal tetani. In one
the oxygen animal had a fatal tetanus, and the control survived. In
the remaining two experiments the oxygen animals died, while the
controls survived their tetani. We fail to see in any of these experi-
ments with rabbits even the shadow of proof that the inhalation of
oxygen can suppress in these animals the increased reflex-irritability
due to strychnine poisoning. Now, all the successful experiments
ever made with artificial respiration were upon rabbits ! If, however,
it be admitted that the results of the above experiments with pure
oxygen do prove that the oxygenation of the blood can neutralize
to some degree the effect of strychnine, they surely do not prove that
the mechanical distention of the lungs has no share in the effects
produced by the classical method of artificial respiration.

There remains the work of Filehne, which was conducted in con-
travention of the claims put forward by Brown-Sequard. The latter
observer, as stated above, was of the opinion that the arrest of res-
piratory movements of the animal (apnoea), as well as the arrest

6 IV i I Ham J. Gies and S. J. Meltzer.

of the spasms in strychnine poisoning, both of which artificial respi-
ration is capable of effecting, are not due to hyperoxygenation of the
blood. The arrest in each case was attributed to the mechanical
irritation of the branches of the vagus, the phrenic " or other dia-
phragmatic nerves," caused by the forced insufflation of air into the
lungs. In support of his view Brown-Scquard states that transverse
section of the spinal cord above the origin of the phrenic nerves or
below their origin, or even section of the vagi, removes the arresting
influence which artificial respiration exerts upon the respiratory
movements.

In contravention of these statements Filehne reports that he
tested these claims in a series of experiments and could not confirm
them. An analysis of Filehne's experiments reveals the fact, how-
ever, that no experiment was made in which he studied the arrest of
strychnine spasms by artificial respiration in animals whose spinal
cord was cut. His attempts in this line were confined to the demon-
stration of the presence of apnoea after the severance of the cord.
Even in these he succeeded in cutting the cervical cord in only one
experiment. In a few other experiments he tried to crush the cord
in young animals by forcibly constricting the cervical column with a
string. The crudity of such a method hardly inspires confidence in
the results attained by it.

Filehne further records a few experiments in which artificial res-
piration arrested strychnine spasms after cutting the vagi. These
experiments, however, as was pointed out by Filehne himself, seem
also to demonstrate that the cutting of the vagi visibly impairs the
favorable effect of the artificial respiration.

We see, therefore, that the experiments to show the effect of inha-
lation of pure oxygen are still far from being decisively in favor of the
exclusive oxygen theory. We find, further, that Filehne's work can-
not be considered a sufficient refutation of Brown-Sequard's mechan-
ical theory. Apparently, much more work must be done before the
questions raised here can be satisfactorily answered.

II. Our Own Experiments with Sections of Cord and Vagi.

From the above analysis it is evident that the claims of Brown-
Sequard have not yet been properly tested, and that they deserve,
therefore, to be investigated anew.

Brown-Sequard believed that the insufflation of air into the lungs

Artificial Respiration and Strychnine Spasms. 7

irritates the endings of the vagus as well as of the phrenic and
" other diaphragmatic nerves," whatever the latter may be. He
might as well have said, also, that the sensory nerves of the thorax
wall and the pleura might be stimulated by the rhythmical pressure
of the artificial respiration. A more suggestive conjecture would be
that the rhythmical pressure upon the contents below the diaphragm
irritates the splanchnic nerves. We now know that stimulation of
the central ends of the splanchnic nerves causes inhibition of inspi-
ration (16). Experiments which exclude only one set of nerves, while
the other paths of innervation remain intact, afford inconclusive
evidence that the mechanical irritation of the nerves has no share
in the inhibiting effect of artificial respiration. Our experiments
were therefore directed in the first place toward the study of the
action of artificial respiration on animals in which the vagi were
cut, and, at the same time, the spinal cord severed at one place or
another.

General method. — The experiments were made on rabbits, which
were kept stretched on a holder, and were under ether anaesthesia
during the operations. The artificial respiration was administered
by bellows through a tracheal tube. The bellows were fastened to
the table on which we operated, and were manipulated by hand. An
average of thirty uniform strokes per minute was maintained, which
caused a pressure rarely exceeding 36 mm. Hg. Each stroke with
the bellows caused a distinct jar of the table upon which the animal
was resting, a fact of importance in our experiments. We employed
strychnine nitrate. An extensive experience has taught us that
white rabbits are more sensitive to strychnine than colored ones.
We have found that 0.45 mgm. of strychnine nitrate per kilo is a
surely toxic dose for a white rabbit, and 0.5 mgm. for a gray one.
Although this knowledge might have sufficed, we employed controls
in almost every experiment. While our main object was the study
of the influence of artificial respiration upon the spasms of strych-
nine, we also made note of the relation of artificial respiration to
apnoea under these conditions.

Abbreviated protocols of our various experiments are given below :

Experiment I. — Gray and white male rabbit, 1920 grams. Tracheotomy.
5.30 p. M. Cord cut between third and fourth vertebra.

5.34. Strychnine injected, 0.6 mgm. per kilo.

5.35. Artificial respiration started, 30 to 35 mm. pressure.

8 Willi am J. Gics and S. J. iMellzer

Experiment I — {coiitinneJ),

5.37. Both vagi cut.

6.07. Artificial respiration discontinued. Animal was watched for ten
minutes longer, and then was removed from board. During the forty-
seven minutes after the injection of the fatal dose of strychnine, tiie animal
did not show even any hyperaesthesia due to strychnine, although the table
was jarred, the rabbit untied and its paws squeezed in testing for reflexes
of the paralyzed hind limbs, and the animal eveti removed from the table.

With the dose administered in I-^xperiment I a normal animal
would have succumbed to a fatal tetanus in less than thirty minutes !
The inhibitory effect of the artificial respiration was distinctly man-
ifest, although the nervous paths of the vagi and the splanchnici
were cut off. However, during the entire period of artificial respi-
ration, there was in this experiment no full suppression of the
animal's own breathing. Further, the concomitant respiratory move-
ments of mouth and nose continued, and became very pronounced
after the vagi were cut. There was no sign of apnoea after stopping
the artificial respiration. In short, artificial respiration produced no
apnoea. Possibly the artificial respiration with only 30 to 35 mm.
pressure was not strong enough to cause apnoea in this large animal.
But it remains a noteworthy fact that in an animal in which the
paths through the vagi and splanchnici were blocked, a certain
degree of artificial respiration was sufficient to influence the strych-
nine spasms, but not to cause apnoea !

Later the same animal was given another injection of strychnine —
0.5 mgm. per kilo. It had distinct convulsions after sixteen minutes.
The noteworthy fact was observed that the convulsions did not appear
simultaneously in the anterior and the posterior parts of the animal,
but occurred in the part above the section of the cord usually before
the part below. Subsequently, when the animal recovered from these
convulsions, it was killed by asphyxia. It again had convulsions,
which appeared in the hind part later than in the front, both sets of
convulsions apparently continuing independently of one another.

Experiment II. — Gray rabbit, 1030 grams. Tracheotomy.
5.37 p. M. Cord cut opposite third dorsal vertebra.
5.42. Injected strychnine, 0.67 mgm. per kilo.
5.44. Artificial respiration started.

5.46. Artificial respiration slackened, signs of convulsive movements
appeared. Artificial respiration immediately increased, perfect rest again.

Artificial Respiration and Strychnine Spasms. 9

Expej-iment II — (contimied) .

6.00. Both vagi cut, no independent respirations, but concomitant
breatliing appears and remains tlirougliout artificial respiration.

6.08. Artificial respiration stopped, soon vibration in upper part, and
gasps.

6.09. Artificial respiration resumed, followed by rest again.

6.14. Artificial respiration discontinued, apnoea for a few seconds.

6.15. Convulsions in front parts, not in hind parts.

6.16. Convulsion in hind legs, none in front; soon, however, opisthot-
onus and death.

In this experiment, with a still larger dose of strychnine, the arti-
ficial respiration could not abolish the convulsions permanently, but
while it was continued, arrested them for thirty-three minutes, the
animal being perfectly relaxed, and even without hypersesthesia
during the entire period. In this smaller animal artificial respira-
tion suppressed the independent respirations of the animal, and
even caused a very brief period of apnoea, but it had no effect upon
the concomitant respiratory movements of mouth and nose after the
vagi were cut.

Experiment III. — White rabbit, 1400 grams. Tracheotomy.

5.03 p. M. Cervical cord cut opposite fifth vertebra, paralysis of hind
and fore legs; no voluntary breathing. Artificial respiration begun.

5. II. Strychnine injected 0.6 mgm. per kilo.

5.31. While handled, slight and brief spasms (?).

5.33. Both vagi cut, gasping and other concomitant respiratory move-
ments cannot be suppressed ; pinching of any part brings out a tetanic
convulsion confined to that part and lasting only as long as the part is
handled ; reflexes.

5.45. Artificial respiration discontinued, brief apnoea, then attempts to
breathe ; convulsions in upper part alone, later in lower part alone.

5.46. Rabbit dead.

In white rabbits 0.6 mgm. strychnine per kilo is a rapidly fatal
dose. For thirty-five minutes, while the artificial respiration lasted,
there were no real convulsions, but throughout the entire experiment
there was a marked reflex hyperaesthesia, upon which the artificial
respiration had apparently only a moderate inhibiting influence.
After the vagi were cut the artificial respiration could not longer
arrest the strong concomitant respiratory movements.

lO Willimn J. Gics and S. J. Meltzer.

Experiment 11' a. — (iray rabbit, 1840 grams. Tracheotomy.

5.17 1'. M, Cord cut opposite fifth cervical vertebra. Rabbit collapsed,
no voluntary respiration, and only faint heart-beat. Artificial respiration,
elevation of rear end of rabbit holder, and compression of abdomen.

5.46. Animal fully recovered.

6.01. Strychnine injected (0.06 mgm. per kilo strychnine sulphate
+ 0.06 per kilo strychnine nitrate).

6.33. Both vagi cut. Extremities squeezed or pulled, board hit, table
thumped, but no convulsions, and even no hypereesthesia. Independent
voluntary respiration continually present.

6.41. Artificial respiration discontinued, no apnoea, breathes well.

6.46 to 6. 49. Short tetanic convulsions either in front extremities alone,
with legs upward, or in the four extremities with front legs downward.

6.50. Tetanus, opisthotonus, and death.

Experiment IV b. — Control, gray rabbit, 1 760 grams.

4.44 P.M. Cord cut between third and fourth dorsal vertebrae.

6.03. Injected strychnine (0.06 mgm. per kilo strychnine nitrate + 0.06
per kilo strychnine sulphate).

6.30. On striking table tetanus in all parts at once, opisthotonus and
death.

Although the animal in Experiment IV a received a fatal dose of
strychnine and was subjected to all sorts of irritations, it manifested
neither convulsions nor hyperaesthesia during the entire time it re-
ceived artificial respiration. The control animal, Experiment IV b,
on the other hand, had a fatal tetanus when the table was struck,
twenty-seven minutes after receiving the strychnine. Six minutes
after discontinuance of artificial respiration the strychnine poisoning
became manifest also in Experiment IV a. The artificial respiration
apparently only inhibited an increase of reflex-irritability but did not
destroy the poison in the body; neither was the strychnine sufficiently
eliminated from the body during the period of artificial respiration to
prevent tetanus.

In this experiment artificial respiration did not suppress the volun-
tary breathing, nor did it produce any apnoea after its discontinuance.

Experiment Va. — Gray and white rabbit, 1 240 grams. Tracheotomy. Ar-
tificial respiration for three minutes, voluntary and concomitant breathing
suppressed. Artificial respiration stopped, apnoea only two seconds. Ar-
tificial respiration resumed, voluntary and concomitant breathing sup-
pressed in one minute. Artificial respiration stopped after two minutes,
apnoea eight seconds.

Artificial Respiration and Strychnine Spasms. 1 1

Experifjient Va — (contimied).

5.45 P.M. Cervical cord cut between fifth and sixth vertebra, animal in
good condition. Artificial respiration resumed, voluntary and concomi-
tant respiration suppressed only after seven minutes. Artificial respiration
stopped, apnoea eight seconds. Artificial respiration immediately begun
again, no voluntary and concomitant movements.

5.57. Strychnine nitrate injected, 0.65 mgm. per kilo.

6.07. Vagi cut; voluntary and labored concomitant breathing set in,
each suppressed after seven minutes.

6.34. Artificial respiration stopped, apncea eight seconds. Animal
observed until 6.52. Although extensively handled during the fifty-five
minutes since strychnine was injected, no sign of hyperaesthesia.

Later on asphyxia was caused by inhalation of hydrogen, and again by
clamping of the trachea. Tetanic convulsions appeared only in the an-
terior part ; the legs were directed towards the head.

Experiment Vb. — Control, gray and white rabbit, 1250 grams.
6.03 p. M. Injected strychnine nitrate 0.63 mgm. per kilo.
6.27. Animal stiff.
6.29. Tetanic convulsion.
6.33. When lifted there was a convulsion which the animal survived.

In this experiment the effect of artificial respiration upon strych-
nine spasms was very plain. They were entirely suppressed during
the fifty-five minutes of observation, although the control animal
began to show a distinct strychnine effect even twenty-four minutes
after injection. The inhibitory effect upon the respiration was
retarded by section of the cord as well as by section of the vagi, but
finally a distinct apnoea was attained.

In the last three experiments the vagi and splanchmici, as well as
the sensory fibres of the pleura and thoracic wall, at least most
of them, were separated from the respiratory centre, etc. The
roots of the brachial plexus were apparently divided in two parts, for
when convulsive movements occurred in the upper part alone, the
front legs were directed toward the head.

Experime7it Via. — Gray and white male rabbit, 1550 grams. Tracheotomy.
Artificial respiration (25-30 mms. Hg) for three minutes. Voluntary and

concomitant breathing soon suppressed. Artificial respiration stopped,

apnoea three seconds.

4.43 p. M. Cord cut "between fourth and fifth cervical vertebrae," breath-
ing stopped. Artificial respiration begun. Heart, lid reflex, etc., all right.
Concomitant breathing soon suppressed. Artificial respiration stopped,

12 William J. Gus and S. J. Meltzcr.

Exptrimeiit VI a — {contiiiueJ).

apncea fifteen seconds, soon " liead breathing." Artificial respiration
resumed again, head breathing soon suppressed.

4.57. Injected strychnine nitrate 0.7 mgni. per kilo.

5.16. Both vagi tied off, concomitant breathing appeared, but disap-
peared again after four minutes.

5.32. Artificial respiration stopped, apna-a fifteen seconds, then "head
dyspnoea" ; no hyperaesthesia otherwise. Artificial respiration again.

5.40. Artificial respiration stopped, apnoea fifteen seconds, then gradual
development of head dyspnoea and asphyxia.
5.43. Heart stopped. No convulsions.

Experiment VI b. — Control, gray and white rabbit, 1050 grams.
5.01 p. M. Injected strychnine nitrate 0.7 mgm. per kilo.
5.07. Convulsions, did not survive.

In Experiment \\ a when artificial respiration was stopped there
appeared now and then a very faint indication of thoracic movement.
Possibly it was produced passively by the dyspnoeic contraction of
the cervical muscles. The autopsy showed that the cord was severed
at the lower border of the fourth cervical vertebra, but the cut was
diagonal and possibly a few fibres of the phrenic escaped. At all
events this experiment is a strong demonstration of the efficiency of
artificial respiration in suppressing strychnine spasms, and in pro-
ducing apnoea, even after the vagi, splanchnici, brachial plexus,
and almost all of the phrenic nerves are excluded. Although the
control animal had a convulsion six minutes after injection (0.7
mgm. per kilo), the animal in Experiment VI a manifested no
sign of strychnine spasms either during the forty-three minutes of
artificial respiration or during final asphxyia. Furthermore, there
was no concomitant breathing during the artificial respiration, and
an apnoeic pause was present after each interruption.

Experiment Vila. — Gray female rabbit, 11 20 grams. Tracheotomy.

5.20 P.M. Cord cut near upper border of fifth cervical vertebra, animal
breathes normally. Artificial respiration for a few minutes, voluntary res-
piration persistent. Artificial respiration stopped, no distinct apnoea.
Artificial respiration begun again.

5.30. Both vagi cut, labored, concomitant breathing; voluntary and
concomitant breathing subsiding slowly.

5.35. Injected strychnine nitrate, 0.7 mgm. per kilo.

6.05. Artificial respiration discontinued, no apnoea. Animal observed
five hours longer. Had no sign of strychnine poisoning. When then

Artificial Respiratioii and Strychnine Spasms. 13

Experiment Vila — (^continued).

given a comparatively large dose of strychnine, it had a number of short
convulsions in either of the two parts, independendy of one another.

Experiment VII b. — Control, gray female rabbit, 1050 grams. For better
comparison had ether anaesthesia for a few minutes.
5.40. Injected strychnine nitrate 0.7 mgm. per kilo.
5.59. Convulsions, succumbed in six minutes.

This experiment again is a classical demonstration of the inhibitory
effect of artificial respiration upon the strychnine spasms even after
section of cord and vagi. The effect upon respiration was less
pronounced.

Experiment Villa. — Gray female rabbit, 1750 grams. Tracheotomy.

3.59 p. M. Artificial respiration begun, only very slight concomitant res-
piratory movements.

4.01. Cord cut between second and third cervical vertebrae, concomitant
breathing gready increased, after a few minutes decreased again.

4.09. Artificial respiration discontinued, apnoea for a few seconds, then
dyspnoea. Artificial respiration again.

4.15. Animal recovered from anaesthesia. Injected strychnine nitrate,
0.7 mgm. per kilo. Heart-beat, lid reflex, etc., normal until 4.30, when
heart-beats became slower and labored concomitant respiratory movements
reappeared.

4.35. The respiratory movements rapidly diminished ; no lid reflex.
4.37. Heart-beats faint.

4.39. Death.

During the fifteen minutes after injection the animal was perfectly nor-
mal, but there was no sign of hypersesthesia.

Experiment VIII b. — Control, gray rabbit, 1 700 grams.

4.18 p. M. Etherized (for comparison) and kept under ether until 4.23.
4.19. Injected strychnine nitrate 0.7 mgm. per kilo.
4.33. Tetanic dance.

4.36. Convulsion terminating fatally at once. Although this animal
was under the influence of ether while strychnine was injected, fourteen
minutes after injection it manifested the unmistakable effects of this drug.

In Experiment Villa the vagi, splanchnici, and all thoracic nerves,
including the phrenici, were excluded. Although the animal died
early, it lived long enough, and was normal long enough, to demon-
strate that the strychnine had no effect so long as the artificial respi-
ration was continued. There was once also a distinct apnceic pause.

14 William J. Gics and S. J. Mcltzcr.

Experiment JX a. — Gray female rabbit, 1750 grams. Traclicotomy.
4.00 p. M. Artificial respiration begun.

4.02. Injected strychnine nitrate, 0.7 mgm. per kilo. The voUmtary
respirations were completely suppressed. Six minutes of artificial respir-
ation, concomitant breathing not completely suppressed.

4.07. Cord cut at third cervical vertebra. Heart-beat, lid reflex, etc.,
normal, concomitant respiratory movements increased ; remained unsup-
pressed throughout entire experiment.

4.12. Vagi cut. Animal normal throughout the remainder of the ex-
periment. There was apparently a hypera^slliesia in the lower part,
pressing or pulling legs was followed by contraction or tremor in legs, but
these continued only as long as pull or pressure lasted. Blowing on
animal, hitting table, no effect. Tremor in abdominal muscles ; they even
seem to contract synchronously with artificial respiration, simulating
superficial independent voluntary breathing.

4.42. Artificial respiration stopped, all contraction and tremor disappear
immediately (are due apparently to the local stimulus of the artificial res-
piration); head dyspnoea appears. Artificial respiration resumed again.
Extremities and tail repeatedly pinched, pulled, etc., response with local,
short reflexes, either during stimulation or immediately after. Pinching
ear or other parts of head produce no reflex, but voluntary motion, moving
away.

5.03. Artificial respiration discontinued ; head dyspnoea, but no other
movement of body.

5.04. Slight movement, and later, vibration only in front legs.

5.05. Sudden tetanus in all four extremities, followed by clonic con-
vulsions.

5.06. Artificial respiration resumed. Lid reflex and heart-beat soon
normal again, no more convulsions.

5.10. Artificial respiration discontinued again.

5.12. Sudden tetanus. Artificial respiration at once, and tetanus
stopped suddenly. This procedure was repeated several times with same
result, but sometimes tetanus stopped even while there was no artificial
respiration.

Experiment IX b. — Control, gray female rabbit, 1600 grams.
4.22. Strychnine nitrate, 0.7 mgm. per kilo.
4.35. Tetanic convulsions.
4.45. Blown on, immediately violent convulsion, succumbs.

In I'2xperiment IX a the section of the cord was above the phrenici,
and the influence of the vagi and all other nerves concerned was pos-
itively excluded. The animal had a dose of strychnine which proved

Artificial Respiration and Strychnine Spasms. 15

fatal to the control rabbit in less than half an hour. Although the
animal in Experiment IX a was continually handled, and the reflexes,
etc., tested, for an hour, while artificial respiration lasted, there was
no reaction which could be ascribed to the effect of strychnine.
Pounding the table or blowing on the animal had no effect at all.
Pinching or pulling a leg brought out a local reflex which was
apparently due only to the increased reflex-irritability caused by the
section of the cord. Pressing one hind leg, for instance, would bring
out a short flexion or extension of the opposite, or of a front leg.
The artificial respiration caused short contractions of the abdomi-
nal muscles. But pinching any part of the head caused no reflex-
response. The strychnine, however, was not destroyed within the
body, nor sufficiently eliminated from it. Soon after stopping the
artificial respiration there appeared convulsions and tetani, which by
their entire character were apparently due essentially to the strych-
nine and not to asphyxia, or at least not to asphyxia alone. But these
convulsions also could be stopped instantly by artificial respiration.

The influence of artificial respiration upon apnoea was not carefully
noted in this experiment, but the concomitant respiratory movements
continued during the hour while the artificial respiration lasted,
although their intensity gradually decreased.

Our first series of experiments brought out one positive result.
The claim of Brown-Sequard, that section of the cord or of the vagi
abolished the arresting influence which artificial respiration exerts
upon strychnine spasms, is entirely unfounded. Not only does sec-
tion of the vagi alone, or of the cord alone, fail to impair this influ-
ence, but even cutting the vagi, combined with such section of the
cord as excludes all influences of the splanchnic, diaphragmatic, and
thoracic nerves, apparently does not interfere with the inhibitory
influence of artificial respiration upon strychnine spasms. There
were no convulsions in any of our experiments as long as suffi-
ciently strong artificial respiration was administered. In many
experiments no convulsions appeared even after the artificial res-
piration was stopped, although in all cases doses of strychnine
were employed which by control experiments were proved to be
surely toxic and mostly fatal. In some experiments artificial respi-
rations arrested instantly the tetanic convulsions which were per-
mitted to break out.

The doses of strychnine which we employed were, however, not

1 6 Williain J. Gies and S. J. Mcltzer.

much above the toxic or fatal minimum. Possibly section of the cord
or vagi does interfere somewhat with the degree of favorable influ-
ence which artificial respiration might exert under such conditions.
Filehne, who admits some impairment due to the section of the vagi,
does not state the weight of his animals. Possibly, however, the
doses which he employed were a trifle too large. Overdosage might
also explain the claims put forward by Brown-Scquard. Ikit the
description of his experiments is too brief to permit any very definite
interpretation. In fact it is not even evident that Brown-Sequard's
conclusions regarding the relations of the sections of cord, or vagi,
to the arresting influence of artificial respiration upon strychnine
spasms were derived from actual experiments, and that they were not
mere inferences from the experiments he made on the production of
apnoea.

Regarding the latter, our own experiments have indeed demon-
strated that section of the cord and the vagi impairs more or less the
production of apnoea by artificial respiration. In some cases after
section of the cord, and especially after additional section of the vagi,
neither the voluntary respirations nor the concomitant respiratory
movements could be suppressed. This was observed in some of the
larger animals. Possibly the degree of ventilation employed in our
experiments was not sufficient to accomplish this end in an animal
with a comparatively large thorax. However, in all the experiments,
section of the cord, or of the vagi, even during artificial respiration,
immediately brought out again the voluntary breathing of the animal
and especially the concomitant respiratory movements. It invariably
took a much longer time to suppress the latter after section than
before it.

Our experiments also showed that while artificial respiration com-
pletely suppresses the increased reflex-irritability due to strychnine-
poison, it does not interfere, at least not strikingly, with the increased
reflex-irritability induced by section of the cord. In all cases we were
able, with little or no difficulty, to produce distinct reflexive move-
ments by pinching a leg, touching an eye, etc., the posterior extrem-
ities responding more readily than the anterior ones. In one case,
with section above the phrenici, each blow of the bellows brought
out a contraction of the abdominal muscles simulating spontaneous
breathing, which ceased on stopping the artificial respiration.

We noticed also, in the cases in which mild convulsions appeared
after artificial respiration was stopped, that the parts lying above the

Artificial Respiration and Strychnine Spasms. 17

line of section of the cord and those lying below it had their convul-
sions independently of one another. They were mostly insynchro-
nous. In the experiments in which section of the cord was near the
fifth cervical vertebra, the interesting observation was made that
when the convulsions occurred in the anterior part, the anterior legs
took part in it by moving toward the head, and that when the pos-
terior part was convulsed the anterior legs moved toward the tail,
pressing against the body. When, however, a violent tetanus broke
out, the spasm convulsed all parts nearly simultaneously.

Thus it is evident that our experiments have established the fact
contended for, but not proved by Filehne, namely, that section of the
cord and vagi does not interfere with the inhibitory influence which
artificial respiration exerts on strychnine spasms. But does this fact
prove that the inhibitory influence of artificial respiration is due to
the chemical influence of the oxygenation of the blood and to this
alone .-' Does this fact indicate that the mechanical act of rhythmical
insufflation has no share in the inhibitory influence .-*

The persistence of the favorable influence observed after section
of cord and vagi could only then serve as an irrefutable proof if the
claim for the mechanical share had been restricted to a hypothesis
that the inhibition acts either through the agency of the respira-
tory centre or through the inhibitory mechanisms of the brain. If
this is what Brown-Sequard meant, his theory is surely disproved
by our experiments. The favorable influence of artificial respiration
against the increased irritability of the spinal cord continues even
after the cord has been severed from the controlling parts above it.
But why restrict our hypothesis .'' We know that any reflex may be
inhibited within the spinal cord by any mechanical stimulation of any
part of the body. We have also seen in our experiments that, in an
animal with a severed cord, artificial respiration caused rhythmical
contraction of the abdominal muscles. This fact shows that the
insufflations into the lungs, and the consequent abrupt increase of
pressure upon the organs within the thoracic cavity, result in stimu-
lating also the dorsal nerves imbedded in the abdominal section of the
body. Furthermore we know that this insufflation causes an inhibi-
tion of centres lying within the medulla (respiratory, vaso-motor,
cardio-inhibitory centres). Why then should it not be assumed that
the rhythmical insufflations into the lungs stimulate all nerves within
the thoracic and abdominal regions and thus inhibit increased reflex-
irritability in all parts of the cord }

i8 William J. Gics and S. J. Meltzer.

The hypothesis formulated by Brovvn-Scquard is certainly untenable.
That the arrest of the spasms can be due to the mechanical stimu-
lation of the endings of the vagi, the phrenic and " other diaphragmatic
nerves " alone, our experiments with section of the vagi and the cord
have proven conclusively. But no cutting of the cord is capable of
disproving the hypothesis that rhythmical insufliation is a mechanical
stimulus for all the nerves within the trunk, by means of which an
inhibition is caused in every section of the spinal cord above a cut as
well as below it.

The question, therefore, is still open : Does the mechanical element
involved in artificial respiration have a share in the arrest of the
strychnine spasms, just as it is now generally assumed that it has a
share in the production of apnoea ?

III. Artificial Respiration with Hydrogen.

For the solution of this question a method presents itself which at
first sight appears to be quite simple. Previous investigators who
desired to prove that it is the chemical factor which causes the arrest
of the spasms have tried to introduce the oxygen without the compli-
cation of the mechanical element. Desiring to test the efficiency of
the mechanical factor, we sought to determine the effect of artificial
respiration with its chemical factor removed ; i.e., artificial respira-
tion with an indifferent gas. It was partly by this method, indeed,
that the value of the mechanical element in the production of apnoea
was ascertained. We have, therefore, endeavored to study the effect
of artificial respiration with pure hydrogen upon the strychnine
spasms.

General method. — The method we employed was comparatively
simple. Bellows were connected on one side with a gasometer con-
taining pure hydrogen, and on the other side with the trachea of the
animal. The tube connecting the bellows with the gasometer con-
tained a valve which permitted the entrance of the gas into the bel-
lows, but prevented it from going back to the gasometer. The tube
connecting the bellows with the trachea contained a valve permitting
the escape of the gas in the direction of the trachea, but preventing
its return to the bellows. The expiratory air escaped through a lat-
eral tube submerged under water (MLiller's water valve), by which
arrangement air was prevented from entering into the trachea through
the expiratory aperture during a voluntary inspiration. All the con-

Artificial Respiration and Strychnine Spasms. 19

nections were carefully made air tight. Each suction of the bel-
lows brought hydrogen into it, and each compression drove the
hydrogen into the lungs. The pressure was regulated by means of
a stop-cock carried by the expiratory tube, and it was registered by
a manometer connected by a T tube with the bellows-trachea tube.

We had, of course, no expectation of being able to continue the
exclusive inhalation of hydrogen long enough to prevent the develop-
ment of the strychnine poisoning, in the same manner as we suc-
ceeded in preventing it by the artificial respiration of air. We had
observed that when once a tetanus broke out in our experiments it
could be suppressed instantaneously by artificial respiration. In fact
this instantaneous effect appeared to us to be in favor of the theory
of a mechanical effect, since an effect due to a sufficient increase of
oxygen in the blood could hardly develop so promptly after the first
few strokes with the bellows. We therefore had reasonable expecta-
tions of witnessing the same instantaneous effect when pure hydrogen
would be insufflated, or at least of observing it, long before the
unavoidable asphyxia would finally compel the discontinuation of
this gas. However, the first preliminary experiment, to determine
the effect of insufflation of pure hydrogen upon the production of
apnoea, brought us a surprise.

Experiment X. — White rabbit, 1700 grams. Tracheotomy, connected with
bellows and gasometer, expiratory tube submerged. Insufflation of hy-
drogen for a brief period, apnoea for a few seconds. Repeated a few
times with same result. Encouraged by the absence of asphyxia, the
insufflation was continued consecutively for eighteen minutes, during
which time there was no voluntary breathing, no concomitant respiratory
movements, and no perceptible cyanosis of visible mucous membranes.
After discontinuing the insufflation of hydrogen an apnoea of fifteen
seconds appeared, but this was followed immediately by rapid superficial
breathing and very rapid, faint heart-beats. Artificial respiration with air
improved this condition, but the animal soon died through an accident.

Eighteen minutes' insufflation of pure hydrogen without asphyxia!
That was surely an unexpected result. Before discussing it, how-
ever, we should quote a few of these hydrogen experiments in which
also toxic doses of strychnine were injected.

Experiment XI a. — White rabbit, 1240 grams. Tracheotomy.
4.30 p. M. Injected strychnine nitrate, 0.6 mgm. per kilo.
4.33. Trachea connected with bellows, etc. Continued insufflation
without incident till 4.58, when tetanic convulsions set in. Continued

20 William J. Gies a7id S. J. Meltzer.

Experiment XI a — (coutiiiutii).

insufflation until 5.01 without favorable effect. Insufflation stopped,
animal thoroughly asphyxiated.

5.05. Artificial respiration with air.

5.07. Discontinued, no apnoea, immediately rapid breathing, a minute
later convulsions, which continued for a few minutes. Animal killed.

Experimetit XI b. — Control, gray and white rabbit.

5.17. Injected strychnine nitrate, 0.5 mgm. per kilo.
5.30. Convulsions broke out.

The animal in Experiment XI a was a white rabbit which, as men-
tioned above, was more susceptible to strychnine than the gray and
white one. It received a larger dose than the gray control animal.
Nevertheless, the convulsions did not break out until twenty-eight
minutes after the injection, while the control had convulsions thir-
teen minutes after the injection. In this experiment the insufflation,
however, could not put off asphyxia longer than twenty-five minutes,
and with the onset of asphyxia the convulsions broke out.

Experwient XII a. — ■ White rabbit, 2600 grams. Tracheotomy.

4.51 P.M. Injected strychnine nitrate, 0.53 mgm. per kilo.

4.55. Trachea connected with bellows, etc.

5.1 1. Some spasmodic twitching (beginning dyspnoea?). Increased
the number and energy of the ventilations, animal quiet again.

5.15. Both vagi cut, " head dyspnoea " sets in.

5.25. Voluntary breathing of the animal appears and gradually in-
creases.

5.28. Insufflation of hydrogen stopped, no apnoea, very labored dysp-
noeic breathing.

5.42. Trachea clamped, death. No strychnine effect at any time.

Experitnent XII b. — Control, white rabbit, 1970 grams.

4.29. Injected strychnine nitrate, 0.45 mgm. per kilo.
5.01. Convulsions, died in two minutes.

In Experiment XII a, the animal received more strychnine than the
control, which succumbed thirty-four minutes after injection, but had
no convulsions for forty-seven minutes; i.e., during the time it was
under observation. The slight twitchings which appeared sixteen
minutes after injection were promptly suppressed by the increase in
ventilation with hydrogen. The inhibitory effect upon respiration,
however, was greatly diminished by the section of the vagi. The

Artificial Respiration and Strychnine Spasms. 21

concomitant breathing set in immediately, and the voluntary breath-
ing appeared soon also, and apparently would have terminated in
asphyxia, if the hydrogen insufflation had not been discontinued.

Experiment XIII a. — White rabbit, 1660 grams. Tracheotomy.
4.48 p. M. Injected strychnine nitrate, 0.54 mgm. per kilo.
4.50. Trachea connected with bellows, etc. At no time voluntary or
concomitant breathing, no sign of hypergesthesia.

5.21. Insufflation of hydrogen stopped, brief apnoea, then normal
breathing. Observed till 5.42, no convulsions.
Expermient XIII b. — Control, white rabbit, 1420 grams.

5.27. Injected strychnine nitrate, 0.49 mgm. per kilo.
5.53. Had convulsions, and died in about two minutes.

The control had a fatal tetanus in twenty-six minutes, while animal
XIII a, with a larger dose, showed no strychnine effect for the fifty-
five minutes it was kept under observation. The insufflation lasted
for thirty-one minutes and exerted a distinct inhibitory effect upon
the respiration.

The results we obtained in these experiments were extraordinary
indeed. Not only could the effects of fatal doses of strychnine be
completely prevented by insufflation of pure hydrogen, but the animal
could be kept by such an uninterrupted insufflation, as was seen in
Experiment XIII a, for thirty-one minutes without manifesting any
signs of asphyxia, dyspnoea, or cyanosis.

We all know very well that spontaneous inhalation of hydrogen
alone results in asphyxia almost immediately. This is an old, well-
established fact, and we have tested it ourselves by the following
direct experiments. When the trachea of an animal was connected
directly with the gasometer, without the intervention of the bel-
lows, the animal thus surely inhaling, spontaneously, pure hydrogen,
asphyxia set in after thirty to forty-five seconds, and rarely as late
as after sixty seconds. Apparently, then, it was the action of the
bellows which deferred asphyxia so long.

The first thought which occurs is that the bellows were, after all,
not perfectly air tight. We have tested them by letting the animal
spontaneously inhale the hydrogen from the gasometer through the
expanded bellows without ventilating them. The asphyxia was then,
indeed, deferred a little longer than when the inhalation occurred
without the intervening bellows. However, the gain was at the
utmost a minute or two, and therefore the amount of air which
could have found access to the bellows must have been at most

22 William J. Gies aiid S. J. Mcllzcr.

very small. Hut even granting that during the sudden and forcible
expansion of the bellows more air was sucked into them than during
the voluntary breathing, the amount of air which was able to pene-
trate the pores must under all circumstances necessarily have been
very small in proportion to the quantity of hydrogen which, under
constant pressure, had free access through the open lumen of a wide
tube. It must also be remembered that the animal not only had no
asphyxia under these conditions, but also that it was constantly in
a state of apnoea, — a state which occurs only, it is assumed, when
the animal receives more air than normally.

We may add, also, that, according to Osterwald (17), a diminution
of oxygen favors the outbreak of strychnine spasms. In our experi-
ments, with surely diminished oxygen there was no sign of convul-
sions even with fatal doses of strychnine.

These experiments brought us more than we looked for. It was
now no longer a simple question whether the mechanical factor of
artificial respiration has a share in the inhibition of strychnine
spasms. The question which confronted us was whether one of the
fundamental and apparently definitely settled principles in the theory
of respiration did not require revision.

Searching through earlier literature on the subject of respiration
we discovered that we had touched upon a long-forgotten chapter in
the discussion whether the absence of oxygen or the presence of
carbon dioxide is the cause of inspiration.

In 1862 L. Traube (18) made experiments with insufflation of hydro-
gen on dogs, in the same manner as we have made them on rabbits,
and found, as we did, that artificial respiration with pure hydrogen
may be carried on for a long period (forty- six minutes in one experi-
ment), the animal remaining all the while in a state of apnoea. On
the other hand the addition of carbon dioxide to the air rapidly
caused dyspnoea. Traube, in consequence of these observations, gave
up his original idea, that the absence of oxygen is the stimulus for
inspiration, and accepted the view that the real cause of respiration
is the presence of carbon dioxide. Heidenhain and Krause (19) soon
contradicted Traube's statement, and explained his conclusion by
assuming that his bellows were not air tight.

Traube (20) repeated his experiments, oiled his bellows, and took
all precautions, as he states, to prevent the entrance of air, and in-
sisted on the correctness of his former results, attributing the failure
of Heidenhain and Krause to some fault in their technique.

Artificial Respiration and Strychnine Spasms. 23

Traube was contradicted also by Thiry (21),^ and finally by
I. Rosenthal (23). Rosenthal did not repeat Traube's experiments,
but connected the trachea of the animal with a gasometer of special
construction containing pure hydrogen, and found that the animals
became rapidly asphyxiated. By special calculations Rosenthal
arrived at the conclusion that air which contains only i per cent of
oxygen is sufficient for the maintenance of the animal, an amount
which presumably found its way into the bellows in Traube's experi-
ment. That was the last word, at least the last we found recorded
in this discussion.

We may add that Traube's technique suffers from still another
objection. In his experiments the opening for expiration had no
valve. The animal, therefore, could obtain, during inspiration, sufifi-
cient air through this opening, even if it were made very small. As
long as it was large enough for expiration it was also sufficient for
inspiration. We have established this fact by experiment. The
trachea was connected directly with the gasometer while the expi-
ratory tube was submerged : asphyxia in forty-five seconds. The
expiratory tube was left free in the air, and the stop-cock turned
so as to make the lumen permissibly narrow : the animal went on
breathing without noteworthy impediment for some time.

Rosenthal's paper appeared in 1864, and at that time there had
not yet arisen the question whether the mechanical distention of
the lungs can cause inhibition of inspiration. The only question in
the minds of the earlier investigators was whether absence of oxygen
or presence of carbon dioxide is the stimulus of respiration. And as
the simple inhalation of hydrogen caused asphyxia, this appeared
to prove that it is the absence of oxygen which causes respiration.
Traube's experiments, therefore, seemed to have no further object.
The value of the mechanical element which distinguishes artificial
respiration from spontaneous breathing had not yet been recognized.
We now know, from the studies of Hering and Breuer, Head, Gad,
Meltzer, and others, that the mechanical effect of the distention of
the lungs has a distinct inhibitory influence upon respiration.

It is now, furthermore, the general consensus of opinion that both

1 That, at least, is what Thiry states in his paper in the Zeitschrift fiir ration-
elle Medizin (iii), xxi, p. 25. It is not stated on what grounds the opinion is based.
MiESCHER-RtJscH (22), however, quotes Thiry from a French paper as saying
that artificial respiration with air and hydrogen causes apnoea. This paper was
not accessible to us.

24 ll^i/liam J. Cics and S. J. Meltzer.

the presence of carbon dioxide, as well as the absence of oxygen, act
as stimuli to the respiratory mechanism. But it is surely not the
actual immediate need of oxygen for metabolic purposes which in
the latter case is the stimulus. The blood and lymph and tissues
are provided with a surplus of oxygen for actual oxidative necessities.
It is the first intimation of a deficit in this sinking fund which acts
as a warning signal, — as a stimulus for increased provision of oxygen,
increased inspiration. Is it, then, inadmissible to assume that this
warning, this stimulus resulting from diminution in the body's in-
come of oxygen, could be overcome for some time by the inhibitory
influence of the rhythmical mechanical effect of distention of the
lungs, if sufficient provision were made for the full escape of the
carbon dioxide.'* Our experiments do not, of course, warrant such a
positive conclusion. The bellows permitted the entrance of air to
some degree, but the amount of air which entered was surely com-
paratively small. If, therefore, our experiments, as well as those of
Traube, do not yet permit positive conclusions in this regard, they
are at least suggestive enough to urge the necessity of a reinvestiga-
tion of this particular question with more favorable methods. In
this relation the necessity of avoiding suction apparatus in the execu-
tion of artificial respiration with indifferent gases seems important.

In this connection, also, we wish to call attention to the statement
of Head (24) that he caused apnoea by insufflation of hydrogen. His
conclusion was that the apnoea was due to mechanical effects. He
used bellows, and does not mention any precaution taken to guard
against the entrance of air into the bellows. Could not the conten-
tion be made against his conclusions also, as it was raised against
Traube's, that it was the air which entered through the pores of the
leather into the bellows that brought about the observed result?

Regarding the arrest of the strychnine spasm, which we observed,
with hydrogen insufflations, it appears very probable that it is due
largely to the mechanical effect of the insufflation, and that it is not
essentially a result of the admixture of small amounts of air. Here
also additional experiments, and by other methods, will have to be
made before the question can be definitely settled.

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Zeitschrift fiir rationelle IMedizin (3), xviii, p. 76.

2. Leube: Archiv fiir Anatomic und Physiologic, 1867, p. 629.

Artificial Respiration and Strychnine Spasms. 25

3. UsPENSKY : Archiv fiir Anatomie und Physiologic, 1868, p. 522.

4. Rosenthal : Comptes rendus, 1867, Ixiv, p. 1142.

5. Schiff: Beitrage zur Physiologie, 1896, iii,p. 211 ; reprinted from I'Imparziale,

1867.

6. RiCHET : Comptes rendus, 1880, xci, p. 131.

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schaften, 1873, No. 24, p. 369.

8. Ebner : Inaugural dissertation, Giessen, 1870.

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11. Brown-Sequard : Archives de physiologie, 1872.

12. Filehne : Archiv fiir Physiologie, 1873, P- 3^1 •

13. Ananoff : Centralblatt fiir die medicinische Wissenschaften, 1874, No. 27,

p. 417.

14. OsTERWALD : Archiv fiir experimentelle Pathologie, 1899, xliv, p. 451.

15. V. CzYHLARZ: Zeitschrift fiir Heilkunde, 1901, p. 160.

16. Graham: Archiv fiir die gesammte Physiologie, i88r, xxv.
I 7- OSTERW ALD : Loc. cit.

18. Traube : AUgemeine medicinische Central-Zeitzung, 1862, p. 296.

19. Krause: Studien aus dem physiologischem Laboratorium zu Breslau, 1863,

ii, p. 31.

20. Traube : AUgemeine medicinische Central-Zeitung, 1863, p. ']'](>.

21. Thiry : Zeitschrift fiir rationelle Medizin (3), xxi, p. 25.

22. MiESCHER-RiJscH : Archiv fiir Physiologie, 1885, p. 355.

23. Rosenthal: Archiv fiir Physiologie, 1864, p. 456.

24. Head : Journal of physiology, 1889, x, p. 40.

'^

Reprinted from the American Journal of Physiology.

Vol. IX. — March 2, 1903. — No. I.

AN EXPERIMENTAL STUDY OF THE SUGAR CONTENT
AND EXTRAVASCULAR COAGULATION OF THE
BLOOD AFTER ADMINISTRATION OF ADRENALIN.

By CHARLES H. VOSBURGH and A. N. RICHARDS. 1

[ Carried out under the auspices of the Rockefeller Institute for Medical Research at the

Laboratory of Physiological Chemistry, of Columbia University, at the

College of Physicians a7id Surgeons, lYew Yorki]

Introduction.

EARLY in 1902 the discovery was made by Herter and Richards^
that the injection of solutions of adrenalin chloride into the
peritoneal cavity of dogs was followed by an intense though transient
glycosuria. It was also found that the application of adrenalin solu-
tion directly to the surface of the pancreas produced a similar effect.
As a result of a number of experiments in this direction, the sugges-
tion was offered that this form of glycosuria was in reality of pan-
creatic origin.

In extending these observations, Herter and Wakeman ^ have
found that the power of adrenalin to produce glycosuria, when applied
to the pancreas, is not specific but is shared with various substances.
The number of such substances is comparatively large, and apparently
the only quality common to the series is a reducing activity. A
seeming exception to this rule was found in potassium cyanide.
When solutions of this substance were applied to the pancreas in
amounts far too small to produce general toxic symptoms, glycosuria
resulted. This substance, like hydrocyanic acid, while it has no
reducing power, exerts a specific action on the animal cells in pre-
venting them from absorbing oxygen.^ It is natural to suppose that,
in the absence of the normal amount of oxygen in the cell, an excess

^ Research scholar of the Rockefeller Institute.

- Herter and Richards : The medical news, 1902, Ixxx, p. 2or.

^ Herter and Wakeman : Virchow's Archiv fiir pathologische Anatomic und
Physiologie und fiir klinische Medicin, 1902, clxix, p. 479; Herter : The medical
news, 1902, Ixxx, p. 867.

* Geppert: Zeitschrift fiir klinische Medicin, 1889, xv, p. 208; Ibid., p. 307.

36 Charles H. Vosbiirgh and A. N. Richards.

of reducing substances may be formed. It is possible that these
substances may act in a manner comparable to those of the above-
mentioned series in bringing about the excretion of sugar. From
the facts brought out by their observations in this regard, Ilerter and
Wakeman are inclined to attribute the production of glycosuria upon
the application of adrenalin and other substances to the pancreas
to a toxic action on the cells of that gland which is closely connected
with the power of reduction.

If this view of the matter is correct, an important relationship sug-
gests itself between this form of experimental glycosuria and condi-
tions in the human organism which may give rise to an excretion of
sugar. The fact that many organs of the body may form reducing
substances capable of easy oxidation which may reach the pancreas
in the blood stream, carries with it the possibility that, if the normal
balance between the amount of these substances and the oxidizing
power of the pancreas be disturbed, the production of glycosuria may
occur.

Concerning the mechanism through which adrenalin brings about
the excretion of sugar no positive statements can as yet be made.
The work of Minkowski ^ and his followers, which has furnished the
basis of the belief in the existence of an internal secretion of the pan-
creas exercising a controlling influence on carbohydrate metabolism,
justifies an assumption that the sugar elimination is the result of an
alteration in the nature, activity, or amount of this secretion. The
glycosuria-producing effect of injury of certain parts of the central
nervous system,- and the increase in sugar formation in the liver
which follows stimulation of the coeliac plexus'^ or of the vagus nerve,*
may lead to the supposition that adrenalin glycosuria results from
the action of a nervous mechanism. Finally, it is known that under
the abnormal conditions which follow the injection of phlorhizin '' or
chromic acid,'' glycosuria may occur, owing to an increase in the
permeability of the kidney cells. The possibility that adrenalin

1 Minkowski : Untersuchungen iiber den Diabetes Mellitus nacli Extirpation
des Pancreas, Leipzig, 1893.

■^ Cl. Bernard : Lecons sur la physiologic et la pathologie du system nerveux,
Paris, 1858, i, p. 401.

" A- and E. Cavazzani : Centralblatt fiir Physiologic, 1894, viii, p. 33.

* Levene : Centralblatt fur Physiologic, 1894, viii, p. 337.

5 V. Mering: Zeitschrift fiir klinische Medicin, 1889, xvi, p. 431.

® KossA : Archiv fiir die gesammte Physiologic, 1901-1902, ixxxviii, p. 627.

Stigar Content and Coagulation of the Blood. 37

glycosuria is the immediate result of changes in the kidney has not
yet been excluded.

Whatever may be the manner by which the effects of adrenalin
are brought about, it is probable that the mechanism involved is one
which is active, though in a different degree, under normal condi-
tions. A determination of the identity of the mechanism is therefore
of importance, not only in explaining the phenomenon in question,
but also from the fact that it may throw light on some of the
processes connected with the normal metabolism of carbohydrate
within the organism.

Before such a determination can be made, however, a more accu-
rate knowledge of the internal conditions antecedent to the excretion
of sugar is necessary. With this end in view we have made a some-
what detailed study of the sugar in the blood, after intraperitoneal
injection of adrenalin, as well as after application of that substance
to the pancreas.

Sugar Content and Coagulation of Arterial Blood
AFTER Treatment with Adrenalin.

It has long been known that the glycosuria produced by extirpa-
tion of the pancreas,! puncture of the floor of the fourth ventricle,^
and poisoning with certain substances, such as carbon monoxide,^
is the immediate result of an increased accumulation of sugar in the
blood. On the other hand, injections of phlorhizin * are followed by
the excretion of sugar due to the effect on the kidney. In the latter
case the percentage of sugar in the blood never rises above normal,
and may even fall below that amount. To determine in which
class adrenalin glycosuria belongs, we have made a number of deter-
minations of the sugar content of the blood of dogs which had been
subjected to treatment with adrenalin. In this series also we have
attempted to ascertain the rapidity with which this substance acts,
and the course and duration of its influence.

Method of collection and analysis of blood. — Healthy, well-nourished
dogs were anaesthetized with pure ether, a cannula introduced into a
femoral artery, and a portion of blood taken. The solution of adrena-

^ Minkowski : Loc. cit.

2 Cl. Bernard : Loc. cit.

3 Senff : Ueber den Diabetes nach der Kohlenoxydathmung, Inaugural dis-
sertion, Dorpat, 1869.

^ v. Merino : Loc. cit.

38 Charles H. Vosburgh and A. N. Ric/iards.

liii chloride ' was then injected by a hypodermic syringe into the
peritoneal cavity or, after an incision through the abdominal wall, was
painted on the surface of the pancreas with a soft brush. Portions
of blood were then drawn from the femoral artery at various
intervals.

Having in mind the possible production of glycosuria by means of
anaesthetics,- as well as by asphyxia,"^ care was taken to keep the
anaesthesia as light and as constant as possible. Moreover, we
believe that this factor may be left out of account in these experi-
ments, since the control portion of blood, taken before adrenalin
treatment, was collected under the same conditions of anaesthesia
as the subsequent portions which are compared with it.

The portions of blood were analyzed according to the following
procedure. The blood was drawn directly into a beaker containing
a solution of phosphotungstic acid in dilute hydrochloric acid."^ The
beaker was counterpoised on a balance and the blood weighed imme-
diately after its withdrawal from the artery. On boiling this mixture
the blood proteids are precipitated in a granular form leaving a
water-clear fluid free from proteid. The precipitate was washed
thoroughly with hot water, a process which is rendered easy by its
porosity and its friable character. The combined filtrate and wash-
ings were nearly neutralized with sodium hydroxide and evaporated
to small bulk on the water bath. The evaporated residue was made
up to known volume (50-100 c.c.) with water, and was filtered. The
reducing power of this solution was determined by the Allihn method.
The results were calculated in terms of dextrose from the weight
of the metallic copper. The figures given represent the averages of
closely agreeing duplicates.

Method of determining coagulation. — In one of our early experi-
ments we noticed that a portion of blood drawn for the purpose of
rinsing the cannula clotted very rapidly. As a result of this observa-
tion, in a number of later experiments we have taken separate por-

^ In all the experiments outlined in this paper, the adrenalin chloride solution
(toVo) prepared by Parke, Davis, &: Co., by the method of Takamine, was used.

■^ CusHNV: Pharmacology and Therapeutics or the action of drugs, 1899,
p. 160.

^ Dastre : Comptes rendus des seances de I'academie des sciences, 1879,
Ixxxix, p. 669.

* This solution contained 70 gms. of phospliotungstic acid and 20 c.c. of hydro-
chloric acid, sp. gr. 1.20, in a litre. About 5 c.c. are sufficient to completely pre-
cipitate the proteids in i gm. of blood.

Stigar Coiite7tt and Coagulation of the Blood. 39

tions of blood to be tested regarding this point. The amount drawn
in each case was 2 c.c, collected in a graduated cylinder of 5 c.c.
capacity. The time which elapsed between the collection of the
blood and the time at which the cylinder could be inverted without
loss of its contents, was noted as the time of the coagulation of the
blood.

The results of our determinations are given in Table I, pages 40, 41.

These experiments show unmistakably that the administration of
adrenalin chloride either by intraperitoneal injection or by painting
it upon the pancreas is followed by a marked increase of sugar in the
blood. The increase is very noticeable within the first five minutes
after the application and reaches its maximum within three hours.
A very gradual fall then ensues, which may continue until the per-
centage of sugar becomes subnormal (Exp. 2). In a dog recently
fed (Exp. i), the blood sugar may be double the normal quantity
fourteen hours after the injection. A marked rise occurred in the
case of a dog (Exp. 7) which had been starved for six days. In Ex-
periment 8 a fatal dose of adrenalin was given. A slight increase in
the sugar content of the blood occurred shortly after. One minute
before death ensued, twenty-four hours after injection, the percentage
of sugar was approximately normal.

Simultaneously with the production of hyperglycaemia, an effect on
the coagulability of the blood is observed. In every case, without
exception, the time of coagulation is lessened after adrenalin is given.
This diminution is equal in some cases to four-fifths of the coagula-
tion time of the control.

Arthus^ has shown that the time of coagulation decreases if the
blood is allowed to come in contact with blood already clotted or with
an exposed tissue surface. Special care has been taken therefore in
these experiments to remove the clot from the cannula before each
collection. Furthermore the portion for the coagulation test was
collected just after that for sugar analysis, a circumstance which
insures the rinsing of the cannula.

The recent observation ^ by the same author that the mere with-
drawal of large amounts of blood from the body hastens the coagula-
tion of subsequent portions, raises the question whether the results
which we have observed may have been due to loss of blood alone.
To test this point, a control experiment was made in which the

1 Arthus: Journal de physiologic et de la pathologic generalc, 1902, iv, p. 283.

2 Arthus: Ibid., ■p. 273.

40

Cha7'les H. J'osburgh atid A. N. Richards.

ice. adren-
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M. ; anacs-
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Sugar Content and Coagulation of the Blood. 41

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42 Charles H. Vosburgh and A. N. Richards.

amount of blood taken was very small. The details of the experi-
ment are as follows :

A small dog, 5.3 kilos in weight, which had received no food for twenty-eight
hours previous to the exi)erinient, was etherized and a cannula introduced
into the femoral artery. 2 c.c. of blood collected at 2.34 p. m., coagulated
in 5 min. 50 sec. An incision was made through the abdominal wall and
2 c.c. of adrenalin solution applied to the pancreas with a soft brush
at 2.43 p. M. Subsequent samples of blood (2 c.c. each) coagulated as
follows :

5 min. after adrenalin application (2.48 i'. M.) coagulated in 1 min. 45 sees.
16 " " " " (2 59 P.M.) " 2 " 0 "

33 " " " " (3.16 i\ M.) " 2 " 32 "

1 hr. " " " (3.45 P. M.) " 3 " 10 "

Other control experiments in which large amounts of blood were
removed, and no treatment with adrenalin given, show no such marked
changes as are seen in the observations in Table I.

The Source of the Excess of Sugar in the Blood in Adren-
alin Glycosuria, as Indicated by Comparative Analysis
OF Blood Collected Simultaneously from the Portal
and Hepatic Veins and the Femoral Artery.

The results just detailed show clearly that the phenomenon of
adrenalin glycosuria is due to an increase of sugar in the blood.
The source of this excess of sugar is of great importance in deter-
mining the mechanism by which this effect is brought about. We
have endeavored to trace the source of the sugar by means of analysis
of blood taken simultaneously from the portal and hepatic veins and
from the femoral artery. In these experiments it was necessary to
collect successive portions of blood at various intervals from the same
blood-vessel without interfering with the normal circulation in that
vessel. The methods which have hitherto been devised for the col-
lection of portal and hepatic blood appeared to be inadequate for our
purpose, as well as somewhat difficult and uncertain of application.^
We have therefore made use of an original method, the description of
which follows. A cannula of special design ^ (see accompanying dia-
gram) is the essential feature of the method.

1 For description of older methods, see Seegen : Die Zuckerbildung im Thier-
korper, 2 Auflage, Berlin, 1900, p. 66. .

2 The special cannulas used in these experiments were made for us very skil-
fully by Mr. John T. Hoyt of the Department of Physiology in this institution.

Sugar Content and Coagulation of the Blood. 43

General method. — The method of fixing the cannula into a vein is as follows :
The vessel is carefully exposed, and the outer connective tissue sheath dis-
sected away. Loose ligatures are passed about the vessel at each end of
the cleared portion, which should be about 2 cm. in length. Before intro-
ducing the cannula, its parts should be so arranged that the flange of the
outer tube is separated by a space of about 0.5 cm. from that of the inner
tube. The brass rod should be in position, totally filling up the bore of
the inner tube. The ligatures about the vessel are then tightened momen-
tarily by an assistant, and a longitudinal slit made in the wall of the vessel
between them. This slit should be a trifle shorter than the diameter of
the flange. The flange of the inner tube is then introduced into the
interior of the vessel through the slit, the outer tube pressed down till the

Explanation of Diagram. — The cannula consists of three parts, viz. : an inner
tube {A), an outer tube {B), and a nut (C). The inner tube is about 6.5 cm.
in length. Its outer diameter is 4 mm., its inner diameter, 2 mm. On it a
screw thread {A') is cut, extending for 3.5 cm. from one end. The other end
is provided with a flange (Z?) 9 mm. in diameter. The end view of this flange
is shown at D' . The outer tube (i9),3.5 cm. long, fitting closely over the inner
tube, terminates in a flange {E) similar to the one on the latter. By means of
the nut the two tubes may be held in such a position that their flanges are in
close contact. All parts of the instrument are made of brass. The cannula
is also provided with a brass rod (not shown in the diagram) about 28 cm.
long, of such a thickness that it fits closely into the bore of the inner tube.
A small shoulder brazed on the rod at the proper point insures the complete
v2> * occlusion of the cannula when desired.

wall of the vessel is held tightly between the two flanges, and the nut
screwed down so that the hold is retained. The Hgatures are then loos-
ened and the normal circulation is resumed. With a little practice the
operation can be accomphshed with no loss of blood and an interruption
of the circulation of only thirty to forty seconds. On connecting a rubber
tube with the inner tube of the cannula and removing the brass rod, blood
can be withdrawn at pleasure.

In our experiments to determine the sugar content of the blood flowing to and
from the liver, we have introduced cannulas of this type into the portal
vein at its juncture with the pancreatico-duodenalis, and into one of the
larger hepatic veins at a point between the liver and the diaphragm.

In order to expose the vessels, a transverse cut was made through the abdomi-
nal wall, following the curvature of the free border of the ribs and extend-
ing for about three inches on each side of a point on the median line just
below the xyphoid cartilage. Bleeding was prevented by ligaturing the
vessels which it was necessary to cut. The abdominal organs were pro-
tected from exposure by cloths moistened with warm saline solution. The
time necessary for the operation and the introduction of the cannulas into
both veins usually amounted to about one hour.

44 Charles H. V'osburgh afid A. iV. Richards.

In drawing blood from the hepatic vein it was necessary to use a suction pump
to overcome the negative pressure, which is very manifest in the venous
circulation at this point. For this purpose, the beaker containing the
phosphotungstic mixture, previously weighed, was placed under a small
bell-jar which was connected by its lower opening with a suction pump.
A small glass tube inserted through the rubber stopper which closed the
upper opening of the bell-jar, and terminating at a point directly over
the beaker, served to conduct the blood into the precipitating fluid. On
connecting the cannula in the hepatic vein with the glass tube, and apply-
ing suction, the estimated amount of blood is easily obtained.

For collecting arterial blood, a glass cannula of the ordinary type was intro-
duced into the femoral artery.

The order of procedure in obtaining the portions of blood simultaneously was
as follows : The beakers which were to hold the femoral and portal blood
were counterpoised on balances placed at the side of the operating table.
Small glass tubes were clamped in a position to lead the blood into them.
The beaker for collection of hepatic blood was placed under the bell-jar
arranged as described above. The brass rods were removed from the
cannulas in the portal and hepatic veins, and connection with the proper
glass tubes made, passage of blood being prevented by clamping the
rubber tubes. The connection between the cannula in the femoral and
the third glass tube was made. At a given signal the clamps were re-
moved and the blood collected. The brass rods were immediately replaced
in the cannulas, and the portions of blood weighed. The time necessary
for collection of all three portions of blood has seldom amounted to more
than ten or fifteen seconds.

The analysis of the blood was carried out in a manner exactly similar to that
previously described.

By means of these methods we have succeeded in obtaining blood
simultaneously from the three vessels mentioned, both before and
after the application of adrenalin chloride to the surface of the pan-
creas. The results of sugar determination are given in the table on
page 45-

The results of Experiments i, 2, 3, and 6 present, we believe, a true
picture of the events in this connection succeeding the application of
adrenalin to the pancreas. The samples of blood taken before treat-
ment with adrenalin agree fairly closely in their sugar content. On
the ground of these figures it cannot be said that the amount of
sugar in the blood issuing from the liver is greater than that of the
femoral artery or portal vein. After treatment with adrenalin, how-
ever, the relations are changed. In Experiment 3. four minutes after

Sicgar Content and Coagulation of the Blood. ■ 45

« <

as

33

Chest opened ' and artificial respiration
employed.

At time of first collection of blood, pan-
creas and intestines normal in appearance.
At second collection, pancreas somewhat
congested, intestines somewhat cyanotic.
At third drawing of blood, the pancreas
was very much congested and small intes-
tine very blue.

At time of first and second collections of
blood, the pancreas and small intestines
were normal in appearance. When the
third and fonrlli series were taken, these
organs were much congested.

At first drawing of blood, the pancreas
and intestines were normal. At the sec-
ond, the small intestine was congested and
cyanotic.

Pancreas and intestines normal at time
of first collections. Pancreas congested and
small intestine congested and cyanotic at
time of second.

Pancreas and intestines normal in ap-
pearance at first collections. Pancreas
congested and intestines congested and
cyanotic at time of second.

0

<4-l

0

c«
bC

Hepatic
vein.
Per ct.

0.08
0.25

0.164

0 284
0.346

0.136

0.201
0.252
0.209

0.316
0.348

0.210
0.240

0.208
0.227

Portal
vein.
Per ct.

0.10

0.08

0.165

0.197
0.245

0.169

0167
0.187
0.207

0.282
0.314

0.227
0.158

0.181
0.223

Femoral

artery.

Per cent.

0.10
0.15

0.160

0.247
0.322

0.159

0.187
0.215
0.208

0.311

0.385

0.221
0.242

0201
0.208

Time of collec-
tion compared
with time of
application.

6 min. before
20 min. after

16 min. before

10 min. after
41 "

19 min. before

4 mill, after
26 "
66 "

13 min. before
27 min. after

10 min. before
21 min. after

10 min. before
25 min. after

Time of
applica-
tion of
adren-
alin.

P.M.

CO

4.39
4.40

5.42
3.25

0

CO

Time

of
collec-
tion.

p. M.

9.31

9.57

4.23

4.49
5.20

^ ^ 0 VO

5.29
6.09

3.15
3.46

5.26
6.01

5

0
0

M-.4

0

5

0

<

Hepatic
vein.
Gms.

31.2
30.0

16.8

16.2

12.3

21.0

13.2
13.3
19.1

13.85
15.45

13.8
12.0

11.5
19.1

Portal
vein.
Gms.

31.4
11.4

14.9

14.4
8.4

13.7

17.6
14.5
13.6

15.5
20.1

16.5
11.7

15.15
12.4

Femoral
artery.
Gms.

CO CO CO

ON OtI-^
CO CO CO CO

0 0 ' CO -h
u-. CO 1 '-^- CO

14.7
16.0

Time
since
last
fed.

His.

CO

■^

s

CO

^

CO

•jqSpAN. 5

0

CO

OS

00

LO

LO

CO

CO
CO

6 M-, d,
>• 0 X ^

CQ

CO

-h

U-;

^

46 diaries //. Vosburgh and A. N. Richards.

the application of the substance to the gland the sugar content of the
arterial blood rises 0.028 per cent, that of the portal blood remains
practically the same, while the increase in reducing power of the
blood emerging from the liver amounts to 0.065 P^^ cent. The same
relation, though on a higher plane, is apparent twenty-six minutes
later. Sixty-six minutes after, the sugar percentage from all the ves-
sels is approximately the same. Precisely similar results are obtained
in Experiments i and 2, and in a lesser degree in Experiment 6.
Judging from the results of these analyses then, a formation of sugar
in the liver must be the cause, in part at least, of the increase of
sugar in the blood.

Experiments 4 and 5 apparently form exceptions to this conclusion.
It will be noticed, however, that the percentage of sugar in the sam-
ples taken before adrenalin treatment are abnormally high, especially
in Experiment 4. It is possible that the mechanism which takes part
in the production of adrenalin glycasmia has already been affected by
the operative disturbance. It is not an unfair assumption that the
additional impulse given by the application of adrenalin is on that
account less effective and its result more transient. Consequently at
the time of the collection of the second portions of blood the secretion
of sugar is lessening. The same reasoning may hold good with
regard to Experiment 5.

Comparison of the sugar content of the portal blood with that from
the femoral artery and hepatic vein in Experiments i, 2, 3, and 5 shows
that the increase of sugar following treatment of the pancreas with
adrenalin is least in the portal vein. While in the control samples
the sugar percentage of the portal is as high or higher than that of
the femoral or hepatic blood, after treatment with adrenalin, it is
lower in every case. In this connection we would call attention to
certain changes in the appearance of the organs of the abdomen. At
the time of collection of the first samples of blood the appearance of
the intestines and pancreas was normal. As the experiment pro-
ceeded, however, the pancreas became congested and the intestines
cyanotic. The latter symptom is due, probably, to a partial obstruc-
tion of the circulation by the formation of a clot at the flange of the
cannula. The effect of this partial obstruction is a partial asphyxia
of the tissues. That the relative decrease in the sugar of the portal
blood is dependent upon increased utilization within the tissues
through which it passes there can be no doubt. Whether this con-
sists in a mere increased oxidation of sugar, owing to the increased

Sugar Content and Coagulation of the Blood. 47

to

S

Appearance of the pan-
creas and intestines normal
throughout experiment.

Intestines became some-
what blue at the time of
second and third collections
of blood. Pancreas normal.

Intestines somewhat blue
at time of second blood col-
lection. Pancreas normal.

Intestines somewhat blue
at second collection. Pan-
creas normal.

CO

to

(P

to
0 ^

E c«

^§

0

B
p
<x^
-a
0
0

.S

bX)

3

m

Hepatic

vein.
Per cent.

0.210
0.242
0 205
0.217

0.166
0.188
0.174

0.216
0.260

0.194
0.190

0.202
0.185

Portal

vein.

Per cent.

0.213
0.193
0.198
0.185

0.160
0.179
0.173

0.174
0.218

0.176
0.198

0.204
0.149

Femoral

artery.

Per cent.

0.236
0.220
0.190
0.217

0.174
0.183
0.194

0.215
0.264

0.188
0.182

0.206
0.175

Time

of_

collection.

% % % %

CU fc Pl) Ol

LO LO 0 LO

Tt- <0 <^ <3
Tt- lH UO V6

4.24 p. M.
4.46 p. M.
5.16 P. M.

12.10 p.m.
12.42 p. M.

11.50 a.m.
12.30 p. M.

ON -i-

LO ro
CM ro

S
0

;-•

-a
0
0

X5
Mh
0

5
0

<

Hepatic

vein.
Grams.

^ NO t-7 !>;
NO ^ CO (^

OD t^ CM

(^ NO (^

(■0 ■+

18.15
20.7

14.9
17.0

" " " "

Portal

vein.

Grams.

i-H On t^ .— 1
ro CM — ^ LO

rH i—l i—l .—1

(Nl LO ON

-f (^ -^

15.1

25.8

(■0 rO
1— 1 1— 1

1— 1 1^

•+ (Na

1— 1 T—i

Femoral
artery.
Grams.

LO ON NO LO

Tt- (NJ (^ rO

,— 1 ,— 1 ,— 1 f— 1

0 (^ OD
CM rf Lr^j

LO LO

(^ •*■

LO ON

ro (^

1— 1 1— 1

Time
since
fed.

Hours.

(Nl
(Nl

ON
(Nl

OD T^
^ CM

(M

Weight.
Kilo.

NO

LO

1— ;

t-.

^

No.
of
experi-
ment.

-

CM

ro

1

48 Charles //. Vosburgh and A. N. Richards.

supply of that substance, or whether there is a decomposition of
another character in increased amount, owing to lack of oxygen in
the tissues, our experiments do not decide.

In referring to Experiments 4 and 5, the idea has been expressed
that the high sugar content found in the control samples was possibly
due to the effects of operative disturbance. The question might
naturally be raised as to whether the eft'ects noted in our other experi-
ments might not be due to that cause rather than to the influence of
adrenalin. To settle this point we have made a series of five control
experiments in which the blood was collected in a manner similar
to that described, the treatment with adrenalin being omitted. The
results are given in Table III, page 47.

These figures show an essential difference from those given in Table
II. In only one case does the blood of the hepatic vein contain con-
siderably more sugar than that of the femoral artery. In only one
experiment (3) is there an essential rise in the sugar of the femoral
blood. The results indicate, therefore, that while in a small percent-
age of experiments carried out according to this method, the opera-
tion may give rise to a hyperglycaemia similar to that produced by
adrenalin, in the majority of cases we are justified in attributing the
results to the action of adrenalin.

Sugar in the Blood of the Pancreatico-duodenal Vein after
Treatment of the Pancreas with Adrenalin.

It has been shown in the experiments of Series II, page 45, that in
the hyperglycaemia which follows the application of adrenalin to the
pancreas, the increase of sugar is least in the blood of the portal vein.
We have attributed this circumstance to an increased decomposition
of sugar in the intestinal tissues, and have suggested that it may be
connected with a partial obstruction in the circulation of the blood
through those tissues. To ascertain whether the congestion of the
pancreas which is regularly observed after treatment of that gland
with adrenalin takes part in this phenomenon, we have tested the
blood from the pancreatico-duodenal vein. Though an increased de-
composition of sugar in the pancreas would be at variance with our
ideas regarding the events taking place there, its possibility has not
been positively excluded.

The method of collecting blood was as follows: A cannula of the
design previously described was introduced into the portal vein at a

Sugar Content and Coagulation of the Blood. 49

•

a;

73

TJ

S

1)
bo

0)

0

(U

CO

tn

(U

(U

0

CJ

lU

c

1)

fcJD
0

to

0

c« 0 '^

^

0

CJ

0
0

0

u

<U '-' C

+j

.

.^j

Ji

^

a rt

J=

^

nj

(U

rS

0

CJ

<

d

"rt

"rt

^
&

'a

a,

^
&

3

s

1

s

_aJ

^

a

>^

(U

s

£

E

•^

>>

0

Oh

s

0

53

0

0

<u

0

<u

53

^

>-

^

>

zn

^

>

X

>

>

s

."

0

? ^ i

<N1

LO

t^

0

r^

r^

0

(Nl

LO,

0

CO

■4-4

<a c f

0

J>.

0

0

i>.

c«

CO

(T)

0

cd

N

ro

c^

(M

CSl

(NJ

(Nl

CNl

CM

ii g I-

S "^ D

0

c5

0

d

d

d

d

d

d

d

d

^

(2 ^

3

2 >> =

NO

0

CM

CM

d

(M

d

c^

<u

tj

a;

<u

S

53

S

collec
n pared
me of
ition.

0

0

4)

6

cJ

(U

c(S

cJ
(U

4h
cj

"13

cJ

m
en

(J

(U
(fl

cJ

73

1 oj 'Jjn'S.

1)

n

10

LO

'rt

LO

(N

0

CM

"rt

^

1—5

0

0

1—1

0
^

Tim-

tion

wit

api

d

S

s

1

s

1

d
<u

in

s

"§

's

■P

s

ro

ro

CO

On

LO

LO

1^

LO

CZ)

NO

Ir-

NO

CO

(Nl

^

1—1

"— '

CM

(Nl

10

C

ly^

0

0

M-H 0 C

P

^

(Nl

0*^ -^

OD

0

'S

S.^o = ^

C^

^f

0

Th

1

0

Tj^

03 ^

<NI

0

-^

0

rf

^

^

5

1-n

0

0

LO

0

LTj

LO

Time

of

ollectii

P. M.

U-5

0

t^

I>.

ro

ro

c>a

0

-t-

ro

t^

(^

CO

,—1

ro

LO

\o

F— 1

0

d

r~^

L^

CM

LO

ro

■^

ro

"^i^

ro

0

■—1

^

■—I

CM

^

fO

•:f

^

^

^

•^

•*■

-rf

•^

a

0

t—

p

S c s

vo

J>;

n-

't-

0

1— 1

00

CM

-+

^

CTn

■a
0
0

h'S rt

0^

-h

d

c^j

d

d

od

c^

Cn

LO

NO

1—1

1—1

■—1

.—1

"^

C-i

_^

0

5 >>!2

^

c r; I-

0

fO

s

vd

NO

m 0 i"

S y-a 3

(Nl

'^

0

.^ S (u 0

(N

tsi

ro

H-S- jc

^ °

•+

-*■

vO

^ 5

1—1

d

CO

>^ ■"

2^ " X P

-^

(M

■0

dj .S

^^^^^^

50 Charles H. J'osburgk and A. N. Richards.

point just opposite the entrance of the pancreatico-duodenahs. Loose
ligatures were placed about the portal vein, one on either side of the
cannula. The cannula was opened at the same time that the ligatures
were tightened. Blood is thus obtained from the desired vein, free
from admixture with portal blood. The portal circulation is inter-
rupted for a few seconds, but the pancreatic not at all.

We have collected blood in this manner both before and imme-
diately after painting the pancreas with adrenalin, and analyzed it for
sugar. The results are given in Table IV.

The results of these experiments are very uniform. In Experi-
ment I, the percentage of sugar in the pancreatic blood rose 0.073
per cent in the first three minutes after adrenalin was applied. In
Experiment 2, an increase of 0.068 per cent occurs within twenty-five
seconds. In Experiment 3, the rise in the first forty-five seconds
amounts to 0.032 per cent. In the last experiment, we have con-
tinued the collection of blood when the gland was very much con-
gested, and have compared these samples with portions taken at the
same time from the femoral artery. The analyses show a continued
rise in the sugar percentage, and only a slight difference in the blood
from the two sources. We are forced to conclude, therefore, that
there is not an increase in the decomposition of sugar in the pan-
creas antecedent to the rise of sugar in the general circulation, and
that the difference observed in the second series, between the reduc-
ing power of the blood of the portal vein and that of the femoral
artery, is not dependent on processes of this nature in that gland.

Summary of Conclusions.

1. The intraperitoneal injection of adrenalin chloride, as well as
the application of that substance to the pancreas, gives rise to a
marked increase of sugar in the blood. This hyperglycaemia makes
its appearance immediately after the administration, reaches its maxi-
mum in from one to three hours, and may continue for over fourteen
hours.

2. Simultaneously with hyperglycaemia occurs a decided diminution
in the time of extravascular coagulation of the blood. This phe-
nomenon appears to be due also to the application of adrenalin to
the pancreas.

3. The cause of this form of hyperglycaemia, as indicated by com-
parative analysis of the blood flowing to and from the liver, is to be

Sugar Content and Coagtilation of the Blood. 51

attributedj in great part at least, to an increased sugar formation in
that organ.

We are indebted to Dr. C. A. Herter for the suggestion of the
subject of this work, and for valuable counsel during its progress.
We also wish to express our obligation to Mr. William D. Cutter for
assistance in a number of the operations.

26

Reprinted from the American Journal of Physiology. '"'■"

Vol. IX. — May i, 1903. — No. III.

ON THE IRRITABILITY OF THE BRAIN DURING

ANEMIA.

By WILLIAM J. GIES.

[From the Physiological Institute of Bern University^

I. Introduction.

DURING the summer of 1899 I had the pleasure of assisting
Professor Kronecker in a study of the irritability of the brain
during anaemia.-^ Our research could not be concluded during my
stay in Bern that summer, but we both looked forward to completing
it together in the following year. Unfortunately for me, return to
the Physiological Institute has been impossible thus far, and the
work which has been delayed on that account has lately been resumed
by Professor Kronecker and Dr. Stumme. At the suggestion of
Professor Kronecker, the results of our investigation are presented
here in some detail though briefly.

In the preparation of these notes I have received numerous sug-
gestions from Professor Kronecker, who has also revised the state-
ments relating directly to our experiments. Throughout practically
all of our research, Professor Kronecker not only directed the work,
but did a very large share of it. His well-known generosity to his
pupils is again shown by his desire that this investigation, which was
chiefly his, shall seem to be wholly mine.

II. Description of Experiments.

In this research we sought especially to determine the order of
cessation, as well as the period of continuance, of certain reflexes dur-
ing anaemia of the brain.

Acute ansemia was brought about by perfusion with the solutions
indicated on the next page.

The animals employed were toads, frogs, rabbits, and dogs.

1 GiES: Report of the British Association for the Advancement of Science,
1899 (Dover), p. 897.

131

132 William J. Gics.

The solutions used were various strengths of pure sodium chloride,
Ringer's solution, and modifications of it, Schiicking's solution (both
of sodium and calcium saccharates), rabbit and horse serum, and
0.7 per cent sodium-chloride solution containing paraxanthin or
chloralbacid.

Experiments on toads and frogs. — Perfusion in the cold-blooded
animals was conducted with the least possible pressure through the
abdominal vein. In this series of experiments we used all of the
various solutions already enumerated, except serum.

Seventeen experiments were made, seven with toads and ten with
frogs, each of which was continued for a period of from one to nine
hours. The total amount of perfused fluid varied from 25 c.c to
1590 c.c. In most cases perfusion was continued until the heart
ceased to beat.

The table on page 133 gives a summary of the more important
results obtained in this connection. The terms "skin," "lid," and
" nose," in the table, refer to the reflex movements caused by pres-
sure on those parts.

During the period of perfusion, the following functions gradually
weakened, and then usually disappeared in this order: (i) respiration,
(2) skin reflex, (3) lid reflex, (4) nose reflex, (5) heart beat.

The relative time of cessation of these reflexes varied considerably,
not only with the character of the solutions, but also with the rapidity
of their perfusion and the amounts used.

Convulsive extension of the limbs occurred in all the experiments
in the earlier stages, but toward the close of each experiment and
before the reflexive movements of the eyelids ceased, no such mani-
festations were observed, nor could they be induced by mechanical
stimulation.

Perfusion of physiological saline solution containing 0.03 per cent
of paraxanthin induced hyperaesthesia at first, but the reflex responses
quickly came to an end, as the perfusion continued. Cumulative
muscular rigor was the most pronounced feature of the experiment.
At the end of the experiment the body was perfectly stiff. With a
solution containing 0.015 per cent paraxanthin, moderate hyper-
aesthesia was observed at first, as in the case of the 0.03 per cent
solution, but the rigor of the former experiment was absent in this.

During perfusions with physiological salt solution containing
I per cent chloralbacid, repeated spasmodic extension of the extremi-
ties was the main feature. With the solution containing 0.33 per cent

On the Irritability of the Braiit during An(2mia. 133

TABLE I.

;_*

0)

0

rt

C

S

c

^

<

1

Toad

2

"

3

"

4

"

5

"

6

"

7

«

8

Frog

9

"

10

"

11

"

12

«

13

"

14

"

15

"

16

"

17

"

Solution used.

NaCl — 0.6%

" -0.8%

Ringer's ^

Ringer's ^

NaCl— 0.6%

f NaCl - 0.7 % ^

;Calcium \

( saccharate — 0.03 % )

(NaCl— 0.7% \

■jParaxanthin — 0.03 % ('

j NaCl — 0.7% I

(Paraxanthin— 0.015% i'

(NaCl — 0.7% )

iChloralbacid — 1% . . }

(NaCl— 0.7%
iChloralbacid— 0.33%

h. m.

6 15
8 10

3 45

8 30

9 10

4 45
3 15
3 00
3 30
3 30
1 20

5 50

8 30

1 25

2 05

0 50

1 05

Cessation of reflexes.

Time after beginning the

perfusion.

Resp.

h. m.

4 15
6 15
2 25

5 25

6 00
2 00
2 15

1 00

2 15
2 00

0 30

1 10

2 00
0 35

0 30

0 24

0 29

Skin,

h. m.

5 25

7 40
3 10
7 25

6 15
3 15
2 30
2 30
2 40
2 20

1 00

2 45

3 20
1 00

1 50

0 35

0 36

Lid.

h. m.

5 30
7 45
3 25
7 45

6 15
3 30
2 35
2 20
2 50
2 50

1 15

2 50

3 30
1 10

1 55

0 35

0 50

Nose.

h. m.

6 00

7 55
3 25

8 05
6 30
3 45
2 35
2 25
2 55
2 50

1 15

2 55

3 25
1 05

1 55

0 33

0 52

Heart
beat.

h. m.

6 15
8 10

3 45

8 30

9 10

4 45
3 15
3 00
3 30
3 30
1 20

1 25

2 05

0 50

1 05

c.c.

475
780
290
740
1590
625
575
600
275
275
180

650

730
95

145

25

120

Red cor-
puscles at
the end of
perfusion
in fluid
from

U

1 White's modification : 0.6% NaCl, 0.01% NaHCOs, 0.01% CaCl.., 0.0075 % KCl.

2 HoweU's modification : 0 7% NaCl, 0.026% CaCL, 0.03% KCl.
^ Not ascertained.

* Heart continued to beat long after the conclusion of the experiment.

1^4 \Villia77i J. Gus.

of chloralbacid, spasmodic twitching in the limbs was the most notice-
able incident.

At the end of the experiments with the solutions containing para-
xanthin and chloralbacid, after the heart had ceased to beat, solution
of calcium saccharate was perfused. In each case this solution
caused the heart to begin beating, and rapidly induced the normal
stroke and rhythm.

Before passing to the next series, it should be stated that in each
of the preceding experiments the animal became oedematous. Even
those animals in which perfusion took place at the lowest possible
pressures, and for the shortest intervals, showed unmistakable signs
of oedema.

It was impossible to remove entirely the blood-corpuscles from the
capillaries in the heart and brain, even when the perfusion was con-
tinued uninterruptedly for eight hours, and as much as 1590 c.c. of
fluid had slowly passed through the body. In all cases the fluid
expressed from the heart and brain contained an appreciable number
of red and white corpuscles.

In most of the experiments, when the heart had come to a stand-
still after continuous irrigation with physiological saline solution, also
Ringer's solution, rhythmical contractions could be promptly induced
by perfusing Schiicking's solution. This result was obtained even
when mechanical and electrical stimulation had failed to restore the
normal beating.

Experiments on rabbits. — We report the results of thirteen exper-
iments on rabbits. In this series we used all of the so-called " in-
different " solutions already mentioned.

Considerable difficulty was experienced in our efforts to devise a
method which would prevent almost instant death of the animals, and
yet which would speedily result in pronounced anccmia.

Ligaturing the arteries to the brain, before or simultaneously with
the beginning of the perfusion, brought on convulsions immedi-
ately. This was the case whether the ligatures were placed about
the arteries in the neck or in the chest. Even when the perfusion
had been begun shortly before the arterial blood was completely shut
off, it still remained impossible to prevent convulsions and quickly
ensuing death.

In Experiments 1-5 (see the table on page 135), the blood-vessels
in the neck were quickly tied as perfusion was begun. In Experi-
ments 6-10, they were tied just above the heart as perfusion was

On the Irritability of the Brain dtuHng Ancsmia, 135

instituted. Experiments 11-13 were carried out by the following
method.

Instead of closing the arteries to the brain, the abdominal aorta,
vena cava, and vena porta were ligated, and the heart's action utilized
to pump the perfusion fluid through the brain. The warm solution
was directed into the heart by way of one jugular, and passed from
the brain by way of the other. With this method, ansemia was
gradually though quickly induced, convulsions were entirely pre-
vented, and life was considerably prolonged.

In all cases, microscopic examination of the fluid pressed from the
brain showed the presence of red corpuscles.

TABLE II.

Solution used.

Cessation of
reflexes. Time
after beginning
the perfusions.

S= 8-

2 =

gms.

1300
1100
1500
1800
1600

1800

2800
1600

1400

1500
1800
2000
1900

Rabbit serum

NaCl — 1%

(NaCl-1% )

jCalc. sach.— 0.035% (

Rabbit serum

(NaCl — 1% \

|Calc. sach.— 0.035% i

NaCl — 1%

Rabbit serum

min.

16

(?)

(?)

8

(?)

min.

14

(?)
12

7
2

2

3

(?)

(?)
14
13
9i

c.c.

110

30

200

250

35

230

450
110

15

40
350
260
150

mm. Hg.

90-120
130-150

90
90-110

90

75-85

110-140
110-150

100

100

70-110
80-120

15
23
17

10-12
8-11

I 36 William J. Gics.

The disappearance of functions in these experiments was not at all
regular in the first ten. The events of each experiment transpired
so quickly that it was extremely difficult to note accurately the time
of cessation of each reflex. In the last three experiments respiration
ceased first in one, second in two; the "lid reflex" disappeared first
in two, second in one. In each of the last three experiments, the
"nose reflex" was the third to disappear. Heart beat was always
fourth in the order of cessation.

Experiments on dogs. — Only two experiments were performed on
dogs. The first was by a method similar to that in the tenth experi-
ment with rabbits. The weight of the dog was 12 kilos. The
pressure of perfusion was 140-150 mm. Ilg. The amount of blood
drawn at the beginning of the experiment was 47 grams. The per-
fusion fluid was a 0.7 per cent solution of sodium chloride containing
0.03 per cent calcium saccharate. Perfusion was continued for forty-
two minutes. The volume of fluid perfused was 1125 c.c. The
amount of haemoglobin present in the fluid leaving the jugular vein
at the end of the experiment was 30 per cent of the normal content
in blood-
Reflex responses failed in the following order: (i) lid and nose
reflexes in twenty-six minutes; (2) respiration in forty minutes; (3)
heart beat in forty-two minutes.

There were no convulsions at any stage of the experiment.
In the second experiment, with a small dog weighing only 5 kilos,
200 c.c. of blood was taken, and an equal quantity of horse serum
immediately afterwards was transfused to take its place. This pro-
cess was repeated three times at intervals of half an hour. After the
fourth blood-letting, the dog ceased to breathe, and did not recover
when the new portion of serum was transfused. Aside from varia-
tions in heart action and respiration, no special functional changes
were observed until the end, when respiration suddenly ceased, and
the other functions came to an end about the same time. Death was
neither preceded nor accompanied by convulsions.

III. Summary of Conclusions.

The more important conclusions of these preliminary experiments
are that when the brain is subjected to anaemia by the process of per-
fusing solutions, such as Ringer's, Schiicking's. serum, etc., its func-
tions soon cease. When the anaemia is induced rapidly, convulsions

On the Irritability of the Brain During Ancemia. 137

ensue. When it is brought about gradually, anaemia may be made
acute without causing the appearance of convulsions.

When anaemia of the brain is produced gradually by the methods
used in these experiments, the functions here referred to cease usually
in the following order :

(A) In cold-blooded animals: (i) respiration, (2) skin reflex,
(3) lid reflex, (4) nose reflex, (5) heart beat.

(B) In warm-blooded animals: (i) lid reflex, (2) respiration, (3)
nose reflex, (4) heart beat.

Reprinted from the Medical Record, Vol. 59, No. 17, April27, 1901

AN EXTREME CASE OF SIMPLE ANEMIA.
By ROLFE FLOYD, M.D.,

AND

WILLIAM J. GIES, Ph.D.,

NEW YORK.

History. — X , female, nineteen years old, single,

mulatto, waitress, was admitted to Dr. Delafield's ser-
vice at Roosevelt Hospital, November 13, 1899. Her
mother was dead of consumption. She had had no
previous illness which could bear on the case. Her
health had always been good. In April, 1899, she
had been delivered of her first child, at term, in an
institution, A normal puerperium followed. She
was set to work again as soon as possible. In August
she began to feel weak and to suffer from headache
and vertigo. About one month later she began to
notice dyspnoea on exertion with marked cardiac palpi-
tation, and slight cardiac pain at times. In October a
persistent diarrhoea began, of six to eight large, fluid,
fecal movements a day, containing no blood or mucus
and not associated with tenesmus or colic. Marked
anorexia but no vomiting accompanied the diarrhoea.
She had to give up work. In November she fainted
once and her ankles became moderately oedematous.
She continued to nurse the infant till the first week in
November. She had not menstruated since her preg-
nancy.

On admission to the hospital she had almost no red
color in her skin or mucous membranes. The moder-
ate pigmentation natural to her race gave the surface
of her body a leaden hue, but there was no tendency
to a yellow cachectic color. She was poorly nour-
ished but not markedly emaciated. There were no
abnormal physical signs over the lungs and no dys-

Copyright, William Wood and Company.

pnoea while she was quiet. The heart was normal in
size and position, its action weak, rapid, and slightly
irregular. There was a hasmic systolic murmur at the
apex and one over the second left space. The pulse
corresponded to the heart action. The arteries were
neither thickened nor contracted. There were no
abnormal signs over the abdomen, which was thin and
retracted. There was slight oedema of the ankles.
The voice was weak, and the general weakness and
apathy were very marked indeed. Temperature, ioi°
F. ; pulse, 140; respiration. 36. The urine was acid,
pale, clear; s. g. i.oii; it contained no albumin and
no sugar. Microscopical examination was negative.
Blood: HE., 12 per cent.; R. C, 750,000; \V. C,

The patient was put to bed and given milk and one
solid meal a day, which was increased to three meals
two weeks later. She was started on gr. xxiv. of sul-
phate of iron and gr, y\y of arsenious acid in twenty-four
hours, and two weeks later the iron was increased
to gr. xxxix. A little codeine was given to control
the diarrhoea.

During the first week she was in the hospital, her
temperature ran between 101° and 103.8°, averag-
ing 102°; during the second week between 98° and
101°, averaging over 100°, and showing a tendency to
reach its maximum about noon and its minimum about
midnight; during the third week about the same as in
the second the breaks being more pronounced and
sustained; during the fourth week between 98° and
100.6°, averaging 99.4°, with the same tendency to rise
about noon; during the fifth week it never reached
100°, and after that time it ran a normal course.

The oedema disappeared in a day or two. After ten
days' treatment her general strength began to improve.
After two weeks her appetite commenced to come
back, her voice and pulse grew stronger. Pulse, 90;
respiration, 20. At this time, also, the diarrhoea began
to yield. During the third week her color began to
return and she was able to leave her bed. The
changes for the better, once instituted, proceeded with
surprising rapidity and she left the Hospital^ two
months after entering it, practically well.

The symptoms, then, which the change in the com-
position of the blood caused in this case, besides the
pallor, were marked general weakness, some headache,
syncope, dyspnoea on exertion, cardiac palpitation,
disturbed heart action, anorexia, diarrhoea, and oede-
ma of the ankles. The absence of menstruation cannot
certainly be attributed to the change in the blood, be-
cause of the nursing. It is noteworthy that there was
neither bleeding, nor vomiting, nor cachectic color of
the skin.

After leaving the hospital she continued to take gr.
XV. of sulphate of iron a day for two weeks, and since
then she has taken practically no medicine. One
month after leaving the hospital she began to men-
struate and has been regular ever since. She has been
steadily employed for over one year now, and, except
for being somewhat prostrated by the extreme heat last
summer, has enjoyed perfect health. She was last seen
February 5th of this year.

Blood. — The blood was examined once a week during
the patient's stay in the hospital, and, after her dis-
charge, at first every two weeks, then every month or
two, and latterly at intervals of three or four months.
The HB. examinations were made by Fleischl's method.
The outlines of the corpuscles were traced with a cam-
era from smeared preparations (A), but, in order to
obtain accurate figures of the small and deformed cells,
at each of the first eight examinations a few of these
were traced from a specimen of fresh blood before cre-
nation had set in (B). Following are the records of
each exaniination :

On admission: HB., 12 percent.; R. C, 750,000;
W. C, 3,300. The red cells varied considerably, but
not extremely, both in size and shape. A number of
red cells were larger than normal (macrocytes), meas-
uring 10.5 i-L in diameter, a number were small and
deformed (microcytes). The HB. was moderately un-
evenly distributed. " Ringing " and extreme pallor
of the cells were not present, except in the small de-
formed cells, at this or any subsequent examination.
A very few nucleated red cells (normoblasts) were
found. Changes in the coloring matter of the red
cells were not observed. The white cells for the most

part were normal. A considerable number of degen-
erating white-cell nuclei were to be seen and an ab-
normally large percentage of small mononuclear leuco-
cytes (over thirty per cent.) were present, a feature
which obtained in the first seven examinations, i.e., as
long as the differential counts were continued. There
were no excess of eosinophiles and no myelocytes at
that time or later (Fig. i).

Second week: HB., 18 per cent.; R. C, 645,300;

0

o

0

o o

O

oo

o

o

09 0

-o^

0

o

o

0

o

00 ^ cToO o^a

Fig. I

W. C, 4,200. The appearances were identical with
those above described.

Third week: HB., 25 per cent.; R. C, 1,016,000;
W. C, 5,600. A distinct improvement in the appear-
ance of the blood was evident. The marked varia-
tions in size and shape were less frequent, the moder-
ate variations were still universal, and there were still
a number of macrocytes. The HB. was more evenly
distributed. One nucleated red cell (normoblast) was
found. The w-hite cells were practically normal (Fig.

2).

Fourth week: HB., 45 per cent.; R. C, 1,568,000;
W. C, 5,600. The universal variation in size and
shape was less marked, but the considerable variation

of a certain number of cells persisted, dividing the
red cells more or less into two classes. The HE. was
pretty evenly distributed except in the small and de-
formed cells in which it was irregular and often defi-

O % ogo
oOOOo

0 0 o 0 «o o

o

O (^^3

Fig. 2.

Q

a

Fig. 3.

5'

o

O O

oOo o

00^^ So o

B

cient. The tendency to form niacrocytes was less pro-
nounced (Fig. 3).

Fifth week: HB., 58 per cent.; R. C, 2,556,000;
W. C, 9,200. The universal change in shape and
size was distinctly less than at the preceding exami-
nation. Marked variation in size and shape persisted
in a small number of cells, and a pretty strict division
of the red cells into two classes could at this time be

0-0 OoOo o

^ O ^ o O r) ^

&

Fig. 4.

made. The macrocytes had practically disappeared.
HB. was evenly distributed in all the cells except the
small and deformed ones (Fig. 4).

Sixth week: HB., 66 per cent.; R. C, 2,900,000;
VV. C, 11,800. The great majority of the cells had
nearly reached the normal limits of variation in size
and shape. Every here and there a markedly de-
formed and small cell was still to be seen. HB. as
week before (Fig. 5).

Seventh week: HB., 85 per cent. ; R. C, 3,520,000;
W. C, 12,400. The appearance of the blood was
practically as in the sixth week, except that the general
appearance of the cells was slightly more even (Fig. 6).

Eighth week: HB., 90 per cent.; R. C, 3,556,000;

W. C, 11,400, The appearance of the blood was now
normal except for the small and deformed cells usually
deficient in HB., which could be found in every sec-
ond or third field (Fig. 7).

Ninth week: HB., 97 per cent.; R. C, 3,796,000;

o

p ^

A B

Fig. 5,

Vy. C, 7,000. The appearance was the same as at the
eighth week, but the variation in size and shape of the
small cells was not so marked. Just after this exami-
nation the patient left the hospital,

o 080

9^0 rP O r, ^

0 "^ c*

Fig. '■.

Eleventh week: HB., 78 per cent,; R. C, 3,500,-
000; W. C, 10,200. The general appearance of the
blood still fell within the normal limits of size and

oooo^oooo ^

O Q

A B

Fig. 7.

shape variation, but there was slightly more variation
than two weeks before. The small and deformed cells
were seen in every third or fourth field (Fig. 8).

0.0 °0
8o„o %

00 o

A
Fig. 8.

Thirteenth week: HB., 82 per cent.; R. C, 3,786,-
000; W. C, s,ooo. The appearance was the same as
at the preceding examination.

8

Fourth month: HB., 76 percent.; R. C, 4,592,000;
W. C, 7,200. The appearance was the same as at the
last preceding examination. Small and deformed
cells were still present.

Sixth month: HB., 87 per cent.; R. C, 4,496,000;

■ A
Fig. 9.

W. C, 8,200. The appearance was the same as
above, but the small and deformed cells were not so
frequent.

Seventh month : HB., 77 per cent. ; R. C, 4,712,000;
W. C, 9,000. The variation in size was still within
but close to the normal limits; the shape was regular.

ogo^o o

Oq o ono <^

o'^Oo^oooO

A
Fig. 10.

Small and deformed cells were growing still less fre-
quent.

Eighth month: HB., 80 per cent. ; R. C, 4,800,000.
The cells -had grown a little more regular in shape.
The small and deformed cells had practically disap-
peared— one of two were found (Fig. 9).

Eleventh month: HB., 73 per cent.; R. C, 4,196,-

ooo; W. C, 5,Soo. The variation in shape was again
within but close to the normal limit. One small and
deformed cell was found after considerable search.

Fifteenth month: HB., 70 percent.; K. C, 4,712,-
000; W. C, 6,Soo. The variation in size had again
become less. No small and deformed cells were
found (Fig. 10).

In the above set of observations the following features
are noteworthy : Starting with about the most depleted
condition that is compatible with life, the blood, under
a maximum dosage of gr. xxxix. of ferrous sulphate and
gr. J^ of arsenious acid in a day, passed to a condition
approximating the normal, with a subsidence of all the
symptoms in a period of seven weeks. Then, the treat-
ment being entirely suspended a little later, the blood
continued in about the same condition for one year and
one month after convalescence had been established.
The variation in size and shape of the cells, at first
very marked though not extreme, steadily diminished
until the normal limits were reached at the eighth
week, never again to be transgressed. An inconsider-
able and diminishing number of the cells, however,
continued to be small and deformed as late as the
eighth month. Nucleated red cells (normoblasts) were
found with difficulty at the first three examinations
and not at all thereafter. Macrocytes were found in
considerable numbers at first but disappeared at the
fifth week. The HB. index, about i at the first ex-
amination, increased till it was a little over i and
remained so throughout the active period of con-
valescence. It then fell slightly below i and stayed
there. The absence of "ringing" and pallor of the
cells was therefore to be expected. Both the HB. and
the number of red cells reached the full normal limit
but at different times. The white cells, reduced in
number at first, rose distinctly above the normal count
at the seventh week, then fell again to normal and re-
mained there. No myelocytes were seen. The accom-
panying chart will make some of these points more clear.

The case is classed as simple anaemia >because of
the rapidity and degree of the recovery. The age, the
medication — almost exclusively ferrous sulphate — the
absence of cachexia, the continued absence of all

symptoms without treatment, are corroborative points
of interest. It is not unlikely that the patient is now
tending toward a relapse of moderate severity as is
frequent in simple anaemia.

Urine and Faeces. — Our chemical examination of the
urine and faeces gave us the following data^ :

Urine: The urine varied in color from a very pale

X,

>

\

~^

\

\

/

i >

/

/

<

\

^ '^

^^

\

\

^-N-^

;^--^

^

V

\>

K

CIS

^1

01 "

■ ^ Analyses of urine and fseces were made daily during the first
four weeks, in the department of physiological chemistry of
Columbia University, at the College of Physicians and Surgeons.

II

to a deep golden yellow. Several samples were more
highly colored than normally. The reaction was
usually alkaline; once or twice amphoteric (litmus).
A slight sediment settled out in each twenty-four
hours' urine. This usually contained epithelial cells,
triple phosphate, and calcium phospliate; now and
then pus cells, ammonium urate, and calcium oxalate
were found. The sediment never contained casts of
any kind. The specific gravity ranged from i.oii to
1. 019. Once it was 1,022. The volume for twenty-
four hours varied between 515 c.c. and 1,011 c.c.

The following substances were invariably absent ' :
coagulable proteid, proteose, dextrose, leucin, tyrosin,
lactic acid, diacetic acid, oxybutyric acid, haimoglobin,
cholesterin, acetone, ptomains, and bile salts.

The substances present in each sample, in
quantities approximately equal to normal amounts,
were: urea, uric acid, chlorides, phosphates, sul-
phates, indican, creatinin, alloxuric bases, oxalic acid,
phenol, and nucleo-albumin (mucin).

Several samples contained unusual amounts of uro-
erythrin. Each of these contained bile pigment also,
but no bile salts. Urobilin appeared to be markedly
increased in some of the urines.

The following table presents the results of a few
quantitative analyses of twenty-four hours' urine
passed during three successive days during the second
week :

First Day.

Second Day.

Third Day.

Urea

iS.t^oogm.

.;Si "
.066 "
I: 38.5

I • 7.2

23.600 gm.
.527 "
.093"
I : 44.8

T • K.6

19.800 gm.
.613 "
.080 "

Uric acid

Alloxuric bases

Ratio, uric acid to urea

Ratio, alloxuric bases to uric
acid

I : 32.3
I : 7.7

Volume

680 c.c. SS4C.C.

862 C.C.

Faeces: Several daily portions were completely
fluid. Usually the daily mixed faeces were partly fluid

' In all of our chemical tests, on both urine and faeces, we em-
ployed the methods our own experience and the work of others
have shown are the most satisfactory.

12

and partly solid. The odor was always very strong,
those of aromatic bodies and fatty acids predominating.
A few samples were yellowish in color; usually they
were greenish-gray to greenish-black. The more solid
portions were never homogeneous, varying much in
color and composition. Small mucous clots were
contained from time to time. The reaction was always
alkaline.

The solid matter, examined under the microscope,
contained starch granules, muscle fibres, connective-
tissue fibres, fat droplets, pigmented particles (yellow),
epithelial cells, triple phosphate, soap crystals, calcium
phosphate, and myriads of bacteria.

We were unable at any time to find albumin, pep-
tone, proteose, urea, ptomains, blood corpuscles, Char-
cot-Leyden crystals, haematoidin crystals, or choles-
terin in crystalline form.

The following substances, in dissolved form, were
readily identified, however, by the usual methods,
some only occasionally: cholesterin, bile salts, fatty
acids, indol, skatol, phenol, tyrosin, alloxuric bases,
stercobilin, hydrobilirubin, nucleo-albumin (mucin),
and bile pigment.

A review of the chemical data of this case brings
out the fact clearly that little was found which may be
attributed to any single line of metabolic disturbance
— practically nothing that may be said to be peculiar
to the anaemic condition.

The specific gravity of the urine was low, its color
usually pale, and the volume somewhat less than the
average, yet the fluctuations were mostly within the
normal limits. The sediment contained nothing of
special significance. The amount of urea was low,
that of uric acid and alloxuric bases at, or somewhat
above, normal; but the ratios of urea to uric acid,
and of uric acid to alloxuric bases were within the
customary fluctuations. Chlorides, phosphates, and
sulphates appeared to be relatively as great in amount
as urea. Deductions from our quantitative results can-
not be very accurate, however, since it was impossible
in this instance to regulate satisfactorily the quantity
and character of the patient's food.

Albuminuria of haemic origin was suspected in view

13

of the fact that albumin has been eliminated in the
urine during anaemia, but coagulable proteid could
not be detected at any time. The uroerythrin we
found several times, along with a little bile pigment,
and the increased urobilin in some of the urines,
rather suggest that the normal pigment metabolism
was somewhat disordered, but as the first two sub-
stances were present only a few times, little impor-
tance can be attached to their occurrence. Although
ptomains, putrescin particularly, have been found in
the urine during pernicious anaemia, they were en-
tirely absent in this case. The absence of lactic acid
is also noteworthy, several observers having assumed
it to be a constant constituent of the urine in perni-
cious anaemia.

There appeared to be little of special significance
in the composition of the faeces.

These results are valuable, we think, chiefly because
of their negative character, and we are inclined to
believe that, up to the present, few if any special
chemical qualities of the excreta have been definitely
established as pathognomonic of the various anaemic
conditions. Unfortunately, practically all of the work
of the past in this direction has been of a very frag-
mentary character; the results differ widely, and most
of them have been recorded with little regard for such
influences modifying the usual course of metabolism
as the quantity, character, and composition of the
food; various secondary pathological conditions; to
say nothing of other incidental factors of importance.'

A Compared Case of Pernicious Anaemia. — In
connection with this case we wish briefly to call at-
tention to another. A woman, sixty-four years old,
had been gradually losing strength but not flesh, and
getting pale and sallow for one year. She had grown
much more rapidly worse for two months. She pre-
sented marked anorexia, no vomiting, constipation, no

' We are indebted to Dr. William A. Taltavall for the follow-
ing facts : He examined the urine and faeces in three cases of
pernicious anaemia ; putrescin and cadaverin were invariably
absent ; urobilin was not increased ; uric acid and nuclein bases
appeared to be diminished somewhat. The latter consisted
mostly of hypoxanthin.

14

pulmonary or cardiac symptoms, no evidence of bleed-
ing anywhere. When admitted to the hospital she
was well nourished, but very pale and sallow. Her
lungs were normal. Her heart was normal in size
and position; its sounds were very weak, its action
was slightly irregular, the pulse corresponding. Ar-
tery was normal. No abnormal signs w^ere elicited over
the abdomen. Slight oedema of the ankles was present.
The voice was very weak. Extreme general prostration

I

c

) r- OO

oQ o

^ o o

■■^

o

p V)

oo

0,0

A
Fig. 12.

was evident. Temperature, 99.4° F. ; pulse, 76; res-
piration, 28. The urine was acid, s. g. 1.012, with a
considerable trace of albumin and a few hyaline casts.

Blood: HB., 22 per cent.; R. C, 756,000; W. C,
4,800. The red cells varied considerably, but not
extremely, both in size and shape. There was some
tendency to the formation of macrocytes, A moderate
number of small deformed cells were present. The
HB. was fairly even in most of the cells, but often very
deficient in the small ones. One normoblast was found
after prolonged search, but no megaloblast. The HB.
index was somewhat over i. There was nothing ab-
normal about the white cells. No myelocytes were
found (Fig. 12).

The patient was put on fluid diet and given gr. xx.
of ferrous sulphate, gr. \ of arsenious acid, and 3 ii,
of carnogen every day. Anorexia and vomiting be-

15

gan very soon. Attacks of syncope occurred. She
grew steadily weaker and became restless. The tem-
perature rose over loo^ F. only on the last two days
of her illness. Death occurred at the end of one
week from asthenia.

The blood on the day before death was HB., i6 per
cent.; R. C, 576,000. The appearance of the blood
was in no way changed from that of the week before.
Although no autopsy was held, the case was considered
clinically typical pernicious anaemia.

The similarity of the blood in the two cases is
striking and the clinical features also are very similar,
with the following exceptions: the age — nineteen in
the first case, sixty-four in the second; the absence of
the cachectic color in the first case, its marked occur-
rence in the second; the rapid and copiplete response
to treatment in the first case, and its complete absence
in the second.

The above comparison seems to demonstrate that
even the crude division of primary anaemias into sim-
ple and pernicious cannot be made, especially in
severe blood lesions, by the examination of the blood
alone, but that clinical features must weigh equally
with it in establishing a diagnosis. We believe that
to obtain a satisfactory classification of anaemias it
will be necessary, besides counting and studying the
peripheral blood, (i) to understand the life history of
the blood cells, which can be accomplished only
through study of the physiology and pathology of the
blood-making and blood-destroying organs; (2) to
investigate more thoroughly the interrelations between
the normal and diseased processes occurring in the
blood and those occurring in the other body tissues;
(3) carefully to correlate the results of such studies
with those obtained by clinical experience.

16

4^0

[Reprinted from American iVledioinc, Vol. IV, IMo. 4, pages
133-138, July 26, 1902.]

A CASE OF PANCREATIC FISTULA OF THREE
YEARS' DURATION, WITH A CHEMIC STUDY
OF THE FLUID ELIMINATED.

BY

FRANCIS W. MURRAY, M.D.,

Professor of Clinical Surgery, Cornell Universitj' Medical College; Sur-
geon to New York Hospital and St. Luke's Hospital.

AND

WILLIAM J. GIES, M.S , Ph.D.,

Adjunct Professor of Physiologic Chemistry, Columbia University ;
Consulting Chemist at the New York Botanical Gaiden.

In about 80 ^ of the operated cases of pancreatic cyst
on record, Gussenbauer's method of incision and drain-
age was employed, and the mortality due directly to the
operation was less than 6fo. While this method is
attended with such favorable results and is safer than
extirpation, which is limited in application, there are
certain disadvantages attending it in that the healing
process is slow, at times most tedious, and there is also
the danger of a permanent fistula. The duration of the
healing process varies from one to several months, and
in a case recorded by Korte, 2^ years elapsed before the
fistula closed.

As to the number of cases resulting in permanent
fistula we possess no definite information, since in many
instances details are wanting as to the ultimate result of
the fistula still existing when the case was reported. It
is probable, however, that in a small percentage of the
cases the fistula was permanent, as not a few were cyst-
adenoma. In such cases the conditions favor perma-
nency of the fistula, and extirpation of the entire cyst
offers the only means of cure. Since duration of healing
after Gussenbauer's method varies from one month to
2 J years (as in Korte' s case), one should wait a long time
before deciding in a given case that the fistula is a per-
manent one. If, however, at the time of operation we
had to do with a cystadenoma, which did not allow of
extirpation, then the question of permanency of the
resulting fistula would be more easily decided.

In the case we report the fistula has existed over three
years, has not diminished to any extent, in depth, and

its secretion, while, as a rule, of .small amount, steadily
continue.s. The clinical history of the case was reported
at the meeting of the New Y(jrk Clinical Society in
December, 1900, and was published, with two other cases
of pancreatic disease, in Ameriean 3redici)ie, January 25,
of the present year. Since that time a very thorough
chemic study has been made of the fluid eliminated.

The significance of the results of this chemic examina-
tion can be appreciated only in the light of the full
clinical history of the case, which is, therefore, again
given in brief detail :

CLINICAL HISTORY,

Before Achnission.—^l. D., female, 19 years old, admitted
May 8, 1899, to St. Luke's Hospital. Family history: Father

Fig. 1.

died of kidney trouble, mother of heart disease. Personal his-
tory: Patient had good health until seven years ago, since then
has suftered from attacks of gastritis, lasting three weeks at a
time. The attacks were attended with vomiting of foul and
greenish material, or coffee-grounds matter; no blood. During

the attacks there was epigastric tenderness and sharp shooting-
pains in the stomach and radiating to the back, pain worse after
eating. No history of clay-colored or fatty stools, no jaundice,
very constipated. Has had no appetite and has lost 10 pounds
in the past month. Six weeks ago her physician discovered a
tumor the size of an egg in the epigastric region ; since then the
tumor has steadily increased in size. The tumor appeared just
after an attack of gastritis, hut there is no history of sharp pain
or collapse. Since then the patient has been free from pain or
stomach symptoms. Last menstruation in March; had pre-
viously been regular.

On Admission. — Patient well nourished, skin dirty yellow,
mucous membranes pale, slight acne about face. Tongue
coated, teeth poor. Lungs normal. Heart negative, excepting
a systolic apex murmur transmitted to left ; no accentuation of

Fig. 2.

second pulmonic. Liver : dulness begins at fourth right inter-
space and extends to free border, edge not felt. Stomach reso-
nance a little higher than normal. There is a smooth, hard,
tense, semifluctuating tumor, about the size of a large cocoanut,
which is a little to the left of the median line in the epigastric
and umbilical regions, and extends into the left hypochondriac
region. Its area of flatness begins just below the stomach and
extends to the level of the umbilicus, where it meets the reso-

nance of the transverse colon. The flatness rnns to the left and
backward to the spine, its upper border l>ehind being li inches
below the angle of the scapula. About two inches below the
most prominent portion of the tumor in front, a free, sharp,
smooth edge can be felt running through the umbilicus to the
left in a curved direction. Right kidney palpable, freely
movable. Temperature 100°, pulse 90, respirations 24. Urine
light yellow, acid, sp. gr. 1,010, no sugar nor albumin, contains a
few vesical epithelia.

Diagnosis, pancreatic cyst.

Operation, May IS, 1899. — Ether anesthesia. Through an
incision in median line beginning just below the ensiform and
continuing downward for four inches, the peritoneal cavity was
opened. No adhesions found, stomach displaced upward, and
colon downward.

Pearl-colored cyst, seen presenting behind the gastrocolic
OQientum, which was adherent to anterior wall of cyst. Peri-
toneal caviry was walled off by gauze packing, large aspirating
needle thrust into cyst, and 30 oz. of clear straw-colored limpid
fluid withdrawn. As the wall of the cyst collapsed, it was drawn
up into the abdouiinal wound aud its cavity exposed through a
thret^inch incision, aud several ouuces of .eimilar fluid removed
by sponges. Cyst was thin-walled and lined with a smooth
membrane, and ran upward behind stomach. Careful probing
failed to reveal any communication between cavity of cyst and
adjacent organs. Incision in cyst wall partially sutured with
catgut, upper half of abdomiu'il incision closed with silk
sutures, cyst wall sutured with silk to parietal peritoneum in
lower half of abdominal incision. Ijarge rubber drainage tube
inserted into cavity of cyst, edges of wound protected with rub-
ber tissue and iodoform gauze ; sterile gauze dressing over all.
Operation lasted about an hour, patient sent to Avard in good
condition. Slight reaction followed operation, and convales-
cence was soon established.

The discharge from the cyst was profuse for about 10 days,
and then it gradually decreased, requiring a change of dressing
several times daily.

Chemic QanUtien of the Cystic Fluid. — Pathologist reported
that the fluid removed from the cyst was alkaline, sp. gr. 1,018,
f)palescent, and contained free fat, cholesterin and leukocytes.
It emulsified fats, changed starch into glucose and digested
albumin.

Convalescence. — The convalescence was uneventful, thp
wound gradually became smaller, the discharge lessened, and
on June 21 the patient was referred to the out-patient depart-
ment with a narrow fistula discharging a small amount of thin
yellowish fluid. The fistula was about five inches in depth, and
passed downward i nto the left hypochondrium. Since discharge
from the hospital the patient has been kept under observation,
and while the fistula lias never healed, there has been a great
improvement. She is strong and able to work, has no more
attacks of indigestion, bowels are regvilar, aud there has been a
great increase in weight.

For a year after the operation a small rubber drainage tube
■was woru, but owing to the steady contraction of the wound in
the abdominal wall, it was necessary to substitute a straight
silver tube three inches in length and of 32 F. caliber. This
prevents any retention of secretion and is worn with no dis-
comfort, and at present one small dressing of gauze suffices for
24 hours. Should, however, the patient become excited or

nervous, the secretion of pancreatic fluid is greatly increased,-
and at such times frequent dressings are needed.

Remarks. — This was a case of pancreatic cyst in a
young adult, and as far as could be ascertained at the
operation the cyst arose from the distal portion of the
gland. The etiology is obscure ; there was no history of
traumatism, and the attacks of severe epigastric pain and
vomiting occurring at intervals during the seven years
probably bore an etiologie relation to the formation of
the cyst. It should be observed that the cyst was first
discovered after one of these attacks, and since then
there has been complete cessation of pain and vomitingi
This fact may suggest pancreatic calculus as the cause,
but as no stone was ever seen in the feces, and as none
was found at operation, although sought for by the
finger and probe, there is no substantial foundation for
considering calculus as the etiologie factor.

The cyst was of large size, had thin walls and was
lined with a smooth glistening membrane, and presented
none of the appearances of cystadenoma. Incision and
drainage was deemed safer owing to the firm adhesions
to the neighboring organs. The diagnosis was made
before operation from the location of the cyst, and its
relations to the stomach and colon when distended with
air, by the history of rapid growth, and finally by the
absence of cystic disease elsewhere in the abdomen.
Aspiration was not resorted to, as it is attended with
danger, and it should be discarded in favor of explora-
tory incision.

Sugar was never present in the urine, and fatty stools
were never seen. Efforts to heal the fistula by local
treatment have been made steadily and at frequent
intervals. Injections of iodin, silver nitrate, carbolic
acid, nitric acid, curetting and packing of the fistula,
have been tried, but so far in vain. The fistula is still
about five inches in depth, and its apparent capacity less
than an ounce, but on two occasions when the drainage
tube was left out, fiuid to the amount of six ounces
accumulated in the cavity. The distention of the cavity
by the retained secretion caused nausea and some epigas-
tric pain, which was quickly relieved by the introduc-
tion of the drainage tube.

The general condition of the patient at present is
excellent, she has gained in weight and strength, has no
trouble with digestion, is able to work and suffers no
inconvenience from the fistula. Local treatment of the
fistula is still continued, and should it be considered

B

necessary for any rt-ason to excise the remaining portion
of the cyst, it will he ap|)roache{l l)y a counter incision
through the lumbar region.

In Fig. 1 may be seen the size of the cyst, and its
relations to the stomach and colon as determined by i)er-
cussion, the stomach and colon being distended with
air. The dotted line below the tumor represents the
position and course of the free, sharp edge felt upon
examination at admission.

Fig. 2 shows the tumor in profile as it presents below
the ribs and between the colon and stomach.

RESULTS OF RECENT CHEMIC STUDY.

As has already been indicated, the chemic exami-
nation of the cystic fluid at the time of the operation, and
of that discharged somewhat later, showed that pan-
creatic constituents characterized it. It seemed desirable
at this late stage of elimination, also, to ascertain by
chemic means whether the continued discharge is
from a permanent pancreatic fistula or whether the fluid
has other than a pancreatic origin.

The daily fiow of fluid has been considerable. Usu-
ally the liquid has been thin, watery, turbid, almost
colorless. Occasionally it is tinged with hemoglobin or
hemoglobin derivative, and is somewhat nuicigenous.

Our analyses were made of fluids collected at wide
intervals and under varying conditions, with a purpose
of securing a representative average of results. In the
examinations referred to below the fluid was collected
either with a syringe or a catheter, the patient lying on
her back or side during the process. Coughing favored
the discharge of the fluid, and was resorted to occa-
sionally, by direction, during the first two periods of
collection, in order to facilitate withdrawal. The meth-
ods of analysis were those commonly in use. Chemic
examination was made immediately or within a few
hours after the fluid had been collected.

A. January SI, 1902. — After the silver tube had been re-
moved from the fistula, a small amount of amber colored fluid
could be withdrawn at frequent intervals with a syringe. The
flow gradually increased, and, in the course of a half hour,
45 cc. of fluid was collected. This is designated below as the
" first portion." With the aid of a catheter an additional quan-
tity of the fluid, 31 cc, which flowed somewhat more rapidly,
was withdrawn in 15 minutes. This was almost colorless,
though slightly turbid, and is referred to below as the " second
portion."

a. FHrst Portion, 4-5 cc. — This was amber-colored, opalescent ;
contained minute flocks and possessed a slight, though distinct

odor, suggestive of volatile fatty acids. A trace of hemoglobin
was present, the blood having come from a very slight wound
of the tissue inside the orifice during the use of the syringe.

The fluid was alkaline to litmus. Acid phosphate was
absent. It contained a slight amount of proteid coagulating at
63° to 65° C. A few erythrocytes were to be seen under the
microscope ; some leukocytes and, here and there in the field ,
epithelial cells also. No crystalline matter was present. A
good biuret reaction was obtained with the fluid and a trace of
reducing substance was detected in it. The phenylhydrazin
test showed that this was due wholly or at least mainly to dex-
trose. Calcium, magnesium, sodium and potassium salts of
phosphoric, sulfuric and hydrochloric acids were present in
minute amounts.

Tested by the methods now in vogue, the fluid was found to
possess only slight tryptic and scarcely any lipolytic action ;
was entirely devoid of milk-curdling and inverting power, but
showed comparatively marked amylolytic effect. Pepsin was
absent. The emulsifying power of the fluid was the same as
that of lymph.

The fluid did not contain fibrin — no sign of coagulation
manifested itself at any time.

The following percentage results for general composition
were obtained :

Water 99.34

Solids 0.66

Oi'ganic matter 0.3-3

Inorganic matter 0.31

Of the total solid matter:

Organic 52.93

Inorganic 47.07

6. Second Portion, SI cc. — Almost colorless. Less turbid
or opalescent than the first portion. Peculiar odor missing.
No hemoglobin present. Qualitative factors otherwise were
the same as for the first portion except that a proteid coagula-
tion was obtained at 69° to 70° C. The fluid did not contain red-
cells and no lipolytic action was induced by it. Further, the
amylolytic action was relatively weaker.

The following percentage results were obtained in quantita-
tive analysis :

Water 99.54

Solids 0.46

Organic matter ' o.l9

Inorganic matter 0.27

Of the total solid matter :

Organic 42 32

Inorganic 57.68

B. February 10, 1903.— The fluid was removed about three
hours after a light breakfast. The fluid was 45 to 50 cc. in vol-
ume, light yellow in color, alkaline in reaction and turbid, con-
tammg small particles. One-half of it was filtered.

a. Filtered Porho?i.— Slightly opalescent. Qualitatively it
was the same as the second portion examined on January 21
except that all ferment tests were negative save that for dias-
tatic enzyme.

The following percentage results for composition were
obtained :

8

Water 9!».lo

8nlids 0.S5

Organic matter 0.52

Inorganic matter O.'H

Of the total solid matter :

Organic 61.63

Inorganic 38.37

b. Unflltered Portion. — Quite turbid with flocculeut material.
Leukocytes grouped in clusters made up the particles visible
to the naked eye. The tluid was the same qualitatively, other-
wise, as the filtered portion, except that very weak, almost
imperceptible, tryptic action was demonstrated in addition to
amylolytic.

The appended percentage composition results were ob-
tained:

Water 99.09

Solid.s 0.91

Organic matter 0.56

Inorganic matter 0.3-5

Of the total solid matter :

Organic 61.24

Inorganic 38.76

The striking features of the results under A and B
are (1) the very weak, practically negative, action of the
fluid so far as typical pancreatic enzymes are concerned,
under conditions which had been made particularly
favorable to them ; and (2) the very slight amount of
solid substance, particularly organic matter, contained
in the fluid. The tryptic action was so slight and uncer-
tion that the enzyme may have come from the leuko-
cytes of the fluid. The diastatic action manifested, while
vigorous, wtis no more pronounced than that shown by
any serous fluid, a statement applying with equal force
to the observed lipolytic effect.

The generally negative results of the preceding tests
for typical pancreatic enzymes led us to examine fluid
collected at a time when pancreatic activity would be
most decided and when, therefore, the probability of
diffusion or direct delivery from the gland into tlie fis-
tula (if either process now occurs at any time) would be
greatest. Any duct, or passage, connecting with the
cyst would naturally empty more fluid into the fl.stula
during such a period of glandular activity than at any
other. Accordingly, the collection was begun at the
patient's home just an hour after tlie completion of the
heaviest meal of the day and continued into the third
hour after the meal had been taken, with the following
results :

C February 18, 1902. — There was a gradual increase of flow
after removal of the tube. At the end of three-quarters of an

9

hour it was comparatively rapid. In one hour and 10 minutes
125 cc. of the fluid passed from the fistula.

This surprising result is worthy of special notice. Although
as much as 20 liters of fluid have been removed from a pancre-
atic cyst (by Stapper) at the time of operation, no such afterflow
as this has been previously observed. Indemans noted a flow
of 120-130 cc. per day for a few days after operation, but this
soon diminished in quantity.

The fluid above mentioned had only a very faint tinge of
yellow, was slightly opalescent, odorless and without appreci-
able sediment. Its specific gravity w^as 1,002.8. Qualitatively
it was the same as the first portion collected on January 21
except that coagulable proteid was separated at 68° C, red cells
wereabsent and crystals of calcium oxalate were obtained on
concentration.

The tests for enzymes wei-e practicallv negative except for
amylopsm, which was present in comparatively active amount
:Notryptic action could be shown even with the aid of dilute
alkali. The emulsifying action was slight and only such as
may be obtained with any serous fluid.

^,^T^®,^o^lo"'i"S results for percentage composition were
obtained m duplicate :

1- 2. Average.

Water 99.520 99.520 99 520

Solids.. 0.480 0.480 0.480

Organic matter 0.329 0.321 0..325

Inorganic matter 0.151 0.157 0.155

Of the total solid matter :

Organic. 68.50 67.72 68.11

Inorganic 31.50 32.28 31.89

. The analyses of February 18 were repeated, after a good
interval wnth fluid collected under similar conditions, ?. e
w^ithm 1 to .3 hours after the heaviest meal of the day. Samples
of the patient's urine, passed on the same and the previous day
were also carefully examined. The results follow •

D. April 1, 190S.-The silver tube had been kept out of the
fistula during part of the day. Later, because of a tendency to
closure and retention, a rubber tube had been inserted In the

flnid wf;hr?v^'t!S°''^?if '""^ """^^^^^ *"^®' *^® fi^st portions of the
To?.. J ^ -^^^^^'^ ^ syringe were amber-colored and con-
tained considerable mucus, leukocytes and some oil globules

latfou"" wHnh f *^^ '''^^r ^''''^ required considerable man pt:
lation, which fact doubtless accounts, in part, for the ereater
^n{^??/"i^i°?' l^^«^s..etc., in the fluid first collected. The flufd
collected m this way is referred to below as the " first portion ''
tho fl!^/^'; '^*!,^!."'^H^ collection a catheter was inserted and
the fluid carried directly to a bottle. The flow appeared to be

iTsome J>fa?'^T,?i"r°^"" ^'^f^-^^ ^°^^' ^^«° it se'S^ed to slow
^^n^^% f^- ^^ ^ ^°^^* ^^^ *^ imnutes, 155 cc. of the fluid was
collected (Compare with result of February 18 ) This !l
referred to below asthe " second portion "

a. Fi7-.st Portimi.-The fluid collected at first possessed dis-

nf°lfr.^w*^'-^^^*^°^' ^^^'y «"^^t tryPtic power and only ftrace
of lipolytic influence. It was composed as follows :

Water „_„

Solids %•'?

Organic matter f-o

Inorganic matter Qgg

10

Of the total solid matter

Organic 70.69

Inorganic 2y.31

Compared with previous analyses, the chief dittereuce to be
noted is the somewhat increased proportion of solids. This
was undoubtedly due to the mucus in the fistula at the time,
and which was taken up by the syringe. The catheter delivered
the fluid of the second portion as it gathered in the fistula. The
comparative analyses given below show- that the mucus is a
variable and a secondary constituent.

b. Second Portion. — This was given more extended analysis
than any of the other portions collected. Its specific gravity
was 1,()03.6. With the exception of enzyme content, the fiuid
j)ossessed all of the qualitative characters of that collected on
February IS, calcium oxalate, however, being more in evidence.

This fluid possessed comparatively vigorous diastatic
action even iu tne cold. At 40 C. it showed tryptic power
very gradxal/y, and had some lipolytic action. Even when
tested with etliyl butyrate and litmus, however, the latter action
was seen to be comparatively slight.

The following substances could not be detected in the fluid:
Bile pigment, proteose, peptone, tryptophan, nuclein base,
urea, leucin, tyrosin, creatin, glycogen. These and previous
negative results indicate that neither the liver nor a kidney is
involved in the production of the fluid.

In addition to the substances already found in each sample
of fluid collected, cholesterin crystals were observed in this.
The coagulable proteid consisted of both albumin and globulin.
On boiling, the fluid gave off an odor suggestive of fatty acid.
In the cold, acetic acid precipitated a proteid insoluble iu a
moderate excess of the ai-id. This substance, which appears to
have been nucleoproteid, was somewhat soluble, howe\er, in a
slight excess of hydrochlori(; acid.

The amount of coagulable i)roteid was accurately deter-
mined. The first separation was made within two hours of the
time of collection, the second 12 hours later. The results are
practically the same. The original fluid, in the meantime, was
kept in a cool place — at 15° C.

First determination = 0.1896 gram coagulable proteid per 100 cc.
Second " =0.2000 " " " "

Average = 0.1948 " " "

The second result for coagiilable proteid content, compared
with its duplicate, .shows, further, how little tryptic action the
fluid was able to exert — though, of course, the conditions dur-
ing the interval were not particularly favorable to such action.
At the same time, if there had been any significant quantity of
trypsin in the fluid, a good proportion of this small amount of
proteid would have been hyd rated beyond the coagulable
stage.

The following results for general percentage composition
were obtained:

Water 99.07

Solids 0.93

Organic matter 0..5S

Inorganic matter 0.35

Albumin, globulin 0.19

11

Of the total solid matter ;

Organic, ., 62.51

Inorganic 37.49

Albumin, globulin 2U.96

c. Urine of March SI and April 1, 1902. — The results of our
examination of the patient's urine may be summed up in the
statement that it was found to be normal for both days. Ex-
cepting mucus and a few epithelial cells, no proteids or pro-
teid elements could be detected. Sugar was absent, as shown
by negative results with Nylander's solution and with phenyl-
hydrazin.

Fatty stools have never been observed, it should be
remarked again — the feces have been normal constantly.

REVIEW OF QUALITATIVE RESULTS.

A general review of our qualitative results shows that
the fluid is similar to a simple transudate. In no previ-
ous case has the specific gravity been as low as that
recorded here— 1,002.8. Gussenbauer found it as high
as 1,610. Qualitatively, the fluid is like many of those
from pancreatic cysts already analyzed. Quantitatively,
it is mucii difft-rent than the fluid from some ; similar,
however, to others. The significant variations from most
of the fluids previously analyzed are the low content of
organic matter, indicating absence of particular inflam-
mation and the large proportion of water. Unlike a
number of such cystic fluids examined previoui^ly, it is,
further, entirely devoid of constituents representing
various stages of tryptic proteolysis. The noteworthy
content of oxalic acid (calcium oxalate) brings to mind
the similar result obtained by Zdarek in Ms examina-
tion of fluid withdrawn soon after operation.

These later examinations empha><ize the deductions
drawn from the results of those of January 21 and Feb-
ruary 10. All of the data indicate that the case under
consideration is not now one of true, permanent pancre-
atic fistula, although they do uot exclude the probability
that diffusion from the pancreas constantly takes place
to some extent, or that pancreatic tissue makes up part
of the wall of the cyst. Certain it is, at all events,
that the fluid is not pancreatic juice in the ordinary
sense.

SUMMARIES OF QUANTITATIVE RESULTS.

The following summary shows the uniformity of our
average quantitative analytic results :

12

Tari.e I.— Genera I. Percentage Composition of the Fluid from
THE Fistula.

Constituents.

Water

Solids

Organic matter...

Inorganic matter
Of the U)U1 solids.

Organic

Inorganic I 47.07

J anus

iry2l.

February 10.

February 18,

a

b

a

b

a

99..52

b

99M

99.54

99.15

99 09

99 52

0.66

0.46

0.85

0.91

0.4K

0.48

0.35

0.19

Ooi

0.56

a33

0.32

0.31

0.27

0.33

0.35

0.15

0.16

02.93

4>.32

61.6:^

6124

68.5U

67.72

47.07

57.68

38.37

38.76

MM

32.28

b*

99.07
0.93
0..5S
0.35

62.51
37.49

Av'r-
age.

99.32
0.68
0.41
0.27

.59.55
40.45

* The results of our analysis of the first portion obtained on April 1,
are not included above, because of the exceptional amount of mucus,
etc.. in the fluid at the outset of its collection.

The significance of the above analytic figures may be
fully appreciated at a glance, on comparison of our aver-
age results with similar data for various lymphatic or
serous fluids given in the appended table ;

Table II.— General Percetntage Composition of Lymph and
Transudates.

_

ee

s

E."-

x-C

c

c-

&

^2 53

O

X,

O

"

99.32

99.17

0.68

0.83

0.41

0.32

O.'/i

0.51

0-3

(U

.3_

■6

01

,,

3

—

^-

a

C

o».

■S-c

A

u

£"0

i«

>5

0

uC

<h

^

a

O.

a

■H

X!

<

98.68

98.63

98.43

97.89

1.32

1.37

1.57

2.11

0.49

0.98

1.13

0.88

0.59

0.97 1

ci —

Water

Solids

Organic...

Inorganic

98.69
1.31
0.51
0.77

96.09
3.91
3.0:}

0.88

93.S9
6.11
.5.18
0.93

1, *, », "Results summarized by Halliburton: Textbook of Chemic
Physiology and Pathology. 1891, pp. 334-*56.

2 Given in Schiifer's Textbook of Physiology, 18J8, i, p. 123.

8. ', 8 To be found in Mandel's translation of Hammarsten's Text-
book of Physiologic Chemistry, 1900, p. 193.

Our results are almost identical with those for cerebro-
spinal fluid. They show clearly, we think, that the
fluid from our patient's fistula has the general characters
of a transudate and that it is very much like ordinary
lymph. The similarity to the cerebrospinal fluid also
suggests that selective cells have somewhat influenced
composition — cells probably situated in the wall of the
cyst.

13

All of the analyzed fluids referred to in the above
table were samples of the fluid obtained on first with-
drawal, which naturally would contain more solid
matter, particularly proteid, than such portions as
might flow from the body immediately on formation.
In the former cases prolonged osmotic influences, par-
ticularly resorption of w^ater, would tend to raise the
percentage of inorganic products, whereas cellular
activity would bring about increase of organic constitu-
ents. Our own patient's fluid on retention, would, for
the same reason, surely contain a somewhat greater pro-
portion of solid matter — as it did at the time of opera-
tion, when its specific gravity was 1,018.

The results summarized in the appended table show
that, so far as general composition is concerned, the fluid
we have examined is not very similar to pancreatic
juice — even such as is collected from a permanent fistula
— a further fact in harmony with our qualitative,
enzyme results. The figures for blood plasma are also
brought into comparison :

Table III.— Cojipositiox of Pais'creatic Juice and Blood
Plasma.

Fluid from
our own
jiatient's
llstula.

Fluid from a
tempornry
pancreatic
fistula.i

Fluid from a

pancreatic
fistula.'-!

Fluid from a
permanent
pancreatic
fistula
(dog).«

Water

Solids

Organic matter...

Inorganic matter

93.32
0.68
0.41
0.27

86.41

13..59

13.25

0.34

97.59
2.41
1.79
0.62

97.68
2 32
1.64
0,68

90.29
9.71
8.86
0.85

1 Zawadsky : Centralblatt fiir Physlologie, 1892. v, p 179

2 Herter : Zeitschrift fiir physiologische Chemie, 18s0, iv, S. 160.

3 Schmidt : Hermann's Handbucla der Physiologie, 1881, v-2, S. 189.
* Halliburton : Textbook of Chemie Physiology and Pathology,

1891, p. 334.

Only a few quantitative analyses of the fluids from
pancreatic cysts have been recorded. The following
summary shows the general chemie relationships of the
fluid we have examined to those analyzed by previous
observers. In each case the analyzed fluid was collected
either at the time of operation or shortly after. Our
results, it will be seen, are more nearly in accord with
those of Zdarek than of any other :

14

Tabi^e IV.— Gexekal Percentage Composition of Pancreo-
CYSTic Fluids.

ii

■3

S

c

9ST0

i.ao

0.36
0.94

c

C

■■0
r5

1

1^

0

Water

Solids

99.32
0.68
0.41
0.27

98 94
l.OtJ
0.19
0.S7

98.05
145
0..55
0.90

98 21
1.79
1.00
0.79

98.14
1.86

92.68 86.41
7..S2 13.59

Organic matter

Inorganic matter

6.51 13.25

0 81 , 0 34

Coagulable proteid

0.19

0.10

0 32

0.27

0.82

1.66

9.21

REVIEW OF CHEMIC OBSERVATIONS IX PREVIOUS CASF:S.

The published results of cheinic analysis of the fluid
of various established pancreatic cysts have shown that
the enzymes are frequently absent, not only from the
fluid withdrawn at the time of operation, but also from
that eliminated during the healing of the wound. As
Korte sugge.sts, stagnation and consecjuent prolonged
contact with the other constituents of the fluid are
doubtless destructive to the enzyme. Analysis has also
shown that occasionally the enzymes have been absent
from the fluid retained in the cyst, but have appeared
for a while in the secretit)n thrown from the drainage
tube, only to again disai)pear, and that permanently. In
such instances it is probable that changes in the cells of
the gland due primarily to drainage, as in cases of per-
manent experimental pancreatic fistula, cause alterations
in the character of the fluid and the complete disappear-
ance of the enzymes. In other cases of pancreatic cyst
the enzymes were detectable in all samples of fluid with-
drawn. In one rather odd case, cited by Korte, the
enzymes were absent from all samples of fluid, but
could be extracted from the wall of the cyst.

These facts, together with the additional observations
by various investigators that lipolytic, proteolytic and
amylolytic enzymes are found in various pathologic and
lymphatic fluids, seem, in considering the qualities of
cystic contents, to lead to the conclu.sions that (1) the
presence of slight amounts of these enzymes does not
necessarily imply a pancreatic source of the fluid ; further,
that (2) the absence of these enzymes does not nece.ssarily
mean that the fluid in question has an extra-pancreatic
origin.

Korte, in summing up, emphasizes the following as
the chief points in our knowledge of the characters of

15

pancreocystic fluid. It is usually tinged with hemo-
globin or its derivatives, reddish to black in color, some-
what slimy, alkaline, rich in proteids, specific gravity
1,010-1,020 and frequently contains enzymes and cellular
detritus. When the enzymes are present, in pa/iicu/ai'ijj
active quantity in the "•puncture-fluid," the presumption
is strong that the fluid is directly derived from the
pancreas. The absence of enzymes from such fluid is no
evidence, however, that the cyst is not truly pancreatic
in nature.

That our own patient's cyst was truly pancreatic was
definitely established at the time of operation. That the
fluid no longer partakes of the characters of true pan-
creatic juice harmonizes entirely, therefore, with obser-
vations of the past.

GENERAL OBSERVATIOXS.

Transudation is mainly a physical, hydrostatic mat-
ter. But the permeability and the character of the
tissues separating the blood and the transudate naturally
determine the selective factors and largely influence
composition. The cyst wall is always very vascular.
In this case it was, at the time of operation, lined also
with a smooth membrane. In retention cysts the wall
is frequently the more or less altered wall of the original
structure. The vessels are thin and no doubt unduly
permeable. Passive conge.-tion has probably become
chronic and has doubtless increased permeability. Under
these conditions the fluid of the cyst has lost its original
qualities and is not easily comparable, except in a general
way, with any other. Degenerate cells may also be respon-
sible in part for the character of the transudate.

The walls of pancreatic cysts are usually composed of
connective tissue, and incision and drainage has in
nearly all cases favored ready granulation and rapid
healing. The cystic membrane in some cases has con-
sisted partly of pancreatic tissue, normal or degenerate
or both, or has been lined with a secreting epithelium.
Such an epithelium naturally interferes with granula-
tion, may entirely prevent closure of the wound and
makes the flow of cystic fluid continuous. In our own
case, epithelial cells are to be found in the fluid along
with leukocytes and mucus, and in all probability the
cyst is still lined, in part or throughout, with a secreting
mucous membrane. Although the interior of the cyst
has been steadily treated with carbolic and nitric aci(N,
etc., healing has appeared to cease and the fistula

16

porsi:?ts. The tube was lately kept out of the fistula
for several days. The aperture narrowed at once and
cumulative retention resulted, niueh to the i)hysieal dis-
tress of the patient. The tube has again been rejdaced,
the patient being thereby relieved, and the flow goes on
as before.

With the exception of the first of K()rte's cases none
other like ours appears in the records. In Korte's ease
a fistula similar to that in our patient remained 2i years
after the operation. Varied treatment repeatedly with
caustic substances, heat cauterization, etc., was without
result. Several times the fistula closed temporarily, but
as often opened up, with continued flow. F'inally,
2J years after operation, complete healing suddenly
occurred spontaneously. Several years thereafter Korte
saw the patient, founcl that the closure was permanent
and the patient enjoying good health.

Riegener has expressed the opinion that in Gussen-
bauer's operation of incision and drainage there is little
danger of a permanent fistula resulting. The facts
Korte recapitulates, as well as the experience our own
case affords, show that this possibility is not as remote
as Riegener imagined.

It has been fretiuently observed that during periods
of excitement or nervousness the flow from the fistula of
our own patient has been particularly abundant. The
patient herself has come to associate special elimination
with such conditions. Since transudation is determined
largely by intracapillary pressure, it seems probable that
such periodic increases in the quantity of fluid are
dependent on vasomotor changes, with augmented blood-
pressure in the splanchnic region in general and the
capillaries of the wall of the cyst in particular, rather
than on special secretory activity of the pancreas. That
the increased flow is not due to formation of true pan-
creatic juice is very evident from our results. Several
of our analyses were made of fluid obtained in abundance
during the before-mentioned periods of nervousness.

CONTCLUSIONS DRAWX FROM THE RESULTS OF CHEMIC
ANALYSIS.

The results of our recent analyses and those made at
the time of and shortly after the operation, seem to be in
harmony -v ith the following conclusions :

Such connections of the pancreatic gland with the
cavity of the cyst as may have existed at the time of

17

operation have closed and direct secretion from the
gland into the fistula has ceased.

The fluid originally contained pancreatic products in
abundance. The fluid still leaving the fistula appears,
however, to be a transudate, resulting probably from
chronic serous inflammation. It is possible, of course,
that lymph from the pancreas contributes to the flow
and that the lining membrane, in part consisting of
abnormal pancreatic tissue, influences the composition of
the fluid.

That the pancreas is no longer seriously involved is
evidenced by the continued vigorous health of the
patient — good appetite, absence of fatty stools, neither
sugar nor proteid in the urine. At least sufllcient nor-
mal pancreas remains to perform all of the observable
functions of the gland.

The case is similar to the exceptional one of K5rte's,
in showing that after incision and drainage of a true
pancreatic cyst (1) general recovery may be rapid, (2)
the functions of the pancreas remain normal, (3) the
patient enjoy excellent health thereafter, (4) with a
persistent permanent fistula eliminating a transudate
containing {a) a minimal proportion of solid matter, (6)
a maximal percentage of water, and (e) little or no pan-
creatic enzyme.

The case is different than any other on record in (1)
the length of time the fistula has persisted, and (2) in
the quantity of fluid steadily eliminated from it.

BIBLIOGEAPHY.

Practically all original papers on pancreatic cysts printed before 1898
are listed by Korte. See his monograph for references to the work of
observers mentioned above. Korte: Deutsche Chirurgie; Die chirur-
gischen Krankheiten und die Verletzungen des Pankreas, 1898, Liefer-
ung4nd, p. XV (Literatur).

Momer, K. A. H. : Skandinavisches Archiv fur Physiologie, 1895, v,
S 274.

Lenarcic : Centralblatt filr inuere Medlcin, 1898, xlx S 773
V. Brackel: Deutsche Zeitschrift ftir Chirurgie, 1898, xlix, S 293.
Payr : Wiener klinische Wochenschrlft, 189S, xi, S. 629
Pollard : British Medical Journal, 1899, ft. I, p 594.
Keitler: Wiener klinische Wochenschrlft, 1899, xll, S 764
Zdarek : Ibid, 1899, xii, S. 767.

Israel : Deutsche medicinische Wochenschrlft, 1900, xxvi S 352
Bessel-Hagen : Verhandlungen der deutschen Gesellschaft fur
Chirurgie, 1900. xxlx, S. 6S3.

Fitz : American Journal of the Medical Sciences, 1900, cxx n 184
Lazarus: Zeitschrift fiirHeilkunde, 1901, xxii, S 165
Seeflsch: Deutsche Zeitschrift flir Chirurgie, 1901, lix, S. 153.
Subbotic : Ibid, 1901, lix, S. 197. = . , ,

Murray: American Medicine, 1902, iii, p. 141.

M1SCKLI.ANEOUS RESEARCHES.
Reprints, Nos. 29-35.

ANTITOXIC ACTION OF IONS.
XOEB Si GIES.

4m %1^

24C Jacques Loeb und William J. Gies:

Separatabdruck aus dem Archiv f. d. ges. Physiologie Bd. 93, 1902.

"Weitere Untersuchungen

iiber die entg-iftenden lonenw^irkung'en und

die RoUe der AVerthig^keit der Kationen bei

diesen Vorg-ang-en.

Von
University of Chicago Columbia University,

Jacqnes Lioeb, l¥llliain J. Oles,

unci

I. Eiiileitnng.

1. Withrend Ringer^) und HowelP) die Meinuiio: aiissprachen,
dass das Calcium der „Reiz" fiir die Herzthatigkeit sei, indem es
die Systole auslose, wiesLoeb^) darauf bin, dass das Calcium nicht
direct fur die rbythmischen Contractionen und die Herzthatigkeit
nothig sei, sondern nur indirect, namlicb urn die giftige Wirkung des
Kochsalzes im Blut oder in den Geweben aufzuheben. Zwei Gruppen
von Thatsachen fuhrten ilin zu dieser Annahnie , namlicb ersteus,
dass ein Zusatz von Calcium zu einer Losung nur dann gunstig wirkt,
wenn die Losung grossere Mengen von Salzen mit einwerthigem
Ration, l)esonders Natriumsalze, entbalt. In einer mit dem lebenden
Gebilde isotoniscben Losung eines Nicbtleiters finden im Allgemeiuen
keine rbytbmiscben Contractionen statt, wie viel Calcium man aucb
zusetzen mag*). Die zweite, entscbeidende Beobacbtung wai- aber

1) Ringer, Journal of Physiology vol. 3 p. 388. 1880, vol. 4 p. 29, 222,
vol. 5 p. 247, vol. 6 p. 154, 361, vol. 8 p. 20, 288, vol. 9 p. 425.

2) Howell, American Journal of Physiology vol. 2 p. 47. 1898.

3) J. Loeb, American Journal of Physiology vol. 3 p. 327, 383, 434. 1900
und vol. 6 p. 411. 1902. Pfluger's Archiv Bd. 80 S. 229. 1900 und Bd. 88
S. 68. 1901.

4) Loeb, Ueber lonen, welche rhythmische Zuckungen hervorrufen. Fest-
schrift fur Fick. Braunschvreig 1899. American Journal of Physiology vol. 3
p. 383. 1900. Pfluger's Archiv Bd. 91 S. 248. 1902.

Weitere Untersuchungen liber die entgiftenden lonenwirkungen etc. 247

folgende: Die Eier von Fundulus, die sieh norraaler Weise im See-
wasser entwickeln, 1)ilden keinen Embryo, sondern sterben rasch ab,
wenn sie in eiuer reinen Kochsalzlosung sich entwickeln von der
Concentration, in der dieses Salz im Seewasser enthalten ist. Fiigt
man einen kleinen, aber bestimmten Betrag eines Calciumsalzes zu,
so entwickeln sich die Eier ebenso gut wie im Seewasser. Dass
a])er in diesem Falle die Calciumionen nicht direct ftir die Ent-
wicklung nothig sind (den „Reiz" bilden), sondern nur indirect (um
die giftigen Wirkungen der Kochsalzlosungen aufzuheben), wird da-
durch bewiesen, dass die Eier in mehrfach destillirtem Wasser sich
vollig normal entwickeln ^).

Die Rolle der lonen in diesen Vorgangen stellt sich Loeb
folgendermaassen vor. Die Ursachen („Reize") ftir die rhythmischen
Contractionen sowohl wie fiir die Zelltheilungs- und Entwicklungs-
vorgange sind nicht die lonen, sondern bestimmte chemische (kata-
lytische) Vorgange und zwar, da fiir Herzthatigkeit sowohl wie fiir
die Zelltheilung geniigende Sauerstoffzufuhr ausnahmslos unerlassliche
BedinguDg ist, anscheinend Oxydationsvorgange. Die Betheiligung
der lonen diirfte sich moglicher Weise darauf beschranken, dass die-
selben die physikalischen Zustande der lebenden Substanz in einer
fiir die Ausfiihrung der nothigen Bewegungen giinstigen (oder un-
giinstigen) Weise beeinflussen. Das ware der Fall, wenn beispiels-
weise in einer reinen Kochsalzlosung Bestandtheile des Protoplasmas
verfliissigt wiirden, welche fest sein soUten, und wenn ein kleiner
Zusatz von Calcium die Verfliissigung verhinderte ^). Wenn das
riehtig ware, so sollte man auch erwarten, dass, wenn die Gewebe
zu viel Calcium enthalten, ebenfalls giftige Wirkungen entstehen. Die

1) Physiologen scheinen im AUgemeinen anzunehmen, dass Kochsalzlosungen
die ungiftigsten Lijsungen iinter den Losungen von Elektrolyten seien. Das ist
nur fiir gewisse physiologische Vorgange riehtig, z. B. Muskelcontractionen. Fiir
die ersten Entwicklungsvorgange von Funduluseiern (und anscheinend auch fiir
andere Fischeier und vielleicht auch Froscheier) ist KCl weniger giftig als NaCl.
Americ. Journal of Physiology vol. 6 p. 411. 1902.

2) Eine ausfiihrlichere Discussion dieses Zusammenhanges zwischen fermen-
tativen Processen und lonenwirkungen findet sich in Loeb's Comparative
Physiology of the Brain and Comparative Psychology p. 17 ff. New York and
Loudon 1900.

248 Jacques Locb iiud William J. Gics:

Beobaehtiingeu iiber die Eiuwiikuuii von zu viel Calcium bei den
Coutnictiouen der Medusen und der Heizthiitiukeit stiitzen diesc An-
schauung. Wenn das Centrum einer Meduse oder das Ilerz in Folge
einer zu starken Dosis von Calcium zuni Stillstand gekomnien ist,
so kann es wieder anfangen zu schlagon, wenn man es in eine
reine Kochsalzlosung oder eine Kochsalzlosung niit weniger Calcium
zuriieksetzt.

2. Wenn es sich hier in der That um antagonistisclie Wirkungen
von lonen (auf die pliysikalischen Zustiinde ge^Yisser Protoi)lasma-
bestandteile) handelte, so war zu erwarten, dass die Kolle von
Calciumionen einer reinen Kochsalzlosung gegeniiber auch durch
andere lonen iibernonimen werden konnte; und dass ferner eine
kleine Dosis von Calciumionen nicht nur Kochsalzlosungen, sondern
auch die Losungen anderer Salze, namentlich mit einwerthigen
Kationen, entgiften miisse.

Hardy's Untersuchungen M iiber die Fallung suspendirter Theil-
chen in fliissigen Medien vermittelst Elektrolyten brachte Loel) auf
die Vermuthung, dass die antitoxischen Wirkungen der Calciumionen
gegeniiber einer reinen Kochsalzlosung vielleicht bedingt seien durch
die Werthigkeit und positive Ladung des Calciumions, und dass es
desshalb moglich sei, dass andere zweiwerthige Metalle ahnliche auti-
toxische Wirkungen ausiiben wis das Calcium. Die Versuche iiber
die Entwicklung von Funduluseiern bestatigten diese Erwartuug auf
das Ueberraschendste -). Von einer geringen Concentration an sind
die Losungen der Chloride mit einwerthigem Kation fiir das Fun-
dulusei giftig, d. h. kein ])efruchtetes Ei kann in einer solchen
Losung einen Embryo bilden, und die befruchteten Eier sterben als-
bald. Fiigt man aber einen sehr kleinen, aber bestimmten Betrag
irgend eines loslichen Salzes mit zweiwertliigem Kation zu (mit
Ausnahme von Hg und Cu), so bilden sich im AUgemeinen ebenso
viel Embryonen wie im Seewasser. Je hoher die Concentration der
Losung des Salzes mit einwerthigem Kation ist, um so mehr Calcium
ist auch zur Entgiftung nothig. Dagegen konnten mit Anionen hoherer
Werthigkeit die toxischen Wirkungen einer reinen NaCl-Losung nicht

1) Hardy, Pioceodings of the Iioyal Soc. vol. 6b p. 110. 190U.

2) Loeb, 1. c.

Weitere Untersuchungen iiber die entgiftenden lonenwirktmgen etc. 249

aufgehoben werden. Zur volligen Entgiftung von 100 ccm einer

5

Q m^) NaCl-Losung waren beispielsweise nothig:

ungefahr 4 ccm ttt CaSOi
64

AM

„ ^ » Qo BaClg (gleiches Anion mit NaCl!)
„ 2 „ -^ZnSO,

„ 2 „ -Q- CoCls (gleiches Anion mit NaCl!)

Wenn man die ausserordeDtlich geringe Quantitat des entgiftenden

Salzes beriicksichtigt, so wird es klar, dass es sich hier nicht um

eine directe Wirkimg des entgiftenden Salzes auf die Kochsalzlosung

5
handeln kann. Die zur Entgiftung von 100 ccm -^ m NaCl nothigen

o

Caleiumionen betragen nur ein Tausendstel der Natriumionen (und
Cl-Ionen). Wenn man aber die Concentration der reinen Kochsalz-

fit t^

losung selbst um 20 "/o verringert (also eine -^ statt einer ^ m

NaCl-Losuug anwendet), so eutwickelt sich in derselben im giinstigsten
Falle vielleicht ein Procent der Eier. Durch Zusatz von 4 ccm einer

Ayr K

^ CaSOi- (oder Ca [N03]2)-L6sung zu 100 ccm einer -^ m NaCl-

Losung entwickeln sich in derselben aber ebenso viele Embryonen
wie im normalen Seewasser, also ca. 90 *^/o oder mehr aller Eier
l)ilden bei gilnstigem Material Embryonen. Es muss sich also wohl
bei diesen antitoxischen Wirkungen darum handeln, dass die ein-
werthigen und zweiwerthigen Kationen einen entgegengesetzten Ein-
fiuss auf eine im Ei entbaltene Substanz ausiiben. Dieser Einfluss
ist zum Theil wenigstens eine Function der Werthigkeit der lonen
und ferner wohl auch eine Function des Vorzeichens der Ladung,

5

1) Eine -5- J?i-L6sung ist eine solche, welche 5 Grammmolektile (oder 5 Mol.)

o

der gelosten Substanz in 8 Litem der Losung enthalt. Das Zeichen m steht fiii'
Mol. Eine m-L6sung enthalt 1 Mol. der gelosten Substanz in 1 Liter der Losung.
Es ist ohne Weiteres einleiichtend , dass diese Bezeicbnungsweise vor der iib-
licben Bezeichnung der Concentration im Sinne von Normallosungen den Vorzug
verdient.

250 Jacques Loeb und William .1. Gies:!!

da durch Anionen keine antitoxischen Wirkunpen hervorgerufen
werden konnten.

Die beschrankte Dauer der Laichzeit erlaubte Loeb nicht, den
Gec;enstand zu erschopfen, und so war es nothip, diese Versuche
dieses Jahr weiter zii fiihren. Die Auswabl der einzelnen Probleme,
iiber die wir ini Folgenden berichten, rlihrt von Loeb her, die Aus-
fiihrung der Versuche fiel Gies zu, dieselben wurden aber von
Loeb genau verfolgt, so dass die folgenden Ergebnisse fast alle von
uns beiden verificirt sind. Die Veisuche wurden in Woods Holl
ausgefiihrt,

n. Ueber die Gegenseifi|?keit der eut^iftenden Wirkuug zweier

Elektrolyte.

In seiner fruheren Mittheilung hatte Loeb bereits die Frage
aufgeworfen, ob es aueh moglich sei, eine giftige Losung eines
Caleiumsalzes durch Zusatz eines Salzes niit einwerthigeni Ration zu
entgiften. Er fand, dass das niit Salzen von K und M^4 gelang,
dagegen nicht niit Salzen von Li und Na'). Wahrend man also
eine giftige NaCl-Losung durch kleine Quantitaten eines Caleium-
salzes entgiften kann, kann man eine Calciumchloridlosung durch
Zusatz eines Natriumsalzes nicht entgiften. Wohl aber ist das durch
Kalium- und Aunnoniumsalze nioglich, aber nur, wenn man ausser-
ordentlich grosse Quantitaten der letzteren anwendet. Loeb fand,

dass in einer -j- Ca(N03)2-Losung die frischbefruehteten Fundulus-

o

eier im Allgemeinen keineu Embryo zu bildeu im Stande sind. Urn
100 ccm einer solchen Losung zu entgiften, waren 2 — 4 ccni einer
2V2 m KCl-Losung noting, d. h. die Quantitat der toxischeu und
antitoxischen Substanz mussten von fast derselben Grossenordnung sein.
Bei der Entgiftung einer Kl-Losung durch Ca(N03)2 konnte die anti-
toxische Substanz weniger als ein Tausendstel der toxisehen betragen !
Auch diese Thatsache ist nur verstandlich unter der Annahme, dass es
sich hierbei nicht um directe Wirkungen der beiden Elektrolyten
auf einauder, sondern um gemeinsame Wirkungen auf eine im Ei
enthaltene Substanz handelt, wobei das zweiwerthige Ration im All-
gemeinen eine viel grossere und entgegengesetzte Wirkung hat wie

1) Loeb, Americ. Journal of Physiology vol. 6 p. 411. 1902.

Weitere TJntersuchungen iiber die entgiftenden lonenwirkungen etc. 251

das einwerthige Ration. Aehnliclie Erfahrungen machten wir in
Bezug auf Magnesiurasalze. Es lag uns daran, diese Erfahrungen
zu erweitern.

Wir wahlten dazu ein sehr giftiges Salz, namlich ZnS04. In

5
einer ^ m NaCl-Losung entwickelt sieh niemals ein Fundulusembryo,

o

wenn die Eier nicht allzulange nach der Befruchtung in die Losung

5
gebracht werden. Setzt man zu 100 ccm einer -^ m NaCl-Losung

4 Oder 8 cem einer ^^ ZnS04-Losung, so entwiekeln sich eine grosse
Zahl von Eiern. In einem besonderen Versuche bildeten in 100 ccm
^ m NaCl -I- 4 ccm ^ ZnS04 26 *^/o aller Eier Embryonen, wahrend

in 100 ccm ^ m NaCl -|- 8 ccm — ZnS04 84 "/o der Eier Em-

O Oil

bryonen bildeten. In normalem Seewasser bildeten ea. 46 '^lo der

Eier derselben Cultur Embryonen. Die antitoxischen Wirkungen

dieser Dosis ZnS04 sind also gegentiber der grossen toxischen Wirkung

5
der -^ m NaCl-Losung ganz erstaunlich. Es lasst sieh nun zeigen,

dass in diesem Falle die Zinksulfatlosung nicht nur die giftige
Wirkung der Kochsalzlosung aufhebt, sondern dass auch umgekehrt
die Kochsalzlosung die giftige Wirkung der Zinksulfatlosung aufhebt.
Die Eier von Fundulus entwiekeln sieh namlich, wie schon erwahnt,
in destillirtem Wasser ebenso gut wie im Seewasser. Fiigt man

aber zu 100 cem destillirtem Wasser 4 ccm (oder 8 ccm) einer -^

ZnS04-Losung, so vermag auch nicht ein einziges Ei einen Embryo
zu bilden. Das Zinksulfat ist also in der Concentration, in welcher
es als Gegengift gegen das Koehsalz angewendet wurde, ein Gift,
das die Entwieklung der Eier des Fundulus absolut unmoglieh
macht und das letztere raseh todtet. Wir suchten nun festzustellen,
was die minimale Dosis vou Koehsalz ist, welche die giftige Wirkung
des Zinksulfats in der oben erwahnten Concentration vollig aufhebt.
Wir verfuhren so, dass wir zu je 100 ccm einer Kochsalzlosung von

versehiedener Concentration 4 cem oder [S ccm einer -^ ZnS04-

Losung zusetzten und den Procentsatz der Eier bestimmten, welehe
Embryonen bildeten.

252 Jacques Loeb and William .1. Gies:

Tabelle I.

rroceutsatz d. Eier.
Natur der Losung welche Embryonen

bildeten

100 ccni destillirtcs Wasser 49 "/o

m
82

100 „ dcstillirtes Wasser 4 8 ccni !^ ZnSO^ 0 °/o

111

100 „ III Natl + 8 ccni ^ Zn804 1 "/o

7 in

m ., -^ m „ + 8 „ 32 . «"'»

100 „ A ,« „ + 8 „ j| , 80/0

100 „ A »< „ ^ '^ . I n 290/0

100 „ -!,« „ + 8 „ ^ „ :^40/o

100 „ 1 m „ + 8 „ I „ 37%

100 „ -|- 7» „ + 8 „ g „ 38«'/o

100 „ Im „ + S „ g „ 440/0

100 ., 1,« „ +8 „ I „ 80/0

100 „ Im „ + 8 „ g „ 30/0

100 „ ^m „ + 8 „ g „ 0»/o

Es ist also klar, dass von einer jiewissen Concentration an NaCl
die Giftwirkunji von ZnS04 aufzuheben ini Staude ist. Das Optiniuni
der antitoxischen Wirkuug des Kochsalzes wurde erreicht in einer

Losung von ]i>() ccm -x- NaCl 4- s ccni . ZnS04. In dieser

Mischung konimen auf ein Molekiil ZnSOi 50 Molekiile NaCl. Uni

aher 1(.)U ccm einer - m XaCl zu entgiften, waren nach den friiheren

Versuchen von Loeb ca. 2—4 ccm einer -7 ZnS04-Losung nothig.

04

Wahrend also 1 Molekul ZnS04 fiir die Entgiftung von 1(»(JU Mole-

kiilen Kodisalz ausreicht, sind iimgekehrt .jO Molekiile Kochsalz zur

Entgiftung von 1 Molokill ZnS04 erforderlich ! Das zeigt schlagend

die Zunahme der antitoxischen Wirksamkeit eines Rations mit seiner

Weitere Untersuchuiigen iiber die eutgifteudeii loneuwirkungen etc. 253
Werthigkeit ^). Unsere Tabelle zeigt ferner, dass, wenn die Con-
centration der Kochsalzlosung hoher wird als -^ m, der Procentsatz

o

der sich entwickelnden Eier wieder abninimt, offeubar, weil jetzt
Kochsalz im Ueberschuss zugesetzt wird und das Zinksulfat die
giftigeu Wirkungen des Kochsalzes nicht mehr aufzuheben vermag.
Das war uach den frtiheren Beobachtuugen Loeb's zu erwarten,
da derselbe gefunden hat, dass die zur Entgiftung von lOU ecm einer
Kochsalzlosung nothige minimale Menge von Ca(N03)2 mit der Con-
centration der Kochsalzlosung zunimmt.

Die in der Tabelle I erwahnten Versuche wurden wiederholt,
und urn die Constanz der Resultate zu zeigen, wollen wir Tabelle II
hier anfuhren:

Tabelle 11.

Proceutsatz der Eiei-,
Natur der Losung welche Embryonen

bildeten

100 ccm destillirtes Wasser^) 58 "/o

100 „ „ „ + 8 ccm ^ ZnSO^ 0 o/o

100 „ -I m NaCl + 8 ccm ^ ZnS04 70 'Vo

100 „ i-m „ + 8 „ I „ 39%

100 „ Im „ + 8 „ I „ 60/0

100 „ ^m „ + 8 . i . O'/o

100 „ Im „ + 8 „ g „ O'Vo

Wir schritten nun zur Untersuchung der Frage, ob die anti-
toxische Wirksamkeit der Salze mit einwerthigem Kation (z. B. Li,
K, NH4) gegen ZnS04 von derselben Grossenordnung sei wie die
von NaCl. Loeb hatte friiher gezeigt, dass zur Aufhebung der Gift-
wirkungen der Chloride, Nitrate oder Acetate von Na, Li, K und NH^
uugefahr die gleiehe, sehr geringe Dosis eines Calciumsalzes nothig ist.
Es zeigte sich in der That eine sehr schone Uebereinstimmung.

1) Dass die Anionen hoberer Wertbigkeit keine antitoxiscben Wirkungen
haben, bat Loeb fruber nacbgewiesen. Pfliiger's Archiv Bd. 88 S. 68. 1901
und Americ. Journal of Pbysiology vol. 6 p. 411. 1902.

2) Wenn nicbt das Gegentheil erwabnt ist, so wiu'de in alien Versucben
und Losungen zwei Mai destillirtes Wasser benutzt.

254

Jacques Loeb und Williimi .1. Gies:

Tabclle III.
Natur dor Losung

Procentsatz der Eicr,

wclcliL' Kmbryonen

l)ildeten

lOU ccin destillirtcs Wassor 68*Vo

lOU
100
100

+ 8 ccm H7J ZnSC),

0»/o

ni
l28

»(
"64

LiCl

8 ccm ^ ZnS04 0 «/o

+ 8

" 32

100 „ -^2 " + ^ » 32

100 ^
100 „

Hi

T6
m

T

+

. + 8 „ ,

0»/o

0°/o

6"/o

21 o/o

100 ccm destillirtcs Wasser 51 "/o

loo

+ 8 ccm jTp Z11SO4

uu „

8 ''

T u

' 32 '^"'

00 „

2
-8'"

n

+ 8

n

32 "

00 „

3

))

+ 8

»

m

32 "

00 „

4

)7

+ 8

n

32 "

00 „

5

J)

+ 8

I)

7H

32 "

00 „

6

8'"

))

+ 8

»

III

32 "

0»/o
33 »/o
64%
46 "/(.
21%
13 <Vo

0%

Talu'Uo IV.
Natur der Losuug

Procentsatz der Eier,

weldio Einbryoneu

bildeten

100 ccm destillirtcs Wasser 58%

100

+ 8 ccm 5j; ZiiS()4.

32

100 „ ^KCl -1 Seen g2 i^nSO^.

100

100

•' 32

16

^^ •' .32

-1 8 —

.00 ,. I , + s „ I

0%
0%
0%
8 "'0
42%
64 «/o

Weitere Untersuchungen tiber die entgiftenden lonenwirkungen etc. 255

Starkere Losungen von KCl wirkten nicht besser, sondern sincl
sehlechter als — - Losungen.

NH4CI war etwas wirksamer. Das Optimum schien erreicht bei
einer Mischung von 100 ccm ^ NH^Cl -f- 8 cem ^ ZnS04, wie die
folgende Tabelle zeigt:

Tabelle IV a.

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten
100 ccm destillirtes Wasser 68 "/o

100 „ „ » + 8 ccm ^ ZnSO^ . Qo/o

100 „ ~ NH,C1 + 8 ccm g ZnSO, O-^/o

100 „ 25g „ + 8 „ 32 „ 00/0

100 „ ^ ;, + ^ " S " I'*/"

100 „ -g^ „ + 8 „ 32 " 4»/o

100 „ -32- „ + 8 >, 32 . 220/0

100 „ -^ „ + 8 „ g „ 67%

100 „ ^ „ + ^ " i " -^Q'^o

Es wurde nun untersueht, ob die giftigen Wirkungen der Zink-
sulfatlosung auch durch Salze mit zweiwerthigem Ration vermindert
Oder aufgehoben werden konnen, und ob in diesem Falle die anti-
toxisehe Dosis nicht kleiner ist, als wenn das antitoxische Salz ein
einwerthiges Ration besitzt. Tabelle V zeigt, dass viel weniger
Ca(N03)2 als NaCl nothig ist, urn die Giftwirkung von ZnSO^
aufzuheben.

Tabelle V.

Procentsatz der Eier,
Natur der Losung welcbe Embryonen

bildeten
100 ccm destillirtes Wasser 49%

100 „ „ „ + 8 ccm ^ ZnS04 0 «/o

100 „ ~ Ca^NOs)^ + 8 ccm g ZnSO, B^lo

1^ " ^ " +8 „ 3^ „ 190/0

05(i Jacques Loel) mid Willinm .T. Hies:

I'rocentsatz der Eier,
Natur der Lbsuug welche Embryonen

bildeten

100 ccm ^ Ca(N03)o + 8 ccm ^ ZnS04 50%

100 „ ^ " + « " S " 50"/"

100 . f « + « " S " 460/0

100 „ ^ „ + 8 „ g „ 390/0

100 „ ^ „ + ^ " S " ^''"^

100 „ -^ „ + 8 „ I Qo/o

Es ist oflfenbar, dass die antitoxische Wirkung des Calciumions
f^egen die Giftwirkung des Ziukions ganz erheblich grosser ist als
die irgend eines eiuwerthigen Kations.

MgClg verhielt sieh dagegeu ganz anders, wie Tabelle VI zeigt.

Tabelle VI.

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten

100 ccni destillirtes Wasser 25 0/0

100 „ „ „ + 8 ccm g ZnSOi O^/o

100 „ ^ MgClg + 8 ccm g ZnS04 0 0/0

100 „ -I- „ + 8 « I . 00/0

100 „ ^ " + 8 . S „ 00/0

100 „ -^ „ + 8 „ I „ 00/0

100 „ ^ « + 8 „ I „ 140/0

100 . -f- „ + 8 „ 3^ „ 10/0

Es ist raoglich, dass MagDesiumsalze mit anderem Anion als CI
audere Resultate geben. Weitere Versuche miissen hieruber an-
gestellt werden. Allein es ist auch zu beachten, dass in alien Ver-
suchen Loeb's an Fundulus nur das Ration fur die antitoxisehen
Wirkungen in Betracht kam, wahrend das Anion keine Rollo spielte.

Was fiir die Aufhebung der giftigen Wirkungen von Zinksulfat
gilt, gilt auch fur Bleisalze. Loeb hatte schon bemerkt, dass die

Weitere Untersuchimgen liber die entgiftenden lonenwirkungen etc. 257

giftigen Wirkimgen von loo ccni einer -^ essigsauren Natriumlosiing

durch Zusatz von ca. 4 ccm ttt essigsaurem Blei aufgehoben werden

konnen. Wir wiederholten diese Versuche mehrfach mit demselben
Resultat und geben hier ein Beispiel:

Tabelle VH.

Procentsatz der Eier,
Natur der Losimg welche Embryonen

bildeten
100 ccm destillirtes Wasser 46 °/o

100 „ -^ CHgCO.Na Oo/o

100 „ destillirtes Wasser + 4 ccm |^ Pb(CH3C02)2 ... 0%

100

))

100

m
" "2"

100

m
" T

64
m
64

+ 8 „ 1^ „ ... 0»/o

CHgCOgNa + 4 ccm ^ Pb(CH3C02)2 23 «/o

+ 8 „ ^ „ 31%

Es ist ricbtig, dass wegen des gemeinsamen Anions die Dis-
sociation des essigsauren Natriums und essigsauren Bleies in den
letzten zwei Losungen der Tabelle verringert ist. Das konnte niog-
licher Weise die Giftigkeit des essigsauren Bleies in diesen Ver-
suchen verringern, kann aber nicht fiir die Giftigkeit des essigsauren
Natriums in Betracht kommen, da die Quantitat des essigsauren
Bleies so verschwindend klein gegen die Quantitat des essigsauren
Natriums ist, dass die winzige Verringerung der Dissociation des
letzteren seine Giftigkeit nicht merklieh beeintrachtigt. Dass das
nicht eine blosse Argumentation oder theoretische Annahme ist,
sondern thatsachlich richtig ist, hat L o e b durch viele Versuche ilber
die Giftigkeit einer reinen NaCl-Losung festgestellt. Es sei daran

erinnert, dass weder kleine noch grosse Mengen von KCl oder LiCl

5
die Giftigkeit einer -^ m - Kochsalzlosung zu verringern im Stande

o

sind , trotz der Verringerung der Dissociation , worauf ja schon in
der Einleitung hingewiesen wurde. Dass aber das gemeinsame Anion
audi nicht fiir die Beseitigung der Giftwirkung des essigsauren Bleies
verantwortlich ist, wird u. A. durch die vorausgehenden Versuche
iiber die Entgiftung von Zinksulfat bewiesen, in welchen die beiden
antagonistischen Salze kein gemeinsames Anion hatten.

258 .lacques Loeb und William J. Gies:

III. Ilahen (li<' Losiin^eii vou Nichtleiteni eiiie antituxisciie

Wirknn^ V

Loeb') hatte in seineu ersten Mittheilunjieii darauf hiiigewieseu.
(lass (lie antitoxischen Wirkunpeu nur von Klektrolyten und wesent-
lich nur von den Kationeu der letzt(Meu ausuehen. Diese Thatsaclio
wai' von Interesse, well sie uioulicher Weise auf eine Bedoutung der
elektiischeu Ladunir der loneu fur die antitoxischen und vielleiclit
audi andere nhysiolo^'ische Vorgiinjie hiuwies. Bei der grosseu Rolle,
welche die P'^lektrolyte in der Constitution und Dynainik der lehenden
Substanz spielen, war es notliig. Niclits unversucht zu lasseu, uin zu
entscheiden, ob die Niclitleitcr thatsachlich ausser Stande siud, die
giftiiieu Wirkuugen eines Salzes zu beseitiuen oder zu verrinaern.
Wenn der Maehweis eines gesetzmjissigen Verlialtens sich auf negative
Hesultate stiitzen muss, wie in diesem Falle, muss die Zahl der
Versuclie viel grosser sein, als wo es sich urn positive Ergebnisse
liiindelt. Wir unternahnien desshalb eine grosse Zahl von Versuclien.
uni sicher zu stellen, dass die giftigen Wirkungen einer Kochsalz-
l(>sung Oder Zinksulfatlosung durch die Nichtleiter Rohrzucker, Ilarn-
stoff, Glycerin und Aethylalkohol nicht verringert werden. Wir
glauben, sagen zu diirfen, dass das zutrifft. Wir woUen
einzelne Versuchsreiheu etwas ausfiihrlicher besprechen.

Wir wahlten als toxische Lbsung luo ccm einer ' m NaCl-

o

Losung und suchten festzustellen, ob Zusatz von Harnstoff diese Liisung
entgiften konne oder weuiger giftig niache.

In der ersten Versuchsreihe werden zu je loO ccm einer ^ ni

o

NaCl-Losung, V'a- 1, -, 4, 8 und 10 ccm einer . - Ilarnstofflosunu'
zugesetzt. Die Giftigkeit der Kochsalzlosung wurde nicht verringert.
Dann wurde statt der .t- Losung eine — - Harnstoff losung gewahlt und

5
V'g, 1, 2, 4, s und 16 ccm derselben zu je loo ccm der ^ m NaCl-

o

Losung zugesetzt, mit wieder ganzlich negativem Resultat. Dann
wurden ^i^, 1, 2, 4, 8, 16 cem einer 3 m-Harnstoff losung zu je 100 ccm

5

g- 1)1 NaCl-Losung zugesetzt, mit wieder vollig negativem Resultate,

1) Loeb, 1. c.

Weitere Untersuchungen iiber die entgiftenden lonenwirkungen etc. 259

und das Gleiche war in einer weiteren Versuchsreihe der Fall, in

der V/g^ 1, 2, 4, 8 und 16 ccm einer 10 *w- Harnstofflosung zii 100 ccm

5

^ m NaCl-Losung zugesetzt wurden. Wir dilrfen also wohl sagen,

dass es unmoglich ist, mit Harnstoflf die giftige Wirkiing einer Kochsalz-

losung zu verringern. Man kann nicht einwenden, dass der Harn-

stoff selbst in den Dosen, in denen er zugefugt wurde, giftig ist.

Denn erstens vernichtete Zinksulfat in einer an sieh giftigen Dosis

die Giftwirkungen von NaCl , und zweitens ist, me Loeb friiher

schon gezeigt hat, das Fundulusei sehr unerapfindlich gegen Harnstoff-

')yh
losung. In einer Yp- Harnstoff losung bildeten beispielsweise ebenso

viele Eier Embryonen wie in normalem Seewasser oder destillirtem

Wasser. Selbst in einer -^ - Harnstofflosung wurden noch einzelne

Embryonen gebildet.

Unsere Versuche, ob Rohrzucker im Stande sei, die toxischen
Wirkungen einer reinen Kochsalzlosung aufzuheben, waren nicht so
voUstandig. Wir stellten nur zwei Versuchsreihen an. In der einen

wurden Vg, 1, 2, 4, 8 und 16 ccm einer -^--Rohrzuekerlosung zu je

o

5
100 ccm -^ m NaGl-Lbsung zugefugt. In keinem Falle bildete sich

o

5
ein Embryo. In einer zweiten Reihe wurden zu je 100 ccm -3- m

o

NaCl V2, 1, 2, 4, 8 und 16 ccm einer 2V2 wz - Rohrzuckerlosung zu-
gefilgt. Auch diesmal wurden keine Embryonen gebildet. Im Hin-
blick auf die sogleich zu erwahnenden Versuche mit Zinksulfat
milssen wir aber die Frage offen lassen, ob mit sehr grossen Dosen
von Rohrzucker nicht am Ende kleine antitoxische Wirkungen zu
erzielen waren.

Es gelang uns auch nicht, durch Zusatz von Aethylalkohol oder

5
Glycerin die toxischen Wirkungen einer -5- m NaCl -Losung abzu-

o

5
schwachen. Wir setzten zu je 100 ccm der -5- m NaCl-Losung V2,

o

1, 2, 4, 8 und 16 ccm einer 095 "o"? 10 m und 20 m - Alkohollosung
zu, ohne jede Spur einer antitoxisehen Wirkung. Glycerin wurde in

E. Pf luge r, ArcMv fur Physiologie. Bd. 93. 18

9j^() Jacques Loeb uml William J. Gies:

— - und 3 »/-Losungen anfjewandt, ohiie dassEDtitoxische Wirkungen

8

beobachtet wurden.

Die Versuclie mit Zinksulfat als toxische Substan/ fielen ebenso
uegativ aiis, mit einer einzigen, aber wic wir glauben, nur schein-
bareii Aiisnahme. Wir benutzten in diesein Versuche als giftige

Losuug 5 ccm einer ~ ZnSOrLosung, welche zu loo ccm HoO zu-

gefiigt wurde. Wir batten ja gesehen und uberzeugten uns von neueni

in jedem der folgenden Versuche, dass, wenn man die ^-ZnS04-Losung

21 fach durch destillirtes Wasser verdiinnt, sie die Entwicklung von
Enibryonen verhindert. Wir batten ferner gesehen, dass, wenn man

statt 100 ccm destillirten Wassers loo ccm einer ^ bis {- »/ NaCl-

o o

Losung zusetzt. die giftigen Wirkungen geriuger werden oder auf-
hdren. Wir versuchten nun, ob auch die giftigen Wirkungen von

5 ccm einer ^-Zinksulfatlosung verhindert werden, wenn man 100 ccm

der Losung irgend eines Nichtleiters zusetzt. Wir setzten in einer

,r , ., , . m m m m m m , ,x . *,-

Versuchsreihe 10(J ccm emer ^^, tjtt, 3—, — , -j-, -r- und >»-Harnston-

t)4 oZ lo o 4 —

zu je 5 ccm der ^ - Zinksulfatlosung. Kein einziges Ei bildete einen

Embryo. In einem analogen Versuch wurden die gleichen Mengen
einer Glycerinlosung statt der Harnstofflosung benutzt. ohne dass
sich ein Embryo bildete. Auch Losungen von Aethylalkohol waren
nicht im Stande, die toxischen Wirkungen des Zinksulfats aufzuheben.
Ganz unerwarteter Weise gab aber ein Versuch mit Zuckerlosung
positive antitoxische W^irkungen, wie aus Tabelle VIII hervorgeht.

Tabelle VIII.

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten
100 ccm destillirtes Wasser 55o/o

100 „ „ , + 5 ccm ^ ZnS04 0 %

100 „ ~ Rohrzucker + 5 ccm ^ ZnSO^ 42 »/o

100 „ 1,» „ + 5 « i " 47<Vo

Weitere Untersuchimgen iiber die entgiftenden lonenwirkungen etc. 261

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten

100 ccm -H- Rohrzucker + 5 ccm ^ ZnSO^ 4 °/o

100 „ ^ „ + 5 „ I „ lo/o

100 „ ^ „ + 5 „ ^ ^ Qo/o

100 ccm einer -^ — -^ - Rohrzuckerlosung waren also im Stande,

die giftigen Wirkungen von 5 ccm einer ^-Zinksulfatlosung fast ganz

aufzuheben. Es ist jedoch zu beriicksichtigen, dass die Rohrzucker-
losung ein Jahr alt war. Wir erhielten aber [mit einer frisch-

bereiteten -^ - Rohrzuckerlosung ebenfalls, wenn auch geringere posi-
tive Resultate. Leider war die Laichzeit von Fimdulus inzwischen abge-
laufen, so dass wir keine weiteren Versuche mehr anstellen konnten.
Wir sind geneigt, anzunehmen, dass der Rohrzucker die Entgiftung
der Zinksulfatlosung durch die Bildung von Zinksaccharaten und da-
durch bedingter Verminderung der Zinkionen zu Stande brachte. Wenn
das richtig ist, so konnen wir allgemein sagen, dass die Nichtleiter
nicht im Stande sind, bei Funduluseiern die toxischen Wirkungen von
lonen aufzuheben, es sei denn, dass sie Verbindungen mit denselben
eingehen und so die Concentration der toxischen lonen vermindern.

IV. Konnen die toxischen Wirknngen eines Elektrolyten durch
H- Oder HO-Ionen anfgehoben werden?

Die Thatsache, dass die antitoxische Wirksamkeit eines Kations
so rasch mit der Werthigkeit desselben zunimmt, bringt diese
Beobachtungen in Beziehung zu den Thatsachen, welche auf einen
ahnlichen Einfluss der Werthigkeit auf die Fallungserscheinungen in
colloidalen Losungen hinweisen. Dieser Einfluss der Werthigkeit
der lonen auf die Fallung suspendirter Theilchen wird von B re dig
anders aufgefasst. Nach ihm ist „der von Lin der und Picton,
Schulze u. A. gefundene Einfluss der Werthigkeit des Kations
wohl auf den grosseren Gehalt an hydrolytisch abgespaltener Saure
mehrwerthiger Metalle zuriickzufiihren" ^). Das machte es nothig,

1) Br e dig, Anorganische Fermente. Leipzig 1901.

18*

2(52 Jacques Loeb und William J, Gies:

zu prUfen, ob nicht die giftigen Wirkungen einer reinen Kochsalz-
losung durch Zusatz von Sauie aufgehobeu werden konnten. Zuiiilchst
wiirden Versuche iii)er die Giftigkeit verschiedener Siluren auf das

Fiindulusei angestellt. Dieselben ergaben, dass in yrr^ und selbst

-^-Losungen von .anorganischen Sauren im Allgenieinen kein
2000 b &

Fundulusei einen Embryo zu bilden vermag. So entwickelte sicli

weder in ^^ HCl noch in ^^ HNO3 ein Embryo. Es macht

den Eindruck, als ob die giftigen Wirkungen der Sauren nicht aus-
schliesslich auf das Wasserstoffion bezogen werden diirften. Wir
woUen eine Versuchsreihe hier anfiihren. Es kam uns in derselben
darauf an, solche Concentrationen zu benutzen, die gerade unter
der in voraufgehenden Versuchen gefundenen Schwelle fiir absolute
Giftigkeit liegen.

Tabelle IX.

Procentsatz tier Eier,
Natur der Lbsung welchc Embryonen

bildeten
100 ccm destillirtes Wasser 33%

100 „ 4^ HCl 270/0

100 . M 340/0

100 „ 4^HN03 270/0

'^ ^ mo " ^^"'°

100 „ 2000 "^'^^^ ^'''

7)1

100 » 4000 " 20/0

100 „ i^HCIOs 30/0

100 « » » ^"/»

100 . 2^H.P0, 00/0

100-4^ " 1"/"

m

100 „ joQ^HgAsO, 20/0

100 " 2^ . 100/0

Weitere Untersuchungen iiber die entgiftenden lonenwirkungen etc. 263

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten

100 ccm Y7^7^K Essigsaure 9 ^/o

100 " » . " • • • • l^"/».

100 „ 2^ Milchsaure 1 o/o

100 « » . » • ■...■ ' i°/o

100 „ jqqq Weinsaure 7 <'/o

100-6^ " 10°^">

10*^ » TTfriA Citronensaure 16 %

4UUU

100-6000 » 210/0

Da bei dem hier angewendeten Grad der Verdiinnimg die

Dissociation ziemlich vollstandig ist, so ist der auffallende Unterschied

in der Giftigkeit z. B. zwischen H3PO4 und H3ASO4 schwer zu ver-

stehen, es sei dann, dass gewisse Anionen bei der Giftwirkung be-

theiligt sind. Allein, da die relative Giftigkeit der Sauren nicht

unser eigentliches Thema ist, so wollen wir uns lieber gleich der

Frage nach den antitoxisehen Wirkungen der Sauren zuwenden. Um

die (allerdings geringe) Moglichkeit einer antitoxisehen Wirkung des

Anions der Saure auszuschliessen, beriutzten wir Salzsaure als auti-

toxische Substanz gegen NaCl. Wir fanden, dass Salzsaure oder

richtiger Wasserstoffionen in den von uns angewendeten Concen-

5
trationen die giftigen Wirkungen einer -^ m - Kochsalzlosung nicht

o

aufzuheben im Stande sind, wie Tabelle X zeigt.

Tabelle X.

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten

100 ccm Seewasser . 47**/o

100 „ |- m NaCl 0«/o

100 „ -|- m „ + V4 ccm ^ HCl 0«/o

100 „ Am „ + V2 , ^ „ 00/0

2()4 Jacques Loeb und William J. Gies:

Procentsatz der Eier,
Natur der LOsung welche Embryonen

bildeten

100 can |- m NaCl + 1 ccm ^ HCl 0 «/o

100 „ |-m „ + ^ " m " ^'''

100 „ I w „ + ^ ^ m " ^"'"

100 „ I m „ + 4 „ — „ 00/0

Loeb hatte bereits mitgetheilt, dass die Hydroxylionen bei
Weiteni nicht so giftig filr das Fuudulusei sind wie die Wasserstoff-

ionen. In ^^-Losungen von KHO bildeten eine Reihe von Eiern

noch Embryonen, wahrend fiir NaHO und Ca(H0)2 die Grenze etwas

niedriger liegt, namlich — . Loeb faud, dass Hydroxylionen die

toxischen Wirkungeu einer Koclisalzlosung nicht aufbeben oder
vermiudern. Wir wiederholten den Versuch ebenfalls mit demselben
Resultat.

Tabelle XI.

Procentsatz der Eier,
Natur der Losung welche Embryonen

bildeten
100 ccm destillirtes Wasser 41 ^lo

100 „ -^ tn NaCI 0 "/o

100 „ -g- m „ + V4 ccm ^ KHO 0 "/o

100 „ Am „ + V2 „ ^ „ 00/0

100 „ A ^ „ +1 „ ^ „ 0%

100 „ |m „ +2 „ ^ „ 0%

100 „ Am „ +3 „ ^ „ Oo/o

100 » |»^ » +4 „ ^ „ 00/0

Wir sehen also, dass die giftige Wirkung einer |- w? NaCl-Losung

o

weder durch HO- nocli durch H-Ionen l)eseitigt werden kann, soweit
uusere bisher angestellten Versuche gehen, imd dass es daher wohleinst-

Weitere Untersuchungen iiber die entgiftenden lonenwirkungen etc. 265

weilen nicht angeht, die antitoxischen Wirkungen, welche durch
Elektrolyte mit mehrwerthigen Kationen erzielt werden, aiif hydro-
lytisch abgespaltene Saure zuriickzufiihren.

V. Weitere Versuche iiber die Entgiftnng von Kochsalzlosuiig
durcli mehrwerthige Metallionen.

Loeb hatte gefunden, dass sehr kleine, aber bestimmte Mengen
irgend eines Salzes mit zwei- oder dreiwerthigem Metall die giftigen
Wirkungen grosser Mengen eines Salzes mit einwerthigem Ration,
z. B. Kochsalz, aufbeben. Die mehrwerthigen Kationen, mit deren
Salzen er bisher antitoxisehe Wirkungen erzielt hat, waren : Mg, Ca,
Sr, Ba, Fe, Co, Zn, Pb, Al, Cr. Negative Resultate erhielt er mit
Kupfer- und Quecksilbersalzen. Wir dehnten diese Versuche weiter
aus und fanden, dass auch die Mangansalze im Stande sind, die
giftigen Wirkungen einer reinen Kochsalzlosung vollig aufzuheben,
dass Nickelsalze nur in beschranktem Maasse derartige Wirkungen
haben. Wir wollen eine Versuehsreihe hier mittheilen.

Tabelle XH.

Procentsatz der Eier,

Natur der Losung welche Embryonen

bildeten

100 ccm Seewasser 48 %

100 „ -I- m NaCl 0»/o

o

100 „ |- m „ +4 ccm ^ MnCla 52 o/o

100 „ |-m „ + 8 „ ^ „ 650/0

100 „ |-m „ + 16 „ ^ „ 340/0

100 „ A m „ + 2 „ ^ NiCla 0%

100 „ A^ „ + 4 „ f „ 50/0

100 „ Am „ + 8 „ f „ Qo/o

Die Versuche wurden wiederholt und bestatigt. Der Umstand,
dass wir Chloride von Mangan und Nickel benutzten, um die Koch-
salzlosung zu entgiften und dass so die Dissoeiaton der Kochsalz-
losung verringert wurde, hat nichts mit dem Resultat zu thun, da, wie
wiederholt erwahnt, erstens der Zusatz irgend eines Chlorids mit

0(;,; Jacqnes Loeb und William Gies;

einwerthipem Kation keine antitoxischen Wirkungen hervorbringt,
und da zweiteus die zugesetzte Menge des Manganchlorids ausser-
ordentlich klein im Verhaltniss zur angewandten Kochsalzmenge ist.
Dieser Puukt ist ubrigens in den friiheren Versuchen von Loeb ein-
gehend gepruft worden. Spuren antitoxischer Wirkung erhielten wir
niit Th(N03)4 und U02(N03)2. In einem Falle wurden zu 100 ccm

I" m NaCl 1 cem ~ U02(N03)2 zugesetzt, und 3"/o der Eier bildeten

Embryonen, Dieser Versuch wurde wiederholt und bestatigt. Durch

Zusatz von 8 ccm von y^ Th(N03)4 zu 100 ccm -^ m NaCl erhielten

wir ebeufalls eine Andeutung einer antitoxischen Wirkung. Aber
alle Versuche, mit Uran- und Thoriumsalzen kraftigere antitoxische
Wirkungen zu erzielen, schlugen fehl.

Es gelang uns auch nicbt, mit Cadmiumsalzen irgend welche anti-
toxische Wirkungen zu erzielen. In Bezug auf Kupfer- und Queck-
silberionen nahm Loeb an , dass dieselben bereits in derjenigen
Concentration todtlich sind , in welcher sie fur die antitoxischen
Wirkungen zur Anwendung gelangen miissen. Ob dasselbe auch fiir
Cadmiumionen zutrifft, vermogen wir einstweilen nicht zu entscheiden.

VI. Schlussfolgerungen.

Die vorliegende Arbeit bestatigt die frtihere Beobachtung von
Loel), dass jede Losung eines Elektrolyten von einer gewissen
Concentration an die Entwicklung des Funduluseies hemmt und das
Ei tbdtet, dass aber diese giftigen Wirkungen im Allgemeinen ganz
Oder theilweise durch Zusatz eines zweiten Elektrolyten aufgehoben
werden konnen.

Die Arbeit bestatigt ferner und liefert neues Material fur die von
Loeb gefundeue Thatsache, dass fiir den Grad der Wirksamkeit
des antitoxischen Elektrolyten die Werthigkeit des Rations derselben
eine grosse Rolle spielt, wenn nicht entscheidend ist; und zwar ist
im Allgemeinen die antitoxische Wirksamkeit zweiwerthiger Kationen
ausserordentlich viel grosser als die einwerthiger. Wahrend bei-
spielsweise ein Molekiil Zinksulfat fur die Entgiftung von loOO Mole-
kulen Kochsalz bei der eben giftigen Concentration des letzteren
ausreichte, waren umgekehrt 50 Molekule Kochsalz fur die Entgiftung

Weitere Untersuchungen iiber die entgiftenden lonenwirkungen etc. 267

von einem Molekiil Zinksulfat bei der eben giftigen Concentration des
letzteren erforderlich.

Unsere Versuche machen es unwahrscheinlich, dass die antitoxischen
Wirkungen von Salzen mit mehrwerthigem Ration durch die in gewissen
dieser Losungen enthaltenen freien Wasserstoffionen bedingt sind.

Unsere Versuche endlich bringen, wie wir glauben, iiberzeugendes
Material dafiir, dass Losungen von Nichtleitern, namlich Harnstoff,
Rohrzucker, Glycerin und Alkohol, keine antitoxischen Wirkungen
auf die Losung eines Elektrolyten haben^ mit der scheinbaren
Ausnahme der Falle, in denen der Nichtleiter (z. B. Rohrzucker)
die Concentration der giftigen lonen durch Bildung schwer dis-
sociirbarer Verbindungen verringern konnte, (z. B. Saccharat-
bildungen).

In Bezug auf die Grundlage fiir die antagonistischen Beziehungen
zwischen zwei Elekrolyten und die besondere Bedeutung der Werthig-
keit und moglicher Weise der elektrischen Ladung der lonen sei an
die friiheren Arbeiten von Loeb erinnert. Derselbe zeigte, dass
zwei verschiedene Annahmen hier zulassig sind. Es ist erstens
moglich, dass die Metalle dadurch wirken, dass sie Verbindungen mit
gewissen Protoplasmabestandtheilen eingehen uud so die Eigenschaften
des Protoplasmas verandern. Oder es ist moglich , dass die lonen,
vielleicht vermoge ihres elektrischen Feldes, auf gewisse colloidale
Losungen in den Zellen wirken und so die Zustande des Protoplasmas
beeinflussen, ohne dass sie chemische Verbindungen mit den Bestand-
theilen einzugehen brauchen, deren Eigenschaften sie andern. Herr
Dr. W. Koch hat neuerdings im physiologischen Institut in Chicago
gefunden, dass (colloidale?) Losungen von Lecithin durch kleine Quanti-
taten eines Elektrolyten mit zweiwerthigem Kation gefallt werden,
nicht aber durch Elektrolyte mit einwerthigem Kation ; und dass sogar
ein Antagonismus zwischen den Salzen mit einwerthigem und zwei-
werthigem Metall besteht, indem Zusatz von Kochsalz (oder KCl etc.)
zu der Lecithinlosung die zur Fallung des Lecithins nothige Menge
eines Elektrolyten mit zweiwerthigem Kation erhoht. Da Lecithin
in allem Protoplasma erhalten ist, so ist immerhin die Moglichkeit
vorhanden. dass die antagonistischen lonenwirkungen zum Theil auf
den Einfluss der Elektrolyte auf den physikalischen Zustand der
Lipoide in den Zellen zuruckzufiihren sind. Was aber auch die
Ursache dieser antagonistischen lonenwirkungen sein moge, das
Wichtigste ist einstweilen der Nachweis, dass sie bestehen, und dass

268 J*cqaes Loeb nod William Gies: Weitere Untersachungen rtc

wir bei alien Veisacben mit Xahrlosungen mit dem von Loeb ein-
^fuhrten BegriflF der pbysiolc^sch aquilibrirten Salzlosungen *) zu
rcdmen haben, d. h. solchen Salzlosungen, bei denen die Giftwirkungen
sich geirenseitig aufheben, welche jeder einzelne Elektrolyt oder jede
einzelne Gnippe von lonen haben wtirde, wenn sie allein in Losung
wazen.

I) Americas Joonial of Physiology vol. 3 p 4M. 1900.

Reprinted from the American Journal of Physiology.

Vol. V. — April r. igoi. — No. III.

30

A NOTE ON THE EXCRETION OF KYNURENIC ACID.

By WILLIAM J. GIES.

\_Fr-07n the Laboratory of Physiological Chemistry, of Colunib-ia University, of the College
of Physicians and Sttrgeons, N'eiif York^

IN their paper on the excretion of kynurenic acid, Mendel and
Jackson showed that substance to be a direct product of proteid
catabolism. They found, further, that excretion of kynurenic acid
accompanied accelerated proteid decomposition, whether this condi-
tion was brought about by fasting, or the ingestion of proteid food in
quantities largely in excess of the needs of the body, or through the
action of drugs. These observers also noted that, in conditions of
ordinary nitrogenous equilibrium, the kynurenic acid in the urine
was greatly diminished or might be entirely absent. ^

The author, in repeating recently some of Mendel and Jackson's
experiments, determined the excretion of kynurenic acid (i) dur-
ing periods of nitrogenous equilibrium; (2) when proteid catabolism
was stimulated, by chemical dosage as well as by excessive ingestion
of proteid substance ; and (3) when proteid catabolism was diminished
by the lack of food. The animal, a healthy mongrel bitch, weighing
15 kilos, was confined in a cage suitable for metabolism work and
given daily, at 9 a. m and 6 p. m., in two equal portions, a diet of 250
gms. of hashed meat,^ 50 gms. of cracker meal, 40 gms. of lard and
700 c.c. of water, containing a total of 9.854 gms. of nitrogen.

The experiment lasted twenty-four days and was divided into three
periods. Throughout the first period, of seven days, normal condi-
tions prevailed and the dog was in almost perfect nitrogenous equi-
librium. During the second period, ten days, the animal was given

1 Mendel and Jackson : This journal, 1898, ii, p. rgo. See also, Mexdel
and Schneujer: Proceedings of the American Physiological Society. This foar-
nal, 1901, V, p. ix.

'^ The hashed meat was prepared in bulk, freed from surplus moisture and kept
in bottles, in a cold storage room, the frozen condition maintaining constancv of
composition.

191

192 William J. Gics.

several large doses of tellurous oxide, a substance which not only
causes slight stimulation of proteid catabolism, but likewise induces
vomiting and loss of appetite.' In the third period, of seven
days, normal conditions were present once more and equilibrium
was restored.

On the morning of the second day of the dosage period, when the
greatest amount of tellurous oxide was administered (0.5 gm. with
the morning meal), all of the food given with it was vomited immedi-
ately. The second half of the daily portion of food was vomited in
the evening also, so that no food was retained that day.- On the
following day twice the usual amount of food was given. All of it
was eaten and retained. Vox the remainder of the dosage period no
gastric disturbances were induced and nitrogenous equilibrium was
restored.

The experimental data^ in this connection are given herewith in
the table on the opposite page.

Nitrogen was determined by the Kjeldahl process ; uric acid with
Ludwig's, ^ kynurenic acid with Capaldi's,^ methods. Uric acid was
determined in combined urines, which were preserved with powdered
thymol ; the figures in the tables were recorded on the last days of
each separate combination. The nitrogen of the daily food was
9.854 gms. The "total nitrogen balance" includes the nitrogen of
the fasces and hair. The nitrogen of the vomit of the ninth day
(10.335 g"is.) was subtracted from the ingested nitrogen of the
period in striking the balance. The total nitrogen in the faeces of
the three periods was 2.374, 5.154 and 3.291 gms., respectively; in
the cast olT hair it was 1.054, 1232 and 1.184 Z"^- The amount of
tellurous oxide given on the first day of the dosage period was 0.5
gm., on the second, o 75 gm. ; on each of the third and fourth, 0.25
gm.; during the remainder of the period, o.i gm. per day. Indican,
determined by the Jafife-Stokvis test,*^ was present in the urine of each
period.

1 -Mead and Gies: This journal, 1901, p. 147.

•' The quantity of nitrogen in the vomit slightly exceeded that of the daily food,
showing that none of the latter had been retained. The excess of nitrogen in the
vomit came from gastric mucus.

^ These results were presented informally at the last annual meeting of the
American Physiological Society.

< Neubauer und Vogel: Analyse des Harns. zehnte Auflage, 1898, p. 820.

'" Capaldi : Zeitschrift tiir physiologische Chemie, xxiii. p. 92.

'' Neubauer und \'ooel : Ibid., p. 166.

A Note on the Excretion of KyniLrenic Acid. 193

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The results of this experiment agree entirely with those obtained
by Mendel and Jackson. It will be seen from the table that, except-
ing traces at the very beginning of the experiment when the dog was
about to enter into equilibrium, kynurenic acid was eliminated only
during the second period and then only on the days when the physio-
logical balance was upset by the circumstances attending tellurium
dosage. When the animal drew upon its own store of proteid, as it
certainly did on the day of vomiting, kynurenic acid in small quantity
was excreted for the first time. On the following day, when fed
more than enough to satisfy its immediate needs, kynurenic acid was
again eliminated. 0\\ the two succeeding days excretion of kynurenic
acid continued ; but it failed to appear when equilibrium was restored.

That the dog was in almost perfect nitrogenous balance during the
second half of the dosage period (five days), when, with the exception
of the trace on the thirteenth day, no kynurenic acid was eliminated,
is evident from the following summary:

Nitrogen excreted :

Urine . . . 46.127 )

Fa.'ces i . . 2.577 / • • • -+9.320

Ilairi . . . 0.616)

Nitrogen ingested 49.270

Nitrogen balance — 0.050

From these figures it is also clear that the increased nitrogenous
catabolism, represented by 3.079 gms. of nitrogen (the " total nitro-
gen balance"), occurred in the first half of the period, during four
days of which kynurenic acid was eliminated in appreciable quantity.
These results indicate, further, that when nitrogenous equilibrium is
completely upset by vomiting, it may sometimes be quickly restored
by proper quantitative feeding.

It seems worthy of note, in this connection, that intestinal putre-
faction, as indicated by the constant presence of indoxyl in the urine,
was normal throughout the experiment. This, since kynurenic acid
was excreted only when metabolism was disturbed, suggests, of
course, that formation of this substance may occur independently of
putrefactive changes in the intestine.- It certainly may be entirely
absent when putrefaction is quite marked.

^ The figures for nitroti;en of faeces and hair represent one-lialf of the totals for
the period. The quantitative elimination of each was constant daily, so that the
al)Ove amounts are almost exact values.

- .See Menuel and Schneider: Loc. cit.

A Note on the Excretion of Kynurenic Acid. 195

In conclusion, attention may be drawn to the fact that uric acid
was eliminated in constant quantity throughout the experiment and
that, therefore, kynurenic acid did not replace it. Excretion of the
latter occurred independently of elimination of the former. The
results recorded here confirm the observations of Solomin,^ and also
those of subsequent workers in this connection.

^ SoLOMiN : Zeitschrift fiir phjsiologische Chemie, 1897, xxiii, p. 497.

Reprinted from the Medical Record, Vol. 59, No.9, March 2, 1901

SOME FACTS REGARDING "UREINE."'
By A. F. CHACE. B.S., A.B.,

AND

WILLIAM J. GIES, Ph.D.,

NEW YORK.

We were amazed, recently, on reading Dr, William
Ovid Moor's account of " The Discovery of Ureine, the
Principal Organic Constitutent of Urine, and the True
Cause of Uraemia," ° to find that such sweeping general-
ities had been based upon methods so defective chemi-
cally. Our faith in the older observations that urea
is the chief organic constituent of urine has been so
complete, and our confidence in the deductions of far-
reaching significance based thereon has been so thor-
ough, that it was impossible, in the absence of real
chemical evidence in favor of Dr. Moor's assertions,
to accept his inferences that "the human urine con-
tains a liquid [!] organic body, in a quantity superior
to urea," and that "this organic liquid (ureine) is the
most characteristic component part of urine."

Dr. Moor began his paper with the statement that
he found the human urine to contain " a large quantity
of some organic substance which gives in a very in-
tense manner " the same characteristic blue reaction
with a solution of ferricyanide of potassium and ferric
chloride that may be observed when morphine and va-
rious alkaloids are treated with a solution of these two

' From the Laboratory of Physiological Chemistry of Columbia
University, at the College of Physicians and Surgeons, New
York.

'^ Communication presented to the Thirteenth International
Medical Congress, Paris, 1900. Published in the Medical
Record, 1900 (Sept. ist), vol. Iviii., No. 9, p. 336.

Copyright, William Wood and Company.

salts of iron, " Numerous and exact investigations,''
he adds, "forced him to the conclusion that none of
the known organic or inorganic components of urine
could account for this intense blue reaction, and that,
therefore, some chemical body, until the present un-
known, must be the cause of this strange phenomenon."
We do not know, of course, how " large " the quantity
of this substance was, nor how " intense" the reaction
it caused ; but the fact that Gautier,' Bouchard,'^ Aduc-
co,' and others have found basic bodies in both nor-
mal and abnormal urine giving this reaction, makes it
seem probable that Dr. Moor's " strange phenomenon "
is to be referred, in part at least, to substances of simi-
lar character dissolved in urine, and not especially to
anything unknown, as he has assumed. There is suf-
ficient reason to believe that each of these observers
was dealing with chemically pure, individual sub-
stances, and that these products were not contaminated
with ureine.

Dr. Moor goes on to say: "It is not surprising that
the existence of a metabolism product of such great
importance should, until the present, have escaped our
knowledge, for every urinary analysis has been made
with the firmly rooted idea that urine is a liquid com-
posed of water and of inorganic solid ingredients"!
In the light of every-day knowledge regarding the
quantity of such organic substances as urea, uric
acid, creatinin, hippuric acid, etc., in normal urine
and their relation to metabolism, this inaccurate state-
ment is wortli considering only because it suggests
Jiow much of fact there is in the ureine story.

In one place Dr. Moor concludes that "this organic
metabolism product of the human body (ureine) be-
longs to the group of alcohols of the aromatic series;
at a temperature of about 80° C. it begins to split into

' Gautier : " Les alcaloides derives des matieres proteiques
sous I'influence de la vie des ferments et des tissus. " Journ. de
I'anat. et de la physiol., iSSi, p. 330.

* Bouchard: '" Ue I'origine intestinale de certains alcaloides
Tiormaux ou pathologiques." Revue demed., 1SS2, p. 12.

^ Aducco : " Sur I'existence de bases toxiques dans les urines
physiologiques." Arch. ital. de Biol., 18SS, p. 203.

several bodies belonging to the class of aromatic oxy-
acids." At another, he states "ureine is a ferment,
which has a potential energ}' of at least 130° C."'
Yet neither of these conclusions is accompanied by
any statement of fact upon which to base them, and
only imagination seems to account for them. Thus,
ureine is said, in this connection, to be ''the princi-
pal [!] cause of the ammoniacal fermentation of urine,
as without its presence urea cannot be decomposed into
ammonia and carbon dioxide." Again, "only a tem-
perature of above 130° C, perhaps 140° C, can split
urea into ammonia and carbon dioxide." All this in
mere dogmatic statement, with no experimental justi-
fication and entirely in opposition to the numerous re-
sults of very laborious work for years by many careful
investigators.

Dr. Moor also says: "Urea, in many respects, is
just as indestructible [does he mean undecompos-
able?] as iron, silver, or any other elements, for the
strongest mineral acids do not decompose [!] it, but
simply combine with it." Is it not true that one of
the properties of all matter is "indestructibility"?
Dr. Moor, however, may have intended to use the
word in a different sense from that customarily ap-
plied to it. If he means to refer to comparative sta-
bility, what of the fact that even dilute hydrochloric
acid decomposes urea? Everybody knows, also, how
quickly urea is broken up when concentrated nitrous
acid, for example, acts upon it.

In all of the unaccountable assumption character-
izing Dr. Moor's paper, there is nothing, however, to
compare with the biological burlesque at the close.
"Without ureine," he says, "all organic matter would
become converted into urea, which would remain in
nature without any use, and thus within a limited
period of time all vegetation and animal as well as
human life would cease."

Truly, as Dr. Moor remarks, "this wonderful or-
ganic fluid," this "mysterious chemical body," ought
to receive very careful study. We ourselves have
been of the opinion, however, that the method by
which it has been made deserves much more attention.

With a view of testing these points experimentally,
we have lately made several preparations of ureine,
not only by the method given in the paper alluded to
at the outset, but also by the improved process out-
lined in Dr. Moor's latest communication.'

Reviewing the method, as improved, the normal hu-
man urine, varying in quantity from 1.5 to 49 litres,
was evaporated at a temperature not above 50° C,
usually at 48°. C, until no more vapor could be seen
arising from the surface of the lluid, even after the
application of Dr. Moor's " indispensable " and "deli-
cate test"; as long as rapid withdrawal of the ther-
mometer from the fluid, following directions, caused
"a puff of vapor ascending from the mercury bulb,"
the evaporation was continued. It stands to reason,
of course, that even though no steam can be seen
arising from the evaporating fluid under these condi-
tions, insensible vapor may continue to form at this
point, and a large proportion of water may be left in
the fluid. Certainly, water cannot be completely re-
moved from a urinary residue at such a low temper-
ature— a matter of importance, bearing not only on the
separation of urea, etc., farther on, but also influenc-
ing the percentage of ureine.

The concentrated fluid, still containing a fairly large
proportion of water, of course, was next kept in an or-
dinary freezing mixture, at 10° below 0° C. for several
hours. (Dr. Moor says: "We reduce the temperature
of the liquid, if possible, to 0° C." The length of
time the mixture is to be held there is not given.)
A heavy precipitate, mostly of inorganic matter,
quickly separated. Absolute alcohol at 10° below
0° C, equal to half the quantity of fluid, was then
added to " facilitate filtration,'"' and this mixture fil-
tered in small quantities (while below the freezing-
point), thus removing most, though by no means all,
of the urinary salts.

The main bulk of urea was thrown out of this fil-

' Moor : " The Discovery of Ureine, the Principal Organic
Constituent of Urine." The Medical Record, 1900 (Sept.
22d), Iviii. No. 12, p. 471.

trate in the form of urea oxalate with powdered oxalic
acid (i gm. for each loo c.c. of urine used), and then
alcoholic solution of oxalic acid — thirty-per-cent solu-
tion— was added " until no further precipitate was
formed." By very careful work at this point we found
that immediately visible precipitation could not be
relied upon as a true indicator of complete separation.
Consequently, in order to avoid excess of oxalic acid
(Dr. Moor says nothing about this) its alcoholic solu-
tion was added in small quantities at intervals of
several hours until the filtrate gave only a delicate
reaction for oxalic acid, with calcium chloride in the
presence of acetic acid. At this stage only a slight
permanent precipitate formed on adding a little more
alcoholic solution of oxalic acid, even after standing
over night. It must not be forgotten in this connec-
tion, however, that urea oxalate is somewhat soluble
even in absolute alcohol. But under these conditions,
with considerable water left in the previously evapo-
rated residue, the alcoholic mixture possessed increased
solvent action. In testing with calcium chloride, the
positive reaction just referred to was doubtless due in
partto the combined oxalic acid in solution in the
form of urea oxalate. This method, even at best,
therefore, certainly does not suffice, as Dr. Moor seems
to assume, for complete and satisfactory removal of
urea.

The filtrate from the urea oxalate was next concen-
trated at 48° C. In a few hours a thick, dark-brown,
oily fluid was obtained. This Dr. Moor has the assur-
ance to speak of as a chemical individual— " ureine "
• — admixed merely with pigment and " some saline mat-
ter." He says that at this point " there is nothing but
ureine, together with coloring matters." He adds that
this ureine " is sufficiently pure to satisfy all exigen-
cies of clinical medicine and of physiology," and then,
inconsistently and with little reason, concludes, "for it
is evident that the presence of some saline matter can-
not influence the qualities of ureine." How, we may
ask, were the other organic substances of the urine
having physiological influence removed, such as indi-

can, aromatic oxy-acids, toxic basic bodies, etc., etc.?
Furtlier, wliat reason is there for believing that the
presence of urinary saline matters cannot influence
the toxicological qualities of ureine— potassium salts
for example?

In the preparation of chemically pure (?) ureine
from this point, following Dr. Moor's recommenda-
tions for making ureine for merely chemical purposes,
the alcoholic solution was "treated successively with
barytes to remove the sulphates and phosphates, with
nitrate of silver to separate the chloride of sodium"!
This addition was made very carefully in eacii case, to
avoid excess of silver and barium, a precaution not re-
ferred to by Dr, Moor. But did all of this result in
removal of inorganic salts contained in the urinary
residue, or did it efifect mainly their transformation?
We think we are safe in saying that the potassium
chloride, for example, still present in ureine was
merely converted into soluble nitrate of potassium
and into insoluble chloride of silver. The latter was
filtered off later, but the nitrate remained in the
ureine, possessing even more toxic influence than the
corresponding chloride. In short, these reagents,
speaking generally, removed radicles, not salts; the
amount of inorganic matter left behind was undimin-
ished, if not increased; and the mixture became in
reality more toxic. We fail to see, therefore, how
chemical purity was approached" by such awkward
steps as these. We purposely followed Dr. Moor's
directions in detail here, however, merely to obtain
results that would be comparable with his. Very
heavy precipitates formed on the addition of the re-
agents, showing how large was the proportion of inor-
ganic substance in the ureine which its discoverer
pronounced " sufficiently pure to satisfy all exigencies
of clinical medicine and of physiology.''

Finally, in the preparation of pure (?) ureine, as
Dr. Moor directs, coloring matters were removed with
mercuric nitrate. Much of the residual urea and other
organic substances, as well as coloring matter, were

precipitated from the mixture by this substance. At
first the precipitate with mercuric nitrate dissolved in
the mixture, but eventually became permanent. In
order to avoid excess of mercury (Dr. Moor gives no
suggestion in this connection), addition of the nitrate
was discontinued when nearly all color was removed
and only very slight precipitation was still obtainable.
The bulkiness of the precipitate at this point, as well
as its appearance, further emphasized the absurdity of
considering the previous fluid anything but a mixture.
The final liquid was decidedly acid in all cases, as all
the filtrates had been from the beginning. According
to the original paper, Dr. Moor neutralized with so-
dium carbonate at this point. " It is advisable then,"
he says, "to add a sufficient amount of sodium carbo-
nate, so as to render the liquid slightly alkaline."
After all this he described ureine as a "very slightly
alkaline, almost neutral," substance. VVas its reac-
tion just what Dr. Moor made it? In his second com-
munication Dr. Moor says, why we do not know, "It
is better not to neutralize subsequently with sodium
carbonate." Consequently, though he does not say
so, ureine would have to be an acid substance, if
purity had been attained by the procedure he has
outlined.

The decolorized ureine was finally evaporated at
48°-5o° C, to remove water, and then was carefully
analyzed qualitatively. Employing customary meth-
ods of separation and detection, "purified" ureine
was found to contain sodium, potassium, ammonia,
phosphate, urea, creatinin, pyrocatechin, phenol, alka-
loidal substances, and nuclein bases. Other urinary
substances were present which we did not attempt
to identify. In spite of the fact that we avoided ex-
ce:s of mercuric nitrate in the process of decoloriza-
tion, we always found mercury in ureine. Doubtless
a soluble organic compound of mercury that had
formed with the nitrate was its immediate source.
Nitrate and oxalic acid, also introduced during Dr.
Moor's process of " purification," were constant con-
stituents. Bluish to brownish amorphous material

separated as the fluid concentrated after decolorization.
Shaken up repeatedly with ether during a period of
three months, ureine separated gradually into four per-
manent layers of different color and varying degrees of
transparency, and the ether itself became slightly yel-
lowish. Samples that had been allowed to stand ex-
posed to the air for three months, deposited crystals
of urea. The mother liquor yielded additional crys-
tals when its temperature was reduced to, and held
at, o° C. Under these conditions the fluid became
semi-solid, so large was the proportion of crystalline
material.

Dr. William A. Taltavall, who has had considerable
chemical experience in this laboratory with urinary
extractive bodies, analyzed several of our preparations
and favored us with some of the qualitative data just
presented. Mr. A. N. Richards, assistant in physi-
ological chemistry, has also given us valuable help in
this connection. We cordially thank both of these
gentlemen for their able co-operation.

In a special series of preparations designed to de-
termine the influence of variations of the method, we
obtained the crude ureine from forty-nine litres of
urine, and then, dividing it into two equal parts, decol-
orized one half with mercuric nitrate, the other half
with plumbic acetate. Since the deductions in Dr.
Moor's first paper were based on the qualities of neu-
tralized ureine, we neutralized with sodium carbonate
one half of each quantity of the ''purified'' ureine.
That is, of the portion decolorized with mercuric ni-
trate, one half was neutralized ('" M — 2 '), the other was
not (" M — I '■), In thesame way, one half of that decol-
orized with plumbic acetate was neutralized (" P — 2 "),
the other remained acid ("P— I "). "M— 2,""P— i,"
and "P — 2 " solidified after evaporation at 45° C. for
about a week, because of crystallization of urea and
inorganic matter. They were unlike in appearance
and hardness; "M — i," a thick oily fluid at this stage,
seemed to contain the least quantity of solid matter,
and to hold the smallest amount of crystalline sub-
stance in suspension.

8

The appended table (results in duplicate) shows the
loss in weight of samples of each of these preparations,
after having been at higher temperatures for prolonged
periods; also the ash of each:

Preparation.

0 s,

Total Number of Days
IN Air Bath at 100-
110° C.

.S-c

3%.

lO^.

2S-

^^

M— I

Grams.
3.90
3.94

6.49
7.63

5.65
6. So

5-12

6.09

Grams.
3-36
3-37

5-51
6.66

5.03
6.02

4.58

5-51

Grams.
2.63
2.64

4-47
5-49

4.16

5-17

3.81
4.70

Grams.
2.25
2.23

4.02

4.82

3-79
4-73

3-53
4-33

Grams.
1. 88
1.96

3.5S
4.10

3-41
4.21

3.08
3.69

Grams.
0 1788

M— 2(neu t ral-
ized).

P— I

.1818

.8137
.9566

.3562
.4231

•7590

.7888

P — 2 (neutral-
ized).

The following figures represent the loss of sub-
stance, at the end of the drying process, both in
amount and in percentage, of original ureine; they
give, also, the proportion of ash in the ureine and in
the final dried residue :

Total Loss of Substance.

Percentage of Ash in :

Preparation.

Amount.

Per Cent.

Original
Ureine.

Final
Residue.

M— I

2.02

1.98

2.gi

3-53

2.24
2.59

2.04
2.40

51.80
50.25

44.84
46.26

39.65
38.09

39.84
39-41

4.51
4.61

12.54
12.53

6.30
6.22

14.82
12.95

9.5I
9.27

22.73
23.33

10.44
10.05

24.64
21.36

M — 2 (neutral-
ized) .

P— I

P — 2 (neutral-
ized) .

These results show at a glance that ureine, as our

qualitative results proved, is not a definite chemical
substance, and indicate that with comparatively unim-
portant modifications of method it varies greatly in com-
position. If it were "a body belonging to the group
of alcohols of the aromatic series," we should expect
to find little or no residue after such vigorous heating
for so long a period. Except that they were viscid
rather tiian fluid, and a little darker in color, some of
the final products were the same in appearance as the
original mixture and seemed little affected by the
heating. If "at a temperature of about 80° C. it
(ureine) begins to split into several bodies belonging
to the class of aromatic oxy-acids," what organic sub-
stance, may we ask, is left behind that is so resistant
to this destructive action of a temperature of ioo°-
110° C? There was, of course, some decomposition
of contained organic products; but, we believe, most
of the loss represented in the above tables was due to
elimination of water. The figures in the tables are
valuable, then, for the suggestion they make that
ureine is a mixture.

In his second communication Dr. Moor says: "If
we add nitric acid to ureine, a solid waxlike mass will
be formed at once, and this is a fact worth remember-
ing, as otherwise one might think that this solid, wax-
like substance was the result of a chemical combina-
tion of nitric acid and urea." In all probability it
was. This waxlike mass could be obtained immedi-
ately with concentrated nitric acid in all of our ureine
preparations. When it was broken up mechanically
in an excess of concentrated nitric acid, and examined
under the microscope, this mass was found to consist
almost solely of urea nitrate crystals. Diluted some-
what, each sample of ureine gave an abundant yield of
crystals of urea nitrate and urea oxalate, with the cor-
responding concentrated acids.

We cannot agree that " it is this constitutent (ureine)
of urine which is the cause of its specific odor." The
longer the period of evaporation in preparing ureine,
we have found, the less distinct is the odor remaining
with it. The ability of ureine "to take up large quan-

titles of oxygen with great facility '' is doubtless
equivalent to the total capacity of its various constit-
uents to do the same. The reaction with potassium
ferricyanide and ferric chloride given by ureine may
be attributed, in part at least, to the reducing sub-
stances we have found in it.

Dr. Moor's statements regarding the amount of
ureine in urine, and also its specific gravity, cannot
be credited. Our own experience in comparative ob-
servations shows that each is determined largely by
the length of the period of evaporation in preparing
ureine. The more prolonged the evaporation, after the
time that vapor no longer can be seen arising from the
fluid, the less is the volume, and the greater the weight,
of residue (ureine).

With respect to the toxicity of ureine, upon which
Dr. Moor lays so much stress, and its consequent in-
fluence in uraemia, little need be said in view of the
chemical facts we have just presented. We tested
this matter, however, in two experiments. In the first,
in a healthy dog weighing 5 kgm,, subcutaneous injec-
tion of 8 c.c. of " purified," concentrated ureine caused
great restlessness, diarrhoea, diuresis, and vomiting
during the first twenty-four hours. Marked local irri-
tation and oedema about the point of injection also
resulted. There were no convulsions at any time; no
suggestions whatever of uraemia. The dog was chloro-
formed on the third day after injection.

In the second experiment, on a lively cat weighing
2 kgm., subcutaneous injection of 4 c.c. of the same
preparation of ureine, after it had been further concen-
trated at 45° C. for forty hours, caused marked local
irritation and was followed at once by restlessness;
later by diuresis, diarrhoea, vomiting, paralysis, and
finally by death in convulsions, nine hours after intro-
duction.

The results of our second agree in the main with
those of the single experiment, reported in detail by
Dr. Moor, on a rabbit with "3^ c.c. of ureine ob-
tained from the urine of a pregnant woman in the ninth
month of her pregnancy." We are unable to say, how-

ever, that any single symptom exhibited in our experi-
ments was due to any one definite chemical compound
in the mixture Dr. Moor terms ureine. The poisonous
action of ureine is doubtless due to the sum of the toxi-
city of the normal urinary compounds contained in it.

The fact has long been known that the normal urine
contains substances of a very toxic character.' Potas-
sium compounds, as all of our readers know, are
prominent among these; but even more poisonous are
the various organic bodies of an alkaloidal nature, pres-
ent in only minute proportion. Dr. Moor's method of
preparing ureine fails to eliminate completely either
potassium salts or the normal basic alkaloidal bodies
giving the typical reaction with potassium ferricyanide
and ferric chloride, and the toxicity ascribed to ureine
must undoubtedly be referred, in part at least, to these
substances dissolved in it. The evaporation process
from the beginning, it is perhaps needless to point out,
causes an accumulation in ureine of these various pro-
ducts possessing toxic influence.

Summing up in a few words: Ureine is not a
chemical individual. It is a mixitire containing
several of the organic substances and a considerable
proportion of inorganic salts ordinarily found in nor-
mal urine. Further, its toxicity can be referred to
some of these normal urinary constituents. Conse-
quently, our knowledge of the cause of uroemia, we
regret to say, has been in no way increased by Dr.
Moor's work on ureine, nor can any of his deductions
regarding the biological significance of this urinary
complex be accepted.

' The latest reference to the matter that we have seen is the
recent paper by Dresbach, confirming the previously accepted gen-
eral fact, without, however, isolating or identifying any active
substances : "On the Toxicity of Normal Urine," The Journal
of Experimental Medicine, 1900, v., p. 315.

Reprinted from American Medicine, Vol. V, No. 5, pages
175-176, January 31, 1903.]

SOME NOTES ON POLLAGCI'S NEW METHOD OF
DETECTING ALBUMIN IN THE URINE.

BY

GORDON LINDSAY, B.S., Ph.G.,

AND

WILLIAM J. GIES, Ph.D.,

of New York Citj".

College of Physicians and Surgeons, New York City.

Methods for detecting "albumen" in the urine have
accumulated so rapidly in recent years that it is fre-
quently a difficult matter to decide which is best adapted
for special clinical purposes. The delicacy of the meth-
ods alluded to is so variable and the number of possible
fallacies connected with the use of each so numerous,
that the difficulties of selection are made all the greater.
Then, too, in the use of the various methods, not a little
confusion results from the fact that many of the tests
show the presence of such amounts of proteid as are of no
clinical importance — such, for example, as are contained
in the normal urinary mucus.

We have recently investigated the utility of Pol-
lacci's new method for the detection of albumin in urine.
The original description apjjeared not long ago in the
Schweizerisclie Wochenschrift filr Cheraie unci Pharmacie
(1901, xl, p, 168). We have not had access to the original
paper but several abstracts ^ agree in giving the follow-
ing facts regarding the method :

Pollacci has made a modified Spiegler reagent with
the composition indicated below :

A. 1 gram tartaric acid "t

5 " mercuric chlorid ^dissolved in 100 cc. water.
10 " sodium chlorid ]

B. Solution A + 5 cc. formaldehyd (40^^ solution).

In applying this solution (B) for the detection of
albumin, Pollacci uses 2 cc. of his reagent and cautiously
adds 3-5 cc. of the urine, as in Heller's test, care being
taken to stratify the solutions and to prevent their
admixture.

"Should a white zone appear at the line of contact

1 Chemist and Druggist, 1902, Ix, p. 82 ; Therapeutic Monthly, 1902,
ii, p 228; Merck's Report, 1902, xi, p. i36, also 237.

of tho two fluids the urine contains pathologic albumin.
If this ring or zone appears slowly, after about 10 to 1 ">
minutes, it indicates the presence of only normal (juan-
tities of albumin." Pollacci established the limits of sen-
sitiveness of the various albumin reagents now in use,
compared with his own, with the following results:

Heat, with acetic or nitric acid 1 in To.WiO

Heller's reagent 1 " V«,0()0

Potassium ferrocyanid and acetic acid 1 " 100,000

Jolles' reagent 1 " 150,000

Roberts' " 1 "300,000

Siilfosalicylic acid 1 " 300,000

SSpiegler's reagent 1 " 305,000

I'ollacci's •' 1 "370,000

It did not appear probable to us that this method
would show only the presence of albumin. We were
inclined to believe that other proteids would be indicated
by it. This belief was fully warranted.

We fintl, as Pollacci states, that the reagent shows the
presence of mere traces of albumin, although it does not
appear to be so delicate as Pollacci's figures would indi-
cate. But we have also observed that the reagent pre-
cipitates minute amounts of other proteids also, such as
globulins, proteoses, mucoids, mucus proteids and even
gelatins. The test has no difl'erential value, therefore,
and the reagent must be regarded as a general proteid
precipitant rather than an albumin detector.

We are also unable to agree with Pollacci that the
proteid normally present in the urine reacts with his
reagent only after a lapse of 10 or 15 minutes. Deduc-
tions drawn from the observed breadth of the " zone "
and from the lapse of time until the ring appears are not
reliable. We have tested numerous samples of urine
from individuals apparently in perfect health and in each
instance, in less time in this connection than that speci-
fied by Pollacci we obtained the white ring at the point
of junction of reagent and urine. That these urines
were normal in this respect was not merely assumed
from the evident good health of the individuals excret-
ing them, but shown experimentally by the fact that
none of them gave positive reactions in Heller's test.

We have not attempted to determine, in this connec-
tion, the responsiveness of alkaloids and other remedial
agents commonly detectable in the urine and frequently
affecting the "albumen" tests. The presence of mer-
cury in the acid reagent makes it probable, however,
that other substances, nonproteid in character, readily
respond to the reagent.

SUMMARY OF COXCLUSIONS.

1. Pollacci's reagent readily precipitates various pro-
teids — simple, compound and albuminoid.

2. The test is too delicate for ordinary clinical pur-
poses, since the normally occurring urinary proteids are
precipitated by the reagent.

3. Various nonproteid substances occurring in the
urine in health and disease are probably also precipitated
by the reagent.

4. The latter possesses little or no advantage over
Spiegler's fluid.

33

Reprinted from " American Medicine," Vol. Ill, No. 10,
page 387, March 8, 1902.

PR0TE0SURIA.1

BY

H. O, MOSENTHAL, B.A.,

AND

WILLIAM J. GIES, Ph.D.,

College of Physicians and Surgeons ;

of JMew York City.

Among the proteid products which occur in the urine
under various conditions, are proteoses. Tlie urinary
proteoses appear to be chemically identical for the most
part with the proteoses formed normally in the gastro-
intestinal tract during the digestion of albuminous
matter. Proteoses frequently appear in the urine when
extensive tissue catabolism occurs, such as takes place in
connection with various fevers, intestinal ulceration,
carcinoma, apoplexy, gangrene, yellow atrophy of the
liver, absorption of pus and of exudates rich in leuko-
cytes, etc. They arise also, occasionally, from spermatic
fluid, and may originate from the food in nephritis, and
as a result of disease of the walls of the digestive tract.
The uriue not infrequently contains proteose during
pregnancy. When proteose passes into the blood from
any cause it is eliminated in the urine.

Before the time of Kiihne's classic researches, pro-
teoses were included in the term peptone. In recent
years, however, more exact chemic differentiation of
primary and secondary proteoses, and peptones, has
taken place and it seems highly probable that the urin-
ary peptone of the earlier observers was in reality
deuteroproteose. Many researches in the last decade
have demonstrated that deuteroproteose is frequently
found in the urine in disease, but that true peptone
occurs only rarely and apparently only in association
with deuteroproteose. In no known case has more than
5 grams of poteose been eliminated in 24 hours. The
quantity is usually much less.

1 We use the generic term " proteosuria " in preference to " albumo-
suria " merely because in these urinary conditions more than one type
of proteose is eliminated. The generic term, therefore, is the more
accurate, unless urine containing only albumose is referred to.

The use of tho term "peptonuria" in conneetion
with the proteoses of the urine is not only inaceurate, in
the ii^lit of our present kno\vled<?e, but eonfusing as
well. It should be restricted to the oeeurrenee of true
peptone as we now under.-tand the term.

In addition to the various proteoses, another substanee
of similar (lualities, known as histon, sometimes appears
in the urine, which was doui)tless also formerly' deteetetl
and desi«i:nated i)eptone. Histon lias been detected in
the urine in eiuses of peritonitis, i)neumonia, erysipelas,
scarlet fever and in lymphemia. " Bence Jones' i)ro-
teid," which repeatedly appears in the urine in associa-
tion with multiple myelomas of the bones, and which
for a lonj; time was regarded as albumose, is in reality a
coagulable substance. Recent researches have shown
tliat it is not a proteose. Its exact nature is still unde-
termined.

Numerous methods for the detection of the proteoses
and other proteids in the urine have recently been sug-
gested. Freund ' has lately communicated a "method
for the detection of peptone in the urine and feces."
Freund shows throughout his paper, however, that he
has taken the usual liberty with the term pei)tone. He
seems to have had proteose in mind, not peptone.

His method for the detection of proteose is very sim-
ple, and may lie summarized as follows: 10 cc. of urine
is tirst acidified with 2-3 drops of tf acetic acid, and
then treated with 20 /c neutral or basic lead acetate — occ.
The milky mixture is thoroughly boiled and the i)recipi-
tate of proteid, inorganic matter, etc., is filtered off.
'I'he tiltrate is next treated with [)()tassiuni hydroxid as
long as a j)recipitate of lead hydroxid continues to
form, when the mixture is again boiled for a moment or
two. The tiltrate, it is claimed, is entirely free from urobi-
lin, and c >ntains a little more than 90^^ of the proteose
originally present in the urine. The presence of the pro-
teose in this tiltrate may finally be detected with the biuret
reaction. The filtrate is always water-clear, says Freund,
jtigments such as uroerythrin, uro-l)ilin, bilirubin and
hematoporphyrin being completely precipitated.

All of these results, adds Freund, are obtainable with
proteose-containing feces. He states that in a large num-
ber of experiments with this method, normal feces were
found to be entirely free from proteoses (" peptone ").

Not only is the title of Freund's paper rather mis-

' Knnind : Ccntralbl:ut (iir innore >redicin, 1901, xxii. p. W7.

leading, but his conclusions, also, are hardly warranted.
The method he uses for preparing the final proteose-
containing filtrate does not exclude peptone, and if
gelatin were present, by accident or otherwise, it also
would be contained in the filtrate.^

We have made numerous experiments with urine
and feces to test the validity of Freund's method.
Moderate amounts of various proteids or their concen-
trated solutions were dissolved in, or mixed with, urine
and feces from individuals wlio had been in perfect
health continuously for a long time. The samples thus
prepared, together with the corresponding normal
urines and feces as controls, were very carefully sub-
jected to Freund's method, and the biuret reaction
applied finally as he directs. The normal feces, and the
feces with proteid admixture, were extracted l^or a few
minutes in hot water and the filtrates treated the same
as the urine. Care was taken to effect extraction speed-
ily, so as to i^revent hydration of any contained proteid.
Basic lead acetate was used for precipitative purposes
with both urine and feces.

Positive results were repeatedly obtained by this
method in samples of normal urine which had been
treated with the following substances :

(1) " Witte's peptone " (containing jn-oteoses).

(2) Pure peptones, made by us from tendomucoid,
fibrin and ligament elastin.

(3) Commercial gelatin (containing gelatose).

(4) Pure gelatins, made by us, from tendon, bone, and
ligament.

(5) Pure primary and secondary proteoses, of our own
make, from tendomucoid, fibrin, and ligament elastin.

(6) Aqueous extract of sheep pancreas (containing
nucleoproteid, proteose, and peptone).

(7) Egg albumen : commercial products, also from
fresh eggs, (containing ovomucoid, Neumeister's "pseu-
dopeptone ").

(8) Ox blood (containing seromucoid).

Among the proteid substances which gave negative
results under similar conditions were :

(1) Mucus from the gastrointestinal tract (containing
nucleoproteid and mucin.)

(2) Mucoids from tendon, cartilage and bone.

1 The frequent use of gelatin in solution in the sick-room makes it
highly probable that sometimes small quantities of it by accident get
into the vessels used for collecting urine. Commercial gelatin con-
tains gelatoses. A very slight quantity of gelatin or gelatose will give
a strong biuret reaction.

(;}) Various aTiinial and vegetable albumins and glob-
ulins.

Many of the final filtrates wei-e quite yellowish to rod
in color, contrary to Freund's experience, although in a
majority of cases all of the urinary pigment was removed.
Wlien large excess of blood was present in the first place,
the final filtrate contained soluble, pigmented derivative
of licmoglobin. Further addition of lead acetate, how-
ever, entirely removed it.

The same positive and negative results with nearly all
of the above proteids were also obtained when these sub-
stances were adn)ixed with dog feces. The latter nor-
mally contained nothing that gave a biuret reaction in
the final filtrate. Every sample of normal human feces
tested by us, however, gave a positive result. The reac-
tion was stronger in the presence of the above substances.
Further, the final filtrates were usually highly colored.
Our biuret tests were made on one-half of each portion ;
the other half serving for comparison. " Pei)tone," it is
said, doi's not occur in the feces normally, although it is
probable that peptone, as well as proteose, occasionally
appears in the feces in health, particularly as a result of
the normal bacterial action on undigested proteid such
as muscle fibers or on mucus. Possibly the coloring
matter present accounted for the biuret reaction in the
fecal extracts we examined, just as urobilin in the urine
may affect it.

These results show, we think, that Freund's method
is not a differential i)rocess, and that it cannot be safely
applied to the urine or feces as a peptone test. They
prove that peptones, proteoses and gelatins in urine and
feces may each give positive results with it. They indi-
cate, further, that seromucoid in the urine might also
affect the final reaction.

Since the foregoing was completed we have seen Ito's
paper on the occurrence of true peptone in the urine.
He gives improved methods of detecting proteose and
peptone in urine in tlie presence of each other. (See
J)ei(fsc/ies Arc/iir/nr k/inisc/ie Medicin, 1901, Ixxi, \). 29.)

34

Reprinted from the American Journal of Physiology.

Vol. VII. — September i, 1902. — No. VI.

ON THE QUANTITATIVE DETERMINATION OF ACID-
ALBUMIN IN DIGESTIVE MIXTURES.

By p. B. hawk and WILLIAM J. GIES.

S^From the Laboratojy of Physiological Chemistry of Columbia University, at the College
of Physicians and Surgeons, New York?^

contents!

Page

I. Introductory 460

II. Experimental 464

Preparation of acidalbumin 464

Acids employed (salts formed) 467

Proteoses and peptones used 468

Quantities of solids and fluids taken 469

Precipitation of acidalbumin 469

Experiments 1-3. — Precipitates obtained on neutralizing acid solu-
tions and on boiling the neutral filtrates 470

Experiments 4-10. — Influence of proteoses and peptones, with

variable amounts of acidalbumin 474

Experiment II. — Effect of volume of solution on the precipitation

of equal amounts of acidalbumin 484

Solubility of acidalbumin in saline solutions 485

III. General summary of average results 489

IV. Summary of general conclusions 489

I. Introductory

IN many of the experiments which have been carried out to deter-
mine quantitatively the proteolytic power of pepsin under various
conditions, the chief deductions have been drawn directly from the
amounts of undigested or residual matter rather than from the pro-
portions of the digestive products themselves. In a majority of these
cases the figures for undigested matter have doubtless suggested
approximately correct conclusions in this regard, but it seems prob-
able that, in some instances at least, quantitative studies of the
albuminates, proteoses and peptones formed would have furnished
more accurate and acceptable data.

The writer has recently been engaged in a study of the action
of pepsin under varying degrees of acidity with a number of acids,
and in the presence of different ions, the results of which will be
reported later. In experiments of such character the increasing

460

The Qjtautitative Dctcrmi7iation of Acidalbiimin. 461

or decreasing amounts of acid associated with the pepsin, to say
nothing of its quality, variously affect the proteid indicator, irrespec-
tive of the influence on the latter of the enzyme. Different propor-
tions of acidalbumin would be formed, also, with variations in the
chemical character and physical condition of the proteid used to
test relative zymolysis. If correct comparative deductions are to be
drawn from the results of such experiments, it would seem that de-
termining the amounts of albuminate present in each case would be
almost if not quite as important as ascertaining the quantity of undis-
solved or undigested substance. It is conceivable that in comparative
cases where, for example, the undigested matter might be decreased,
the proportion of acidalbumin formed by the mere solvent action of
the acid might be correspondingly larger. To assume from the fact
of diminished quantity of original proteid, in such an instance, that
zymolysis had been greater in the one case than in the other obvi-
ously would be unwarranted.

In the first of the writer's ion experiments, previously alluded
to, purified fibrin was used as the indicator. At the end of the di-
gestive interval the residue was filtered on a weighed paper and a
given portion of the filtrate carefully neutralized for the precipitation
and quantitative determination of the albuminate. After standing
from twelve to twenty-four hours the precipitate was filtered on a
weighed paper and, after washing and drying, estimated in the cus-
tomary manner. Later, however, it was discovered that boiling the
digestive fluid from which the neutral precipitate had been filtered,
caused a further precipitate, presumably of albuminate, which was
not separable by neutralization in the cold. The amount of this
precipitate seemed comparatively small, but of course, for accuracy's
sake, could not be ignored. The boiled fluid was either permanently
turbid or minute flocks separated from it. The precipitate was
obtained on boiling, in spite of the most careful neutralization of
the digestive fluid. It likewise occurred independently of the char-
acter of the alkali used in neutralizing, the acid associated with the
pepsin, the length of time between neutralization and filtration, and
the volume of the digestive fluid. ^

These facts led us to make a special inquiry into the accuracy of

the neutralization method for directly precipitating and determining

the quantity of acidalbumin in digestive mixtures. This simple

method is desirable and convenient not only for the special experi-

1 See page 4S5 for furtlier reference to the influence of volume.

462 p. B. Hawk a7id William J. Gies.

ments in progress in this laboratory, but also for various other
proteid studies. Further, such separation by neutralization alone is
particularly advantageous in digestive experiments because it can
be made without affecting the associated proteid products. Direct
determinations of any substance, when they can be made accurately,
always possess advantages over indirect determinations.

Very little attention has been given to the quantitative determina-
tion of acidalbumin. In those cases in which its approximate
determination when present among other proteids has been desired,
neutralization in the cold has been effected and then the precipitate
has been filtered as in the writer's experiments just referred to. In
most instances, however, acidalbumin has been determined as a part
of albumin or globulin in the form of coagulated proteid; or, by
reason of small amount or relative unimportance, has been ignored
altogether.

As an example of direct determination quantitatively the process
recently referred to by Effront ^ may be cited. In a general way this
method has been in occasional use for years. In the experiments
by Effront the acidalbumin (" syntonin ") in a fluid mixture of
proteoses, peptone, etc., was precipitated by careful neutralization.
The neutral mixture was allowed to stand for two hours and the
flocculent precipitate which had then separated was filtered on a
weighed paper. Boiling was not a part of the process.

As we have already indicated, boiling the filtrate from the acidal-
bumin obtained in the cold fluid usually yields an additional
flocculent proteid precipitate, an occurrence suggesting that mere
neutralization is not sufficient for effecting separation if particular
quantitative accuracy is desired."^ Several theories to account for
this fact suggest themselves.

It is usually stated that albuminates are insoluble in neutral salt
solutions, although not all observers are agreed on this point.'^ It

1 Effront: Chemisches Centralblatt, 1899, ii, p. 457.

^ Umber, among others, has noted, in cases where only a slight amount of
acidalbumin, or none at all, could be precipitated on neutralizing, that the neutral
filtrate remained clear on boiling, but additional " acidalbumin " separated on
evaporation of the fluid to one-half its volume. See Zeitschrift fiir physiologische
Chemie, 1898, xxv, p. 263.-

^ Heynsius : Chemisches Centralblatt, 1876, p. 807; Morner : Jahresbericht
der Thier-Chemie, 1877, p. 10; Savix : Ibid., 1887, p. 2; Nikoljukin : Ibid.,
p. 5 ; Halliburton : Text-book of chemical physiology and patholog}^, 1891,
p. 128.

The Quantitative Deterniiuation of Aeidalbiiiuiti. 463

may be admitted that these derived proteid substances, and particu-
larly the dried products, are for the most part insoluble in neutral
saline media, but the moist, fresJdy precipitated digestive albuminate
is clearly soluble in solutions of various salts, as we ourselves have
definitely ascertained.^ Consequently, on neutralization of its acid
solutions, a portion of the acidalbumin remains in the salt solution
formed in the process. On boiling the filtrate, however, some of
this last residual portion is precipitated because albuminates are
coagulable by heat in neutral saline fluids.-

It appeared probable, also, that in the experiments in mind the
associated proteoses and peptones exerted solvent action on the
albuminate, thus increasing the retaining power of the solution and
thereby helping to prevent complete precipitation on simple neu-
tralization.'^

We were inclined to believe for a time that carbon dioxide in the
fluid, which might have influenced the indicators (litmus and lacmoid
papers), was driven out on boiling and its possible solvent action
done away with, so that the rest of the albuminate was then thrown
down.*

That the precipitate obtained on boiling was not due to earthy
phosphate impurity in the reagents was definitely ascertained.

As the neutral point is approached in such experiments as these,
it is possible that portions of the albuminate which have already
been precipitated are redissolved and perhaps modified by the dilute
alkali, added drop by drop to the nearly neutral fluid. These dis-
solved portions are not precipitated again in the cold, possibly,

^ See page 487. Most of the statements regarding insolubility of acidalbumin
refer to the dried product prepared from muscle tissue. By many writers these
statements have been accepted as including the acidalbumin formed during pepsin
proteolysis. Acidalbumin prepared from muscle tissue begins to diminish in
solubility in dilute acid, even after standing under water for only a few minutes.
Acidalbumin formed during peptic digestion is very different in this respect, for
its solubility in dilute acid remains essentially the same, no matter how often it
is wa.shed, nor is its solubility altered by drying at 40° C. Even then it is soluble
to a certain extent in dilute salt solution. Myosin albuminate under these con-
ditions is quite insoluble. See pages 464, 472, and 486.

2 We do not say above that all of the albuminate is precipitated on boiling, for
the reasons given on page 474.

8 See page 479.

* The alkaline fluids used in neutralizing were dilute KOH and NaOH. These
naturally introduce some carbonate, no matter how carefully the pure solutions
are handled in such experiments. See page 487.

464 P' B. Hawk and William J. Gies.

because of the lack of acidity or on account of the solvent action of
the increasing quantity of salts formed in the neutralization process.

We are not aware of any combinations of albuminate with proteose
or peptone, nor of transformations of these substances under the con-
ditions of these experiments, which would account for the precipitate
thrown down when the neutral fluid is boiled.^

It seemed desirable, then, to determine the influence of the various
factors referred to and, particularly, to ascertain the proportions
of albuminate lost on neutralizing, as well as the proportion thrown
down on boiling the filtrate from which the neutralization precipitate
had been removed. The facts we have ascertained, bearing on the
sufficiency of the precipitation method of direct determination of
acidalbumin, are indicated in the summaries of our experiments on
pages 470-484.

11. Experimental.

Preparation of acidalbumin. — Two varieties of acidalbumin were
used in these experiments. One was prepared from muscle with 0.2
per cent HCl at room temperature, the other from fibrin with pepsin
— HCl (0.2 per cent) at 40° C.

Acidalbumin /rof?i muscle. — A. Several pounds of fresh, lean meat was finely
minced in a meat chopper and the hash thoroughly washed in running
water for thirty-six hours. After straining the last washings through cloth
the hash was placed in an excess of 0.2 per cent HCl and kept there for
twenty-four hours. At the end of that time the acid extract was filtered
and the " syntonin " separated by neutralization with dilute KOH. The
separated precipitate was redissolved in 0.2 per cent HCl and reprecipi-
tated three times with dilute KOH for the complete removal of impurities.
The final precipitate obtained from the filtered solution was frequently
washed during twenty-four hours by decantation in seven to eight litres at
a time, at first with ordinary water, at last with distilled water. All of the
washings contained substance yielding the biuret reaction and causing very
faint turbidity on boiling. A trace of this substance persisted in the
washings, indicating a slight solubility of the freshly precipitated material

^ The solutions were not sufficiently concentrated for the separation of hetero-
proteose, nor was there any acidity for the precipitation of acroproteose. The
precipitate bore no resemblance to " coagulated " heteroproteose. There is no
reason for believing that d} sproteose separated under these conditions.

The Qua7ititative Determination of Acidalbuniiii. 465

even in water. The precipitate was finally filtered off, spread on a glass
plate in a thin layer and dried in a few hours in warm air at a temper-
ature slightly under 40° C The dried material was eventually ground
to a very fine powder before using. About 50 gms. were prepared.

B. A second preparation from washed meat was made by essentially the
same method as that used for the separation of the first. This prepara-
tion was not dried, but the moist substance after thorough reprecipitation,
washing, filtration, etc., was used in the sixth experiment, as indicated
on page 478. In this preparation, also, the washings, in spite of their
volume and frequency, contained, to the last, a trace of substance separ-
able by boiling. Phosphates were absent from the later washings.

Acidalbumin from fibrin. — C. A considerable quantity of fibrin, which had
been kept in 95 per cent alcohol for some time, was put through a meat
chopper and the alcohol thoroughly washed out in running water. After
the completion of the washing process the fibrin was placed in a mod-
erate amount of HCl (0.2 per cent) containing only a very small propor-
tion of pepsin and was kept at 40° C. for about an hour — until practically
all of the fibrin had dissolved. The amount of pepsin selected was small,
and the period of digestion short, so that the proportion of albuminate at
this stage should be large. The digestive mixture was now brought quickly
to the boiling-point, to destroy the pepsin ; was kept at the boiling-point
for a minute or two, and then immediately cooled to about 25-30° C.
The cold filtrate was next neutralized with dilute KOH, and the iieavy
flocculent precipitate redissolved in 0.2 per cent HCl and reprecipitated
once with dilute KOH. after which it was repeatedly and very thoroughly
washed by decantation in large excess of ordinary water and, finally, in
distilled water. The substance settled quickly and could be washed
repeatedly in twenty-four hours. Even to the last, the washings gave
biuret reactions and became turbid on boiling, just as with the product
obtained from muscle. It seems necessary to conclude that in this case
also the freshly precipitated material was slightly soluble in the water.

The freshly precipitated substance was used in the first experiment, as staled
on page 470.

D. The second preparation of acidalbumin from fibrin by digestive process
was made in essentially the same manner as the previous one. Fibrin
boiled in water, and then extracted in alcohol and ether was used. The
precipitate was washed in about fourteen litres of water frequently during
twenty-four hours. Even to the last, the washings again became slightly
turbid on boiling and on the addition of picric acid. On warming, the
turbidity with the latter appeared to diminish somewhat and to increase

1 The time required for the drying was too short for any perceptible bacterial
changes to have occurred.

466 p. B. Hawk and William J. Gies.

again on cooling, facts indicating the presence of proteose with acid-
albumin.^
The moist substance was finally dried in a thin layer ^ in warm air at a tem-
perature below 40° C. The dried substance was finely powdered. It
weighed 2.5 gms.

E. A third preparation of acidalbumin was made from fibrin by enzyme

action. The fibrin had not been boiled, although it had been thoroughly
washed in alcohol. This sample also was made by the general method
just outlined. The neutralization precipitate was redissolved in 0.2 per
cent HCl four times and as frequently reprecipitated with dilute KOH.
Extreme care was taken to wash thoroughly and frequently. The precip-
itate was whipped up repeatedly in as much as fourteen litres of water
at a time. The final washings in distilled water were almost entirely free
from substance giving the biuret reaction and yielding turbidity on boiling
or on treatment with picric acid. At this point the substance was divided
roughly into two portions.

a. The first portion was dissolved in 0.2 per cent HCl, the solution
diluted with an equal volume of water and filtered. The filtrate was pre-
cipitated with dilute KOH and the proteid, after thorough washing for a
few hours, was spread on a glass plate and quickly dried below 40° C.
as usual. Twelve grams were obtained.

b. The second portion vi^as dissolved in 0.2 per cent HCl, allowed to
stand several hours, without dilution, and then precipitated, washed and
dried as was the first portion. It weighed about 30 gms.

The washings of both portions at first showed an increased content of acid-
albumin. Merely a trace was present in the final washings in distilled
water.

F, The fourth preparation of acidalbumin from fibrin was made from several

hundred grams of the proteid which had not been boiled in water, but
which had been very thoroughly extracted in alcohol and in ether. The
method of preparation was the same as that for the previous products.
The acidalbumin was thoroughly washed in fourteen litres of water eight
times during forty-eight hours. At the end of the process only a mere
trace of coagulable substance was detectable in the distilled water wash-
ings, and in the last two washings no satisfactory biuret reaction could

^ The washing was done frequently during twenty-four hours, and there was
hardly time enough for bacteria to develop and form proteose. It is possible that
in the course of twenty-four hours slight bacterial changes did occur without our
knowledge. The water used in washing all these preparations was kept free of
antibacterial substances so as to prevent possible transformations of the desired
products through such chemical agencies. See page 486.

- Higher temperature was avoided to prevent possible transformation into a
less soluble product.

The Qjiaiititative Dcterminaiioii of Acidalbumin. 467

be obtained without concentration. The moist substance, after it had
been allowed to drain, and after excess of moisture had been expressed
from it through hard filter paper, was used in the experiment referred to
on page 487.
G. A mixture of residues of D and E was used in the eleventh experiment,
described on page 484.

Acids employed (salts formed). — In order to test the precipitation
method as thoroughly as possible the following acids were used :
hydrochloric, nitric, chloric, sulphuric, arsenic, phosphoric, acetic, lac-
tic, oxalic, tartaric and citric. All these were carefully titrated with
standard alkali and appropriate indicators, and made equivalent to
{'^ NaOH.i

In our experiments the acidalbumin was transferred to the acid and, after
solution of the substance, most of the fluids were carefully made neutral
to litmus. Mixtures in which acid salts were formed were tested with
lacmoid paper. Alkali was added to these until all free acid was exactly
transformed to acid salt.

In all cases permanent, bulky precipitates were formed even while some free
acid still remained in the fluid, a fact in harmony with previous observa-
tions by various investigators.'- The maximum effects were obtained at
the neutral point, however, or when free acid was present only in inap-
preciable traces. '

^ ^'^ HCl contains 0.36 per cent HCl, ^'^ HgSO^ contains 0.49 per cent HgSO^,
3" H3PO4 contains 0.33 per cent H3PO4. These strengths of acid are approxi-
mately equivalent to those used in representative peptic digestive experiments.

•^ The salts formed on neutralization help precipitation. The more saline
matter present in the fluid the greater the acidity may be without the exertion of
solvent action on the part of the acid. This fact accounts for the heavy turbidity
observed in some of the fluids, while free acid was still detectable in them. This
precipitation occurred earlier in some than in others, doubtless because of the
different influence of the anions. It appeared in the sulphate solution as quickly
as in any, SO4 seemingly being helpful to the precipitation of acidalbumin.

^ In reprecipitating several of our main products it was observed that when
the solution was carefully carried from acid to exact neutral reaction, the
supernatant fluid over the main bulk of the precipitate remained somewhat milky.
On adding a little more dilute alkali the substance causing the turbidity became
flocculent and settled out ([uickly under a perfectly clear fluid. The latter still
remained neutral to litmus. In our quantitative experiments neutralization was
carried to the point of flocculation in a perfectly clear fluid. This point corre-
sponds very closely with the point of neutralization of acid and alkali. The
filtrates from the neutral precipitates were '• water-clear." See footnote, page 469.

468

P. B. Hawk and William J. Gies.

Proteoses and peptones used. — Witte's peptone was used in all of
the experiments in which we determined the influence of the digestive
products on the precipitation of acidalbumin.

In such experiments weighed amounts of dry Witte's peptone and our acid-
albumins were dissolved in given quantities of each of the acids above-
mentioned. With most of the acids all of the substance comprising
the " peptone " completely dissolved. In others, however, a perma-
nent precipitate was formed, either immediately on admixture or later
on neutralizing. In each case we determined accurately the amounts
of the precipitates of such extraneous matter and made corresponding
corrections in the data subsequently obtained.

The following summary gives our results in this connection, no precipitate as
bulky or as heavy as that in HCl having been obtained with the acids not
mentioned below.

Summary. — loo c.c. of each acid was taken. All were equivalent to f^ NaOH.
Weighed peptone (Witte's) was dissolved in each.-'^ The solutions were
left standing several hours, and then neutralized with dilute KOH, litmus
or lacraoid the indicator — in some cases before boiling, in others while
the fluid was at the boiling point. After neutralization each mixture was
allowed to stand over night and then was filtered. Subsequent boiling of
the neutral filtrate failed to cause turbidity ; neither did longer standing
result in further separation of solid matter.

Amount of

Acid.

Amount of pep-
tone dissolved.

Time of
neutralization.

neutralization pre-
cipitate.

Gram.

Gram.

A. Oxalic

a 0.5

Before boiling

0.0214

b 0.5

After

0.0201

c 1.0

Before "

0.0392

d 1.0

After

0.0376

B. Phosphoric

a 10

Before "

0.0146

b 1.0

After "

0.0158

C. Citric

a 1.0

Before "

0.0091

b 1.0

After

0.0058

D. Tartaric

a 1.0

Before "

0.0118

b 10

After

0.0076

E. Hydrochloric

a 0.5

Before "

0.0032

b 1.0

u a

0.0058

^ Samples of the preparation used in the succeeding experiments. See page 469.

The Quantitative Determination of Acidalbumin. 469

Compounds with calcium antl with phosphoric acid doubtless account for the
greater portion of the above precipitates. Witte's peptone contains both
of these. The lioihng process does not appear to have increased the
(juantitv of tlie precipitated matter, but rather decreased it in most cases.

Quantities of solids and fluids taken. — Unless otherwise stated,
100 c.c. of acid was used for each experiment of a series. The quan-
tities of acidalbumin were usually between 0.05 and 0.5 gm.; of
peptone, between 0.5 and i.o gm.

We pur[iose]y used small amounts of both solids and fluids, believing that the
mctiiod could be tested most effectively by so doing. With comparatively
large quantities of the proteids and fluid, defects of manipulation are apt
to cause appreciable errors. Small quantities are more easily and
accurately handled. The amounts and proportions employed were such
as have figured in the past in typical digestive experiments. In our main
series of experiments we used portions of the same general supplies of
the acids and Witte's peptone throughout. No variations were intro-
duced, therefore, by reason of differences in the character of the mate-
rials used. In all cases where the dry acidalbumin was used we refer
to the product dried below 40° C The amount of water in the " air-
dried " preparations was accurately determined by drying to constant
weight at 100-105° C. in the usual manner and due correction made
as indicated below.

Precipitation of acidalbumin. — The albuminate was first dissolved
in the acids alone as already indicated, or in the acids with their con-
tent of Witte's peptone. The mixtures v.-ere usually allowed to
stand in this condition for an hour or more, when careful neutraliza-
tion was begun and completed as soon as possible.^

The neutralized fluids with their precipitates were allowed to stand
undisturbed until the following morning, when the solid matter was
filtered on weighed papers, washed with water until free of soluble
matter- and eventually dried in the air-bath in the customary
manner. In the summaries farther on we give the corrected final

' The combining power of the "peptone" for the acid did not. as will be seen,
appreciably influence the effect of neutralization. Acid combined by the peptone
was doubtless too slight in amount to be of significance in this connection.

- In only a few instances were the filtrates turbid. By repeated filtration the
solid matter was retained. The washings, also, on several occasions manifested
initial turbidity, but the solid substance of these was likewise held after several
filtrations. As these washings were always neutral it is difficult to account for
the turbiditv on anv other than mechanical grounds.

470 P' B. Hawk ajid William /. Gies.

weights in each case.^ Other matters of method are indicated with
the summaries of each experiment.

The completeness of precipitation of acidalbumin from its acid solutions by
neutralization depends largely on the amount of saline matter present.'^
In our own experiments the acid was dilute and the final content of salt
in the fluid on neutralizing was small, though sufficient for the precipita-
tion of the amounts used. We were careful to carry the addition of acid
to the point of exact neutrality or disappearance of free acid, which
method, by cautious manipulation, was found to give the maximum
amounts of precipitate.^ Various observers, among them recently Spiro
and Pemsel,* have noted the difficulty of completely precipitating
acidalbumin on neutralizing and it is, perhaps, a fact not generally
appreciated.

First experiment. — In this experiment we endeavored primarily to
get an accurate idea of the relative proportion of substance thrown
from the various neutral filtrates on boiling.

Sujumary. — Fibrin acidalbumin of preparation C was employed. Several
grams of the latter was dissolved in a few c.c. of fl^ HCl and 3 c.c. of
this solution added to each of the acids — 100 c.c. {■^ or equivalent
thereto — and also to 100 c.c. HoO. Neutralization was made with dilute
KOH in this and the four succeeding experiments. The neutral filtrate
was brought to the boiling-point and maintained there about a minute.
The gravimetric results are shown in the table on page 471.

All the above filtrates that were obtained after boiling gave the
biuret reaction and yielded slight amounts of proteid substance with
alcohol. '5 This precipitate dissolved in water and gave the proteose
reaction with picric acid. This fact suggested that proteose was con-
tained in the substance in spite of the thorough washing to which it
had been subjected.^ Possibly, however, some of the proteose was
derived from the albuminate on boiling.''

^ See facts regarding precipitates of extraneous matter from the peptone,
page 468. No allowance was made for the ash of the neutralization precipitates.
The quantities of ash were entirely too slight to affect the figures given.

^ See footnote 2 on page 467.

^ The formation of alkaline salts was avoided. See page 467.

* Spiro and Pemsel: Zeitschrift fiir physiologische Chemie, 1898, xxvi, p. 236.

^ A large proportion of this precipitate was inorganic matter.

^ See page 486.

■^ See page 489. The amount of saline matter present in the fluid was quite
sufficient for complete precipitation of the acidalbumin.

TJic Quantitative Determination of Acidalbumin. 471

In this experiment the precipitate obtained on boiling the neutral
filtrate amounted, usually, to from 5 to 25 per cent of the quantity
separated in the cold with dilute alkali. It is to be noted that no
precipitate was obtained, on boiling, from the "control" nor from the
solution of citrate. Further, it will be observed that the amount of
albuminate recovered from the "water control" was greater than
from any other solution. In this slightly acid mixture the amount
of saline matter was comparatively small and solvent action on the
acidalbumin greatly reduced therefore.^

Acid.

Amount of neutraliza-
tion precipitate.

Gram.

Quantity of precipi-
tate on boiling the
neutral filtrate.
Gram.

Sulphuric

Hydrochloric

Nitric

Lactic

Chloric

O.xalic

Phosphoric

Tartaric

Acetic

Arsenic

Citric

Water (control)

0.031
0.031
0.033
0.033
0.035
0.035
0.036.i
0.036
0.036
0.037
0.039
0.040

0.003
0.004
0.003
O.OOS
0.003
0.004
0.003
0.004
0.005
0.002
none

Average

{a) 0036

(,b) 0.003

Ratio, a \ h - \1\\

Second experiment. — In the preceding series we did not know the
e.xact amount of substance taken to begin with. Further, by dissolv-
ing the albuminate in HCl, and distributing it in such solution, we

' It will he remembered that 3 c.c. of f^ HCl solution of acidalbumin was added
10 the water. The very slight proportionate acidity resulting thereby was sufficient
to keep all of the substance dissolved.

472

P. B. Hawk and William J. Gies.

introduced a small amount of the HCl into all of the acids and in the
water, and thus, perhaps, tended to complicate matters. In this ex-
periment we began with accurately weighed amounts of dry substance.

Summary. — 0.2 gm. portions of fibrin albuminate of preparation D were
weighed carefully and transferred to the acids. The weight of this
amount of albuminate at 100-105° ^- ^'^^ found to be 0.181 gm.^

Acid.

Amount of

neutralization

precipitate.

Gram.

Quantity of precipi-
tate on boiling the
neutral filtrate.
Gram.

Total amount

of acidalbumin

recovered.

Gram.

Sulphuric

Oxalic

Chloric

Nitric

Tartaric

Hydrochloric

Lactic

Acetic

Citric

Phosphoric

Arsenic

0.109
0.114
0.114
0.117
0.123
0.126
0.133
0.134
0.135
0.139
0.143

0.018
0.007
0017
0.009
none
0.008
0.001
0.011
none
0.003
none

0.127
0.121
0.131
0.126
0.123
0.134
0.134
0.145
0.135
0.142
0.143

Average

[a) 0.126

[b) 0.007

[c] 0.133

Average total quantity of acidalbumin lost, 0048 gm. = 265 per cent.

Average quantity of acidalbumin lost on neutralizing, 0.055 gm. = 30.4 per cent.

Ratio, a : b = IS : 1.

b — 3.87 per cent of the original acidalbumin and 5.26 per cent of c.

Each final filtrate gave the biuret reaction and, when treated with
95 per cent alcohol, yielded a slight amount of substance, which
appeared to be proteose in part. These and the above results seem
to indicate that some of the original acidalbumin remains in solution.
The proteose probably came from some of the dissolved albuminate
on boiling. The average proportion of substance separable on boil-

^ See footnote 2, page 466.

The Quautitativc Determination of Acieialbnmin. 473

ing was somewhat less in this experiment than in that preceding.
In several cases, however, it was unusually large, probably because
of inaccurate neutralization in the first place. The variations in the
total amounts recovered are slight, and within the limits of unavoid-
able experimental errors, which shows that there are only insignifi-
cant differences in the precipitative influences of the various salts
formed from the acids under these conditions.^

7 /lird experiment. — The preceding experiment was repeated, but
with myosin albuminate. Preparation A was used for the purpose.

Amount of
. . , neutralization
■ ])recipitate.
Gram.

Quantity of precipi-
tate on boiling the
neutral filtrate.
Gram.

Total amount

of acidalbumin

recovered.

Gram.

Hydrochloric 0216

0.006

0.222

Tartaric 0.222

0.008

0.230

Oxalic 0.228

0.009

0.237

Acetic ;. 0.229

0.002

0.231

Nitric 0.229

0.004

0.233

Citric 0232

0.005

0.237

Sulpiiuric 0233

o.oo.s

0.238

Chloric 0.233

0.007

0.240

Arsenic , 0.237

0002

0.239

Lactic 1 0.239

0.008

0.247

. Phosphoric j 0243

0.003

0.246

Average (a) 0.231

(b) 0.005

(0 0236

Average total quantity of acidalbumin lost, 0.039 gm. = 14.2 pei
Average quantity of acidalbumin lost on neutralizing, 0.044 gm.
Ratio. (7 : /5 = 46 : 1.

cent.

= 16. 0 per cent.

{< = 1.82 per cent of the original acidalbumin, and 2.12 per cent

of ^.

Summary. — 0.3 gm. portions of '' syntonin " were carefully weighed and
transferred to the acids. The weight of this quantity of substance after
drying in the air-bath was 0.275 8™- Much of the material failed to
dissolve in the acids, even after twenty-four hours with frequent stirring.

^ This will be found the case in all our experiments. See footnote, page 467.

474 P. B. Hawk and William J. Gies.

Tlie drying had materially affected its solubility. The freshly precipitated,
inoist substance, however, is very easily dissolved in acids much more
dilute than those used here. The sulphuric acid seemed to have the least
solvent action. The washings from the precipitates obtained on neutral-
ization of the hydrochloric and oxalic acid solutions gave respectively
3 and 6 mgms. of substance on boiling. These amounts were included
in those for the neutralization precipitates, given in the table on page 473.

It will be observed that the average amount of precipitate obtained
on boiling is very nearly the same as in the previous experiment, but
that its proportion of the neutralization precipitate is less than be-
fore. The acidalbumin prepared from muscle is less soluble in
neutral saline solution than that obtained from fibrin through the
action of pepsin. The proportion of total substance recovered is
large, 85.8 per cent, but, nevertheless, a slight loss resulted — a fact
doubtless due, in great part at least, to transformation on heating.^

The final filtrates gave typical biuret reactions and precipitates
with alcohol which, when dissolved in water, responded faintly
though distinctly to the proteose reactions with picric acid, potassio-
mercuric iodide, etc. This fact emphasizes the conclusion stated
above, and further convinces us that in the boiling process some
of the albuminate held in solution is converted into proteose.

Fourth experiment. — The preceding experiments made it clear
that a small though appreciable quantity of acidalbumin remains in
solution when the acid holding it is neutralized ; further that, on boil-
ing the neutral filtrate, a part of this remaining albuminate is precip-
itable, whereas the larger portion appears to be converted into
non-coagulable material. We next endeavored to ascertain the in-
fluence of proteoses and peptone on the precipitability of acidalbumin.

Summary. — 0.2 gm. samples of fibrin albuminate, preparation E, portion a,
were u^ed. This amount was equivalent to 0.180 gm. of substance dried
at 100-105° C. to constant weight. The weights of Witte's peptone in
this and subsequent experiments are for substance as it was received in
the original package. The fluid in the first of each pair of experiments
with oxalic and hydrochloric acids (a) was neutralized at the boiling
point, after nearly all of the acid had been previously transformed to
salt ; that in the second .(b) was neutralized as usual before boiling.
Neutralization in all of the others was made as before in the cold.

^ We do not overlook the fact that the usual errors of manipulation might
account for the observed difference between the quantity taken and that recovered.
Our weighings, filtrations, etc., were very carefully conducted, however, and such
errors were reduced to an inappreciable minimum.

The Ouanfitative Determination of Aeidalhimin. 475

Acid.

A. Hydrochloric

B. Oxalic

C. Tartaric

D. Phosphoric

E. Sulphuric

F. Nitric

G. Citric
H. Lactic

1<"

3^^
\b

Average -

Weight of
peptone.

Gram.

0.5
0 5

1.0
1.0

0.5
05

1.0
10

0.5'
1.0

'o.V

1.0

0.5'
1.0

0.5"
1.0

0.5*
1.0

0.5"
1.0

05
1.0

Amount of
neutraliza-
tion
precipitate.*
Gram.

0133
0154

0.129
0.127

0.130
0.127

0.146
0.145

0141
0.146

0.129
0.143

0.145
0.138
0.137

0.149
0.139
0.155

0.153
0.15S
0.147

0148
0.156
0.144

0.161
0.159
0.175

0.152
0.151
0.140

0.151
0.147
0.146

Quantity of
precipitate on

boiling the

neutral filtrate.

Gram.

0.006

none
0.007

0.005

none
0007

0.004

none
0.007
none

0010
0.005

0.008

none
0.006
none

0.001
0.001

(II) none
0 004
0.004

Total

amount of

acidalbumin

recovered.

Gram.

0133
0.154

0.129
0.133

0.130
0.134

0146
0.145

0.141
0151

0.129
0.150

0.145
0.138
0.141

0.149
0.146
0.155

0.153
0.168
0.152

0.148
0.156
0.152

0.161
0.165
0.175

0.152
0.152
0.141

(111)0.151
0151
0.150

Average total quantity of acidalbumin lost, 0.029 gm. — 16.1 per cent.

Average quantity of acidalbumin lost on neutralizing (cold), 0.032 gm. = 17.8 per cent.

Ratio. I : II (for 2 and 3) = 37 : 1.

II (for 2 and 3) = 2.22 per cent of the original acidalbumin and 2.65 per cent of III.

1 Due correction has been made, as indicated &n page 468.

2 These averages do not include any of the figures for a m A and B. They
represent, therefore, the average precipitation under uniform conditions throughout.
See references in this connection on page 474.

47^ P. B. Hawk and William J. Gies.

The final filtrates from those fluids into which peptone had not
been introduced gave the biuret reaction, faintly though distinctly.
It was strongest in the chloride and oxalate fluids. The delicate pre-
cipitate obtained on treatment with alcohol was composed in part of
proteose'. The alcoholic turbidity also was greatest in the chloride
and oxalate filtrates.

These results are in harmony with the preceding in showing slight
losses of albuminate.^ The peptone appears to be without any par-
ticular influence. The quantities of acidalbumin recovered seem to
be below the average in the chloride solution and somewhat above it in
the citrate. These data accord with the facts, however, that acidal-
bumin is fairly soluble in chlorides and less soluble in equivalent
amounts of citrates. The quantity recovered from the citrate solu-
tion has been relatively high in the preceding experiments, also.

A singular occurrence in this experiment, one rather difficult to
account for in the light of the results of succeeding series, was the
fact that all of the cold neutral filtrates which were free from peptone,
failed to yield a further precipitate on boiling. Most of the cold
filtrates containing peptone, on the other hand, gave appreciable
quantities of coagulum.

Fifth experiment. — In this experiment we repeated parts A and B
of the fourth experiment. Fibrin albuminate from the second portion
of preparation E was used.

Summary. — 0.2 gm. portions of the substance ( E, b) were weighed into each
beaker. This quantity corresponded to 0.179 g"^""- ^^ substance dried
to constant weight at 100-105° C.

The general results and conclusions of this experiment are the
same as those of the fourth. It will be observed that second precip-
itates were obtained in only those fluids which had not been previously
boiled. There is no particular difference in the action of the
chlorides and oxalates. The proportion of unrecoverable substance
in this experiment is practically the same as that of the preceding,

^ The amount of saline matter contained in the original albuminate was small.
Portion b of Preparation E, for example, contained only 0.86 per cent ash. It is
hardly possible, therefore, that the loss of substance was due to removal of in-
organic admixture on reprecipitation. All of our albuminate preparations, it will
be recalled, were originally reprecipitated several times and frequently washed
before drying, in which process inorganic matter was very thoroughly removed.
See footnote 2, page 478.

The Ojiantitalive Detcrviiiiatiou of Acidalbiunin. 477

although different preparations of acidalbumin were used. The final
filtrates gave the usual proteose reactions.

Acid.

. f f Quantity of Total
Weight of , '^"^0""' f'f j precipitate on ; amount of
peptone. : "^"'"'.'f^/T '^oil' ng the 1 acidalbumin
V V , prec.pitate.i | ,^^^j^^, ^^^^^^ , recovered.

Gram. Gram. Gram. Gram.

A. Hydrochloric 1 ,

B. Oxalic 1 { 'I

0.5
0.5

1.0
1.0

0.5
0.5

1.0
1.0

0.133
0.128

0.141
0.164

0.154
0.131

0.146
0.145

0.149
0.148

0.152
0.144

none
0.004

none
0.005

none
0.014

none
0.002

none
0.002

none
0.009

0.133
0.132

0.141
0.169

0.154
0.145

0.146
0.147

0.149
0.150

0.152
0.153

Average 1 a

2 ix

3 a

1 b

2 b

3 b

o.s'

1.0

6.6
1.0

(1)0.140
0.145
0.153

0.136
0.156
0,137

(II) none

0.003
0.003
0,012

(111)0.140
0.145
0.153

0.140
0,160
0 149

Average total quantity of acidalbumin lost, 0.029 gm. = 16.2 per cent.

Average quantity of acidalbumin lost on neutralizing (cold), 0.035 gm. = 19.5 per cent.

Average ratio I : II (ii^) = 24 : 1.

The average for II b (1-3) = 3.35 per cent of the original acidalbumin and 4.0 per cent

of the average for III.
Averages. A. 1,0.142; 11,0 004; III, 0.146 gm.
B. 1,0.147; 11,0.002; III, 0.149 gm.

^ See footnote, page 475.

2 As in the previous experiment, a signifies after boiling ; b, before boiling. See page 474.

SixtJi experiment. — The preceding experiment was repeated.
Muscle albuminate was used, instead of the product from fibrin. The
freshly precipitated substance was taken because of the insolubility
of the " air-dried " product.

478

P. B. Hawk and WiUiafn J. Gies.

Summary. — Preparation B was used. A little over 4 gms. of the moist sub-
stance was dissolved in 425 c.c. of each acid, giving about i gm. of the
freshly precipitated material to each 100 c.c. Of this solution, 100 c.c. was
taken, as usual, for each of the four tests of a series. The amount of sohd
substance in the moist syntonin was not determined directly. Dilute NaOH
was used to neutralize the acids ^ in this and all subsequent experiments.

Acid.

Weight

of
peptone.

Gram.

Amount of
neutralization
precipitate.!

Gram.

Quantity of

precipitate

on boiling the

neutral filtrate.

Gram.

Average total

amount of

acidalbumin

recovered.

Gram.

A. Hydrochloric 1 '^
2
3
4

....

0.5*
10

0.054
0.051
0.064
0.066

0.002
none

0.059

B. Tartaric 1
2
3
4

0.5'
1.0

0.066
0.070
0.077
0.079

0.001
none

0.073

C. Phosphoric 1
2
3

4

0.5'
1.0

0.074
0.074
0.081
0.079

0.002
none

0.077

D. Oxalic 1
2
3
4

0.5'
1.0

0.080
0.084
0.089
0.095

0.001
none

0.087

1 See footnote, page

475.

2 The first of each s

aries was neu

tralized before be

iling, the rest aj

?«- the boiling

1 point had been reached.

Each of the final filtrates from the fluids which had not received
Witte's peptone gave delicate biuret reactions and slight precipitates
in alcohol. These possessed proteose qualities.'-^ The biuret reac-
tions, as usual, were strongest in the chloride and oxalate solutions.

! No differences were observed in the effects of the alkalies used in the
neutralization process. The anions of salts of the alkali metals vary somewhat in
their effects. In these experiments, however, their influences have not been
particularly appreciable. See page 467.

2 We cannot believe that a trace of active pepsin adherent to the original acid-
albumin caused the appearance of proteose at this point in all these experiments.
The boiling of the digestive mixture before the first precipitation of the acid-
albumin surely sufficed for the destruction of all of the enzyme. See methods of
preparation, page 465.

The Qua7ititativc Determination of Acidalbuniiii. 479

The comparatively high results for acidalbumin precipitated from
the phosphate and oxalate fluids are doubtless due in great measure
to phosphate and calcium impurities in this particular preparation of
the proteid. The amount of precipitate obtained in this experiment
from the neutral filtrate on boiling is perceptibly less than in any
heretofore. The failure to obtain such turbidity in the peptone
mixtures may mean that the peptone has actually aided complete
precipitation. On the other hand, there is just as much reason for
assuming that the peptone holds the slight quantities referred to in
solution. Only the first fluid of each series — neutralized before
boiling — yielded a second precipitate.

It might be assumed that the peptone aids precipitation from the
fact that the precipitates from the peptone mixtures are slightly
greater here in each case than the precipitates not associated with
peptone. We have just suggested a reason for this. Aside from the
explanation already offered, the extreme difficulty of washing out last
traces of peptone makes us still more doubtful that these slightly
higher figures should be regarded as particularly significant.

We are justified, we think, in concluding from this and the fore-
going experiments that the peptone has little if any constant, appre-
ciable influence. Our further results harmonize with this deduction.

Expcrii)ie7its 7-10. — These experiments were carried out to
ascertain the influence of increase and decrease in the quantities of
digestive albuminate present in the fluids to begin with, the volumes
of the latter remaining the same. The methods of the previous
experiments were followed in detail.

Summary (7). — o.i gm. samples of fibrin albuminate of portion a, preparation
E were used. This amount of substance at 100-105° C. = 0.090 gm.
Results are tabulated on page 480.

Although the quantity of albuminate was reduced in this experi-
ment, compared with the results of those in which 0.2 gm. was used,
little proportionate difference is to be noted in the amount of preci-
pitate obtained on boiling. Appreciable loss of acidalbumin was
observed as usual. Preliminary boiling here did not seem to favor
the highest quantitative precipitation- The process of first separating
the neutralization precipitate and then boiling the filtrate appears to
be best. In all probability preliminary boiling results in increased
hydration.

48o

P. B. Hawk and William J. Gies.

Acid.

Weight

of
peptone.

Amount of

neutralization

precipitate. 1

Quantity of
precipitate on

boiling the
neutral filtrate.

Total

amount of

acidalbumin

recovered.

Gram.

Gram.

Gram.

Gram.

A. Hydrochloric I > ,

0.065
0.068

none
0.003

0.065
0.071

^\%

0.5

0.067

none

0.067

05

0.072

0.010

0.082

Al

1.0
1.0

0.065
0.063

none
0.006

0.065
0.069

B. Oxalic 1 j J

0.071
0.074

none

0.071
0.074

M?

0.5
0.5

0.063
0.068

none
0.004

0.063
0.072

3{?

1.0
1.0

0.067
0.059

none
0.004

0.067
0.063

Average. 1-3-^

B

(I) 0.067
0.067

(II) 0.003
0.001

(III) 0.070
0.068

Average total quantity of acidalbumi

n lost, 0.021 gm.

= 23.3 per cent.

Average quantity of acidalbumin lost

on neutralizing (c

old), 0.023 gm. =

:25.5 per cent.

Average ratio. I : II (5 = 34 : 1.

The average for II b (1-3) = 2.22 pei

cent of the orig

nal substance an

d 2.90 per cent

of the average for III b

1 See footnote 1, on page 475.

2 Se

e footnote 2, on page 477.

Summary (8). — 0.4 gm. samples of fibrin albuminate, portion b, preparation
E, were used. Dried to constant weight, this amount contained 0.358 gm.
substance.

The Ouanfitaiivc DctcTmination of Acidallminiu. 481

1 Weight

Amount of

neutralization

precipitate.!

(Juantity of

precipitate

on boiling the

neutral filtrate.

Total

amount of

acidalbumin

recovered.

Gram.

Gram.

Gram.

Gram.

A. Iljdiochloric 1 | ',

0.2S1
0.284

none
0.009

0.281
0.293

2 S a 0.5
^ 1 /' 0.5

0.2SS
0.321

none
0.005

0.288
0.326

Ka 10
^ \ b 1.0

0.296 ' none
0.280 ' 0.017

0.2%
0.297

/?. Oxalic 1 { J

0.315 none
0.305 0.008

0.315
0.313

,U 1 0.5
^)3 I 0.5

0303 1 none
0312 0.009

0.303
0.321

3 ) .7 1.0
•^ 1 ^ 1.0

0.309 none
0.291 0.014

0.309
0.305

1

Averae;e \-7,-A

B

1

(1)0.292 (11)0.005
0.306 0.005

(111)0.297
0311

Average total quantity of acidalbumi

n lost, 0.054 gm. = 15.1 per cent.

Average quantity of acidalbumin lost

Dn neutralizing (cold), 0.059 gm. =

= 165 per cent.

Average ratio, I : II ^ = 60 : 1.

The average for II /' (1-3) = 1.40 per cent of the original .substance an

d 1.64 per cent

of the average for III b.

^ See footnote 1, on page 475.

2 See footnote 2, on page 477.

In this experiment four times as much acidalbumin was taken as
in the previous one, yet the actual amount of precipitate obtained
on boiling was only slightly increased ; its proportion decreased. As
in the preceding and some earlier experiments, the precipitate
thrown down on boiling was obtained only from those fluids which
had not been heated previous to their neutralization. In all the
final filtrates, biuret reacting substance could be detected — doubtless
proteose formed in the boiling process.

The amount of albuminate recovered from the chloride solutions
was slightly less than from the oxalate, although in the previous ex-
periment, and before that, little difference between the two was
noted. Such differences as have been observed have not been at

482

p. B. Hawk and William J. Gies.

all constant, probably for the reason that the variations are within
the limits of unavoidable experimental error.

The conclusions drawn from experiments 7 and 8 will be found to
hold for the results of the two following^ ones.

Summary (9). — o.i gm. samples of acidalbumin from fibrin were used, por-
tion a, preparation E. This quantity of substance was equivalent to
0.090 gm., dried at 100-105° C.

Acid.

Weight

of
peptone.

Amount of

neutralization

precipitate.!

Quantity of

precipitate

on boiling the

neutral filtrate.

Total
amount of
acidalbumin
recovered.

Gram.

Gram.

Gram.

Gram.

A. Oxalic 1

0.069

0.003

0.072

2

0.069

none

0.069

3

0.5 '

0.067

0.006

0.073

4

1.0

0.057

none

0.057

B. Tartaric 1

0.064

0.004

0.068

2

0.067

0-004

0.071

3

0.5 "

0.068

none

0.068

4

1.0

0.064

"

0.064

C. rhosphoric 1

2

0.072
0.070

0.003
none

0.075
0.070

3

0.5'

0.058

0007

0.065

4

1.0

0.068

0.006

0.074

D. Hydrocliloric 1
2

0.066
0.070

0.004
0.003

0 070
0 073

3

0.5

0.068

0.006

0074

4

1.0

0.068

none

0.068

Average (l-A)-A
£

(I) 0.065
0.066

(II) 0.002
0.002

(III) 0.067
0.068

C

0.067

0004

0.071

D

0.068

0003

0.071

Average total quantity

of acidalbumin lost, 0.021 gm.

= 23.3 per cent.

Average quantity of aci

dalbumin lost on neutralizing (c

old), 0.023 gm. =

: 25.5 per cent.

General average ratio.

I : 11 = 22 : 1.

General average for II

= 3.33 per cent of the origin

il substance anc

. 4.35 per cent

of the gen

eral average for III.

^ See footnote, on page 4

75.

The Quantitative Determination of Acidalbnmin. 483

Summary (10). — 0.340 gm. samples of fibrin albuminate, portion b, prepara-
tion E, were used. This quantity of substance corresponded with
0.340 gm. substance dried to constant weight at 100-105^ C

Acid.

Weight
of

Amount of
neutralization

Quantity of

precipitate

on boiling the

Total

amount of

acidalbumin

peptone.

precipitate.!

neutral filtrate.

recovered.

Gram.

Gram.

Gram.

Gram.

A. Tartaric 1

0.275

0002

0.277

2

0.2-S9

none

0.259

3

d..s"

0.257

0.007

0.264

4

10

0.256

0.009

0.265

B. Oxalic 1

0.257

0.005

0.262

2

0.266

none

0.26^)

3

0.5 '

0.265

0.005

0270

4

1.0

0.264

0.008

0.272

C. Hydrochloric 1

0.274

0.002

0.276

2

....

0.261

0.002

0.263

3

0.5

0.267

none

0.267

4

1.0

0.264

**

0.264

D. Phosphoric 1

0.284

0.004

0.288

2

'.'.'.'. 1 0.279

0.006

0 285

3

0.5 0.272

0.006

0.278

4

1.0 0.268

0.006

0 274

Average {\—\)-A

(1)0.262

(11)0.004

(111)0.266

B

0.263

0.005

0.268

C

0 266

0001

0.267

D

0.267

0.005

0.281

A%erage total quantity

of acidalbumin lost, 0.033 gm.

= 10.9 per cent.

Average quantity of aci

dalbumin lost on neutralizing (c

old), 0.037 gm. =

: 12.2 per cent.

General average ratio.

I : II = 67 : 1.

General average for II

= 131 per cent of the origina

1 substance and

1.85 per cent

of the gen

eral average for III.

1 See footnote, page 47.S.

The percentage of substance recovered on boiling was unusually
low in this and in the eighth experiment, in which larger quantities
of acidalbumin were taken, a fact suggesting that the loss is pro-
portionately greatest with the least amounts of substance.^

' See tables, pages 488 and 490.

484

P. B. Hawk and William J, Gies.

Eleventh experiment. — The foregoing results show that in these
determinations a small though appreciable amount of albuminate in-
variably was lost. The quantity of substance separated on boiling
was slight and approximately the same throughout. Such differ-
ences as were perceptible appeared to depend mainly on the quantities
of albuminate present to begin with. Thus, the proportion of this
precipitate in the hot fluid to that on neutralization in the cold
was usually greater the smaller the amount of albuminate originally
taken.

This result would indicate that the method of neutralization in the
cold is the more satisfactory the larger the quantity of albuminate
involved. On the other hand, because the volumes and quantities of
acid were uniform in all of these experiments, it might be assumed,
that the solvent action of the salts formed was much the same, even
though the amounts of substance used did vary somewhat. For this
reason, also, the total loss of material noted may have been uniformly
slight.

In order to test these points the following special experiment was
carried out.

Summary. — 0.5 gm. samples of preparation G of fibrin albuminate (0.450 gm.
substance dried at 105° C.) were dissolved in different amounts of f'^
HCl, varying from 50 -cc. to 800 c.c. The solutions were allowed to
stand as usual for an hour or two, then were exactly neutralized with
dilute NaOH, as before, and the bulky precipitate permitted to settle
until the following morning. The weights of the acidalbumin recovered
are given below. The filtrates were then brought to the boiling point and
kept there a moment or two. Each became turbid. The turbidity was
least in the smallest volume of fluid and most pronounced in the largest
quantity. The precipitates soon settled under perfectly clear fluid and
were easily filtered off, with the gravimetric results appended.

Volume of
wHCl.

c.c.

Amount of

neutralization

precipitate.

Gram.

Precipitate obtained on
boiling the neutral filtrate.

Total

amount of

acidalbumin

recovered.

Gram .

Total

quantity of

substance

lost.

Gram.

Gram.

Per cent.

50
100
200
400
800

0.399
0.346
0.348
0.312
0.303

0.005
0.012
0.014
0.035
0.041

1.1

2.7
3.1
7.8
9.1

0.404
0.358
0 362
0.347
0.344

0.046
0.092
0.088
0 103
0.106

TJic Quantitative Determination of Acidalbtimiu. 485

The results of this experiment show quite conclusively that, other
conditions being equal, an increasing proportion of acidalbumin is
lost as the volume of neutral fluid ( NaCl here) becomes larger. We
have no doubt it increases somewhat, also, with a rise in the propor-
tion of saline matter and, vice versa, falls in amount with a decrease
in the proportional content of neutral salt. Although the albumi-
nate here was the same in amount throughout the series, an in-
creasing quantity of coagulum was separable from this neutral filtrate,
a result still further emphasizing the fact of solubility of acidalbumin
in cold neutral saline solution.

Each of the filtrates gave the usual biuret and proteose reactions.
The increasing loss of acidalbumin above was seemingly due to the
greater hydration, inevitably induced by boiling, in the larger
volumes.^

In considering the value of this method, therefore, the volume of
the digestive mixture as well as the percentage content of albuminate
and neutral salts cannot be overlooked.

It appeared quite clear from this and each of the previous series of
experiments that at least a small amount of acidalbumin was soluble
in the cold neutral fluids containing it. Further, it was impossible
to recover all of the albuminate used at the beginning of the experi-
ment. It seemed desirable at this point, therefore, to ascertain
definitely the solvent power of the various saline fluids made through-
out these experiments in the process of neutralizing the acids.

Solubility of acidalbumin in saline solutions. — In the first of OUr
special tests of this matter we ascertained merely the solubility of the

1 The larger the volume the longer the time required, with a given flame of
course, to raise the fluid to the boiling-point, and, therefore, the greater the expos-
ure of the soluble substance to hydrating influence. Some hydration must occur
before the solution reaches the boiling-point. In all probability the material
which separates earliest and causes the initial turbidity is hydrated in part as the
precipitate increases with the rise in temperature. Doubtless some of the mate-
rial in solution is also hydrated before it can be precipitated. Perhaps heating
to only 70-80° C. would have resulted in diminished loss of acidalbumin.

It would be natural to inquire in this connection why, on boiling, a small,
fairly constant amount of substance usually remained as a coagulated precipitate,
although hydration of the larger proportion, dissolved in the neutral filtrate,
invariably occurred. The fact, however, that occasionally no such coagulation
was observed, although loss of substance occurred, would indicate that all of the
substance in the neutral filtrate was transformable into hydration products, and
that, perhaps, the sameness of conditions attending the boiling process accounted
for the similarity in the quantitative results. The very short boiling period was
sufficient now and then to effect complete hydration of the dissolved residue.

486

P. B. Hawk and William J. Gies.

dried fibrin albuminate in water and in 0.5 per cent NaCl, with the
following results.

Summary (A). — Finely powdered samples of fibrin albuminate of portions
a and b, preparation E, were used. 0.2 gm. was weighed for each test.
This amount, dried to constant weight at 100-105° C., was equivalent to
o.iSo gm. for portion a, 0.179 gm. for portion b. 100 c.c. of fluid was
used as throughout all but the previous experiment. The mixtures were
frequently stirred. They were allowed to stand over night, then filtered,
etc., as in the previous experiments.

Solution used.

Weight of substance recovered.
Gram.

Portion a.

Portion b.

A. Distilled water a
b

Average

0.1792
0.1779

01786

0.1738
0.1747

0.1743

B. 0.5% salt solution a
b

Average

0.1767
0.1701

0.1734

0.1663
0.1655

0.1659

1
Total substance taken in each

of A and B 0.180
Average loss in A 0001
Average loss in B 0007

0.179

0.005
0.013

On boiling, the aqueous filtrates remained clear; but the saline
fluids became opalescent. Practically nothing seems to have dis-
solved in water. In salt solution, however, a slight loss resulted in
each test. The results with water show, if we grant that the dry acidal-
bumin is practically insoluble in water, that our preparations contained
at most the merest traces of soluble salts or proteoses^- — obviously
not in sufficient quantity to account to any extent for the loss of sub-
stance noted throughout all of our experiments. Consequently this
experiment is particularly valuable in showing that such disappear-
ance of substance as has been noted in all our previous tests has
been due to loss of albuminate itself and not merely to removal of
soluble impurity.

1 See footnote, page 476.

The Quantitative Determination of Aeielalhumin. 487

We next tried the solvent action of the salts formed in the
neutralization of the various acids previously employed ; also the
solubility in water alone and in water containing peptone.

Summary (B). — Preparation F of our fibrin albuminate (moist substance) was
taken. 100 c.c. of various acids used in the preceding experiments
were carefully neutralized to litmus and lacmoid with dilute NaOH, as
already described. As indicated below, some of these neutral fluids were
thoroughly boiled for a few minutes, without material loss by evaporation,
for the removal of carbonic acid gas. Weighed amounts of our moist,
freshly precipitated acidalbumin were transferred to the neutral fluids
(the boiled ones had been cooled). The mixtures were repeatedly
stirred and allowed to stand over night as in all of the experiments.
At intervals samples from the main bulk of the moist precipitate were
weighed into crucibles for the determination of dry solid matter, as
indicated below.'

It seems obvious, from the results on page 488, that acidalbumin is
somewhat soluble in the salts formed on neutralizing acid fluids for
its precipitation. Although practically insoluble in distilled water
the acidalbumin appeared to be slightly soluble in water to which
proteose and peptone had been added. We are not sure, however,
that this result is not due to the solvent action of the saline matter
present as impurity in Witte's peptone. The proportion of the
amounts which dissolved in the cold neutral saline fluids to the total
quantities originally taken is slight, however. With more decided
acidity to begin with, and therefore more salts formed on neutraliza-
tion than was the case in these experiments, doubtless the more
decided would be the solution of substance, and the greater the
quantity recoverable by coagulation.

The data just obtained also indicate that such slight amounts of
carbonic acid gas as remained in the fluids on neutralization had
little or no measurable influence on the results. The proportionate
amounts of substance soluble in and recovered from the fluids which
had been thoroughly boiled before the albuminate was put into them ^
and from v^^hich, therefore, the carbon dioxide had been removed,

' The moist substance was kept in a covered mortar. Before each sample was
removed, the whole mass was thoroughly mixed. Errors caused by the slight
evaporation of water under the circumstances were thus greatly minimized, and
probably made inappreciable.

'^ The second of each series in the summary on page 488.

488

P. B. Hawk and William J. Gies.

Weight of sub-
stance taken.

Dry substance recovered.

Total
average

amount

Precipi-

of sub-

Solution.

Dry

Insol-
uble.

tate from

Ratio

stance

Fresh.

(calcu-

the fil-

Total.

of

lost

lated).!

trate on

b to a.

(calcu-

boiling.

lated).

Gm.

Gm.

Gm.

Gm.

Gm.

Percent.

Gm.

1. Distilled water 2

3.163

1.232

((z) 1.2184

[b) none

1.2184

3.415

1.330

1.3397

none

1.3397

0.004

2. Distilled water with

0.5 gm. peptone

4.250

1.656

1.6822

0.0076

1.6898

Distilled water with

1 gm. peptone

4.456

1.736

1.7685

0.0121

1.7806

-f 0.078 s

3. Chloride

2.491

0.971

0.7834

0.0138

0.7972

1.76

3.240

1.262

1.0552

0.0066

1.0618

0.63

0.187

4. Oxalate

4.574

1.782

1.5796

0.0276

1.6072

1.75

3.178

1.238

1.0634

0.0212

1.0846

1.99

0.164

5. Phosphate

2.701

1.052

0.9008

0.0152

0.9160

1.69

2.482

0.967

0.8618

0.0148

0.8766

1.71

0.113

6. Tartrate

2 480

0.966

0.8350

0.0188

0.8538

2.25

2.682

1.045

0.9275

0.0216

0 9491

2.33

0.104

7. Nitrate

2.911

1.134

1.0176

0.0168

1.0344

1.65

3.678

1.434

1.3136

0.0286

1.3422

2.18

0.095

8. Lactate

3.447

1.343

1.2398

0.0124

1.2522

1.00

3.743

1.458

1.3834

0.0220

1.4054

1.59

0.072

Average-'

1.221

{a) 1.080

{b) 0.0180

1.098

1.67

0123

Average amount of substance lost =

10.07 per cent of that originally taken (a\

^erage)

and 11.39 per cent of th

; insoluble portion (average).

The average amount of precipitate

Dbtained on boiling = 1.47 per cent of th

e orig-

inal albuminate (averag

e) and 1.67 per cent of the average q

uantity

insoluble in the neutral

fluids.

Ratio, a : b = 60 : I.

1 Portions of the moist substance

were taken at the beginning of the expe

riment

and after the third, fifth, and seventh

series. The quantities of fresh materij

d used

for this purpose varied between 17

334 and 3.9908 gms. The percentages

of dry

matter were found to be 38.38, 38.97,

39.20, and 39.29. See footnote, page 476

- The second fluid of each pair th

roughout the series had been thoroughly

boiled

before receiving the albuminate.

3 This figure represents a gain of

substance ; peptone not completely wasl

led out.

See page 479.

* The averages do not include the

figures for the first two pairs of determii

ations.

The Quantitative Determination of Acidalbiimiii. 489

were slightly greater than the others in some cases, but the same or
less in others.

The results above likewise show an appreciable loss of substance
even after the addition of the precipitate obtained from the boiled
filtrate to that previously filtered off. This loss is doubtless due to
formation of proteose in the process of boiling, as seems to have been
the case in all of our previous experiments. It will be seen from the
tabulated data, that this loss occurs in all of the tests, excepting the
water alone and the water with peptone. The actual increase in
amount recovered in the latter case is very probably due to adherent
peptone which was very difficult to wash completely from the bulky
precipitate. Since, also, the amount of solid matter in each quantity
of the moist precipitate was calculated from special determinations of
the dry substance contained in the fresh material, and not ascer-
tained directly, we cannot lay too much stress upon it. All the
results for " dry substance taken " may be a little high or low
by reason of the unavoidable errors which usually accompany
calculated data under such conditions, no matter how careful the
experimenter may be to attend to every detail of manipulation in
the comparative determinations. That appreciable loss occurred
as usual, however, was clearly shown by the proteose content of
the final filtrates.

The greatest losses appear, from the figures, to have been asso-
ciated with the chlorides and oxalates. In our previous experiments,
also, we noted that the biuret reactions in the final filtrates were
usually strongest in the chloride and oxalate fluids.

III. General Summary of Results.

The table on page 490 summarizes the more important average
data of nearly all of our experiments.

IV. Summary of General Conclusions.

We conclude from these experiments that acidalbumin may be
almost completely precipitated from acid digestive mixtures at ordi-
nary temperatures by careful neutralization. The later stages in the
neutralization process should be conducted with particularly dilute
alkali.

The absolute quantity of acidalbumin remaining in such fluids after
neutralization in ordinary experiments is small, its proportion to the

490

P. B. Hawk and William J. Gies.

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The Quantitative Determination of Acidalbumin. 491

main bulk of the albuminate depending largely on the amount of the
latter, also on the volume of the fluid containing it and on the per-
centage of associated saline matter.

Some of this residual portion of acidalbumin may be obtained on
boiling, although in this process the larger part, sometimes all, is
retained permanently in solution, apparently because of its hydration
into noncoagulable forms.

Proteoses and peptones, even when admixed in comparatively large
proportion, do not materially affect the quantitative separation of
the albuminate.

Neutralization at the boiling point does not insure the greatest
quantitative precipitation of albuminate because of the increased hy-
dration thereby resulting. The largest yield is obtained by neutral-
izing in the cold, heating the neutral filtrate and combining the two
precipitates.

Acidalbumin, particularly that formed through the action of pepsin
on fibrin and in the freshly precipitated condition, is somewhat sol-
uble in various saline fluids.

The sodium and potassium salts formed on neutralizing various
common acid solutions appear to exert approximately equal quan-
titative solvent action on the contained albuminate. Only insignifi-
cant differences in solvent power were noted.

Such quantities of carbon dioxide as are present in fluids neutralized
with freshly prepared solutions of potassium or sodium hydroxides
containing ordinary, minute amounts of carbonate, do not appear to
materially affect the quantitative separation of acidalbumin by the
neutralization process.

Reprinted from the American Journal of Physiology.

Vol. VII. — July i, 1902. — No. IV.

EXPERIMENTS TO DETERMINE THE POSSIBLE AD-
MIXTURE OR COMBINATION OF FAT OR FATTY
ACID WITH VARIOUS PROTEID PRODUCTS.^

By E. R. POSNER and WILLIAM J. GIES.

]_Frotn the Laboratory of Physiological Chemistry of Columbia University, at the College
of Physicians and Surgeons, New York^

IN one of the recent papers from this laboratory on the qualities
of connective tissue mucoid, attention was drawn to the " lack
of particular uniformity in percentage composition " of osseomucoid
preparations. Since the analyzed products had been made with the
greatest care, we were led to the deduction " that the mucin substance
of bone varies in composition just as does the glucoproteid from other
sources ... a conclusion not only in accord with our analytic results,
but in harmony, also, with the deductions drawn under similar condi-
tions, for other tissues and products, by various observers." ^

Later, and for the same reasons, we came to identical conclusions
regarding tendomucoid.^ In discussing the suggestion of Chittenden
and Gies,* that possibly " mucin obtainable from tendon is prone to
carry with it a certain amount of some other form of proteid matter
which the ordinary methods of purification are not wholly adequate to
remove," we indicated that there is " no longer any reason to believe
that proteid impurity is responsible for the observed variations." It
was further stated, at that time, that " we know of no other substance
in tendon which would resist the washing treatment and, by mechani-
cal admixture or chemical combination, account for the orderly varia-
tions observed in the analytic series."

After the latter account of our experiments had gone to the printer,^
Nerking's paper on " fat-proteid compounds " reached us. His results

^ PosNER and GiES : Proceedings of the American Physiological Society,
This journal, 1902, vi, p. xxix.

- Hawk and Gies : This journal, 1901, v, p. 415.

3 Cutter and Gies: Ibid., vi, pp. 167 and 169.

■* Chittenden and Gies: Journal of experimental medicine, 1896, i, p. 194.

5 Cutter and Gies : Loc. cit., p. 169 (foot-note).

2,2,2 E. R. Posner ami William J. Gics.

and views made it seem possible that the variations noted in analytic
results for mucoids as well as for other proteids, may have been due to
combinations or intimate admixtures of the proteid substance with fat
or fatty acid.^

Recalling Dormeyer's- physical explanation for the retention of
that portion of fat which can be removed from tissues only after their
digestion, Nerking suggested that there is quite as much reason for
concluding that such fat is chemically united in the tissue as that it is
held mechanically, and, therefore, that it is removable with difficulty
merely because of such intimate combination. Bogdanow's-^ obser-
vation, that the fat obtained in the later tissue extracts contains an
increased proportion of free fatty acid, might seem to give strength to
such a view, were it not for the probability that the increased amount
of free fatty acid under such circumstances results by hydration of fat
in the long-continued extraction process in boiling ether.

That fatty or fatty acid radicles are combinable with proteid is
clearly evidenced in the example of the so-called lecithalbumins,*
which do not yield their fatty radicles to ordinary extraction with
ether, but can be broken up into fatty and non-fatty matter by appro-
priate methods.

Obtaining results which seemed to point to the conclusion that blood
serum contains combined fat, non-extractable with ether until after
digestion in pepsin-hydrochloric acid, Nerking, at Pfliiger's sugges-
tion, looked for similar combinations in various proteid products as
they are now commonly prepared.

His results indicated that several proteid substances, which had been
prepared and purified by the usual methods, contained varying amounts
of fat or fatty acid in close combination. Further, this fatty radicle
could be broken off and determined quantitatively by Dormeyer's
method. No such combination with ovomucoid was shown, but about
three per cent of extractive matter was separated from sub-maxillary
mucin, among other products. Most of the proteids examined gave
negative results. Albuminoids were not studied.

The quantities of substance extracted, and the amounts of extract
obtained in the process, were comparatively small in each of the posi-

^ Nerking: Archiv fiir die gesammte Physiologic, 1901, Ixxxv, p. 330.
■^ DORMKYER : Ibid., 1895, Ixi, p. 341 ; 1896, Ixv, p. 102.
3 BOGDANOW: Ibid., 1S96, Ixv, p. 81 ; 1897, Ixviii, p. 408.

•* A rdsumti of the literature concerning the.se bodies is given by Cohnheim :
Chemie der Eiweisskorper, 1900, p. 203.

Combination of Fat with Proteid Products. 333

tive cases. When the ordinary unavoidable sources of error in work
of this kind are kept in mind it is difficult to lay very much stress
upon extractive quantities as slight as those obtained in Nerking's
experiments. It should be noted, further, that in no case was more
than one sample of each particular proteid analyzed.

Nerking does not make it clear to the reader of his paper that his
products were given the great care in preparation, particularly the
extended extraction in hot alcohol-ether, which is necessary for their
complete purification. He does not state that he was careful to use
anhydrous ether, nor, indeed, that the samples of ether he employed
had even been distilled by him previous to their use. Possibly he
was not certain, therefore, that the extractive fluid itself would not
sometimes yield residual matter on evaporation. Hd states nothing
regarding the quantity of fatty material contained in the samples of
enzyme used in his digestions. Preparations of pepsin such as he
employed contain appreciable proportions of ether-soluble material.

Toward the end of his paper Nerking himself comments on the
obvious weakness of his experimental evidence on the existence of
"fat proteid compounds." He adds, also, that all his efforts to effect
special combinations of proteid with fat have resulted negatively.

With such doubts in our minds as were raised by the omissions
above referred to, and at the same time appreciating the suggestive-
ness of Nerking's results, particularly in connection with the mucoids,
we set to work to ascertain the facts regarding the proteids referred
to below.

Proteid Products Investigated.

Preparation. — All of the proteids worked with in these experiments,
with the few exceptions to be noted, had been prepared and purified
very carefully by improved or accepted methods for special research
in other connections, some of the data of which have already been
published. This fact is emphasized at this point to show that such
results as were obtained in these experiments were not dependent on
unusual care in this particular instance, in the separation of the pro-
teids, but are typical for these substances as we are accustomed in
this laboratory to prepare them.

Method of estimating extractive substance. — Care was taken to fol-
low Nerking's general extractive procedure. The substance, dried at
100-105° C. to constant weight, was extracted for fifteen to twenty
days continuously in a Soxhlet apparatus with anhydrous ether pre-

334 ^- ^- I^osner a7id William J. Gies.

pared in bulk by us and freshly distilled in glass apparatus before use.
On evaporation, large quantities of the ether completely disappeared
without leaving a residue.

After preliminary extraction, the proteid was digested in a moderate
excess of 0.2 per cent hydrochloric acid containing 0.8 gram of com-
mercial pepsin scales per litre. Digestion in this fluid was rapid and
complete. This quantity of the pepsin preparation (0.8 gm.) con-
tained 2 to 4.8 milligrams of extractive material. After the digestion
the extractions were conducted as in Dormeyer's method.

All ether extracts, those obtained before as well as after digestion,
were filtered, the papers thoroughly washed with ether and the wash-
ings added to the main filtrate. Separation of the ethereal extract
from the fluid digestive mixture was always made exactly, in a sep-
aratory funnel. There was no tendency to persistent emulsion at this
point in any of our experiments. The amount of indigested matter
was at most very slight, even with the mucoids.

The ether extracts, after filtration, were evaporated in vacuo in
small beakers. Care was taken entirely to exclude dust particles after
filtration. A very small amount of water was left behind on evapor-
ating the ether which had been in contact with the acid fluid. The
amount of solid matter dissolved in it must have been very slight in
absolute quantity, although forming an appreciable proportion of the
weight of the extract. See table, pages 338 and 339.

Mucoids. — It has been known for a long time that a certain amount
of ether-soluble matter is admixed with connective tissue mucoid
when the latter is first precipitated from its solution in alkali by
acids. ^ The difificulties in the way of removing this admixture have
been appreciated by various observers, but no one has determined
the chemical nature of the extractive substance. These glucoproteid
products therefore appeared to offer particularly interesting objects
for study in this connection also.

Toidomucoid. — Our mucoids from tendon were prepared for the
experiments recently described by Cutter and Gies.- We used
samples of their analyzed preparations Xos. i to 5 inclusive. Our
extractive results were practically negative for each of these.

A portion of preparation No. i, which by accident had been left in
the air-bath for a few days — a somewhat longer period than was
necessary to carry it down to constant weight — had become slightly

1 Loebisch: Zeitschrift fur physiologische Chemie, 1S86, x, p. 58.
- Cutter and Gies : Loc. cit.

Combination of Fat with Proteid Prodticts. 335

brownish (oxidized?) just as filter paper does, for example, under
similar circumstances. On extracting this material the ether became
yellowish at first, then reddish yellow in color.^ The extracted sub-
stance was very slight in quantity, however, the high tinctorial effect
having suggested a greater amount of solid matter in solution than
was actually found.^

A sample of the same preparation when dried to constant weight
in vacuo, instead of in the air bath, gave essentially the same negative
results. Of course, no pigment was developed.

The results with purified tendomucoid having been negative, we
determined next the amount of extractive matter in the crude ma-
terial, which various observers, as we have already noted, have found it
very difficult to remove in the purification process. For this purpose
we used a sample of crude tendomucoid prepared originally for diges-
tive experiments now in progress. This product was obtained in the
usual way from the Achilles tendon of the ox. After its first precipi-
tation from lime-water the substance was washed free of acid, then
partly dehydrated in 50 per cent alcohol and dried in the air in thin
layers on plates. 10.8 grams of this finely powdered product, in spite
of the treatment with alcohol in its preparation, yielded 0.3 gram of
extractive matter, a large part of which persisted in the substance
even after two weeks' extraction.^

The extractive matter thus obtained was yellow in color and oily
in consistency, but did not contain any fat crystals. Even when dis-
solved in ether and allowed to evaporate spontaneously at room tem-
perature, crystals failed to develop. That it contained fat, however,
was shown by the yield of fatty acid. It did not contain cholesterin
or lecithin in sufficient quantity to respond to the familiar tests.

Our result in this connection emphasizes the need of thorough
extraction in the purification of tendomucoid.

^ The previous extracts were colorless. So were all subsequent ones except
that from crude mucoid.

^ That the usual treatment in an air-bath at 100-110° C. for the removal of
water from proteids is an unsatisfactory method has long been recognized. Such
results as the above, which indicate gradual decomposition, also emphasize the
desirability of an improved method of drying proteid products for analysis.

3 This fact may have been due to the compactness of the powder particles,
since the product had been dried before it was completely dehydrated. It was
not light and fluffy, as is the dry, purified, dehydrated mucoid. All of this extrac-
tive substance, it will be remembered, can be eliminated from the freshly precip-
itated rqucoid without the aid of the digestive process.

336 E. R. Posncr and JVilliam J. Gies.

Osscflvincoid and choiidromucoid. — These preparations had been
analyzed by Hawk and Gies.^ The former was their preparation
No. 6; the latter, preparation " b." Like the tendomucoid, these
products were found to be free from fatty material.

Albuminoids. — Each of our albuminoid products was prepared by
improved method. All were found to be practically free from extractive
material.

Collagen. — One sample of collagen from the femur of the ox had
been made by us from ossein shavings for other experiments not yet
reported. Osseomucoid, etc., had been removed with lime-water and
the albumoid- eliminated by digestion in alkaline trypsin solution.^
A sample of tendon collagen from the Achilles tendon of the ox had
been made in the same way, for the same purpose, and was available
for these experiments.

Gelatin. — Products prepared from bone, for other experiments in
progress for some time, were used. They were made from ossein
shavings obtained from the rib and the femur of the ox, after removal
of the mucoid and albumoid as above. The ligament gelatin used by
us was analyzed by Richards and Gies."* Through the kindness of
the writer's former colleague, Dr. W. G. Van Name, we were able,
also, to use samples of two of his preparations of tendon gelatin —
C and D.-^

Elastin. — Our samples of elastin were prepared and analyzed by
Richards and Gies.*^ Their preparations Nos. 7 and 8 were used.

Simple proteids. — These also gave practically negative results in
the two experiments with purified products.

Globulin. — We used a sample of cocoa edestin obtained by Kirk-
wood and Gies ^ — their preparation No. 5. The endosperm of the
cocoanut, from which this preparation of edestin was made, contains
large proportions of fat and fatty acids, a condition particularly favor,
able to admixture or combination with proteid, if such had occurred.

Alkali albuminate. — This product had been made by Fried and
Gies^ from a mixture of myosin and muscle " stroma substance." It

1 Hawk and Gies : Loc. cit.

■^ Hawk and Giks: This journal, 1902, vii, p. 340.

3 pywALD and KOhne: Jahresbericht der Thier-Chemie, 1877, vii, p. 281.

•» Richards and Gies : This journal, 1902, vii, p. 128.

5 Van Name: Journal of experimental medicine, 1897, ii, p. 124.

^ Richards and Gies : Loc. cit., p. 104.

' KiRKWOOD and Gies: Bulletin of the Torrey Botanical Club, 1902, xxix, p.
343-

•" Fried and Gies: Proceedings of the American Physiological Society. This
Journal, 1901. v, p. xi.

Combination of Fat with Proteid Prodticts. 337

had not been thoroughly extracted with ether in the purification
process.

Commercial products, — These substances were dried egg albumen,
Witte's peptone, somatose, and chloralbacid. An appreciable quantity
of extractive matter was separable from the albumen, but the propor-
tion of such substance obtained from it was not as great as that from
crude mucoid.

Discussion of Results.

The table on pages 338 and 339 summarizes the data obtained in
these experiments. It will be observed that the figures for composi-
tion of the purified products agree with the accepted average data
for each class of substances. Further, it is seen that the absolute
amounts of extractive substance are very slight — so minute, in fact,
as to be practically nothing except for the crude products with their
usual extractive impurities. The influence of ordinary, unavoidable
defects of manipulation on such small quantities of residual substance
is obvious.

The perceptible decrease in the weight of many of the extracts
during the drying process in the air-bath might be interpreted as
indicating a loss of volatile fatty acid. This decrease, however, is
seen to be very slight in absolute amount. It is much more prob-
able that the loss was water only. The small beakers in which the
ethereal extracts were evaporated were light in weight but of a
capacity of 80 c.c. While even this size was somewhat disadvan-
tageous as far as drying and weighing were concerned, smaller ves-
sels could not have served very well in other respects. It is probable
that, in their stay in the desiccators over sulphuric acid, not all of the
moisture was removed from them. In the air-bath it was, of course,
driven off and the total weight thereby reduced somewhat.

Conclusions.

We conclude from the data of these experiments that the above pro-
teids of the simple, compound and albuminoid types, which were
prepared by the best methods now in use, are not " fat-proteid
compounds."

It is obvious, also, that these substances bear no resemblance to
products of the lecithalbumin type.

13^^

E. R. Pos7ier and William J. Gies.

Proteid substance

examined.

Nature.

Percen

age composition.

Amount
used.

C

H

N

S

0

Grams.

Tendomucoid — 1 . . .

47.47

6.68

12..S8

2.20

31.07

4.8327

2 . . .

47.46

6.56

11.78

1.81

32.39

2.2376

3 . . .

47.80

6.60

12.66

1.85

31.09

4.8879

4 . . .

48.92

6.83

12.64

2.80

28.81

2.7916

5 . . .

48.54

6.68

12.69

2.34

29.75

2.0688

Tendomucoid — 1 . . .

47.47

6.68

12.58

2.20

31.07

a. Slightly oxidized

....

4.0910

/'. Dried in vacuo . .

2.8149

Tendomucoid — crude .

12.82

10.8250

Osseomucoid ....

46.53

6.81

11.99

2.55

32.12

4.3211

Chondromucoid ....

45.58

6.80

12.38

2.55

32.69

5.7899

lione collagen ....

....

18.39

4.1949

Tendon collagen . . .

18.01

3.8240

Tendon gelatin — 1 . .

50.16

6.63

17.83

0.21

25.14

4.3821

2 . .

50.15

6.50

17.71

0.26

25.38

4.9536

l>one gelatin — rib . . .

18.20

2.9991

femur. .

18.12

3.51.50

Ligament gelatin . . .

50.49

6.71

17.90

0.57

24.33

5.7211

Klastin — 1

54.47

7.30

16.64

0.14

21.45

5.6747

2

53 84

7.31

17.00

0.14

21.71

8.7429

Cocoa edestin ....

18.24

4.2191

Albuminate (myosin) . .

16.39

4.8249

Egg albumen ....

....

8.2194

Witte's peptone . . .

....

8.1876

.Somatose

....

13.2002

Chloralbacid

12.3439

Combination of Fat with Proteid Products. 339

Preliminary extraction
in etlier, 15-20 days.

Extraction for 7 days in

ether after digestion

in pepsin — HCl.

Total extract.

Extract

Extract dried

Extract

Extract dried

dried

over

HoSO^

24 hrs.

in air-bath at
100° C.

dried

over

H2SO4

24 hrs.

in air-bath at
100° C.

Dried over
HpSOi
24 hrs.

Dried in air-bath
at 100° C.
24-48 hrs.

24 hrs.

48 hrs.

24 hrs.

48 hrs.

Millig]

'ams.

Mgms.

%

Mgms.

10

1.0

OS

1.8

0.037

1.2

0.7

O.S

0.9

0.7

06

2.1

0.09

1.4

0.062

0.7

0.7

1.7

1.1

1.3

2.0

0.041

l.S

O.S

05

1.5

1.0

1.4

3.3

0.12

1.9

0.070

1.1

0.2

0.3

0.7

0.4

0.6

1.8

0.08

0.9

0.044

5.9

2.5

2.3

3.6

2.0

1.8

9.5

0.23

4.1

0.100

2.3

1.1

1.3

0 5

0.0

2.8

0.10

1.3

0.046

114.2

108.9

107.5

202.6

195.7

316.8

2.93

303.2

2.800

0.7

0.6

0.7

0.7

0.1

0.0

1.4

0.03

0.7

0.016

2.3

1.5

1.7

0.8

0.2

0.0

3.1

0.05

1.7

0.030

2.7

0.7

1.7

0.7

4.4

0.10

1.4

0.033

1.2

O.S

0.5

0.3

1.7

0.04

1.1

0.029

1.7

1.3

0.8

1.8

1.0

3.5

0.08

1.8

0.041

1.6

1.2

0.4

2.2

1.3

3.8

0.08

1.7

0.034

0.4

0.1

0.0

1.2

0.5

1.6

0.05

0.5

0.017

1.5

1.1

1.2

0.4

0.0

1.9

0.06

1.2

0.034

1.5

1.0

0.9

1.5

0.6

3.0

0.05

15

0.026

1.5

0.7

0.1

1.7

O.S

3.2

0 06

0.9

0.016

1.3

0.7

0.0

13

0.5

2.6

0.03

0.5

0.006

1.2

O.S

O.S

2.7

l.S

3 9

0.09

2.6

0.061

1.1

0.5

0.7

6.S

—

5,4

7.9

0.16

6.1

0.127

24.1

12.7

0.7

17.6

17.2

41.7

0.51

17.9

0.21s

9.3

6.5

4.6

15.6

14.7

24.9

0.30

4.6

0.056 '

2.4

1.6

0.0

1.0

0.0

3.4

0.26

0.0

8.0

3.2

30

6.5

4.1

3.9

14.5

0.12

6.9

0.056

r>. BOTANICAL STUDIES.
Reprints, Nos. 36-40.

<it>

Vol. 29 No. 6

BULLETIN

OF THE

TORREY BOTANICAL CLUB

JUNE 1902

Chemical Studies of the Cocoanut with some Notes on the Changes
during Germination '^

By J. E. KiRKWooD and William J. Gies

(With Plate 19)

[From the Laboratory of Physiological Chemistry of Columbia University, and
the New York Botanical Garden, New York.]

Synopsis Page.

I. Introduction 322

II. Chemical composition of the ungerminated cocoanut 325

A. Proportions of milk, endosperm and shell in the husked nut 326

B. Composition of the milk 328

C. Composition of the endosperm 331

a. General composition 332

b. Fat 335

c. Crude fiber and carbohydrates 340

d. Proteids 340

e. Inorganic matter (ash) 345

f . Enzymes 345

g. Average composition 345

D. Composition of shell and husk 346

E. Cocoanut pearls 348

III. Changes in the cocoanut during germination 349

A. Morphological changes 349

B. Chemical changes 352

C. Enzymes 358

* Preliminary accounts of some of the results of this research were given in the
Proceedings of the American Association for the Advancement of Science, — : 275.
1900, and in the Proceedings of the American Physiological Society. 1900 : American
Journal of Physiology, 5: 14. 1901,

The term ' ' coco ' ' appears to be derived from ' ' coc " or ' • cocus, ' ' a local name
for the " Indian nut," the fruit of Cocos nucifera, given to it on account of a fancied
resemblance of the base of the endocarp, with its three circular impressions, to the
face of a monkey when it utters a cry having a sound like the word. See f. i,
pi. ig. The term "cocoa" should be carefully distinguished from "cacao," the
product of Theobroma cacao, from "coca" the derivative of Ei-yihroxyloii coca, from
"coco," the coco-kola of commerce, and from "cocco" or "cocoa root" [Colocasia
esculenta).

321

322

KiKKwoon AND GiEs: Chemical Stldies

Introduction

" Of the whole class of

seeds having the character

of luxuries rather than of

necessaries, the cocoanut is

by far the most important to

mankind, whether considered

as a delicious and nutritious

food or as supplying valuable oil and

many other articles useful in social

life." *

The common cocoanut is derix^ed
from Cocos iiiicifcra, a species of palm
growing in practically all tropical coasts
and islands. The cocoa palm grows
naturall)- on the seashore or in its im-
mediate vicinity and does not bear well
when at a great distance from salt
water. (See analyses, p. 335.) At
maturity it has a cylindrical stem about
2 feet in diameter. At its apex the
tree carries a tuft of leaves, wliich are
about 12 feet long. These have num-
erous narrow, rigid and long leaflets.
The leaf, which may attain to 20 feet in
length, consists of a strong mid-rib,
whence numerous long acute leaflets'
spring, giving the whole the appearance
of a gigantic feather. The flowers which
produce the nut are yellowish-white.
They are arranged in spikes, branching
from a central axis, and inclosed with
a tough spathe usually a meter or more

Fic;. 1. Inflorescence of the cocoanut showing
spathe inclosing the spikes, each with numerous
male flowers above and a single female flower near
the base. X i- Winton.

* Smith : Food, 226. 1873.

OF THE COCOANUT DURING GERMINATION

323

in length. Their appearance and arrangement is shown in Fig. i,
on the opposite page. The tree grows to a height of about 60—100
feet and usually bears 80-100 nuts arranged on the tree in bunches
of 10—20. It continues to bear during two generations of men.

The fruit is subtriangular-ovoid in form,
about 1 2 inches long and 6 inches broad. It is
composed of a thick, fibrous "husk" (exocarp)
and thin, hard "shell " (endocarp), containing a
white fleshy seed, the " meat" (endosperm), with a
thin integument (testa). (SeeivV j, p. 324.) The
thick husk is remarkably adapted to the preser-
vation of the seed, whilst the nut is tossed about
by the waves until it reaches some shore, it may
be, far distant from that on which it grew.
While immature the nut is without the solid
endosperm, but is filled with a milky fluid. As
it ripens, however, the endosperm gradually de-
velops and the milky juice diminishes in quan-
tity. The temperature of this juice when fresh
is always comparatively low. (See page 349 for
further reference to the parts of the nut.)

Figures i, 2 and 3 are from cuts loaned to
us by Dr. A. L. Winton, who used them lately in the account of his
very valuable histological study of " The Anatomy of the Fruit of
the Cocos nuciferay^ We are greatly indebted to Dr. \\'inton
for his kind assistance.

The cocoanut forms the chief food of the inhabitants of Ceylon,
the South Sea Islands, the coast of Africa and other tropical coasts
and islands. t The flesh is not only eaten as it comes from the
tree, both ripe and unripe, but it is also prepared and serv^ed in
various ways. In India the " copra" is much used as an ingre-
dient of curries. It forms an accessory part of the diet, and is found

P^IG. 2. Half-
grown cocoanut fruit
with calyx, and axis
from which the male
flowers have fallen.
X 4. Winton.

* Winton : American Journal of Science, IV. 12 : 265. 1901.

t The cocoanut is agreeable to the taste of various domestic and other animals, and
is eagerly eaten by them. The cocoanut-crab [Birgiis latro, suborder Macritra ; an-
omalous form, approaching the Brachytira and closely related to the hermit crab) feeds
almost entirely on the kernel of the cocoanut. Its powerful claws enable ft to easily
peel off the husk and open the hard shell.

324

KiRKWOOD AND GiES : Chemical Studies

in many of the confections, of civilized man all over the globe.*
Immoderate use of the fruit, which according to the people of
the tropics is highly refrigerant, causes, it is said, rheumatic and
other diseases.! The milk is considered an agreeable cooling
beverage in the tropics. It has been known for some time that
irritation of the mucous membrane of the bladder and urethra is
caused by drinking too freely of the cocoanut milk.| It is strongly

K —

Alb-

Fig. 3. Ripe cocoanut. S, lower part of axis forming the stem ; y1, upper end of
axis with scars of male floweis ; if/?, epicarp ; A/es, mesocarp with fibers ; End, endo-
carp or hard shell ; T, portion of testa adhering to endosperm ; A/6, endosperm sur-
rounding cavity of the nut ; A', germinating eye. X \- VVinton.

diuretic. Parisi has u.sed the cocoanut therapeutically as an an-
thelmintic with uniformly satisfactory results. § He states that the
meat of the nut is a powerful taenicide, the milk sharing the prop-

* In the Annals of the New York Academy of Science, 13: 490, 1900-1901,
the following may be found : " Dr. Gies in answer to a question stated that the food
content of the cocoanut is small." This answer is quoted incorrectly. The question
referred to the nitro^ttious food content. It was stated on that occasion that the
" content oi proteid food-stuff is small." See page 340.

t Pavy : A Treatise on Food and Dietetics physiologically and therapeutically
considered, 488. 1878.

J Curtis: Annals of the New York Academy of Sciences, 13: 490. 1900-1901.

^ See Liebreich : Encyklopaedie der Therapie, i: 744. 1896.

OF THE COCOANUT DURING GERMINATION 325

erty.* The cocoanut has been used as a vermifuge in India for
probably forty generations by the beef eaters of that country and
is there well known as a means of expeUing the flat worm.f The
small, green and immature nut is grated fine for medicinal use,
and when mixed with the oil of the ripe nut becomes a healing
ointment.

The fibrous husk (coir) is widely used for the construction of
ropes, brushes, bags, matting, etc. The compact fleshy edible
portion (copra), closely lining the hard shell and which is entirely
fluid or gelatinous when young, contains a large proportion of fat,
which is extracted and used for various commercial purposes, such
as the manufacture of fine soaps and candles and frequently as a
substitute for butter. Cocoanut oil and resin melted together
yield a substance capable of being used with success in filling up
the seams of boats and ships, and in tropical countries for cover-
ing the corks of bottles as a protection against the depredations
of the white ant. A quart of the oil may be obtained from six to
ten nuts. The hard shell is easily polished and lends itself to the
formation of various utensils and ornaments. It also has a 'high
fuel value.

Although considerable is known of the constituents of the
cocoanut, of its nutritive value and commercial uses, little has
been done to ascertain the nature of the changes which the nut
undergoes during germination. At the suggestion of Dr. Mac-
Dougal we have undertaken such a study, more especially from
the chemical standpoint, and although our work in this particular
connection has not been quite as fruitful as we had hoped it might
be, our results are not without some interest.

II. Chemical Composition of the ungerminated Cocoanut
Before beginning our work on the germinating seed we felt it
desirable to make ourselves thoroughly familiar with the chemical
qualities of the ungerminated nut. This seemed all the more
desirable because of the incomplete as well as the disconnected
chemical data thus far recorded in this connection. This purpose
was accomplished in a large number of analyses of numerous

* United States Dispensatory, 1619. 1899.

t American Journal of the Medical Sciences, 67 : 281. 1889.

3l'<! Kirkwood and Gies : Chemical Studies

samples. We record the more important of these results, with
comparative data from the work of others, on the following pages.

Most of the nuts subjected to the analyses referred to farther
on were furnished to us for this work by Hon. VVm. Fawcett and
the United Fruit Co., who sent them in their husks from Jamaica.
They were ripe, fresh and of about the average size. A few de-
terminations were made with material from nuts bought in the
markets in this city — source unknown, though doubtless of West
Indian origin. These were of ordinar)' size, appeared to be ripe
and fresh, and gav^e essentially the same analytic results as those
obtained directly from Jamaica.*

We wish at the outset of this paper to thank Dr. MacDougal
not only for the supply of material with which he favored us, but
also for the suggestions which led us to undertake this work and
for the kind encouragement he has given us from the beginning.

Proportions of Milk, Endosperm and Shell in the husked
Nut. — The weights and proportions of the main parts of the nut
without its husk were carefully ascertained in special observations,
as well as incidentally in other experiments. f The milk was
removed as indicated on page 328. The empty nut was quickly
broken with a hammer, the endosperm and germ, with the thin
seed coat, carefully and promptly removed with a knife, and the
fresh moist parts weighed at once. The results given on the
opposite page were obtained in this connection. |

The only results recorded on these gross relationships that we
have been able to find were those obtained in a single experiment
by V. Ollech, and those by Bachofeu.§ The parts of a single
cocoanut, except the milk, were dried in the air by v. Ollech. ||

*A few showed signs of deterioration, such as "popping" on opening, free acid
in the milk, etc. These were, of cour-e, discarded.

t The weight of the fibrous husk varies considerably, as the amount of moisture in-
creases by absorption or decreases by evaporation. The weights of the other parts are
ordinarily not subject to such fluctuations.

i The weights of the germ and the thin seed coat enveloping the endosperm were
included with the latter.

?See also Berzelius : Lehrbuch der Chemie. Translated by Wohler, 7: 533.
1838.

I V. Ollech : Quoted by Konig, Die menschlichen Nahrungs- und Genussmittel,
etc., 2: 495. 1893.

OF THE COCOAXUT DURING GERMINATION

327

Freshly imported >.uts (from Jamaica).

Weights

in Grams.

1

Percentage of total Weight 1
of husked Nut.

Milk.

/3

Endosperm

Endosperm

3 j

Fruit

Specific

-^

without
Husk.

Shell.

and
Integument.

:Miik.

Shell.

and
Integument.

JMilk.

i

Vol.
c.c.

Gravity.

I

«45

255

437

153

30.2

i 51-7 !

18.I

150

IOI8

2

771

198

379

194

25 -7

49.2

25-1

I go

1017

3

658

168

371

119

25-5

564

18. 1

117

1020

4

718

199

351

168

27.7

48.9

23-4

164

ICI9

5

597

152

327

118

25-5

54.8

IQ.7

"3

1022

6

463

127

251

85

27.4

54-2

IS4

83

IOI9

7

622

195

334

93

2>^Z

53-7

15.0 ;

T

1023

8

563

144

329

90

25.6

58.4

10. 0

87

1027

9

633

166

374

93

26.2

59-1

14.7 '

90

1027

ID

530

^S^'

282

92

29.4

53-2

17-4

90

102 I

II

6^7

150

363

124

235

57.0

19-5

121

1024

12

497

144

267

86

29.0

53-7

17-3

85

IOI4

T3

5^8

162

283

93

30. 1

52.6 ;

17-3

90

1021

14

413

123

256

34

298

62.0 i

8.2

T:,

1030

15

5"

158

309

44

30- 9

60.5

8.6

43

1037

i6

578

190

320

68

32.8

55-4

II. 8

67

IOI6

17

568

142

350

76

25.0

61.6

13-4

74

1O26

i8

495

140

293

62

28.3

592 ,

12.5

60

1024

19

813

221

392

2CO

' 27.2

48.2 i

24.6

194

I02I

20

7S8

2c8

393

157

27.4

5^9 i

20.7

150

1022

21

584
609

1 48

339

97

254
27.8

58.0 1
55-2 '

166
17.0

94

1028

Aver.

169

333

107

104

1023

Nuts from the Market (New York City).

I

1070

250

558

262

23-4 1

52.2

24.4

254

1018

2

1009

251

506

252

24.9

50.1

25.0

246

1015

3

728

202

417

109

27.7 1

57-3

15.0

106

1027

4

800

226

450

124

28.2 '

5&-3

15-5

120

1026

5

688

191

385

112

27.8

56.0

16.2 1

no

1015

6

565

131

316

118

23.2

56.0

20.8 !

116

1017

7

639

210

382

47

32-9

59-8

l^ \

46

1024

8

638

210

311

117

32-9

48.8

18.3 i

"5

1017

9

480

125

304

' 51

26.0

63-3

10.7

48

1034

ID

561

158

307

: 96

28. 2

54-7

17. 1

92

1024

II

733

204

414

115

27.8 :

56.5

15-7

no

1024

12

762

176

380

206

23.1 ,

49-9

27.0

202

1020

Aver.

722

194

394

134

27.1

55-1

17.8 1

130

1021

They represented the following proportions of the total weight,
which was 1,133 grams :

Fibrous Husk. Shell. Endosperm with Germ.* Milk.

30.45 per cent. 19.59 per cent. 37.78 per cent. 12.18 per cent.

Of the total weight of the husked nut, which, by calculation, must

* Including, doubtless, the seed coat as well.

328 KiRKWooD AND GiES : Chemical Studies

have been jSS grams, the percentages of the parts were (calcu-
lated by us) :

Shell. Endosperm with Germ. Milk.

28.17 per cent. 54.32 per cent. 17.51 per cent.

These results, it will be observed, harmonize closely with the aver-
ages of our own determinations.

The data obtained by Bachofeu in this connection will be
found in the table on page 335.*

Composition of the Milk. — The milk was poured from the
nut through an opening made in the "eye" of the fertile carpel
(see page 350) with a cork -borer. Extraneous matter could easily
be kept out of the milk by this procedure and, besides, the fluid
could be obtained when desired in a perfectly fresh, unevaporated
condition.

The milk was found to be faintly turbid and opalescent in each
case, and always contained a few oil globules and occasionally
crystalline matter. It was acid in reaction to litmus although, as
shown by lacmoid, no free acid was present in the normal fluid.
The reaction is due to acid phosphate. Both alkali and earthy
phosphate are present. The latter can be precipitated, in part at
least, on boiling. An abundant precipitate of phosphate is obtained
when the milk is made alkaline. The average specific gravity,
determined with the aid of a hydrometer, was, as already noted on
page 327, 1,023 and 1,021. The average specific gravity of the
mixed milk of i 5 nuts not included in the table on that page was
1,023. Of eight additional nuts not referred to there, and ex-
amined at another time, the figures for the mixed milk were
1,022.

The milk reduces Fehling's and Nylander's solutions and it fer-
ments. It contains some monosaccharide which, from the char-
acters of the phenylosazone derivatives, appears to consist of
either dextrose or galactose, probably of both. Disaccharide in
the form of cane-sugar is also present in good quantity, as might
be inferred from the sweet taste of the milk.

* Results having some relation to these are given by Atwater : Report of the
Storrs (Conn ) Agricultural Experiment Station, 123. 1899. Hamraerbacher ( Land-
wirtschafilichen Versuchs-Stalionen, etc., 18: 472. 1875) found that the endosperm
of two nuts weighed 835.8 grams ; the milk, 303.95 grams. See also, pages 331 and 356.

OF THE COCOANUT DURING GERMINATION

329

On standing the milk turns sour, becomes thicker, and has
■much the odor and physical appearance of soured cow's milk.
The milk ferments readily. As it does so the acidity increases
with a production of acid from the sugar. Alcohols are also pro-
duced in the process. The distillate from the fermented milk has
an agreeable taste and an alcoholic odor.*

Chlorides are prominent with phosphates among the inorganic
substances of the milk. It contains only a very small quantity
of proteid, coagulating above 8o° C, and also traces of a proteose-
like body. Very faint biuret and xanthoproteic reactions were
obtainable with the fresh fluid. A snow-white precipitate consist-
General Composition of the Milk

Milk Used.

Percentage of Fresh Milk.

Percentage of Solids.

No.

Specific
Gravity.

Grams.

Water.

S
Total.

olid Matter
Organic.

Inorganic.

Organic
Matter.

Inorganic
Matter.

i-a

b

IOI9

28.815
27.280

95
95

52
43

4.48
4-57

3-98
4-05

0.50
0.52

88
88

84
60

II. 16

11.40

2-a
b

1020 •

25.403
27-837

95
95

28
44

4.72
4-56

4-27
4.14

0-45
0.42

90
90

58
70

9.42
9-30

'-",

1022

36.382
28.528

94
94

73
62

5-27
5-38

4-78
4.90

0.49
0.48

90
91

57
02

9-43
8.98

4-a
b
c

IO16

25-958
25-823
26.298

95
95
95

73
65
68

4.27
4-35
432

3-88
3-96
3-91

0-39
0.39
0.41

90
90
90

81
92

57

9.19

9.08

9-43

5-«
b
c

I02I

29.416
29.467
24.667

95
95
95

II

23
24

4.89
4-77
4.76

4-47
4-36
4-35

0.42
0.41
0.41

91
91
91

38
39
31

8.62
8.61
8.69

.b-a
b

1024

23.119

23.886

95
95

44
■hZ

4.56
4.67

3-82
3-92

0.74
0.7s

83
84

68
04

16.32
15-96

b

1028

22 540
26.690

94
94

80

94

5 20
5.06

4.18

1.02

80

38

19.62

2,-a
b

1027

28.722
28.409

95
i 94

02
97

4.98
5-03

4.21
4.26

0.77
0.77

84-45
84.69

15-55
15-31

Aver.

1022

26.847

i95

23

4-77

4.21

0.56

88.47

11.53

ing in part of earthy phosphate is obtained on warming the milk
on the water-bath at 70° C. The filtrate from this product when
boiled yields a delicate turbidity of coagulated proteid which be-
comes flocculent on addition of a slight excess of acetic acid.
The filtrate from this coagulum gives only a very faint biuret re-
action. Cocoanut milk is said to contain malate of lime.f

* Cocoa beer, containing 3.4 per cent. "Extractive," has been made by Calmette ;
Chemisches Centralblatt, 2 : 394. 1894.

t Harley and Harley : Proceedings of the Royal Society of London, 43 : 464.
1887-88.

Organic

Inorganic

Water.

Solids.

.Matter.

Matter.

91. sot

8.50

7-31

1. 19

9i-37t

8.63

7.50

I 13

Nitrogen-free

Carbo-

Fat.

E.\tractives

hydrates.

0.07

6.78

0. II

7.01 §

330 KiKKWooD AND GiE.s: Chemic.-xl Studies

On evaporation to a small volume on a water-bath the fresh
milk becomes darker in color, takes on an odor characteristic of
sugar syrups and looks not unlike molasses. Cane-sugar crystal-
lizes from it in abundance on cooling.

The analytic data given on page 329 were obtained in our
study of the general composition of the perfectly fresh milk of the
Jamaican nuts.*

Percentage results in this connection had been obtained pre-
viously as follows :

Nitrogenous
Substance.

0.46

0.38

These results were obtained with milk from nuts grown in the
eastern hemisphere. The milk from the Jamaican nuts appears, as
we have seen, to contain less solid matter, both organic and inor-
ganic. This difference is emphasized by Hammerbacher's ^ ob-
servations on the specific gravity of cocoa-milk. He describes the
milk as a colorless, slightly opalescent fluid, with a specific gravity
at 20° C. of 1044.** The milk from two nuts weighed 303.95
grams. From the nitrogen-free extractive substance in 77.8 grams
of milk contained in a third nut, 0.8504 gram of dextrose was ob-
tained. When milk was warmed with dilute sulphuric acid an odor
of volatile fatty acid became perceptible. A crystalline barium salt
was prepared from the distillate of the acidified milk which was found
to consist of barium propionate.

The milk contains a small amount of diastatic ferment and also
oxidase.ft We were unable to detect any other enzymes.

The following results were obtained by van Slyke^]; in his com-
parative studies of the milk of six unripe nuts and of one ripe one :

* The methods of analysis used for this and similar purposes, throughout our
work, were those commonly employed in the laboratory. See Vandegrift and Gies :
American Journal of Physiology, 5 : 287. 1901.

t Hammerbacher : Landwirtschaftlichen Versuchs-Stationen, etc., 18 : 472. 1875.

t Konig : Menschlichen Nahrungs- und Genussmittel, etc., 2 : 308. 1893. See
also Hizio : Pharmaceutisches Centralblatt, 756. 1833.

g Including 4.42 per cent of cane-sugar. See page 328.

*[ Hammerbacher. loc. cii.

**See our large number of determinations of specific gravity on page 327. Also
references on pages 328 and 329.

tt Hunger: Journal of the Society of Chemical Industry, 20 : 1030. 1901.

JjVanSlyke: Chemisches Centralblatt, i : 595. 1891. Compare with results on
page 329.

OF THE COCOAXUT DURING GERMINATION

J31

Constituents.

Milk of unripe Nuts.

Weight in grams. . .
Specific gravity . . .
Water (per cent. )
Total solid matter { % )

Inorganic substance.

Glucose

Cane-sugar .

"Albuminoid " . .

Fat . .

230.5
1,024.6

94-37
5.63
0-575
458
Trace
0.120
0.084

378.6
1,023.0

94.48
5-52
0.635

3.83
Trace.
0.126
o.ioo

347- o 383.7
1,022.3 1,023.0
94 59 94-89
5-41
0.675

3-45
Trace.
0.114
0.138

5 "

0.611

4.06

Trace.

0.205

o 131

350-0
1,022.1
95-27
4-73
0.658

4-36
Trace.
0.140
0.T45

3300
1,021.5
96.43
3-57
0.602

3-56
Trace.
0095
0.120

Average 1-6.

336.6
1,022.8

95-OI
4-99
0.626

3-97
Trace.

0-133
O.I 20

109.6
1,044.0
91.23

8.77
1.06
Trace.
4.42
0.291
0.145

The chief chemical differences induced by growth, as indicated
by the above results, are an increase in the proportion of solid
matter, including ash, fat and nitrogenous substance. Glucose
almost entirely disappears from the milk of the ripe nut, cane-
sugar replacing it — a fact evidencing synthetic production of disac-
charide from monosaccharide.

Hammerbacher, believing that the endosperm develops directly
from the milk, determined the quantitative relationships of the
saline matters contained in each part from the same nut. He gives
the following as his percentage results :

Ash of the Milk.

Ash of the Endosperm.*

Potassium,

55.200

43.882

Sodium,

0.728

8.392

Calcium,

3-679

4628

Magnesium

6.606

9-438

Chlorine,

10.373

13-419

Phosphoric

acid,

20.510

16.992

Sulphuric acid,

5-235

5.091

Silicic acid,

0.500

102.331

102.342

Minus oxygen

for chlorine, 2.338

3.024

99-993 99-318

The above results indicate a particular increase of the content of sodium chloride
in the ash of the developing endosperm and a corresponding decrease of potassium
phosphate. See pages 322 and 335. The amount of silicic acid in the endosperm
is also noteworthy. See page 335.

Endosperm. General Composition. — The pure white kernel

* Compare with results of Bachofeu's analysis, given on page 335. Our own re-
sults were the same as these qualitatively. Sea also Schaedler. Technologic der Fette
and Oele des Pflanzen- und Thierreichs, 840, 1892, who found 3.60 per cent, of iron
in the ash of the endosperm in addition to the above constituents.

332

KiRKwooD AND GiES : Chemical Studies

or "meat" of the nut is fibrous in structure, closely lines the
shell, is from i to 2 cm. thick, and contains a very large propor-
tion of fat. It is the part used most frequently for dietetic pur-
poses. It possesses a characteristic and pleasant odor and is
very agreeable to the taste. The endosperm cells do not contain
starch granules, but fat needles and proteid lumps are present in
them. The proteid particles are partly crystalline.*

After the kernel has been finely divided in a meat chopper, the
resultant hash may be subjected to increasing pressure, when an
General Co.\iposition of the Endosperm

1
1

Endosperm
used.

Percentage of fresh Endos) <

;rm.

Percentage of Solids.

Organic
Matter.

No,

Grams.

Water.

solid Matter

1

Inorganic

Total.

Organic.

Inorganic.

I-a

b

c

8.467

9.728

10.900

47 70
42.10
46.60

52.30
57.90
53-40

SII9
56-79
52-34

I. II
I. II

1.06

97.88
98.09
98.01

2.12
1. 91

1.99

2-a
b

c

11.885
12. 151
11.707

48.31
48.90
52.29

51-69
51.10

47-71

50.65
50.01
46.61

1.04
1.09
1. 10

98.01
97.87
97.69

1.99

2-13
2.31

3-«
b
c

8.762
8.185
8.923

4390
47-73
46.31

56.10
5227
53-69

55 20
51.20
52-71

0.90
1.07
0.98

98-39

97-95
98.18

I. 61

2.05
1.82

4-a '
b !
c

ii.S'i

9501
9.244

47.89
46.90
4750

52 II
53-10
52-50

51.05
52.05
51-43

1.06
1.05

1.07

97-97
98.02

97-96

2-03
1.98
2.04

5-«

8.942
9312

42.80
43-79

57.20
56.21

56-17
55 21

I 03
1. 00

98.21
98.23

1-79
1.77

6-a
b

10.214
10 624

50-30
48.70

49-70
51-30

48.68
50. 28

1.02
1.02

97-95
98.02

2.05
1.98

1-a
b

10.746
10. 142

10.052

42.21
39.60

45.31

57-79
60.40

53.69

56-83
59-46

52.66

0.96
0.94

98.34
98.45

1.66
1-55

Aver.

1.03

98.07

1.93

oily juice is obtained from it. The filtrate from this turbid mixture
has a higher specific gravity than the milk of the nut, is acid in
reaction, reduces Fehling's solution, contains a dextrin-like body
and the milk salts, gives the proteid color reactions, yields coag-
ulable proteid, and on dilution with water becomes turbid from
precipitated globulin.

The data given above were obtained for general composition of
the endosperm immediately after the nuts were opened. f

* See pages 342 and 352.

t The methods were the same as those employed with the milk. The thin seed-
coat was trimmed off and the pieces of kernel cut into small, thin pieces with a knife.
The material was taken (rem all parts of the nm.

OF THE COCOANUT DURING GeRMINATIOxX

333

Comparison of the averages given on the opposite page may
be made with the following previously recorded results for the
fresh endosperm from nuts of eastern origin : *

Fresh Endosperm

Water. Total Organic

Solids. Matter.

46.64 53.36 52.39

Inorganic

Matter.
0.97

Dry Endosperm

Organic Inorganic

Matter. Matter.

98.20 1.80

The agreement is seen to be very close.

By reason of the dietetic and commercial values of the various
constituents of the endosperm of the cocoa fruit, numerous prod-
ucts of the kernel have been made and analyzed. The air-dried
endosperm, or so-called " copra," is shipped in large quantities from
the tropics. Cocoa-oil is obtained from the copra by various
methods in countries distant from the tropics, the solid residues
remaining after extraction serving various purposes. This residue
makes up the so-called "cocoa-cake" obtained in the process of
expressing the oil at various degrees of temperature. It is also
ground into "cocoa-meal." In both forms, the residual substance

Water.

Total
Solids.

Organic Matter.

Products Analyzed.

Nitrog- 1 ^^ -
enous 1 „ ^ N-free
g^]^_ I I'at. 1 txtrac-
stance. . "^e.

Crude
Fiber.

organic
Matter.

Air-dried endosperm or copra. f
Endosperm, perfectly dried. J
Endosperm, free from fat and

water, j
" Cocoa- cake." |
" Cocoa meal." ||
" Cocoa-ireal," after extraction

of oil. *[

5.81

10.30
II. 12

4-55

94.19
100. CO

100.00

89.70

88.88
95-45

8.88 67.00 12.44
10.29 , 67.35 , I5-II

31.49 1 46.25

19.70 11.00 38.70
17.94 10.88 35.34

23.20 1.85 64

4.06
5.42

16.69
14.40
17.40

45

1. 81
1-83

5-57
5-90
7-32

5 95

* Hammerbacher : Landwii-tschaftlichen Versuchs-Stationen, etc., 18: 472. 1875.
See also Bizio : Pharmaceutisches Centralblatt, 757. 1833.

•j- Konig : Menschlichen Nahrmrgs- und Genussmittel, etc., 2 : 652. 1893. Also

p. 308-

j Hamrnerbacher : Landwirtschaftlichen Versuchs-Stationen, etc., 18 : 472. 1875.

§ Dietrich und Konig : Zusammensetzung und Verdaulichkeit der FuitermitteU
2 : 1031. 189I.

II Dietrich und Konig : i/nd., I : 725.

^ Schaedler : Technologie der Fette und Oele des Pflanzen- und Thierreichs,
624. 1892. (rt) For references to digestibility and nutritive value of cocoa-cake see
results of experiments on pigs and sheep given by Dietrich and Konig, 2 : 1031, 1036,
1040, 1123. ((^) Compare above results with the table for general composition oa
the opposite page.

3;)4 KiKKWooi) AND GiEs : CmiMicAL Studies

is used as food for cattle and as a fertilizer, having special \alue in
both these connections.* It is sometimes also used illicith' as a
food adulterant.

The analytic percentage results on page 333 have been reported
by various agricultural chemists for such products from nuts grown
in the eastern hemisphere.

The following summary of facts connected particularly with food
value was given several years ago by W'oods and Merrill : t

27.9

1-7

14-3

•9

I7-.S

I.O

4.6

.8

24.1

1.4

:>9.o

1.2

16.S

1.8

6.8

1.2

Edible portion, I ' 14.1 5.7 50.6 27.9 1.7 ' 2,986

As purchased, 48. 8 7.2 2.9 25.9 I 14.3 .9 1,529

Without milk, as purchased, 37.3 8.9 3.6 31.7 ; 17.5 l.o 1,872

Cocoanut milk, as purcliased, 92.7 .4 1.5 ' 4.6 .8 97

Shredded cocoanut, 4.3

Shredded cocoanut, 2.8

Edible portion, 5.8

Cocoanut milk, 91.5

Through the kindness of Dr. MacDougal we have been able
to examine an account of " The cocoanut and plant vitality " in the
Bulletin of the Botanical Department of Trinidad (July, 1900, p.
249). Reference is therein made to the report of Bachofeu in the
Queensland Agricultural Journal for April, 1900. Bachofeu says:
"Though there exist several analyses of parts of the cocoanut, no
one seems to have undertaken the task of getting a complete
analysis made with the view of ascertaining the actual demand
made by the cocoanut upon the mineral constituents of the soil."

The results obtained by Bachofeu for a single nut are so com-
plete, and so general in their interest and application that we quote,
on page 335, his general summary in its entirety, t

Bachofeu's results indicate that sodium chloride and potassium
phosphate are the chief inorganic matters drawn upon in the de-
velopment of the cocoanut — chemical data in harmony with the fact

*v. Knieriem : Chemisches Centralblatt, 2 : 672. 1898.

+ Woods and Merrill : Bulletin, Maine Agricultural Experiment Station, No. 54 ;
81. 1899.

I The analyses were made in Ceylon. Native nuts were used.

OF THE COCOANUT DURING GERMINATION

335

that the cocoa palm does not thrive away from the coast or where
salt is lacking in the soil. See second table, page 331.

Bachofeu's Analysis of the Cocoanut

Husk

Total weight in lbs.

" " in per cent.

^ j Moisture in per cent.

\ Dry matter in per cent.
Pure ash in per cent., containing viz :

Sihca, SiOj-

Oxide of iron and alumina, FcjOjAl^Oy

Lime, CaO.

Magnesia, MgO.

t Potash, KjO.

Soda, NajO.

f Potassium chloride, KCl.

Sodium chloride, NaCl.

Phosphoric acid, PjOj.

Sulphuric acid, SO3.

f Containing total potash, Kfi.
* Containing nitrogen, N.

30.71
0.137

Thus of the more important ingredients of the soil 1,000 nuts remove the following :

In Lbs.

Husk.

3.7017
0.8456

13.5255

1.8234

20.2375

Shell.

Kernel.

Milk.

Total Lbs.

Nitrogen, N.

Phosphoric acid, P.^O^.

Potash, K,0.

Lime, CaC3.

Sodium chloride, NaCl.

0.5460
0.0735
0.7127
0.0991
0. 2464

4.4100

1.4053
3.7362
0.2143
0.3563

0.1279

0.77X3
0.1674
0.5431

8.6577
2.4523

18.7527
2.3042

21.4233

Fat. — The striking chemical characteristic of the endosperm is
its large content of oil. This may readily be extracted with fluids
like ether. It can also be obtained in large proportion by pres-
sure, particularly at the tropical temperatures. The fat has the
consistence of butter in northern countries and possesses, when
fresh, a fragrant and characteristic odor and an agreeable taste. It
is snow white, sometimes cream-colored and readily crystallizes in
large rosettes from the molten condition or from its alcohol or
ethereal solutions. These crystals closely resemble those of pal-
mitic acid. They melt at about 20-23° C, and congeal again several
degrees below the melting point. They are fairly soluble in cold
alcohol. Although cocoa-fat differs somewhat in composition in
different countries, it has been found that the variations are compara-
tively slight. The temperature at which the oil is expressed influences

83<3 KiKKwoon and Gies : Chemical Studies

these variations by increasing or decreasing the proportion of fats
mehing only at higher temperatures. These facts account for the
variations in the figures given for melting point. Its specific grav-
ity is 0.9 + .

On heating to about 170° C, the oil gives off the odor of lactic
acid ; at a temperature of 300° C. acrolein may be detected. On
long-continued heating with nitric acid the following dibasic acids
are formed : succinic, adipic, pimelic, suberic and azelaic. Nitro-
caproic acid is also formed. * The oil is very soluble in all of the
well-known fat solvents. It contains some free fatty acid, but con-
sists chiefly of glycerides of caprylic, lauric, myristic and palmitic
acids, t Glycerides of caproic and capric acids are present in ap-
preciable quantity ; also a trace of stearin and some olein. | The
fat dissolves readily at a comparatively low temperature in an equal
quantity of glacial acetic acid. Such a solution becomes turbid at
40° C. § By reason of its content of lovver fatty acid radicles
cocoa-oil has a high saponification value. Cocoa-oil is particularly
resistent to the hydrating effect of superheated steam. ||

The following data were obtained for the percentage fat-content
in the fresh endosperm. The method of determination used was
Dormeyer's :^

Gen'l
123456 7 Average.

Fresh endosperm, a. 38.27 40.01 36.71 35.10 34.60 38.90 38.60
b. 36.14 40.54 35.02 34.90 34.10 40.70 38.40
Average, 37.20 4028 35.87 35.00 34.35 39.80 38.50 37.29

The ether extracts containing the oil were free from lecithin

*Schaedler: Technologic der Fette und Oele des Pflanzen- und Thierrichs, 843.
1892.

+ lhe presence of palmitin (tri) is doubted by Ulzer : Chemisches Centralblatt,
II : I143. 1899.

j The so-called " cocinic acid" or " cocostearic acid" derivable from " cocin "
or " cocinin " is, like the latter, a mixture. The former is a mixture of some of the
above fatty acids ; the latter of their glycerides. See Oudemans : Chemisches Central-
blatt, 192. 1861.

§ Valenta. Quoted by Vaubel : Physikalischen und chemischen Methoden quan-
titativen Bestimmung organischer Verbindungen, i : 162. 1902.

II Klimont : Journal of the Society of Chemical Industry, 21 : 126, 1902.

•^ Dormeyer : Jahresbericht iiber die Fortschritte der Thier-Chemie, 26 : 42. 1896.
The fresh tissue was finely divided and weighed, then dried to constant weight at
100-105"^ C, and all of it extracted with anhydrous sulphuric ether. The usual amounts
of tissue were used.

Grams.

Per Cent

I5.I4S8

60.595

9.5282

38-113

0.5596

2.238

OF THE COCOANUT DURING GERMINATION 337

and could be almost entirely saponified. Hammerbacher * in the
saponification of 25 grams of the pure oil obtained the following
results :

Fatty acids convertible into insoluble lead salts,
Fatty acids convertible into soluble lead salts,
Glycerin,

Total, 25.2366 100.946

The excess in weight of products is explained by the addition
of hydroxyl groups in the cleavage of the triglycerides. Konig
had previously found the glycerin content of cocoa-fat to, be 2.08
per cent. Hammerbacher therefore concludes : "It follows from
these results that this vegetable fat consists in greatest part oi free
fatty acid."

That there is some error in this conclusion, however, is evident
from the results of later work. Benedikt f reports the glycerin
content of cocoa-oil to be 13.3— 14. 5 per cent. Crossley and Le
Suer found that the content oi free fatty acid in terms of oleic acid
varied between 2.50 and 8.86 per cent.|

Stellwaag || studied the fat extracted from cocoa cakes. This
oil was rancid, of course. He found the quantity oi free fatty
acid to be only 9.84 per cent. The fat from the ether extract
melted at 23° C. The saponification figure was 244.4. The
extract contained 81.14 per cent, of neutral fat. The amount of
unsaponifiable matter was 0.51 per cent. The molecular weight
of the fatty acids was given as 207. T[

Studied through the oleo refractometer of Amagat and Jean,
cocoa-oil is found to rotate to the left like an animal fat.**

The composition of cocoa-oil as determined by Konig ff is :

c. H. o.

74. 15 per cent. 11.73 per cent. 14.12 per cent.

* Hammerbacher : Landwirtschaftlichen Versuchs-Stationen, etc., 18: 472. 1875.

f Benedikt und Zsigmondy : Chemiker Zeitung, 9 : 975. 1885.

\ Crossley and Le Suer. Quoted by Hopkins : Oil-Chemists' Handbook, 38, table
iv. 1900.

jl Stellwaag : Landwirtschaftlichen Versuchs-Stationen, etc., 37 : 135. 1890.

■\ See also Konig, Menschlichen Nahrungs- und Genussmittel, etc., 2 : 389. 1893.

** Blyth : Foods, Their Composition and Analysis, 359. 1896.

If Konig : loc, cit., 2 : 3S5. See also, Brandes, Pharmaceutisches Centralblatt,
751. 1838.

338 KiRKWOoD AND GiES : Chemical Studies

The follo\vin;4 facts regarding cocoa-oil have been compiled
from various sources. They may be compared with similar data
for other fats and oils given in the standard works of Konig,
Staedeler, Lewkowitsch and others :

A. The heat of combustion of cocoa-oil is 9,066 small calories
per gram.* It is as low as that of any other fat ; slightly lower
than butter. This is due to the fact that it contains a large pro-
portion of fatty acids of low molecular weight.

B. Melting point is at 24° C. Congealing temperature is 22-
23° C. Fatty acids from it melt at 24.6° C. They congeal at
19° C.t

C. Saponification value = 257. 3-268. 4^

D. Iodine number = 9.0-9.5 ; same for its fatty acids = 8.5-
9.0. §

E. Specific gravity = 0.91 i 5 at 40° C.||

F. Acid value = 9.95-35.21.

G. Reichert-Meissl figure = 7.4 ; Hehner = 88.6-90. 5.
H. Barium figure (Konig-Hart) = i 17-120.

I. Molecular weight of the mixed fatty acids = 196-21 1.

The use of cocoa-fat and other cheap vegetable oils as a
substitute for butter among the poorer classes has been in-
creasing. Cocoa-fat is better adapted for cooking than for table
use. It is frequently employed as an adulterant of ordinary
butter. Prepared cocoa-fat makes a fairly good substitute for com-
mon butter. The fresh material becomes rancid after a time,
because of its accumulating content of free fatty acid resulting from
bacterial agency. Volatile acids are formed. Its tendency to
rancidity is not as great, however, as that of animal fats. The
fatty acid present in the fat to begin with can easily be removed
with insoluble basic compounds, such as magnesia. By this means

* Merrill. Quoted by Sherman and Snell : Journal of the American Chemical
Society, 23 : i66. 1901.

f Konig : Menschlichen Nahrungs- and Genussmittel, etc., 2: 322. 1893.

X Konig : //>ii/.

^ Benedikt. Quoted by Vaubel : Physikalischen und chemischen Methoden
quantitativen Hestimmung organischer Verbindungen, 2 : 235. I902.

II Values' given after E-I inclusive are quoted by Hopkin.s : Oil-Chemists' Hand-
book, 38, table iv. igio. See .ilso Lane : Journal of the .Society of Chemical
Industry. 2o : 1 033 19^1.

OF THE COCOANUT DURING GERMINATION 339.

a "butter" is made from this oil which has the merit of enduring
hot cHmates without becoming rancid. This product has been
recommended for military and naval uses.*

Among the prominent commercial products is the cocoa-butter
made in Mannheim, Germany, f Konig]}: found this product to
have the following percentage composition :

Nitrogenous
Water. Solids. Organic Matter. Inorganic Matter. Fat. Fatty Acid. Substance.

0.15 99-^5 99 848 0.002 99.848 trace. trace.

It has been stated that cocoa-butter is not very easily digested
and that it does not agree with sick people. § The recent re-
searches of Bourot and Jean, || however, show that a specially
prepared cocoa-butter melting at 3 1° C. and containing only a trace
of free fatty acid, is quite as easily and completely digested as
ordinary butter.^

We have already alluded to some of the commercial uses to
which cocoa-fat is put. Soaps made from it combine with or hold
an unusual amount of water while still retaining special hardness,
one pound of the oil yielding three pounds of soap.** It is thus
well adapted for the preparation of toilet soaps. The soaps made
from cocoa-oil are characterized by great solubility in salt solution
and can be precipitated from such fluid only by the addition of a
very large excess of salt. The so-called "marine" or "salt water
soap" has the property of dissolving as well in salt water as in
fresh water and is made of cocoa- oil and soda.ft

*Rusby : Reference Handbook of the Medical Sciences, 3 : 164. 1901.

f See Leffman and Beam : Select Methods in Food Analysis, 182. 1901.

J Konig : Menschlichen Nahrungs- und Genussmittel, etc., 2 : 309. 1893. See also
Schaedler, Technologic der Fette und Oele des Pflanzen- und Thierreichs, 1340. 1892.

^ Liebreich : Encyklopaedie der Therapie, i : 744. 1896.

II Bourot und Jean : Jahresbericht uber die Fortschritte der Their- Cheniie, 26 :
58. 1896. See also v. Knieriem, Chemisches Centralblatt, 2 : 672. 1898.

II "Cocoanut cream," a dietary product much used in the tropics, is made by grat-
ing the endosperm and squeezing through cloth the fluid from the finely divided
material. In a warm climate the resultant mixture contains much oil and is a very
delicious accessory food. Besides the oil, the "cream" contains chiefly carbohydrate
and proteid. See page 332 for references to similar fluid obtained from the endospenn
by pressure in our own experiments.

** Ebermayer : Physiologische Chemie der Pflanzen, 344. 1882. See also Joss,
Pharmaceutisches Centralblatt, 449. 1834.

It See Schaedler, Technologic der Fette und Oele des Pflanzen- und Therreichs,
II78-I188, 1892, where may be found the results for percentage composition of the
sodium soap, given at the bottom of the next page :

340 KiRKwooD AND GiES : Chemical Studies

The harder fats of the oil make excellent candles. They are
used also as constituents for suppositories and related therapeutic
products. Medicinally the oil is employed repeatedly as a substi-
tute for lard, olive oil and cod-liver oil. It is also made the chief
substance by bulk in various salves and in cold cream, pomade and
similar cosmetic preparations. In ointments and cerates it is
especially valuable because of its ready absorption when rubbed
on the surface of the body ; further, it takes up an unusual amount
of water — a useful quality when it is desired to apply saline solu-
tions externally. It shows little tendency to produce chemical
changes in substances with which it may be associated.

Cnide Fiber and Carbohydrates. — Cellulose is a prominent con-
stituent of the endosperm. Associated with the fibrous elements
is a polysaccharide, present in comparatively large quantity. This
substance is only slightly soluble in water, is insoluble in alcohol,
but readily soluble in salt solution. It is precipitated along with
globulin when saline extracts of the kernel are dialyzed ( page
341). The gum is readily transformed into sugar by the action
of diastase or ptyalin.

The fluid pressed from the finely divided endosperm contains
a slight amount of reducing sugar — dextrose. Galactose appears
to have been identified also."^ Cane-sugar is also present.

The following results were obtained in our determinations of
the percentage content of crude fiber in the fresh tissue : t

12 345 General Average.

Fresh endosperm, a 3.96 3.20 2.98 3.40 2.78

b 4.21 3.80 3.12 3.52 2.98

Average, 4.08 3.50 3.05 3.46 2.88 3.39

Proteids. — That the meat of the cocoanut contains at most
only a very small amount of proteid matter is seen at a glance
from the following percentage results for content of nitrogen.;];

Water. Fatty Acid. Sodium Oxide (combined). Sodium Oxide (free). Other Salts. Residue.
58.74 32.82 4.26 1.50 2.26 0.42

See also the Dispensatory of the United States of America, 1899 : 1619, for ref-
erences to objectionable chemical qualities of some cocoa-soaps.

* Green: Soluble Ferments and Fermentation, 100. 1899.

t Determinations were made, after the fresh weighed material had been dried and
thoroughly extracted with ether, by the method adopted by the Association of Official
Agricultural Chemists : Bulletin, Division of Chemistry, U. S. Department of Agricul-
ture, 46 : 26.

% In these determinations the Kjeldahl method was employed.

OF THE COCOANUT DURING GERMINATION 341

General
123456 7 Average.

Fresh endosperm, a 0.657 0.734 o.8c6 0.738 0.766 0.776 0.701
b 0.740 0.781 0.756

Average, 0.657 0.734 0.806 0.738 0.753 0.778 0.729 0.742

The fresh endosperm contains an average of 0.74 per cent, of
nitrogen which, multiphed by the usual factor, 6.25, would indi-
cate 4.63 per cent, of "albuminoid." Some of this nitrogen,
however, is undoubtedly closely associated with the fibrous ele-
ments. Much of it probably is in the form of nitrogenous ex-
tractive.* Some of the nitrogenous substance is soluble in 95 per
cent, alcohol.

The proteid present in the endosperm appears to consist chiefly
if not exclusively of globulin and proteose (globulose ?), the globu-
lin predominating in quantity. f We have made several samples of
cocoa globulin by the method used by Osborne for the preparation
of edestin — in general as follows : % The kernel was run through
a hashing machine and the finely minced substance freed from fat
by repeated extraction in ether for several days. The ether ad-
herent to the tissue was evaporated at room temperature and the
ether-free tissue then extracted in lo-per-cent. salt solution for 24—
48 hours. The saline extract was then filtered off and globulin
thrown from its solution either by the dilution process, by dialyz-
ing for several days in running water, or by treatment with am-
monium sulphate to complete or half-saturation. The deposit of
globulin resulting thereby always contained an appreciable amount
of gummy carbohydrate. The carbohydrate admixture was elimi-
nated by subjecting the deposit to the action of diastase or ptyalin
for 24—48 hours, in the presence of thymol at 45° C. in neutral
fluid, during which time it was transformed into soluble reducing
sugar. § The globulin residue left behind after this treatment was

*The factor 6.25 is here too large, also, because the proteids present contain about
18 per cent, of nitrogen. See pages 343 and 344. Stutzer found that, of the total
nitrogen of cocoa-cakes, from 1.8 to 6.9 per cent, was contained in non-proteid substance.
Quoted by Dietrich and Konig : Zusammensetzung und Verdaulichkeit der Futtermittel ;
2 : 987, 1380. 1891.

•(" The amount of nucleoproteid must be very slight.

J Osborne : See various papers in the Journal of the American Chemical Society
since 1894.

\ Similar diflSculty was experienced by Osborne, who got rid of the gum by repeated
dialysis and precipitation with ammonium sulphate. Journal of the American Chem-
ical Society, 17 : 429, 539. 1895.

;"142 KiKKwoon and Gies : Chemical Studies

further purified by re-solution and re-precipitation. For (juantita-
tive analysis some of the final product was washed in water, alco-
hol and ether, and dried at I00°-I05° C. to constant weii^ht.

Sometimes the globulin prepared in this way was both crystal-
line and amorphous. At other times it was entirely crystalline.
Triangular, hexagonal and rhombohedral forms were frequently
seen, although octahedra predominated.* The crystals s(^ closely

Fk.. 4. Crystals of cocoa edestin.

resemble those we have repeatedly made from hempseed and lin-
seed by the same method, and are so like those given by Osborne
for edestin.t that we felt satisfied from the beginning our globulin
would prove to be of the edestin type. Careful study of the re-
actions of the substance convinced us of this fact, for it gives all of
those attributed to edestin by Osborne.

* The large proportion of gum extracted by the saline solution made it difficult not
only to prepare the proteid in pure form but to obtain it quantitatively. Besides, the
edestin passed in part into an insoluble modification during the manipulations. An
appreciable loss resulted, therefore, in each preparation. We obtained as much as 25
grams of the purified product from the kernels of twelve nuts.

1 0.sborne : Journal of the American Chemical Society. See also his pajier or*,
crystalline vegetable proteids in the American Chemical Journal, 14 : 28. 1893.

OF THE COCOAXUT DURING GERMINATION 343

On the opposite page we give a microphotographic view of
edestin crystals from our second preparation. Although not the
purest, we have selected this preparation for this purpose because
its crystals are mostly rhombohedra. These forms rarely occur in
abundance in edestin precipitates, octahedra being more commonly
obtained. Most of the larger masses among the crystals shown
here are "rounded" octahedra ; not in perfect focus because they
are thicker than the rhombohedra. The smaller particles consist
of globular matter and crystal pieces.

The crystals given in Fig. zf. were photographed for us by the
writer's colleague, Dr. Edward Teaming, who cordially gave us
the benefit of his large experience. We wish here again to extend
to Dr. Teaming our sincere thanks for his valuable assistance.

That the substance under discussion is edestin is further shown
by the results of analysis. We append our results for nitrogen
content, as determined by the Kjeldahl method, calculated for ash-
free substance :

Percentage of Nitrogen in Cocoa Edestin

Preparation . i

I

2

3

4

5

Analytic results.

17.87
17.77
17-79

17.8S
17.96

17.91

17.66
17.69
17-78

18.14
18.21
18.18

18.23
18.20
18.28

Average.

17.81

17.91

17.71

18.18

18.24

Ash.

0.41

0.13

1. 12

1.90

1.84

Preparations I, 2 and 3 contained amorphous material, possibly some of the
gummy matter referred to on page 342, in spite of our efforts to completely remove it.
Preparations 4 and 5 were obtained from 1 and 3 by further treatment with diastase
and by recrystallization by dialysis from lo-per-cent. salt solution. They were practi-
cally wholly crystalline.

The above results show that the globulin separated from the
cocoanut by the methods here employed is edestin.*

This same proteid of the cocoanut was examined by Ritt-
hausen, f who termed it conglutin without really identifying it with
that substance. His analyses gave it a content of nitrogen of
1 7. 87-17. 91 per cent. Chittenden and Setchell % referred to it by

* The edestin from barley contains 18.10 per cent. N. That from maize 18.12
per cent.; from rye, 18.19 per cent.; wheat, 18.39 P^"" cent. Osborne : Journal of the
American Chemical Society, 17 : 547. 1S95.

f Ritthausen : Jahresbericht iiber die FortschrittederThier-Chemie, 10 : 18. 1880.

X Chittenden and Setchell : Quoted by Chittenden, Digestive Proteolysis, 32. 1895.

344 KiRKwooL) .\ND GiES : Chemical Studies

the name of phyto\'itellin. The composition they gave for it is in
general accord with that of edestin (nitrogen content = 18.40 per
cent.), and as they obtained it partly crystallized in octahedra, Os-
borne * has lately suggested that the substance is edestin. The
results we have obtained confirm Osborne's deduction.

The proteose to which we have already alluded was obtained
from the globulin filtrate. The latter was freed from traces of
globulin by the coagulation method, the hot filtrate evaporated to
a small bulk on the water-bath and the proteose precipitated and
purified by the usual method, t About four grams were obtain-
able from fifteen nuts. The product contained both proto and
deutero forms. Some heteroproteose was also detected in the
products formed on dialysis and a trace of dysprotose was
obtained.

The following results for nitrogen content in the ash-free sub-
stance were obtained b}- the Kjeldahl method :

Fkrcf.ntac.e of Nitrogen in Cocoa Proteose

Preparation.

I

2

3

18.57
18.61
18.54

1 General Average.

Analytic results.

18.67
18.50
18.58

18.48
18.46 ,
18.40

Average.

18.58

18-45

18.57

18.53

Ash.

1. 71

1.08

1. 21

i-33

These results differ only slightly from those reported by Chit-
tenden and Setchell-t This difference may be explained by the
fact that mixtures of proteoses hav^e been analyzed in each case
by Chittenden and Setchell.and by us. Their preparation of pro-
teose contained 18.25 per cent, of nitrogen.

In liis volume entitled Digestive Proteolysis, Chittenden gives
the analytic results for eleven different proteids and the proteoses
derived from them (page 67). For seven of these the nitrogen of
the corresponding proteose is somewhat higher than that of the
original proteid. Analysis of our own preparations has shown the
percentage of nitrogen to be greater in the proteose than in the
globulin, a result in accord with the majority rule just noted.

♦Osborne : Journal of the American Chemical Society, 18 : 13. 1896.

fMacDougal: Practical Text-book of Plant Physiology, 164. 1901.

i Chittenden and Setchell : Quoted by Chittenden, Digestive Proteolysis, 32. 1895.

OF THE COCOANUT DURING GERMINATION 345

There appeared to be only a very slight amount of an albumin
in our extracts — a coagulable substance which was not precipitated
from its neutral solution when the latter was half-saturated with
ammonium sulphate.*

Osborne's methods of extracting glutenin and gliadin f in dilute
alkali and acid, and in dilute alcohol, after the removal of globulin,
proteose and albumin as above described, gave mere traces of pro-
teid substances in solution, derivatives, doubtless, of the proteids
already referred to, which perhaps had not been completely re-
moved from the residual tissue ; or possible nucleoproteid.

Peptone could not be detected in any of our extracts. J

Ash. — Composition is referred to on pages 331 and 335.
Qualitatively our results were the same as those there given.

Enzymes. — Water, salt solution and glycerin each failed to
extract appreciable quantities of either proteolytic or adipolytic
enzymes from the endosperm of the fresh, ungerminated nut,
although an active amylolytic ferment was extracted by all of
these fluids. The large quantities of fat and fatty acid in the endo-
sperm suggest that an emulsifying ferment maybe present. This,
however, may be localized in the germ, increasing to physiolog-
ical quantity and activity only in the process of germination (see
page 358). The proteoses present in the endosperm seem to
imply the presence of a proteolytic ferment. Possibly, however,
the proteoses represent a residue from which the globulin was
derived by reverse process. §

We have already referred to the fact that oxidase has been de-
tected in the milk. Traces of it are also contained in the endo-
sperm.

Average Composition. — The average results of our analyses of
the endosperm are summarized in the following table, which pre-
sents the data obtained for the composition of the fresh tissue and
the dry solid matter derived from it (constant weight at 100—
105° C.).

* Cohnheim : Chemie der Eiweisskorper, 150. 1 900.

t Osborne and Campbell : American Chemical Journal, 15: 392. 1893.

% Small quantities of non-proteid nitrogenous substances were detected by Ritt-
hausen : Chemisches Centralblatt, 230. 1880. Compare, also, with recent results
respecting proteoses obtained by Bokorny : Chemisches Centralblatt, i: 1167. 1902.

§ See recent papers in the Zeitschrift fiir physiologische Chemie by Schulze and
Kutscher and their associates.

346 KiKKwoon and Gies : Chemical Studies

Percentage Composition of the Endosperm

Constituents.

Water.

Solids.

Inorganic matter.
Organic matter.

Fat (substance soluble in ether).
Crude fiber (cellulose).
Proteid (NX 5-5)t
.Soluble carbohydrate, non- nitrogenous
substance, extractive, etc. (by differ-
ence).
Nitrogen.

Fresh Endosperm.

Dry Endosperm.*

46.31

5369

I 03

1-93

52.66

98.07

3729

69-45

3-39

6.31

4.08

7.60

7.90

14.71

0.742

1.382

The previous results obtained by Hammerbacher;|: for the
fresh endosperm from nuts of eastern origin were as follows:

Non-nitrogenous
Water. Solids. Inorganic Matter. Organic Matter. Fat. Crude Fiber. Proteid. Extractive.
46.64 53.36 0.97 52.39 35.93 2.91 5.49 8.06

Composition of Shell and Husk. — We have already alluded
to some of the uses to which the shell and husk of the cocoanut
are put by reason of the chemical and physical qualities they pos-
sess. Some facts regarding their chemical composition were given
in the table on page 335.

The following percentage results of elementary analysis of the
powdered shell were obtained by Baumhauer;§ all samples having
finally been thoroughly extracted in alcohol and ether, and then
dried at 120°- 150° C:

Cocos nuci/era. Cocos lapidea.

' _, J • 3- Extracted in

T J • ^L ^?'''^*^'™ '" boiling water, 4. Extracted in 5. Same 6. Same

I. Extracted in boiling water concentrated alkali and in treatment treatment

boiling water. dilute alka.i and alkali and acetic chlorine water. as i. as 4.

acetic acid. ^cid.

c. '

52.99

H.

5.88

Ash.

1-43

47.19 ; 46.27
6.09 5.81
1. 00

43-72
6.11

52.20
5.80

44.20
6.24

0.22

0-55

* According to Dietrich and Konig (Konig, Menschlichen Nahrungs- und Ge-
nussmittel, etc., i : 612. 1893) the air- dried %v!a%K.zx\c^ contains the following in per-
centage of the total dry weight :

Total Substance Soluble in Water. Proteids Soluble in Water. Sugar (Sugar-Yielding Substance).
15.16 2.27 9.25

t The factor 5-5 is used because the proteids of the endosperm contain 18 per cent,
of nitrogen. See references in this connection on page 341, footnote.

+ Hammerbacher : Landwirtschaftlichen Versuchs-Stationen, etc., 18 : 472. 1875.
§ Baumhauer : Pharmaceutisches Centralblatt, 601. 1844.

OF THE COCOAXUT DURING GERMINATION 347

Nitrogen was detected in small amount in the powders which
had not been treated with alkali. The alkaline extracts contained
substance, precipitable by acetic acid, with the following percentage
composition: From Cocos mtciftra, C= 50.04, H= 5.81, ash =
4.45 ; from Cocos lapidea, C = 52.1 5, H = 5.93, Ash = i.oo.

Tromp de Haas and Tollens * were able to show the presence
of a large amount of pentosane (xylan) in the endocarp, the pow-
dered material yielding an abundance of xylose on hydration in 4
per cent, sulphuric acid. Mannose was absent from the acid solu-
tion from which the xylose had been crystallized. After xylan
had been completely removed from the shell-powder by the above
method, dextrose was derived from the residue on treatment with
sulphuric acid in the usual manner.

In his very complete histological studies of the cocoanut, Win-
ton t recently called attention to the fact that both the husk and
shell contain a brown substance which is quickly changed to a red-
dish color by caustic potash, but is unaffected by alcohol, ether or
any of the specific reagents for proteids,^ fats or resins. He also
states that no immediate effect is produced by ferric chloride solu-
tion, but on long standing the color is changed to olive green.
Winton has pointed out the presence of minute silicious bodies
among the fibers of the husk.

Winton, Ogden and Mitchell % give the following percentage
data for the composition of the shell :

Water,
Solids,

Organic matter,
Inorganic matter.
Soluble in water.
Insoluble in HCl,
Ether extract.
Non-volatile,
Volatile,

During germination
soon begins to decay. See page 351.

* Tromp de Haas and Tollens : Chemisches Centralblatt, 2 : 359. 1895.

•j- Winton: American Journal of Science, IV. 12: 265. 1901. Facts are also
given regarding the use of powdered cocoa-shell and the husk as adulterants of ground
spices.

\ Winton, Ogden and Mitchell : Report of the Connecticut Agricultural Experi-
ment Station, 2 : 210. 1898.

7.36

Alcohol extract.

1. 12

92.64

Reducing matters calculated

as

9946

starch.

20.88

0-54

Slarcli,

0.73

0.50

Crude fiber,

56.19

0.00

Nitrogen,

0.18

0.25

Albuminoid (NX 6 25),

113

0.25

Quercitannic acid.

1.82

0.00

: shell remains unaltered.

The husk

348 KiRKwooD AND GiEs : Chemical Studies

CocoANUT Pearls. — Within the nut there is occasionally found
a small stony substance of a bluish white color, a kind of vege-
table bezoar, called in India calappa, which is eagerly purchased
by the Chinese, who ascribe great virtues to it as a sort of amulet
to preserve them from diseases. The cause of its formation in the
nut is unknown.

According to Harley and Harley * these pearls, like those of
molluscan origin, appear to consist almost entirely of calcium
carbonate, with water and organic matter in smaller proportion.
Riedel, quoted by Harley and Harley, states that in 1886, while
in North Celebes, he found a pearl in the endosperm of the cocoa-
nut. One such a pearl was pear-shaped in form and 28 mm. long.

We are greatly indebted to Dr. D. Morris, Imperial Commis-
sioner of Agriculture for the West Indies, for the following very
interesting quotation from a letter to Dr. MacDougal :

" More than two hundred years ago Rumph, an eminent bot-
anist in the East, sent as a present to the Grand Duke of Tuscany
a ring in which a cocoanut pearl had been set. Further, Rumph
himself described cocoanut pearls in his great work with consid-
erable minuteness and gave illustrations of two of them. One
was perfectly round, the other was oval or egg-shaped. * * *
Travelers in the Philippine Islands have heard of cocoanut pearls,
but seldom or ever have seen them. The natives, it is said, keep
" cocoanut stones " as charms against disease and evil spirits.
The rajahs, we were told, highly prized them and wore them as
precious stones. It was only a few years ago that real cocoanut
pearls were at last brought to England. One is now at the Mu-
seum at the Royal Gardens at Kew, brought by Dr. Hickson.
It is almost egg-shaped, perfectly white, and composed almost en-
tirely of carbonate of lime. It has, in fact, a somewhat similar
composition to the pearl of the oyster, and yet there is little doubt
it is a purely vegetable product." f

* Harley and Harley: Proceedings of the Royal Society of London, 43: 464.
1887-88.

t '• Besides these cocoanut pearls," quoting further from Dr. Morris' letter to Dr.
MacDougal, " Rumph describes what he calls ' Melate ' pearls taken from the flowers
of a Jasmine; and a ' Champake ' pearl taken from the flower of a Michelia. If we
had not already seen the pearl of the cocoanut it would have been impossible to believe
that there were such things as Jasmine and Michelia pearls * * * Of their composition,
mode of occurrence and true nature we have yet to learn." See the article by Harley
and Harlev referred to above.

OF THE COCOANUT DURING GERMINATION 349

See also, on the subject of cocoanut pearls, the Proceedings of
the Boston Society of Natural History, 1861 and 1862 ; The
Tropical Agriculturalist, 1887; Nature, 1887.

III. Changes in the Cocoanut during Germination

The nuts for our studies of the changes occurring during
germination were obtained fresh, fully developed and with their
husks still on them, directly from Jamaica. Immediately after their
arrival at the New York Botanical Garden they were imbedded in
earth until they were nearly covered. The earth was kept saturated
with water and a tropical temperature was maintained. These con-
ditions closely approximated those attending normal germination.

Morphological Changes. — Nearly four months elapsed before
the shoots began to appear through the husks, the fibers of the
husk having been pressed aside in their upward progress. At
this stage the stem of the shoot was an inch or more in diameter
at the " root-crown," sharply tapering toward the point of pene-
tration at the surface of the husk. As the growth proceeded it
seemed to gradually become more and more rapid, and by the end
of a year the plants had attained the height of two or three feet,
with a stem about an inch in diameter throughout most of its length.
By this time the part of the husk under the earth had decayed
considerally; it became softer and more porous, and several stout
roots had developed through it and penetrated the soil to the
depth of a foot or more.*

The appearance of the nuts and their plants at this period of
their growth is shown in the cut on page 350. Unless other-
wise stated, the chemical analyses reported farther on were made
of the parts at this stage of their development.

It may not be amiss, in describing the morphological changes
induced in the nut during the process of germination, to also
briefly review, at the same time, the more important facts regard-
ing structure of the nut as it exists in the ungerminated condition.

The entire fruit is, strictly speaking, intermediate between a
nut and a drupe — a "drupaceous nut." The outer covering,

* For facts regarding germination and cultivation see Bailey : Cyclopedia of
American Horticulture, I : 341-343. 1900. Also, Wittmack, L. : Die Keimung der
Cocosnuss. Ber. d. deut. bot, Ges. 14 : 145. 1896.

350 KiRKwooD AND GiEs : Chemical Studies

Fig. 5. Germinating cocoanut at the end of a year, showing plumule and roots,
with husk little altered except where it was m contact with the earth.

usually removed before the nut appears upon the market, is a
thick fibrous layer comprising the exocarp, the epicarp consist-
ing of a smooth, thin, tough coat of a brownish or grayish color.
(See pp. 3 23 and 3 24). The endocarp, or what is commonly known
as the shell of the nut, is composed of three carpels whose lines
of fusion are always apparent. Tlie nut lies in the husk with the
end containing the " eyes "" toward the pedicel. Each carpel con-
tains an "eye," so-called, and under one of these three eyes, the

OF THE COCOANUT DURING GERMINATION 351

softest, is the germ imbedded in the endosperm. The fertile carpel
may be recognized from the fact that it has the greatest degree of
divergence between the longitudinal fusion lines of the carpels.
The true integuments of the ovule are reduced to a thin brown
coat closely adhering to the abundant endosperm.

The embryo is a cylindrical body about 8 mm. in length lying
below one of the natural openings of the endocarp and in a line
perpendicular to the exterior surface of the endosperm. When
germination begins the embryo elongates and, having pushed
through its thin coverings, begins to enlarge at both ends.
From the outer end arises the plumule and the roots ; the inner
end is an extension of the true cotyledon and is developed into a
special absorbing organ. See //. ig.

The absorbing organ is of a soft spongy texture and all
through it are the ramifications of vascular strands which converge
to the narrow " neck, " which connects the absorbing tissue with
the stem. The cotyledon, and by this term, hereafter, we shall
mean the part of that structure specialized for absorption, can
attack only the part of the endosperm to which it is contiguous.
In the earlier stages of growth this absorption is confined to the
part nearest the young shoot, which we may hereafter refer to as
the proximal end of the nut. Finally, however, the cotyledon
fills the entire cavity of the nut and somewhat thins the endosperm
distally, also.

The milk may persist in the nut until the cotyledon has almost
filled the cavity. After germination has proceeded for some time
the milk becomes insipid to the taste, and contains fragments of
cellulose and large drops of floating fat. In nuts in which germi-
nation has continued for a year the cotyledon has entirely filled
the cavity, but usually there is still left a third to a half of the endo-
sperm undigested. This residual portion in normal cases is little
affected, except that it is softened superficially, and to the taste
suggests nothing different from the meat of the ordinary ungermi-
nated nur.

In its natural development the roots of the plant soon take
firm hold of the soil and, long before the endosperm is com-
pletely absorbed, junction between the shoot and the absorbing
organ is broken, the husk decays and the plant enters an inde-

i]r>2 KiKKWooD AND GiES : Chemical Studies

pendent career. Neither the husk nor the shell appears to serve
any other than passive mechanical function, and only the constitu-
ents of the endosperm and milk, so far as the nut is concerned,
nourish the young plant before it finds in the soil the elements
provided there in abundance for its growth to maturity.*

In our microscopic studies, particularly of the cotyledon,
pieces of the fresh part were " fixed " in a mixture of glacial
acetic acid {}^) and yofj alcohol (^). After remaining in this
fluid for a few hours the pieces were transferred to 70^^ alcohol
and later to 85^ alcohol, in which they were kept. Sections were
cut with a razor and mounted in glycerin. Treated with iodine,
such sections of the cotyledon showed an abundance of starch in
all cells except those of the outermost layer or epidermis. This
outer layer stained yellow with iodine. That it contained an abund-
ance of oil was shown by its deep black reaction with osmic acid.
Large globules of oil are, however, distinctly visible in the epidermis
under the microscope without the aid of osmic acid. Oil may
also be found in the subepidermal layers, but it rapidly diminishes
in quantity as the distance from the outside increases and as the
starchy deposit accumulates. Needle-like crystals may be very
readily found in the epidermal cells. These resemble crystals of
tripalmitin, but the fact that so much oil appears in globules, and
that the breaking down of fats must occur to a large extent in
these cells, would suggest that they are palmitic acid rather than
the fat itself.

The above facts make it appear that the starch is formed indi-
rectly at lea.st from the oil.f See references under enzymes,
pages 345 and 358.

See />/. ig for drawings of parts mentioned above.

Che.mical Changes. — The following summary gives briefly
the effects of germination on the individual nuts examined :

I. Not Analyzed. — A. Development had proceeded for nearly
six months. The plumule protruded six inches above the husk.
Roots had developed through the husk — two were about a quarter
of an inch in diameter. The stem was very thick at the " root-

* Note references on page 357 to the functions of the husk in holding water and
possibly furnishing nutrient matter in its decay.

tEbermayer : Physiologische Chemie der Pflanzen, 347. 1882.

OF THE COCOANUT DURING GERMINATION 353

crown" ; sharply tapered to the point of surface penetration. The
fibers about the stem were tightly pressed together. The neck of
the absorbing organ was very woody and fibrous in character.
Absorption of the endosperm at the proximal end was quite
marked ; distal portion undiminished. Milk cavity largely filled by
the absorbing organ. A small space at the distal end remained,
containing viscid white material full of large oil globules ; quantity
less than lO c.c. — doubtless concentrated milk. It was strongly
acid in reaction from acid phosphate, reduced Fehling's solution,
gave only a faint biuret reaction and was free from starch. The
inner surface of the endosperm in the distal portion was soft, having
the consistency of lard.

The weight of the whole shoot, minus the roots, in the fresh
condition was 28.1 grams. Dry, the weight was 4.25 grams or
1 5. 1 per cent, of the fresh substance, indicating a presence of 84.9
per cent, of water in the original plant.

B. This nut, although germinating for the same period of time,
was not quite as far advanced as the previous one, having shoots
that were just emerging from the husk. The stem was thicker,
however. In most respects its internal condition was exactly the
same as that of the first. The fluid in the distal cavity was less in
quantity, not as turbid, contained less oil — otherwise was the same
as that of the previous nut.

The weight of the entire plumule was 38.8 grams. Dried,
it weighed 5.75 grams. Thus it contained 14.8 per cent, of solid
matter and 85.2 per cent, of water.

II, Analyzed. — i. Growth continued for eight months. The
cotyledon entirely filled the cavity. About half of the endosperm
was absorbed ; practically all of that proximally except a thin
layer. The distal residue of endosperm appeared to be normal in
taste and appearance except on the surface, where the soft layer
previously referred to — one fourth the entire thickness — could
again be seen. The outer surface of the cotyledon, that part in
contact with the endosperm, was much corrugated ; the whole
organ, solid but spongy, sweet and agreeable to the taste, pyriform.
In the tables on page 354 and 355 the results of our analyses for
this nut are indicated by the numeral i.

354

KiKKwoon AND GiES : Chemical Studies

General Composition of the Parts of the germinat^^ Cocoanut

Percentage of fresh Tissue.

Parti of the Nut and its Plant.

Solid Matter.

Water.

I. Cotyledon.

A. Central, vascular portion :

Central, between center
and surface :

Total.

Or- I In-
ganic. I organic.

Percentage of
Solids.

Or- In-

ganic I organic

' Matter. Matter.

a--l

C — 2

d—2
'^— 3

/-3

Average.

B. Outer, corrugated portion :

Outer jMjrtion — neck :

a — I

h—2

89.10

87.71
91.62
91.41

88.99

86.07
89- 1 5

84.95
82.79

So. 83
78.98

10.90

99.14

0.86

12.29

99.10

0.90

8.38

99.00

1. 00

8-59

99.11

0.89

II. 01

99.20

0.80

13-93

98.94

1.06

10.85

99.08

o.ga

150S

98.69

1.31

17.21
19.17

21.02

9893
98.26

98.58

1.07

1.74

1.42

92.07
92.25
88.10

89.71

92- 77

92.36

gi.2i

91.30
93-85
90-93
93-24

7-93

7-75

11.90

10.29

7-23

7.64

8.79

8.70
6.15

9.07
6.76

Average.

81.89 I 18.IX 98.62 ' 1.38 I 92.33 I 7.67

II. Residual end ■spenn.

C. Provimal portions after
much absorption :

a — 2

19.09
23.42

80.91

76.58

99-13
99.16

0.87
0.84

98.92
98.90

Average.

21.25 j 78.75 99.15 0.85 I 98.91 ! I

D. Medial portions :

a — 1

t>—i
c — 2

Average.

£. Distal, normal portion : a — 3

III. Stem of the plant.

F. Base, " root crown," with

petioles at lowest parts : a — I

Average.

31-65
3036
28.68

2577
29.12
46.08

68.35
69.64

7132
74-23

70.88
53-92

99-03
99.06
99.25
99- 23

99-14
99.02

0.97
0.94
0.75
0.77

0.86

0.98

98.58
98.65
98.95
98.96

98.78

98.12

08
10

09

G. Parts above the base, with
more petioles :

a — 2
^-3

86.21 13.79 98.95 1.05

86.51 13.49 98.70 1.30

85.15 14.85 98.84 1. 16

84.31 15.69 98.68 I 1.32

8555 14-45 98.79 ! I-2I 91.60 8.40

92-37
90.38

92.06
91.60

763

9.62

7-94
8.40

Average.

IV. Petioles.

H. Alone, or with young
leaves :

a—\
b—\
^—3

82.47 1753 98.80' 1.20
79.87 20.13

81.17 I 18.83

93.20

6.08

83.63 16.37 98.57

82.55 17-45 98.63

82.13 17.87 98.75

82.17 i 17.83 98.63

1.43 91-27 8.73

1.37 92.15 7.85

1.25 9301 6.99

1.37 i 92.34 1 7.66

Average.

82.62 17.38 I 98.65 ; 1.35 ' 92.19 7.81

OF THE COCOANUT DURING GERMINATION

355

General Composition of the Parts of the germinated Cocoanut. — Cotiiinued

Percentage of fresh Tissue.

Percentage of
Solids.

Parts of the Nut and its Plant.

Water.

Solid Matter.

Or- Inor-

Total.

Or-
ganic.

In-
organic.

ganic
Matter.

game
Matter.

V. Leaves.

I. Mature or nearly so : a — i
b—i

C 2

d—-2

e~7,
/-3

74.66
71.99
72.60
72.51
68.45
70.65

25-34
28.01
27.40
27.49
31-55
29-35

98.35
98.10

98-34
98.41
97.96
98-39

1-65
1.90

1.66

1-59
2.04
1. 61

93-49

93.20

93-93
94.19
93-52
94-52

6.51
6.80
6.07
5.81
6.48
S-48

Average. i 71.81 ■ 28.19 98.26

1.74

93.81

6.19

J. Very youngest : a — 3

87.22 12.78 98.50

1.50

86.67

13-33

VI. Roots.

K. Short, not dereloped outside

of husk, with soft tips : a — I
b—2
c — 2

87.08 12.92 98.46
89 89 I 10. II 98.67
86.41 13.59 98.43
87.46 I 12.54 i 98.83

1-54

1-57
1. 17

88.09
86.89
88.44
90.70

II. 91

13.ll

11.56

9-30

Average.

87.71 j 12.29

98.60

1.40

88.53

1 1 .47

L. Parts of longer roots, taken

near the stem : a—\

b~2

1
1

77.92 22.08

82.65 17.35

81. oq 18.91

79.47 i 20.53

98.80
98.85
98.50
98.77

1.20

1.15
1.50
1.23

94.59
93-34
92.09
94.00

5-41
6.66
7.91
6.00

Average.

80.28

19.72 ! 98.73

1.27

93.51

6.49

M. Parts of longer roots, taken

outside the husk : a — i
b—\

C 2

81.70
84.64
82.79

18.30
15-36
17.21

97-94
98.47
98.40

2.06

1-53
1.60

88.76
90.05
90.74

11.24

9-95
9.26

Average.

83.04

6.96

98.27

1-73

89.85

10.15

2. This nut represented germination after ten months and
was essentially like the preceding in all respects. The roots
were thicker and a number of good sized ones had not yet pro-
ceeded through the husk. Their ends were soft and watery,
rounded and blunt. Analyses of this nut are referred to in the
tables on pages 354-355 by the numeral 2.

3. Essentially the same as No. 2, both in stage of develop-
ment and conditions of parts, although the time of germination
was about two months longer. Analyses of the parts of this nut
are referred to in the tables on pages 354-355 by the numeral 3.

4. This nut had germinated for just about a year. The follow-

3")6

KiRKWooD AND GiES : Chemical Studies

ing weights of the fresh parts were very carefully taken ; the data
for nitrogen were determined by the Kjeldahl method.*

Weight
in Grams.

Colyleilon,

196

Central part,

Cortical layer,

EnJosperin,

155

Distal portion.

Proximal portion.

Shell,

161

Stem,

16

Lower part,

" Root crown,"

Roots,

41

Inside of husk,

Outside of husk,

Petioles and young leaves,

rJi

Petioles,

Old leaves and petioles.

71

Total weight,

698

Shell and contents.

512

Whole plant.

186

Cotyledon and endosperm.

351

Percentage of
Total Weight.

Percentage
of Nitrogen.

28.1

0.14

0.31

22.2

0.65

0.93

23.0

2.3

0.70

053

5-9

0.27

0.54

8.3

0.29

0-39

10.2

045

73-4
26.6

50.3

The tables on pages 354-355 give all our results for general
composition of the parts of the germinated nut.f Numerous
deductions may be drawn from these results, as to growth and
metabolism.

The central part of the cotyledon, with its vascular network,
contains more water and less solid matter than any other part of
the germinated nut. The proportion of solid substance in it in-
creases toward the corrugated epithelium, being greatest in the
" neck," where the structure is fibrous and woody.

That the absorbing organ completely takes up the milk is very
evident from the way it fills the cavity and from its own composition,
but it is likewise apparent from our results that water is also with-

* The husk was not weighed because it was decayed underneath and water-logged.
Nitrogen was not determined in the shell because its substance remains unaltered dur-
ing germination.

t The methods of determination were the same as those used previously. The
roots, outside of the husk, which had been in the wet soil, were hurriedly rinsed with
water to remove inorganic matter, then wiped dry with a towel and at once cut into
thin cross sections for analysis.

OF THE COCOANUT DURING GERMINATION 357

drawn from the residual endosperm, this absorption being greatest
at the proximal end of the nut, where absorption was begun in the
first place, and least at the distal end, where it had hardly com-
menced. The roots also are seen to have absorbed considerable
moisture.

The lowest part of the stem contains almost as little solid
matter and is nearly as watery as the cotyledon. The percentage
of water in the stem diminishes as the distance away from the " root
crown" increases. The watery condition of the lower part of the
stem is increased, doubtless, by the fact that the surrounding husk
is impregnated with water, thus favoring direct absorption by
osmosis and at the same time preventing evaporation from the sur-
face of the growing tissue.

The amount of solid matter in the petioles is also compara-
tively slight, little more than in the lower part of the stem. In
the leaves the water is greatest in the youngest, as would be ex-
pected ; least in the oldest — those most exposed to the air.

The roots at the tips are soft and watery, but the older they
become the more solid matter they develop and the more woody
material they accumulate.

In the distribution of the inorganic matter in the fresh parts it is
noticeable that the proportion of saline substance increases with a
decrease of water and vice versa, as in the cotyledon, in the residual
endosperm and throughout the plumule. This condition is such as
might be expected. The relation of the inorganic to the organic
matter in each part, however, is variable. The substance of the
cotyledon and the stem contains a greater proportion of salts than
that of the endosperm and the leaves, the roots likewise holding
a fairly large amount of saline matter. The substance of the endo-
sperm contains least of all, from which fact it is quite clear that
the inorganic matter of the plumule has been absorbed, not only
by the cotyledon from the milk, but also by the roots from the
fluid in the husk and the surrounding earth.

At the beginning of germination the inorganic matter and
water of the milk are doubtless sufficient for the changes that
occur, the organic matter coming chiefly from the endosperm.
Some time before the cotyledon fills the milk cavity and completely
absorbs the milk, the roots have begun to take water and inorganic

358 KiKKwooD AND GiES : Chemical Studies

matter from the fluid in the husk — possibly also organic substance
from the disintegrating husk fibers — and thus they absorb new
nourishment from a large supply. Growth of the plumule is conse-
quently favored. The plumule soon reaches such a height and
development as to enable it to make increasing contributions to
the plant metabolism from the gaseous products the air affords.
By this time the whole growth has become practically independent
of the reserve material of the seed.

Enzymes. — We made only a few preliminary studies of enzyme
distribution. Extracts were made in water, dilute salt solution
and glycerin. The indicators used in nearly all the experiments
were prepared from the materials in the nut itself.

The extracts of the cotyledon were acid to litmus (phosphates),
though, as indicated by lacmoid, they contained no free acid.
Diastatic ferment was found to be distributed in abundance in all
parts of the cotyledon. Oxidase was also present. Only the very
slightest proteolytic action was manifested by the cotyledon ex-
tracts, even when they were obtained in particularly concentrated
form. In some experiments the results were entirely negative, how-
ever. Cellulose-dissolving and fat-splitting enzymes were not
detected in either the cotyledon or the residual endosperm, al-
though we cannot be sure that in our few experiments they have
not escaped us.* Germination progresses so slowly that possibly
some of the enzymes are present in only very minute quantity at
any one time — in such amount, perhaps, as to be undiscoverabie
by the methods commonly employed for ferment detection. We
did not examine the parts of the plumule in this connection.

At this point, before we were able to come to any very definite
conclusions as to the enzymes present and before we could de-
termine the distribution of proteids, fats, carbohydrates, etc., in the
parts of the plant, we were obliged to discontinue our work. The
writer hopes to extend these experiments on the germinated cocoa-
nut to a consideration of related problems of nutrition.

* See our references to enzymes on page 345. Lipase seems to have been found
in the germinating cocoanut by Lumia : Jahresbericht tiber die Fortschritte der Thier-
Chemie, 28 : 724. 1898.

OF THE COCOANUT DURING GERMINATION 359

Hxplanatlon of Hlate 19

Fig. I. An end view of the cocoanut, without its husk, showing the three car-
pels and the "eyes." The fertile carpel is the one included in the largest angle.

Fig. 2. A sectional view through the end of an ungerminated nut, with its husk
removed, showing the form and location of the germ imbedded in the endosperm
(under the micropyle). The shell is indicated by the heavy outline.

Fig. 3. Another sectional view similar to that of Fig. 2 showing development of
the absorbing organ after germination had proceeded for a few weeks. The incipient
stem and roots are to be seen. The cotyledon has enlarged within and without the
shell.

Fig. 4. A longitudinal section through the whole nut somewhat to the side of the
median line. It shows the nut imbedded in its fibrous husk and the conditions found
after germination had gone on for about five months. The absorbing organ has filled
about two thirds of the cavity of the nut but has absorbed comparatively little of the
endosperm. The stem has proceeded upward through the husk, the roots downward
through the husk into the soil. The leaves have been cut off above the husk. (See
page 350 for appearance of full plumule. )

Fig. 5. A section through the layer of the cotyledon normally in contact with
endosperm, showing the corrugated, villiform arrangement of the absorbing epithelium.
This section having been treated with osmic acid shows the localization of the fat
globules.

Fig. 6. A section through that part of the cotyledon given in Fig. 5. This sec-
tion, treated with iodine, shows the localization of starch.

Fig. 7. A few cells from the absorbing epithelium of the cotyledon after enlarge-
ment. This figure shows the large clear oil globules and the darkly staining starch
grains in the subepidermal cells. The crystals seen in most of the cells appear to con-
sist of fatty acid, possibly palmitic.

Fig. 8. Enlarged starch granules from subepidermal cells.

COCOS NUCIFERA.

tR«printed from the Bulletin of the Torrey Botanical Club, 30: 390-402. July, 1903.]

On the Physiological Action of some of the Heavy Metals in Mixed

Solutions'^

BY

Rodney H. True _,_ William T. GieS

AND •'

U. S. Department of Agricztlture N'ew York Botanical Gardeti

It has been shown by several investigators that, in mixed so-
lutions containing the lighter metals, the physiological action of
the electro-positive elements may in a degree antagonize each
other, and a mixture of several toxic solutions of these compounds
may be much less harmful than any one of the constituents taken
singly. Researches concerning the physiology of sea- water have
shown this to be conspicuously the case. Work on soil-solutions
by Kearney and Cameron f has developed a similar situation.

The neutralizing action of the various ions on those of the
heavy metals has been less studied and the present paper presents,
in a preliminary way, results gained from a series of experiments
performed during the summer of 1902 in the Plant Physiological
Laboratory at Wood's Hole, Massachusetts.

As a test object, the primary radicle of seedlings of Litpimis
■ albus was used. The method of procedure consisted essentially
in suspending the seedHngs on glass rods for at least 24 hours in
each experiment, in such a manner as to immerse the radicles in
the solution under study. In most cases the seedlings were under
observation for 48 hours. At least four seedlings were used in
each experiment of a series, and our deductions were drawn from
the figures for average growth. With only a very few exceptions
each member of the quartette manifested the same tendency. The
solutions were carefully prepared from pure chemicals and were
believed to be very accurate. The experiments were made in
beakers.

In order to get a basis for comparison, we made a number of
solutions of different compounds of each of the heavy metals used,

* Read by title at the meeting of the Botanical Society of America, held at Wash-
ington, D. C, December, 1902.

f Kearney, T. H., and Cameron, F. K. Some Mutual Relations between Alkali
Soils and Vegetation. U. S. Dep. Agric. Rep. No. 71. 1902.

390

391

True and Gies : Heavy Metals

and determined the strongest concentration in which the plants
were able to make appreciable growth. This point of undoubted
growth furnished a point of departure in making comparisons. The

Table I
Simple SonrriONS.* Heavy Metals. Average Growth-rates in Millimeters f

Cone. Gm. Mol.

CUSO4

CuCl,

Cu(CH3COs),

AgNO,

HgCU

ZnS0«

W//8192

2.0

6.0

f/ij 1 6384

0.5

1.0

1.0

9.0

9.0

w 32768

I.O

1.0

2.0

13.0

18.0

'",65536

3-5

30

30

2.0

16.0

18.0

W//131072

II. 0

10.5

11. 0

6.0

16.0

w/2b2l44

16.0

I5-0

135

W//524288

22.0

Check.

14.0

14.0

12.0

12.5

150

155

* All control experiments in this and subsequent series were made in water which
had been very carefully distilled several times. All of the solutions were prepared from
distilled water obtained under constant conditions.

f The growth-rates recorded in these tables are those for the first 24 hours.

accompanying tables give the average growth-rates at the end of
twenty-four hours.

Table I summarizes the growth-rates obtained in a series of
solutions of salts of copper, silver, mercury and zinc. In order

Table II

SiMi'LE Solutions. Light Metals and Urea. Average Growth-rates

IN Millimeters

Cone.
Gm. Mol.

NaCl

NajS0«

KCl

KNO3

CaCNOa),

CaCls

CaSO^'

MgClj

Urea

mjH

IS

0.5

mj 16

2.5

9.6

1.0

w/32

3-5

1-3

0.7

3-4

20.0

16.0

3-5

w/64

7-5

1.0

2.0

4.0

20.5

350

8.0

w;i28

13-5

2.8

6.6

8.0

21.0

;// 256

5.8

7.0

9.0

21.0

Ml SI 2

5-5

W,I024

6.2

;//;2048

9-4

W//4096

12.5

Check.

iS-5

10.0

lO.O

14.0

lO.O

12.5I

13.0

lO.O

15-2

to test the possible antagonizing influence of compounds with
other bases, a number of salts of sodium, potassium, calcium and
magnesium were used.

A similar point indicating the maximum concentration per-

IN Mixed Solutions 392

mitting growth was obtained for each of the salts of the lighter
metals taken. This was necessar}'- in order to get some idea of the
permissible concentrations in which the latter might be used.
Table II presents in brief the growth-rates made by the lupine
roots in solutions of the salts of the second group of metals (and
of urea).

Simple Mixtures

Knowing now the effect, on the growth-rate, of the heavy-
metals under study, also that of the salts of the lighter metals
which were made use of, we have a basis for ascertaining the action
of these compounds when their solutions are mixed. The method
of procedure in this connection consisted briefly in mixing, with a
series of graded solutions of the heavy metals, a definite quantity
of the salts of the lighter metals. Two general classes of salts
could be chosen for this purpose ; first, those in which a common
anion occurs in combination with the different cations made use
of; second, salts in which also the anions differed. By making
up solutions molecularly, as was done in every case, a comparison
of results obtained from the same cations, combined in the two
ways indicated, would enable us to judge of the action of the anions.

Table III summarizes growth-results obtained by growing
lupine radicles in a series of solutions of copper salts mixed with
salts of one lighter base in vaiying degrees of concentration. The
growth-rates obtained are directly comparable to those resulting
from the action of the simple solutions of the heavy metals. It
will be noted that when copper sulphate is mixed with calcium
sulphate, the latter containing ;;z/i44 grams per liter, a corre-
sponding growth-rate is found in the case of the simple solution
(Table II) at a concentration of copper sulphate indicated by
7;z/65536, and in the case of the mixed solution (Table III) at a
concentration of 7/^/16384. These facts indicate that the presence
of the given amount of calcium sulphate enables the plant to with-
stand four times as much copper as it was able to withstand when
the latter occurred in simple solution. A further inspection of the
tables shows that when calcium sulphate is present in weaker solu-
tions, the antagonizing action is still strong, even when calcium
sulphate is present in a concentration of 7;^/5i2.

When to copper chloride, magnesium chloride is added in a

393

True and Gies : Heavy Metals

series of graded concentrations, an inspection of the tables shows
that in general little, if any, diminution in the toxic action of the
copper follows. This seems to indicate that magnesium is not
able to diminish the poisonous action of the copper under the
conditions present.

When to copper chloride, calcium chloride is added in the
proportion of w/128, a decided decrease in the killing power of the
copper is seen. When to copper chloride, sodium chloride, nil 16,
is added, a strikingly reversed situation appears. Not only is the
harmful action of the copper not diminished, but the mixture seems
to be slightly more poisonous than the simple copper solution or

Mixed Solutions.

Table III

Heavy Metal and Light Metal. Common Anions.
Growth-rates in Millimeters

Average

Concent, of
Solution
of Heavy

CUSO4 + CaSO*

CuCt, + MgCIa

CuCl,

+
CaCU

CuCl,

AgNOa

HgCI,

ZnS0«
CaSO*

CaSO*

CaSO*

CaSO«

MgCl,

MgCU

MgCl,

NaCl

KN03

CaCl,

Metal

iw/144

»»/256
0.0

m/513

0.0

W//128

wi/256

W//512

m/128

»i/i6

w«/2s6

ml2s6

w/2048

33-5

w/4096

0.0

0.0

0.0

0.0

390

/;//8l92

0.0

0.0

I.O

0.0

0.0

37-5

;«/l6384

3.0

3-0

1-5

1.0

2.0

2.0

2.0

0.0

1.0

37-0

w/32768

8.5

7.0

3-5

1.0

3.0

1.0

6.0

0.0

1.0

2.0

w/65536

14.0

17.0

14.0

4.0

4.0

30

10.5

0.5

3-0

S.o

;/;/l3i072

19.5

50

15.0

1.0

5-0

Check in

12.0

II. 0

II. 0

lO.O

14.0

12.0

14-5

12.0

"5

12.0

13.0

water

29.0

26.5

26.0

4.0

9.0

4-5

20.0

1.0

24-5

Check in

35-0

Solution

of Light

Metal

the simple sodium solution. This would seem to indicate that to
the poisonous action of the copper that of the sodium chloride
itself is added.

When to a solution of silver nitrate a solution of potassium
nitrate, w/256, is added, no very marked change in the action of
the heavy metal is noticeable, the growth-rate coinciding approxi-
mately with that seen in the solution of the pure silver salt.
What difference there is seems to be in the direction of greater
toxicity.

When to mercuric chloride, calcium chloride, m/2S6, is added

IN Mixed Solutions

394

no diminution in the poisonous action of the corrosive sublimate
is seen. Indeed, the mixture is markedly more poisonous than
the solution of the simple salt. Zinc sulphate gives a very differ-
ent result when calcium sulphate, ;// 256, is added. Whereas
growth is much retarded in a ;;f'8i92 solution of zinc sulphate,
in the mixture at 7/2/2048 growth is more than twice as rapid
as in the control grown in water. We have here a very marked
stimulation in the growth-rate, resulting from the addition of the
lighter metal to the zinc.

When to salts of the heavy metals compounds of the lighter

Table IV

Mixed Solutions. Heavy Metal and Light Metal. Different Anions.
Average Growth-rates in Millimeters

Concent, of

CUSO4

CuSO^

CuCCHsCO,)^

HgCl^

CUSO4

Solution of

+

+

4-

-1-

-^

Heavy

CaCU

CaCU

Ca(N03)2

Ca{N03)2

Urea

Metal

W//I28

z«/i28(dup.)

OT/32

ml^z

"','64

w/4096

0.0

w/8192

2.0

I.O

w/ 1 63 84

1-5

30

5.0

6.0

w/32768

3-0

4.0

9-5

16.0

0.0

W65536

7.0

10. 0

0.5

w/l 31072

16.S

4-5

Check

12.0

15-5

13.0

14.0

II. 5

metals are added, in the form of salts in which the anion differs
from that in the copper compound, a condition of things is found
which is not essentially different from that just cited. In Table
IV results bearing on this point are presented.

It will be noted that when calcium chloride, inji2^, is added

Table V

Mixed Solution. Copper Sulphate with Cane Sugar. Average Growth-
rates in Millimeters

Concent, of
Copper
Solution

Cane j Cane
Sugar , Sugar

2711 1 7Jl

Cane ! Cane
Sugar j Sugar

mJ2 \ ml^

Cane
Sugar

wz/8

Cane
Sugar
wz/16

Check

in
Water

^65536

0.0 1 2.7

4-0 7-5

5-5

6.5

15-5

to copper sulphate, a marked diminution in the poisonous action
of the copper compound takes place to a degree equal to that
seen when the anions are similar. When to copper acetate cal-

,'Jf).') True and Gies : Heavy Metals

ciuni nitrate, ;// 164, is added, a similar situation results. In the
case of solutions of mercuric chloride to which calcium nitrate has
been added, we find no amelioration of the poisonous action of
the corrosive sublimate, the growth being, if anything, less in the
mixed solution than in that of the mercuric chloride alone.

In view of what has been said, the question naturally arises as
to the effect of non-electrolytes in solution with the heavy metals.
In this connection but two compounds were studied : cane sugar
and urea. Tables IV and V present the results obtained. It
will be seen that in a solution of copper sulphate, w, 65 536, to
which cane sugar in concentrations varying from vi to ;;f/i6 has
been added, the growth-rate in general increases as the concen-
tration of cane sugar diminishes. The growth-rate is markedly
greater in the solution of copper sulphate containing cane sugar
varying in concentration from ;///4 to w/i6 than in the copper
solution alone. This growth-rate was not a persistent feature, how-
ever, since in all the mixtures except that containing cane sugar at
the least concentration, Jni6, no growth took place in the second
twenty-four hours. It appears, therefore, that when cane sugar is
added in proper proportions, as in these experiments, the poisonous
action of copper is somewhat diminished. This is probably due
to the formation of copper saccharate and a consequent lessening
of the number of Cu ions.*

As regards the effect of the addition of urea, w 64, it appears
that the inhibiting action of the mixed solution is greater than
that of the simple copper salt, the addition of the urea seeming
to increase the total poisonous action.

Complex Mlxtures

In order further to test the effect of additions of lighter metals
to salts of the more poisonous elements, more complicated syn-
theses were made. These were of two classes : one mixed solu-
tion in which all of the salts present had a common anion ; a
second mixed solution in which the anion of the salt of the heavy
metal did not appear in any of the compounds of the lighter

* See Loeb, J., and Gies, W. J. Weitere Untersuchungen iiber die entgiftenden
lonenwirkungen und die Rolle der Werthigkeit der Kationen bei diesen Vorgangen.
Archiv fur die ges. Physiologic, 93 : 261. 1902.

IN Mixed Solutions

396

metals. Mixtures were made in which, in addition to a copper
salt, salts of sodium, magnesium, calcium and potassium, succes-
sively, were added. In every case, Ca excepted, the concentra-
tion in which each compound was present was that which, while
distinctly retarding growth, still permitted it. We have, there-
fore, in every case, a salt entering into the combination in a con-
centration sufficiently great to be a distinctly toxic agent. The
concentrations and other data in this connection appear in Table VI.
It will be noted in each case that the copper salt permits a
slight growth. When the sodium salt is added, the mixture be-

Table VI

Complex Mixtures of One Heavy Metal and an Increasing Number of Lighter Metals.
Average Growth-rates in Millimeters

With common anions

With different anions

(a)

Contents of

Solutions

Av.
Growth
24 hrs.

(6)

Contents of

Solutions

Av.
Growth
24 hrs

Contents of
Solutions

Av.

Growth
24 hrs.

Contents of Solu-
tions

Av.

Growth
24 hr3.

CuClj

mol
65536

5-5

CuCU

mol
32768

I.O

CuCU

mol
65536

2.5

Cu(CH3.C0,),

mol
65536

8.0

CuCl2

NaCl

65536
64

2.5

CuCU
NaCl

32768
64

1-5

CuCU
NaCl

65536
128

1.0

Cu(CH3.C02)2
Na,SO^

65536
128

3-0

CuCU
NaCr
MgCl,

65536

64

512

7.0

CuCl.,
NaCf
MgCU

32768

64
512

2-5

CuCU
NaCr
MgCU

65536

128

1024

60

Cu(CH3.C02)2

Na^SO^
MgCU

65536
128
512

8.0

CuClj
NaCl
MgCl,
CaCU

65536

64

512

32

14-5

CuCU
NaCl
MgCU
CaCl^

32768

64
512

32

7.0

CuCU
NaCl
MgCl,
CaCl^

65536

128

1024

64

9.0

Cu(CH3.C0.3),
Na,SO^
MgCI,
CaCU

65536

128

512

32

17.0

CuClj

NaCl

MgCl,

CaClg

KCl

65536

64

512

32

128

19.0

CuCU

NaCl

MgCU

CaCl^

KCl

32768

64

512

32

128

5.5

CuCU

NaCl

MgCU

CaCU

KCl

65536

128

1024

64

256

lO.O

Cu(CH3.C0,),

NasSO^

MgCU

CaCU

KNO3

65536

128

512

32

128

22.0

Check

13-5

10.5

10.5

13.0

comes somewhat more harmful than the copper salt alone. The
addition of magnesium to the mixture raises the growth-rate to a
point beyond that reached in the copper solution, indicating a
slightly beneficial antagonistic effect. When to these the calcium
salt is added, the growth-rate immediately assumes practically
normal proportions. This neutralizing or antitoxic effect of the
calcium is very marked. When to the combination just referred

;307 True and Gies : Heavy Metals

to the potassium salt is added, the growth-rate is still further in-
creased. In the last mixture we have five salts, each, with the ex-
ception of the calcium compound, in a concentration strong enough
to interfere distinctly with growth. As a result of their presence
-together, not only is there no addition of poisonous effects, but a
neutralization of toxicity to such degree as to permit in the mixed
solution a growth-rate equal to or greater than that seen in the
check culture.

When the concentration of copper solution was doubled and
the concentration of the other salts left as before, we found that
the action of the copper was more slowly overcome, and even in
the most complex mixtures studied, the growth-rate was still be-
low that of the check. Apparently, the poisonous activity of the
copper in these cases was greater than such as could be neutralized
by the quantities of other salts added to it. When, on the other
hand, the concentration of the copper solution was kept as in the
first instance and the concentration of the lighter salts added was
diminished by half, the neutralizing action of the latter was mark-
edly less. In the most complex mixtures under these latter con-
ditions the observed growth-rate only equaled that of the control.
Apparently this fact was due to the unneutralized copper action,
since each of the other salts present were below a harmful con-
centration.

Returning, now, to mixtures in which the anion of the copper
salt is not duplicated in any of the other salts present, we see a
result essentiall}^ like that just noted. When to copper acetate,
for example, salts of the metals used before are added in quantities
equal to those indicated in Table VI, a similar result is seen.
The growth-rate in the pure copper salt in this case is somewhat
greater, since the CH3.CO2 anion is slightly less poisonous than the
CI or the SO^ anion. The addition of the sodium salt again in-
creases the toxicity of the mixture. The further addition of the
magnesium salt diminishes the harmful action somewhat, the activ-
ity of the mixture being, roughly, the same as that of copper ace-
tate alone. The entrance of the calcium salt, as before, produces
a marked acceleration of growth, the rate jumping to a point con-
siderably above the control. The final addition of the potassium
salt still further increases this stimulation. As a result of this ex-

IN Mixed Solutions 398

periment it appears that it is immaterial here, as before, whether
the lighter metals enter the solution in compounds containing an
anion common to that of the heavy metal, or whether the anions
be different.

Discussion of Results

From the evidence at hand in these experiments it appears that,
in solutions of salts, the conspicuously effective component of the
molecule is the cation or the metal. This presumption, raised by
the similar physiological effects produced by the cation of various
salts of the heavy metals in equimolecular quantities, is strength-
ened by the action of mixtures containing a salt of the heavy metal
with salts of lighter metals.

In case several salts having the same cation are mixed in solu-
tion the same lack of conspicuous influence on growth on the part
of the anion is to be seen."*" It is clear that the effect exerted upon
the lupine roots by the salts of the heavy metals tested, differed ac-
cording to the concentration of the salts. When sufficiently di-
luted, solutions containing copper, silver, mercury or zinc ions ex-
erted a more or less clearly marked stimulating effect on growth.
At a greater concentration, perhaps double that causing stimulation,
a retarding influence was usually seen, and in a concentration ap-
proximately doubling this, growth was much interfered with ; and
on again doubling the concentration, little or no growth took place.

The effect of adding solutions containing Ca, Mg or Na ions
was seen to vary with the character of the cation introduced. In
mixtures containing but two salts (Tables III and VI) sodium
seemed to show an increased poisonous action as though that of the
sodium were added to that of the cation of the heavy metal. When
to a solution of copper, a salt of magnesium was added, the mix-
ture seemed to act with nearly the same intensity as the simple solij-
tion containing the copper in like quantity, exerting, therefore,
little influence on the poisonous activity of [the copper. When
calcium was added, a marked reduction of the poisonous activity
of copper ions was observed, a result seen even more strikingly in

* The physiological action of every dissociated salt in solution is doubtless an expres-
sion of the resultant biological effect of its component cations and anions. In these
experiments the influence of the cations was predominant.

399 True and Gies : Heavy Metals *

the case of zinc. Investigations by Swingle,* Clark, f Rumm and
others on the action of Bordeaux mixture, although concerning
very different proportions from those here involved, all testify to
this power of calcium to neutralize the poisonous action of copper.
Whereas the presence of calcium reduced the harmful effects of the
copper to about one fourth of that seen in the simple copper solu-
tion, the antagonizing action of the calcium reduced the toxic ac-
tion of the zinc to, at most, one sixteenth of that of the simple zinc
solution. In the case of silver, the addition of calcium seemed to
exert no ameliorating action. As far as the evidence at hand goes,
it appears that such ameliorating action as was observed and would
be expected stands in an inverse relation to the poisonous activity
of the heavy metal.

From the above, as well as from the work of others, it appears
conclusive that certain cations in mixed solutions exert a physio-
logical action antagonistic to that exerted by other cations. The
question next arises as to the nature of this modification and its
seat. Does a mixed solution of calcium sulphate and copper sul-
phate or copper chloride, for example, produce the change (which
brings about this physiological result) by affecting the condition
of the copper in the solution outside of the cell, or does it bring
about modifications within the cell itself? Is this antagonism an
extracellular chemical change or an intracellular physiological
change ?

We have two sorts of cases to deal with. In the one case the
salts have a common anion and in the other case the anions differ.
We may set aside any such changes as the formation of double
salts or the setting back of molecular ionization in the former case,
since it has been shown that like results are seen when the mixed
salts have common anions. This would seem to be a probable situ-
ation from a priori reasoning also, since in most cases the solutions
of the salts of the heavy metals were so dilute that practically
complete ionization took place. In that event, no matter what its
associated anion was, the metal acted as free ions.

* Swingle, \V. T. Bordeaux Mixture. U. S. Dep. Agric. Div. Veg. Path, and
Physiol. Bull. No. 9. 1896.

t Clark, J. F. The Toxic Properties of some Copper Compounds with special
reference to Bordeaux Mixture. Bot. Gaz. 33 : 26. 1902.

IN Mixed Solutions 400

In the cases of our mixtures of salts having different anions,
chemical reactions might be regarded as possible, with a conse-
quent change in the forms of molecules. Here again, however,
the great dilution of the salt of the heavy metal in our most im-
portant mixtures produced complete or nearly complete ionization,
the heavy metal acting practically as free ions. We can then
hardly regard changes of an ordinary chemical nature as being
responsible for the differences in the physiological results. We
think that interior physiological modifications are responsible for
the observed differences in growth rate. This belief implies that
the simple salt of the heavy metal and the mixture of this salt with
that of a lighter metal, after penetration into the cell, affect the
processes there being carried on in such a way as to bring about
different results on cellular growth. In studying the effect on
growth of simple solutions of copper and calcium salts, for example,
we see that at the concentrations employed copper retards growth
whereas the calcium salts greatly stimulate it. With each we
have, in all probability, to do with antagonistic phases of physio-
logical action. When we examine the results in cases like the
above, it seems highly probable that the so-called antitoxic action
of ions is due to different interior physiological modifications, and
that the growth-rate observed in such experiments as these repre-
sents the physiological sum of oppositely acting stimuli, or of
antagonistic protoplasmic changes.*

It has been shown that when salts of heavy metals are suffi-
ciently dilute they exert a stinmlating effect on growth, and when
solutions of calcium and similar salts are concentrated enough,
they hinder or entirely prevent growth, and may, in the case of
the more soluble chloride and nitrate, prove fatal. Coupin t has
shown that at different dilutions compounds exhibit tliree distinct
phases of physiological action. When the solution is sufficiently
dilute it seems too attenuated to produce any effect on growth.
As the concentration increases, a stimulating phase is seen, which,
on further concentration, passes over into the retardation phase —
pronounced in proportion to the concentration.

*Loeb and Gies, /. c, 267.

t Coupin, H. Sur la toxicite du chlorure de sodium et de I'eau de mer e I'egard
des vegetaux. Rev. Gen. Bet. 10 : 177. 1898.

401 True and Gies : He.wv Metals

In the mixtures of copper and calcium employed in our experi-
ments, we may have had concentrations of each salt in different
phases of action due to the degree of concentration. In CuCl.,,
;// 65536, we see that the Cu concentration is in the phase hinder-
ing growth, the resulting elongation of the root being about 3 mm.
When CaClj, ;// i 28, was tested, it was found to be in a concentration
markedly stimulating when referred to the control in water, grow-
ing 20 mm. in the former case, against 14.5 mm. in the check.
These opposite tendencies were brought together in the mixed solu-
tion with the result that the concentration (in terms of the copper
salt permitting the growth-rate seen in the simple copper solution)
moved up to approximately four times that observed in the
simple copper solution. The stimulating action of the calcium
seems to have operated against the retarding action of the copper,
and the result is a marked diminution in the poisonous action of
the copper.

The opposite result is seen in the mixture containing CuCl,
and NaCl. The latter is in its growth-retarding phase until more
dilute than ;;/'i28. Hence at ni 16 it is in its growth-retarding
phase, and when added to CuCl, at 7//, 65 5 36, likewise in this
phase, the result is a sum of toxicity and an increased depression
of growth-rate follows the combined action of the two. This also
applies to the mixtures containing magnesium.

In considering the more complex mixtures of salts, indicated
in Table VI, the chemical nature and influences of the resulting
solutions are not readily determined. Much more concentrated
solutions result in such mixtures with consequent decrease in dis-
sociation. The probability that we are dealing with various kinds
of non-ionized molecules, as well as with an indefinite number of
ions, makes it impossible to speak definitely with confidence of the
significance of our results in this connection. In general one may
say that here, as in sea water, another complicated mixture of
molecules and ions, the entrance of the calcium salt into the mix-
ture is the stage in the synthesis at which the growth-rate ap-
proaches that seen in the check, and the final addition of the
potassium salt seems further to increase the growth-rate. Or, in
other words, all the salt solutions except the calcium entered the
mixture in a concentration at which singly they would cause a

IN Mixed Solutions 402

retardation of the growth-rate without bringing it to a standstill.
Of the compounds present, therefore, the calcium salt only entered
in a concentration representing the stimulus phase. The marked
effect following the entrance of the calcium and the potassium
may, in part, be due in these experiments to the cumulative in-
crease of concentration of the solution, with the corresponding
decrease in the rate of ionization and the diminished number of
active ions. The fact that the potassium salt, although added in
a concentration hindering growth when taken singly, increased
the growth-rate when added to the mixture as its last member,
seems to strengthen this supposition. Of course, changes of this
nature represent changes in the solution itself and He outside of
the cell, and should not be confused with the mutually antagonis-
tic intracellular action of ions in the case of very dilute solutions.

38

Reprinted from the Journal of the New York Botanical Garden, 1902, iii, p. 169.

ON THE NUTRITIVE VALUE AND SOME OF THE
ECONOMIC USES OF THE COCOANUT.

By William J. Gies.

Few if any vegetable products furnish so many useful articles
as the cocoanut. It forms the chief food of the inhabitants of
most tropical coasts and islands, where the kernel is not only
eaten in the ripe and unripe conditions, but is also prepared and
served in various ways. It forms an accessory part of the diet,
and is found in many of the confections of civilized man all over
the globe. The milk is considered an agreeable cooling beverage
in the tropics, although it is diuretic in its effect, and causes irrita-
tion of the mucous membrane of the bladder and urethra Avhen
taken too freely. Immoderate use of the fruit is said to cause
rheumatic and other diseases.

Experiments recently published in the Bulletin of the Torrey
Botanical Club by Professor Kirkwood and the writer (see Garden
Contribution No. 26), conducted in part in this Garden and with
the cooperation of Dr. MacDougal, indicate that the nutritive
value of the endosperm of the cocoanut resides mainly in its high
content of oil and moderate amount of carbohydrate. Of the
former, the fresh endosperm contains 35—40 per cent.; of the latter,
approximately 10 per cent. The amount of proteid is very slight,
being little more than 3 per cent. The quantity of inorganic
matter is i per cent. The water amounts to nearly 50 per cent.
The chief constituent of the milk, aside from water (95 per cent.),
is sugar, nearly all of the solid matter being thus composed, as
the very sweet taste amply testifies. Various alcoholic beverages
have been made from fermented cocoanut milk.

The endosperm is very agreeable to the taste, and, with the
exception of the cellulose (3 per cent.), is readily digestible.
Domestic animals eat it eagerly, and the cocoanut crab feeds on it
almost exclusively. The residue left over after the fat has been
expressed from the " copra " is widely used in Europe as food for
cattle ; also as fertilizer.

715

ji6 William J. Gies.

The use of cocoa fat as a substitute for butter among the
poorer classes has been increasing, and it is frequently employed
as a butter adulterant. The tendency of cocoa fat to rancidity is
not as great as that of animal fats, and for this reason "butters"
made from it keep well, and have been recommended especially
for military and naval uses. Recent researches show that " cocoa
butter" is quite as agreeable to the taste, and as easily and com-
pletely digested, as ordinary butter. Its heat of combustion is
9.066 small calories per gram.

" Cocoanut cream," a dietary product much used in the tropics,
is made by grating the endosperm and squeezing through cloth
the fluid from the finely divided material. In a warm climate the
resultant mixture contains much oil and is a very delicious acces-
sory food. Besides the oil, the "cream" contains chiefly carbo-
hydrate and proteid.

Soaps made from cocoa oil combine with, or hold an unusual
amount of water, while retaining special hardness, and are char-
acterized by great solubility in salt solution. The so-called
" marine " or " salt-water " soap has the property of dissolving as
well in salt water as in fresh water. The harder fats of the oil
make excellent candles. Cocoanut oil and resin melted together
yield a mixture capable of being used with success in filling up
the seams of boats and ships, and in tropical countries for covering
the corks of bottles as a protection against the depredations of
the white ant.

The fibrous husk (coir) 'is widely used for the construction of
ropes, brushes, bags, matting, etc. The hard shell is easily
polished and lends itself to the formation of various utensils and
ornaments. It also has a high fuel value. The powdered shell
and husk are occasionally used as adulterants of ground spices.

The milk of the nut, as has already been pointed out, is
strongly diuretic. The endosperm shares with the milk the prop-
erty c( a taciiicide, and has been used as a vermifuge in India for
many years, where it is regarded as an excellent means of ex-
pelling the flat worm. The harder fats of the oil are used as con-
stituents for suppositories and related therapeutic products.
Medicinally the oil is employed repeatedly as a substitute for lard,
olive oil and cod-liver oil. It is also made the chief substance by

Nutritive Value of the Cocoanut. 717

bulk in various salves and cold cream, pomade and similar cosmetic
preparations. In ointments and cerates it is especially valuable
because of its ready absorption when rubbed on the surface of the
body, and on account of its ability to hold an unusual amount of
water or saline fluid. It shows little tendency to produce chemical
changes in substances with which it may be associated.

Reprinted from the Yale Scientific Monthly, 1898, iv, p. 204.

ON THE DECOMPOSITION AND SYNTHESIS OF PROTEIDS
IN LIVING PLANTS.*

By William J. Gies.

Few substances are so widely distributed in nature as proteids
and certainly none are of more consequence from a biological
point of view. The tissues of all plants and animals contain these
substances in large proportion, and of the invariable organic con-
stituents of every living, functionally active cell the albuminous are
undoubtedly the most important. That proteids are probably the
most complex compounds with which the chemist has to deal, and
therefore, also, the most elusive in chemical research, are deduc-
tions to which the experiences of all investigators seem to point
conclusively. In spite of the fact, however, that they have long
been the subjects of persistent and carefully conducted chemical
investigation, our knowledge of their molecular arrangement still
remains decidedly indefinite and all attempts thus far completely to
unravel the constitution of the proteid molecule have resulted
negatively. Each of the various theories which have been pro-
posed in regard to structural formulae depends entirely upon the
products obtained in proteid decompositions, since all of the numer-
ous attempts to prepare albuminous material artificially have
invariably resulted in failure. Since the decomposition products
of proteid matter are so multitudinous and, under different condi-
tions so various, it is not at all difficult to comprehend why the
biological chemist is so much in the dark as to the real configura-
tion of the albumin molecule and why, in the total absence of data
afforded by artificial synthesis, he is able to form only hypotheses
as to the manner in which these analytic residues are held in the
undecomposed substance.

Although proteid matter has never been prepared in the labor-
atory from any of its decomposition products, its synthesis is con-

* Being an abstract of a paper upon this subject recently presented before the
Chemical Clab of Yale University.

719

720 William J. Gies.

stantly taking place in plants and, to a certain extent, in animals as
well. The ultimate origin of proteids may be traced to the veg-
etable kingdom, however, for plants are constantly transforming
inorganic into albuminous matter, as a part of the process of their
development, whereas in animal metabolism, albuminous syntheses
are wholly dependent upon proteid residues assimilated after diges-
tion of the food. Consequently, plants are preeminently the pro-
ducers in the biological economy, whereas animals are essentially
the consumers, and in view of the power of the vegetable cells to
build up the simple elements into the organic matter upon which
animal life is either directh" or indirectly dependent, the trans-
formations taking place in plants must ever be matters of prime
interest to all chemists and biologists.

In recent years considerable attention has been given to quanti-
tative determinations of the chemical changes taking place in vari-
ous species of living plants, with results adding very materially to
our knowledge in this relation. Preeminent among those who
have attempted to solve the problems of the proteid transforma-
tions in growing plants is Professor E. Schulze of Zurich, who for
nearly thirty years has been steadily at work upon various phases
of the physiological chemistry of vegetation. Most of the experi-
ments conducted by him to ascertain directly the facts in regard to
proteid decompositions and syntheses were carried out upon care-
fully developed seedlings. In these, of course, the transformations
of the reserve material of the seeds could be accurately followed
and the facts learned by a very simple as well as accurate rnethod
— one, also, which obviates the great difficulties encountered in an
investigation of the changes occurring in larger vegetable forms
and which, besides, does away with the complications arising from
the extraneous influences attending the development of plants
under normal conditions and in the usual environment. Conse-
quently, the results in regard to proteid transformations obtained
by this method must necessarily be referred to the matter original!}-
stored up in the seeds, since the utilization of all other nitrogenous
substances is carefully precluded, and thus a more satisfactory idea
of the steps of cleavage and construction is afforded than could
possibly be derived from a study of ordinary plants under perfectly
normal conditions.

Metabolism in Seedlings. 721

Of the most important nitrogenous compounds, aside from the
proteids, commonly found in plants, glutamin, arginin, phenyl-
alanin and vernin were discovered by Schulze and his pupils.*
Tyrosin, amido-valerianic acid, allantoin and guanidin, though
long known to be constituents of the animal body as products of
proteid catabolism, were first found in the vegetable kingdom by
Schulze t and those working under him, and were separated from
seedlings in which considerable transformation of albuminous ma-
terial had taken place. Leucin was first isolated from plants by
Gorup-Besanez J and Borodin § long since demonstrated that
asparagin is widely distributed as a plant constituent. The latter
substance occurs most abundantly ; the etiolated seedlings from
one species of lupin seeds, for example, having been found by
Schulze to contain as much as 28 per cent, of the crystalline com-
pound, calculated in terms of the dry plant substance. Glutamin
and arginin are sometimes the predominating crystalline constitu-
ents in seedlings, though they rarely mount higher in quantity
than from 2 to 3 per cent, of the dry tissue. The amido acids
may be separated from practically all seedlings, although as a rule
in only comparatively small quantities.

That these important nitrogenous constituents of growing
plants bear a very close relation to those formed by hydrolytic
cleavage of proteids will be seen at a glance. Thus glutamin
and asparagin are the amides of glutamic and aspartic acids.
Both of the latter may be readily formed from proteids by
hydrolysis. Tyrosin, leucin, amido-valerianic acid and arginin
may be prepared without any special difficulty in artificial digestion
of proteids. By the action of mineral acids vernin readily decom-
poses into guanin, a substance which is found in the animal boyd
and is closely related to uric acid. Arginin may be broken down
into urea in the laboratory and guanidin is the imide of urea, the
chief end product of proteid catabolism is the human organism.
Allantoin breaks down into urea and may be formed from uric
acid, both of which bodies are common products of proteid cata-
bolism in the animal system. When albuminous substances are

* Schulze. Zeitschr. f. physiol. Chem. , 1897, xxiv, p. 18 et seq.

f Schulze. Ibid.

\ Gorup-Besanez. Berichte der Deutsch. chem. Gesellschaft, vol. vii, p. 146.

I Borodin. Botan. Zeitschr., Nos. 51 and 52.

722 William J. Gies.

decomposed with nitric acid, phenylalanin is among the resultant
products.

It should not be inferred, however, from the comparisons just
drawn, that the plant constituents named above are to be looked
upon as decomposition products only. Whilst there is abundant
evidence that they ensue as a result of cleavage of the plant por-
teids, and, as has just been shown, afford a suggestive analogy
with the products formed in the hydrolysis of albuminous sub-
stances, both in the animal organism and in the chemical labora-
tory, some of them appear to play important parts in constructive
processes as well. The similarity just noted certainly offers direct
evidence in favor of the view to which modern research has been
steadily leading us, that, with reference to general chemical and
nutritional processes, there is not so much difference between
plants and animals as has been supposed. Not only are there
lower plants, entirely free from chlorophyll, which, so far as
chemical processes are concerned, represent intermediate steps
between higher plants and animals, but the differences existing
between the higher plants and animals themselv^es are more of a
quantitative than a qualitative kind. In the animal organisms the
processes of oxidation and cleavage prevail, whilst in the plant
those of reduction and synthesis predominate.

This apparent similarity in the general nutritional processes of
animals and plants has indeed been the guiding influence in recent
investigations of the physiological chemistry of vegetation and
perhaps much of our information in this connection is partly
based upon deductions drawn almost wholly from such analogy.
Thus Prof. Schulze, who has long been an acknowledged author-
ity in this particular domain of biochemical science, assumes that
the preliminary metabolic changes taking place in plants are
dependent in large part upon the action of unorganized ferments.
This theory, for the reason just suggested, seems so thoroughly
plausible that it has come to be generally regarded as an expres-
sion of absolute fact and is readily accepted in explanation of the
transformations of proteids, for example, because the chief ni-
trogenous plant constituents, exclusive of the albuminous com-
pounds, are closely related to, and in part are identical with, those
formed by the animal enzymes during proteolysis in the alimentary

Metabolism in Seedlings. 723

canal. Although this view seems very probable, and consequently
may be accepted provisionally, it must not be forgotten, neverthe-
less, that exact knowledge of the occurrence and distribution of
enzymes in plants is decidedly limited and that whilst such ferments
as diastase, myrosin, papain, bromelin and others are known to occur
— all of them being comparable in their activities to the various
enzymes of the alimentary tract — the results of experiment and
research have afforded little insight into their real relation to the
changes going on within plants.

As has just been indicated, Schulze's conclusions rest upon the
assumption that seeds and plants contain enzymes capable of
transforming the vegetable proteids into crystalline nitrogenous
products, and occurring in sufficient quantity, besides to make
them permanent as well as important agents in the plant nutritional
processes in general. The hydrolytic action of these agents is
considered to account only for the catabolic products, however,
so that they are the essential factors in proteid decomposition.
According, then, to Schulze's view of the proteid transformations
in growing plants, the decompositions are practically the same in
character and manner as those taking place in the animal organism.
In the animal, proteids, as is well known, are broken down by
hydrolysis in the alimentary canal into proteoses and peptones,
w'hich products may subsequently be converted into amido acids
of the fatty and aromatic series, organic bases, ammonia, hydrogen
sulphide and other bodies. Of the organic bases formed, lysin
and arginin are among the more important. Plants also contain,
as constant products derived in presumably the same general
manner, amido acids of both the fatty and aromatic series and
also arginin, and in them, likewise, the initial products of hydrolysis
appear to be proteoses and peptones. In both plants and animals
the sulphur of the decomposed proteid is eventually found for the
most part in salts of sulphuric acid.

Schulze has found that asparagin, glutamin and arginin ac-
cumulate in seedlings in consequence of proteid transformations
and that they unquestionably result partly from albuminous de-
composition on the one hand, partly as products of synthesis on
the other. His data indicate that this is true also of tyrosin, leucin
and the other amido acids already mentioned, and that the proc-

724 William J. Gies.

esses of decomposition and synthesis in relation to plant proteids
in general go hand in hand. In all of these instances en/.ymc ac-
tion is held responsible for the cleavages.

This investigator has carried out man}' experiments to deter-
mine the relative occurence, in the different parts of germinating
plants, of the various nitrogenous extractives and has obtained very
interesting results. His work indicates a very unequal distribu-
tion of the metabolic products. Thus, experimenting with etio-
lated seedlings rich in asparagin, such as those from lupin seeds,
he observed that in the cotyledons, in which of course the decom-
posing reserve proteid was contained, the proportion of asparagin
to the other non-proteid nitrogenous compounds was much less
than in the h\-pocotyl, and he looks upon this accumulation of as-
paragin in the developing stem as part of the evidence in favor of
the view he entertains that asparagin is built up from nitrogenous
residues resulting from proteid decomposition and that it is directly
concerned in synthetic operations. In another series of experi-
ments of essentially the same character, the figures obtained in
quantitative estimations also tend to confirm the view that aspara-
gin is built up at the expense of non-proteid nitrogenous sub-
stances, for in these instances, whilst the proportion of proteid
matter was diminished very little, the percentage of non-proteid
nitrogenous matter aside from asparagin was greatly reduced,
whereas the latter substance was relatively greatly increased.
Similar results were obtained in connection with glutamin.

Quantitative experimental results with reference to the distri-
bution of the different substances throughout the seedlings sug-
gest, further, that the crystalline nitrogenous bodies such as as-
paragin and tyrosin are in part secondar}- products of the proteid
cleavages and that the primary are proteoses and peptones. The
same data indicate, also, that both the primary and secondary
products arising during germination are, or may be in part at least,
broken down eventually into very simple substances, and that the
resultant nitrogenous residues, probably ammonium salts or per-
haps ammonia itself, together with others, are built up, first into
asparagin, glutamin and related compounds, and then into pro-
teids. It seems entirely probable, further, that the proteid regen-
erated in this way is soon subjected to the usual decomposition

Metabolism in Seedlings. 725

and that this cycle of analysis and synthesis proceeds continually
as the plant develops. According to this theory, then, the ob-
served accumulation of both asparagin and glutamin is due in great
part, if not entirely, to a synthesis of the ultimate decomposition
products of the plant proteids.

Kinoshita and Suzuki * have found that in plant metabolism
asparagin may be synthesized from ammonia and non-nitrogenous
organic substances. Schulze has separated ammonium salts from
fresh germinating plants and, as has just been pointed out, is con-
vinced that ammonia is an important factor in proteid synthesis.
It is well known that ammonia appears here and there in different
animal tissues and fluids, and considerable significance has recently
been attached to this fact. Ammonia under these circumstances
must have been formed as a result of proteid catabolism. More-
over, physiological investigation has clearly shown that bodies
such as urea are frequently the results of the synthesis of simpler
decomposition products. Thus in the formation of urea from am-
monium carbonate in the body — a process well known to occur
— we have a type of this same synthetic decomposition, once more
obtaining proof of the similarity of the qualitative chemical reac-
tions in plants and animals. Here again analogy with events in
the animal system may be taken to strengthen deductions with
reference to facts in vegetable chemistry.

Of what use to the plant, it may be asked, is this transforma-
tion of simple nitrogenous substances into asparagin and glutamin ?
Hansteen's f recent work suggests the answer. This observer
found that, when plants of the duckweed species were given
glucose together with asparagin, an abundant formation of proteid
resulted in them. The close relation of glutamin to asparagin
leads Schulze to include it in the general assumption, therefore,
that the synthesis of some of the products of proteid decomposi-
tion into asparagin and glutamin is really a process which may be
looked upon as a stage of proteid regeneration and which, for that
reason, is of great consequence to the plant.

As to the relation of carbohydrates to proteid transformations
in seedlings, Schulze offers numerous experimental results to

* Kinoshita and Suzuki. Zeitschr. f. physiol. Chem., 1897, xxiv, p. 73.
f Hansteen. Berichte der Deutsch. botan. Gesellschaft, Vol. xiv, p. 362.

726 William J. Gies.

strengthen his conclusions. It is well known that a diet rich in
carbohydrates has the effect of sparing proteid in animal metab-
olism and that it generally results in increasing the quantity of
albuminous matter contained in the system. Quite analogous with
this effect, Schulze finds that, in seedlings rich in proteid, the
accumulation of amides is greatest as a rule in those poorest in
non-nitrogenous material, and conversely, that, in seedlings con-
tainmg the most abundant non-nitrogenous reserve material in pro-
portion to the albuminous matter, the proteid decomposition is
least. This circumstance is in harmony, also, with the conclusions
to be drawn from the work of Kinoshita, Hansteen and others,
viz., that certain of the simple nitrogenous substances resulting
from albuminous decomposition in plants are regenerated into
proteid by becoming united with non-nitrogenous substances such
as glucose.

With reference to the proteid transformations going on in full
grown and normally developed plants little is to be said because
little has been done to determine the facts. The difficulties in the
way of such experimental work are obvious. While our knowl-
edge in this regard is decidedly limited, no facts can be advanced
in opposition to the assumption that in ordinary green plants these
transformations are practically identical in quality with those
known to occur in seedlings.

S:hulze's e.Kperiments with tubers and upon the changes ocur-
ring in roots afford confirmatory testimony to all that has been
concluded in regard to proteid transformations in seedlings. It
seems quite probible that in th^ synthetic processes taking place
in roots, crystalline nitrogenous substances are formed which are
essentially the same in character as those formed in seedlings, and
that the nitrogen taken from the soil in inorganic combination, as
he assumes, is transformed in the roots, for the most part, into
asparagin and glutamin. Tubers show niuch the same results as
those obtained with etiolated seedlings.

Whatever uncertainty, in regard to the experimental data
adduced by Schulze and others, e.vists as a result of the weaknesses
of this greatly abbreviated review of them, may perhaps be cleared
up by the following summary of the results of Schulze's experi-
ments and the conclusions to which they have led him : During

Metabolism in Seedlings. 727

the development of seedlings, and probably also of plants under
normal conditions, the proteids are in part decomposed by
hydrolysis, presumably through the intermediation of enzymes.
The preliminary products of this hydrolytic cleavage are proteoses
and peptones, and among the constant products resulting from the
continued hydration of these, occur amido acids of both the fatty
and aromatic series and nitrogenous bases such as arginin.
Whether in this process asparagin and glutamin are formed
directly, or whether aspartic and glutamic acids first result, and
these in turn are formed into the amides, Schulze does not pretend
to say. The greater part of these nitrogenous products are further
decomposed in the plant catabolic processes, from which less com-
plex nitrogenous residues result — probably some simple com-
pound of ammonia or perhaps even ammonia itself These ulti-
mate nitrogenous decomposition products, then, become the
important factors in the building up of asparagin and glutamin, and
possibly other related compounds, which synthesis is necessary in
order to transform the simpler bodies into substances better
adapted for utilization in the construction of new proteid matter.
In the alterations of the non-nitrogenous reserve materials such
insoluble substances as starch and fat are transformed into soluble
bodies. A part of the latter are made " functionally active," to
use Schulze's phrase, by being converted into glucose or related
carbohydrate. Finally, according to this investigator's view, the
amides eventually unite with the carbohydrate radicals to form the
albuminous compounds characteristic of the plants, completing in
this way the cycle of proteid decomposition and regeneration.

^u

Reprinted from the Journal of the New York Botanical Garden, 1903, iv, p. 37.

CHEMICAL STUDIES OF THE PITCHER PLANT,
SARRACENIA PURPUREA.

By William J. Gies.

At the suggestion of Dr. MacDougal, a few weeks ago, I
began an investigation of the digestive powers of the pitcher plant,
Sarracenia purpurea. Two previous references to this matter will
serve to indicate the desirability of such a study.

A few years ago Vines, in referring to the mode of secretion
of the digestive juices of insectivorous plants, had the following
to say regarding Sarracenia :

"In . . . Sarracenia there are, according to Batalin, no
specialized glands, but the effect of the contact of organic matter
(insects, meat, etc.) with the cells of the lower part of the pitcher
is to cause the excretion of some substance {^probably tJie diges-
tive secretion) between the cuticular and the deeper layers of the
cell-wall of the cells which have been touched, and this is fol-
lowed by the rupture of the cuticular layer. This rupture has
the effect not only of bringing the excretion into relation with
the introduced organic matter, but also of enabling the cells
which have thus lost their cuticle to absorb the organic matter."
(Lectures on the Physiology of Plants, 1886, p. 247.)

More recently Green has written of the pitcher plants as
follows :

" Insects attracted to the plants are enticed into entering the
pitcher and are drowned in the liquid they contain. Some of
these plants, particularly Sarracenia . . . liave nothing but zvater
in the pitchers and the insects drowned therein undergo ordinary
putrefaction, the products of which are absorbed by the plant."
(The Soluble Ferments and Fermentation, 1899, p. 210.)

Thus far my experiments in this connection have been directed
to the detection of an enzyme or zymogen in the tissue of the
pitcher. Plants now growing in the Garden will be utilized later
for a study of the properties of the pitcher fluid.

729

730 William J. Gies.

Two quantities of Sarraccnia from different localities have thus
far been placed at my disposal by Dr. MacDougal. Glycerin
extracts of the thoroughly macerated tissue of one set of plants
showed moderate though distinct digestive action on fibrin at
38° C. in the presence of slight amounts of hydrochloric or
oxalic acids, the control experiments giving negative results.
All of the extracts of the second set of plants, however, were
entirely without digestive action.

In view of the negative results in the second series it is im-
possible at present to draw a satisfactory conclusion in this con-
nection. It may be that the positive results in the first case were
due to a bacterium specially favored by the medium furnished by
the constituents of the glycerin extract, or to enzyme in unob-
served diseased portions of the plants. Again, the negativ^e re-
sults may have been due to a less favorable degree of acidity,
or the secreting cells of the pitchers may have been in a " rest-
ing condition," without either enzyme or zymogen. Further
experiments, with these matters controlled and on pitchers
gathered at a more favorable season, will surely settle these
questions.

The growing plants in the Garden will also be used for direct
determinations of the influence of putrefactive products introduced
into the pitcher fluid.

A NEW PIGMENT.

In the course of the digestive experiments I had occasion to
try the activity of the extracts under neutral, acid and alkaline
conditions. Observing that the diluted neutral extract was prac-
tically colorless, the acid mixture crimson and the alkaline fluid
green, I made a few tests to determine the significance of the
colorific effects.

These tests resulted in showing that Sarraccnia purpurea con-
tains a pigment which in concentrated glycerin extract has a
reddish color, but which when diluted is practically colorless.
At such dilution, when scarcely any color is to be seen, a drop
of dilute acid produces a bright pink throughout the whole fluid ;
alkali in minute amounts turns it green. The pink is converted
to green by alkali, vice versa by acid. Even in crude glycerin

Constituents of a Pitcher Plant. 731

extract the pigment appears to be very sensitive and may be used
to advantage in titrimetric work.

I have named the pigment alkaverdin, because of the beauti-
ful green produced on treatment with alkali, preferring to reserve
the term sarracin for any digestive enzyme which later may be
found to exist in the pitcher.

Excellent "test papers" have been made with the pigment in
glycerin extract. Ordinary filter paper dipped into the red, con-
centrated extract is colorless, wet or dry. The dry paper turns a
bright pink when dipped into acid, a deep green is produced when
in contact with alkali.

The pigment of Sarracenia bears superficial resemblance to the
coloring matters in red cabbage, the purple flowers of mallow,
buckthorn berries, elderberry, dahlia and alkanet root, but vari-
ous tests, thus far, indicate that it is unlike each in fundamental
qualities. Preliminary observations indicate that its solutions are
without special influence on the spectrum, A chemical study of
the qualities of alkaverdin is now in progress.

The aqueous and saline extracts of Sarracenia contain an
abundance of dextrorotary, reducing and fermentable substances,
the characters of which, together with other bodies as yet unde-
termined, I hope to report in due time.

INDEX.

Introductory Pages. Pa.ge.

Table of contents lO

List of illustrations lo

Bibliography Ii

Divisions il

Complete list of reports and papers 13

Reports 13, 14, I5, 16, 18, 19, 20

Research papers 14, 15, 17, 18, 19, 20, 21

Miscellaneous publications ,15; i7, 22

Reprinted Papers and Abstracts of Reports. '

List of reprints of papers and of abstracts of reports, in the order of their arrange-
ment in this volume 23

List of abstracts 25

List of papers 26

[Publications which were not reprinted are referred to on page 28.]

Reprints of abstracts of reports 29

Names of societies before which the reports were made 29

Abstracts of reports which have been followed by publications giving the re-
sults in detail, a-r : ^i

Abstracts of reports of researches which have not yet been published in greater

detail, aa-mm 5 ^

Reprints of papers 65

Titles of journals in which the papers were published 65

Chemical investigations of animal tissues and tissue constituents, reprints 1-15 69

Pathological and toxicological, reprints 16—28 325

Jiliscellaneous researches, reprints 29-35 5^5

Botanical studies, reprints 36-40 659

Research Data.

The isolated letters and the numerals on the succeeding pages I'^fer to the corresponding
reprints, not to the pages of the voliune. See page 26 for further explanation.

The main references when more than one is given are indicated by the heavier letters or
numerals.

Some of the papers have a table of contents on the opening page and many of the articles
give a summary of conclusions at the end.

733

734

Index.

Only the results of our own investigations are indexed. H storical reviews, for
example, are not included.

AbsTirbing organ in gt-rminated cocoanut,
I. 36-

Absorption : absorption of connective tissue
matrix in hone not complete, a, 2, 3 ; in
cocoanut, r, 36 ; influenced by borax and
boric acid, 16, by tellurium, j, 20, 21.

Acetone absent from urine in a case of sim-
])U' anemia, 27.

Acidalbumin: influence of heat, CO.,, pro-
teose and pepton on quantitative separa-
tion ; not completely precipitated by neu-
tralization ; preparation ; quantitative
determination in digestive mixtures .

not " fat-proteid " compound, See acid-
albumin, alkalialhuminate.

Albuminoids not "fat-proteid" com-
jiounds, <), 35.

Albuminuria : albumin absent from urine
after treatment with borax and boric
acid, 16, and in a case of simple anemia,
27 ; examination of Pollacci's method of
detecting, 32 ; result of tellurium poison-
ing, 20, 21.

Albumoids. See chondro- and osseoalbu-
nioid.

Alcohol devoid of antitoxic power, n, 29.

solubility in neutral saline solution, 34; Alkalialhuminate, aa : Goodman's nnicin-
not " fat-proteid " compound, 35. See like substance from muscle.

myosni.

Acids : action of pepsin, kk, mm ; com-
bining power with mucoid, cc. See
amido, boric, carminic, chinic, chondroi-
tin sulphuric, CO,, fatty, glycuronic, H
ion, HCl, kynurenic, lactic, lepidotic,
oxybutyric, sulphuric (ethereal), uric.

Adenin from cerebro-nucleocompounds, 12.

Adrenalin : effects after treatment of pan-
creas, ee, 23, 25 ; effects compared after
subcutaneous, 23, and intraperitoneal in-

Alkaverdin, 40 : a new pigment ; proper-
ties, resemljlances, "test-papers."

Amido acids : from elastin, f, 4 ; in eggs
during development, i, 15 ; in growing
plants, 39.

Ammonia : from elastin, f, 4 ; in growing
plants, 39.

Amylolytic enzyme : in cocoanut, p, q,
r, 36 ; in fluid from pancreatic cyst and
fistula, 28. See enzymes.

Anabolism. See metabolism.

jections, 23, 25 ; effect of boiling on its Analytic data. See "summaries."
influence, 23 ; effect on urine after phlor- Anemia: cases of simple and pernicious.

hizin treatment, 23 ; extravascular clot-
ting of blood, 25 ; glycemia, ee, 25 ;
glycosuria, 23, 25 ; glycosuria not due
to diastatic ferment, 23 ; increased sugar '
formation in liver, 25 ; lesions produced !
in gastro-enteric tract, pancreas and '
other organs, 23 ; no effect on glycogen,
23 ; postmortem appearances, 23 ; source
of sugar excess in blood, ee, 25 ; sugar
in blood from various parts of circulation
(portal, hepatic and pancreatico-duodenal
veins and femoral artery), ee, 25 ; toxi-

with histories, symptoms, condition of
blood, composition of feces and urine,
27 ; cessation of reflexes, convulsions and
irritability of the brain during experi-
mental anemia, m, 26 ; classification of
anemias, 27 ; method of producing
gradual anemia, m, 26 ; value of " blood
count," 27.
Anions : hydroxidion, n, jj, 29 ; in peptic
digestion, kk, mm ; proteid chromate
reaction, jj ; toxic and antitoxic action, n,
29, 37. See ions.

cology, 23, 25. See blood, pancreas, Anthracosis. Sec lung.

liver, intestine.

Albumin : absent from cocoanut endo-
sperm, p, q, 36; not "fat-proteid"
compound, 35. See albuminuria, co-
agulable proteids.

Albuminate, 35 : myosin albuminate (acid)

Antihydrotic action, j, 20, 21 : tellurates.

Antitoxic action of ions, n, 29, 37 ; non-
electrolytes negative.

Apnopa, 24 : during hydrogen insufflation ;
influence of section of cord and vagi on
apnoea produced by artificial respiration.

Index.

735

Appetite : loss after administration of adre-
nalin, 23, borax, 16, tellurium, 20, 21.

Arginin : from elastin, 4 ; in growing
plants, 39.

Arsenic on lymph-flow after administra-
tion of lymphagogues, k, 1, 19.

Artificial respiration, 24 : effect on hyper-
sesthesia after strychnin poisoning and
section of cord ; influence on respiratory
movements and strychnin spasms ; me-
chanical influence on reflexes ; relation
to apnoea ; with hydrogen.

Ash : difficulty of removing from osseo-
albumoid, h, 6. See all materials under
head of " composition."

Asphyxia : after tellurium poisoning, 20,
21 ; during artificial respiration with
oxygen, 24.

Assimilation. See metabolism.

Autopsy after death from adrenalin, 23,
selenium, gg, tellurium, 20, 21.

" Bence Jones' body," dd : properties,
Boston's test.

Bile, j, 20, 21 : regurgitation and tel-
lurium content after poisoning with that
element.

Bile pigment : absent from fluid from pan-
creatic fistula, 28 ; in urine in a case of
simple anemia, 27, and after tellurium
poisoning, 20, 21.

Bile salts absent from urine in a case of
simple anemia, 27.

"Bismuth breath" caused by methyl-
telluride, 20, 21, 22.

Bladder, 21 : tellurium content after
subcutaneous injection.

Blood : absent from feces in a case of simple
anemia, 27 ; adrenalin glycemia, ee, 25 ;
analysis and cannula for collection, 25 ;
characters in simple and pernicious
anemia, 27 ; after administration of
adrenalin — extra vascular clotting, 25,
sugar content and source of the sugar, ee,
25, and sugar in the blood of various
parts of the circulation (hepatic, pan-
creatico-duodenal and portal veins and
femoral artery), 25; no " Rabuteau
crystals" after selenium poisoning, gg ;

sugar from blood to lymph, k, 1, 19 ;
tellurium content after poisoning, j, 21 ;
value of "blood count" in anemia, 27.
See adrenalin, hemorrhage, hemoglobin.

Bone : chondroitin sulphuric acid, a, b, 3 j
chondromucoid-like substance, b, 3 ;
collagen, b ; connective tissue matrix not
completely absorbed during ossification,
a, 2, 3 ; digestibility, b, 6 ; elastin-like
substance, b, h, 6 ; fat, b ; gelatin, b ;
new proteid constituents, a, b, c, 2, 3, 6 ;
nucleoproteid, b ; separation of ossein
constituents, b ; paramucin-like sub-
stance, b ; pigment, b. See osseoal-
bumoid, osseomucoid.

Borax, 16 : effect on digestion, absorption
and assimilation ; elimination ; influence
on feces, urine, intestinal putrefaction,
and on nuti^ition with special reference
to proteid metabolism ; in feces and
urine ; toxicology.

Boric acid, 16 ; effect on digestion, ab-
sorption and assimilation ; influence on
nutrition, with special reference to proteid
metabolism, on urine, feces and intestinal
putrefaction ; toxicology.

Botanical studies. See alkaverdin, cocoa-
nut, germination, ions, pitcher plant.

Brain : cerebron, 11 ; composition, 11, 12 ;
distribution of P-containing substances,
1 1 ; irritability during experimental ane-
mia, m, 26 ; nucleocompounds (cerebro-
nucleic acid, cerebronuclein, cerebro-
nucleoproteid), 12; "protagon," 11 ;
tellurium content after poisoning, j,
21.

Breath. See "bismuth breath."

Breathing, 24 : effect of artificial respira-
tion on concomitant breathing ; influence
of severance of the cord.

Cadaverin absent from feces and urine in
three cases of pernicious anemia, 27.

Cage, improved, for metabolism experi-
ments, 11.

Cane sugar : in cocoanut milk, 36 ; no
antitoxic power, n, 29.

Cannula : for collecting blood, 25 ; for
modified Eck fistula, ee.

73<^

Index.

Capillary pressure as a factor in lymph-
flow, k, 1. 19.

Carbohydrate : in cocoanut, p, q, 36, 38 ;
in pitcher plant, 40 ; in proteid synthesis
in seedlings, 39. See reducing substance,
sugar.

Carbon : in knilV-grinder's lung, fl". De-
termined in most of the substances under
the head of " composition."

Cat minic acid (carmin), 14.

Cartilage proteids : e, 3, 4, 5, 6. See
clioniiroall)Uiiioid, chrondromucoid.

Catabolism. See metabolism.

Cations -. alkali, earthy and heavy metals,
n, 29, 37 ; antitoxic action and valency,
n, 29, 37 ; H and rare metals, n, 29 ; H
ion in peptic digestion, kk, mm ; proteid
chromate reaction, jj. See ions.

Cellulose in cocoanut, p, q, 36, 38.

Cerebron, n : properties; reducing sub-
stance from it ; obtained from " pro-
tagon."'

Cerebronucleic acid, 12 : preparation,
composition, properties ; P content.

CerebrODUclein, 12 : preparation and com-
position ; P content.

Cerebronucleoproteid, 12 : preparation,
composition, properties ; derived purin
bases ; P content.

Chinic acid, hh : influence on uric acid
output.

Chloralbacid, Ml, 26: influence during per-
fu>ion.

Chlorocruorin. 14.

Chlorophyll, 14.

Cholesterin : absent from urine in a case of
.simple anemia, 27 ; in fluids from a pan-
creatic cyst and fistula, 28 ; in knife-
grinder's lung, ff.

Chondroalbumoid ; preparation, composi-
tion, properties, reactions, 6 ; no P, re-
lative i|uantity, h, 6.

ChoDdroitin sulphuric acid : from mu-
coids, bb ; in bone, a, b, 3 ; interferes
with coagulation of proteids by heat,
4-

Choadromucoid : chondromucoid-like sub-
stance in bone, b, 3 ; composition, 5 ; heat
of combustion, 2, 3, 5 ; interferes with co-

agulation of proteids by heat, 4 ; not
" fat-proteid " compound, 35; prepara-
tion, a, 5 ; reactions, bb ; relations to
otiier nuicnids. g, 2, 3, 5.

Chromate reaction for proteids, jj : gela-
tin and i^roteose.

Chr. mogens, 14.

CO2, 34 : influence on quantitative deter-
mination of acidalbumin.

Coagulable" proteids : absent from urine
after borax and boric acid treatment,
16, and in a case of simple anemia, 27 ;
coagulation temperature affected by
chondromucoid and chondroitin sulphuric
acid, 4. See cartilage, cocoanut, cyst,
ligament, tendon, fistula.

Coagulation of blood after administration
of adrenalin, 25.

Coagulation temperature of jiroteids. See
coagulal)le proteids.

Cocoa edestin : preparation, properties,
composition, p, q, 36 ; not " fat-pro-
teid" compound, 35.

Cocoanut : chemical and morphological
changes during germination, r, 36 ; com-
position of parts, p, q, r, 36, 38 ; com-
position of parts after germination, r,
36 ; economic and therapeutic uses, 36,
38 ; nutritive value, p, q, 36, 38 ; rela-
tive weights of the parts, q, 36 ; carbo-
hydrate, fat, proteid, ash, water, en-
zymes, p, q, r, 36, 38 ; pearls, 36.

Co'lagen, 35: not "fat-proteid" com-
pound. See bone, ligament and tendon.

Coloring matters. Animal : bone pig-
ment, b ; bile pigment in urine during
anemia, 27 ; chemical and physical quali-
ties, classification, distribution, 14 ; com-
position of pigment in knife-grinder's
lung, flf; see bile pigment, pigmentation,
hemoglobin, urobilin, uroerythrin. Vege-
table : see alkaverdin.

Composition : ash of cocoanut, 36, lig-
ament, 7, tendon, 8, "ureine," 31 ;
bone, b; blood, 25; brain, 11, 12;
cerebron, li ; cerebronucleic acid, 12;
cerebronuclein, 12 ; cerebronucleopro-
teid, 12 ; chondroalbumoid, 6 ; chon-
dromucoid, 5 ; cocoanut, germinated and

Index.

737

ungerminated, 36 ; cocoanut proteids,
36 ; coloring matters, 14 ; eggs during
development, 15 ; elastin, 4 ; fluid from
pancreatic cyst and fistula, 28 ; gastric
juice, 2t ; gelatin capsules, 16 ; gelatin
from ligament, 4 ; hair, 21 ; ligament,
7 ; ligamentomucoid, 4 ; lung of knife-
grinder, jfif; lymph, 19; muscle, aa ;
nucleocompounds of brain, 12 ; nucleo-
proteid in ligament, 4 ; ossein, b ; osseo-
albumoid, 6 ; osseomucoid, 3; pitcher
plant, 40; prepared meat, i ; " prota-
gon," II; spermatozoa, 9; tendomu-
coid, 5; tendon, 8; "ureine," 31 ;
vomit, 21. See hydration products,
feces, urine.

Compound proteids which are not " fat-
proteid "compounds, 35.

Conclusions (summaries). See summaries.

Congestion : caused by adrenalin, 23, 25 >
by tellurium, 20, 21.

Connective tissues. See bone, cartilage,
ligament, tendon.

Connective tissue mucoids, glucopro-
teids. See mucoids.

Constipation caused by tellurium, 20, 21.

Constituents. See composition.

Contents of papers. See summaries.

Convulsions : caused by strychnin, 24, by
tellurium, 20, 21 ; in gradual and acute
anemia, m, 26 ; strychnin spasms inhib-
ited by artificial respiration, 24.

Cord. See spinal cord.

Corpuscles (blood) : in simple and per-
nicious anemia, 27 ; not completely re-
movable from body by perfusion, m, 26 ;
value of "blood count" in anemia, 27.

Cotyledon of the cocoanut, r, 36 : char-
acters and composition.

Creatin : absent from fluid from pancreatic
fistula, 28 ; in ligament, d, e, 4, 7, and
tendon, d, 4, 8.
•Crustaceorubin, 14.

Cyanosis in intestine during experiments
with adrenalin, 25 : effect on sugar con-
tent of blood in portal vein.

Cyst, pancreatic, 28: history of case, symp-
toms, operation, composition of fluid and
its enzymes, recovery, excretions.

Death : caused by adrenalin, 23, perni-
cious anemia. 27, selenium, gg, strych-
nin, 24, tellurium, 20, 21.

Depression. See prostration.

Dextrin-like substance in cocoanut en-
dosperm, 36.

Dextrose. See sugar.

Diacetic acid absent from urine in a case
of simple anemia, 27.

Diarrhoea : caused by adrenalin, 23, borax,
16, tellurium, 20, 21.

Diastatic ferment not a factor in adrenalin
glycosuria, 23. See enzymes.

Digestion : influence of borax and boric
acid, 16, tellurium, j, 20, 21 ; H ion, kk,
and acids of equal conductivity, mm, in
peptic proteolysis ; quantitative determin-
ation of acidalbumin in digestive mix-
tures, 34. See bone, elastin, enzymes,
feces, gastric fistula, gastro-enteric tract,
hydration products, metabolism, mucoids,
ossein, osseoalbumoid, proteose, pepton.

Disintegration of cells and tissues caused
by adrenalin, 23, tellurium, j, 20, 21.

Distribution : coloring matters, 14, selen-
ium, gg, tellurium, j, 20, 21.

Dyspnoea, 24.

Eck fistula, ee : cannula.

Edema : in anemia, 27 ; in lungs during
perfusion experiments, m, 26 ; in pan-
creas from injected adrenalin, 23.

Edestin. See cocoa edestin.

Eggs : chemical changes during develop-
ment ; distribution of N in amido acids,
purin bases and proteid ; ash ; proteid
decomposition and synthesis ; water — i,
15. Development influenced by ions, n,
9, 10, 29 ; not influenced by extracts of
spermatozoa nor of fertilized ova, 9, 10.

Elastin. In bone : elastin-like substance ;
see osseoalbumoid. In ligament : im-
proved method of preparing, composition,
derived amido acids and ammonia, and
sulphur content, f, 4 ; distribution of N
in acid-hydration products, hexone bases,
f, ii, 4 ; digestibility, heat of combustion,
proteose and pepton, reactions, peculiar
reaction of proteoses, 4; not "fat-proteid "

738

Index.

compound, 4, 35 ; quantity, d, 7. In
tendon : (luantity, d, 8.

Electrolysis of mucoid salts, bb.

Electrolytes. See ions.

Elimination. See breath, epidermal se-
ciilion, feces, urine.

Embryochemical studies. See eggs, ger-
itiinalion, ions.

Endosperm of the COCOanut : ash, carbo-
hydrates, enzyme, fat, proteids, general
composition — p, q, r, 36, 38 ; changes
during germination, r, 36.

Enzymes : in cocoanut, p, q, r, 36, 38,
fluids from pancreatic cyst and fistula,
28, growing plants, 39, pitcher plant,
40 ; extracts of .sperm and fertilized ova
devoid of fecundative (enzyme) action,
g, 10 ; suprarenal glycosuria not due to
diastatic ferment, 23. See amylolytic
enzyme, lipase, pepsin, ptyalin, tryp-
sin.

Epidermal secretion : containing methyl
selenide, gg, methyl telluride, j, 20, 21,
22.

Ether-soluble matter. See brain, cocoa-
nut, fat, " fat-proteid " compounds, feces,
ligament, lung, tendomucoid, tendon.

Excitability of the anemic brain, m, 26.

Excitement : caused by adrenalin, 23 ;
produces increased flow of fluid from
pancreatic fistula, 28.

Excretion. See elimination.

Extirpation. See liver.

Extractives : ligament, d, e, 4, 7 ; seed-
l'"Ss. 39 ; tendon, d, 4, 8.

Extracts. See adrenalin, eggs, enzymes,
pitcher plant, spermatozoa.

Fat : digestion and absorption diminished
by borax and boric acid, 16, by tellurium,
20, 21 ; in bone, b, cocoanut, p, q, r,
36, 38, feces, 16, fluid from pancreatic
cyst, 28, knife-grinder's lung, ff, liga-
ment, 7, tendon, 8 ; not combined with
proteid to form ' ' fat-proteid ' ' com-
pounds, o, 4, 35. See ether-soluble
matter.

"Fat-proteid" compounds, o, 4, 35.

Fatty acid. See " fat-proteid " compounds.

Fatty stools always absent in a case of
pancreatic cyst and tistula, 28.

Feces : cage for collection, 11. Qualities
and composition under abnormal condi-
tions : see adrenalin, anemia, borax,
boric acid, pancreatic cyst and fistula,
selenium, tellurium. See also, Freunds'
pepton method, gastro-enteric tract, in-
testine.

Femoral artery, 25 : sugar in blood.

Ferments. See enzymes.

Fertilization, 9, 10: nature of process.
See eggs, spermatozoa.

Fistula : cannula for Eck fistula, ee ; pan-
creatic fistula — condition, character and
quantity of fluid, symptoms, treatment,
excretions, etc., 28.

Food for metabolism experiments, hh, i,
16, 21. Sec nuat.

Freund's " peptone " method, 3;^ : criti-
cism.

See

' bismuth

Garlic odor of breath.
Vjreath.''

Gastric digestion. See digestion.

Gastric fistula, j, 20, 21.

Gastric juice, j, 20, 21 : influence of tellu-
rium on secretion.

Gastro enteric tract : elimination of strych-
nin into it, 17, 18 ; influence of contents
on detection of small amounts of strych-
nin, 17, 18; influence of .selenium, gg,
and tellurium, j, 20, 21 ; lesions caused
by injected adrenalin, 23, 25 ; reduction
of tellurium compounds, j, 20, 21. See
absorption, digestion, feces, gastric
fistula, intestine, vomiting.

Gelatin. From ligament : preparation,
composition and heat of combustion, 4 ;
quantity, 7. From ossein, 6. Precipi-
tated by acid and chromate, jj.

Gelatin capsules, 16 : nitrogen content.

Germination : chemical and morphologi-
cal changes in the cocoanut, r, 36 ;
chemical changes in seedlings, 39 ; in-
fluence of ions on seedlings, 37. See
enzymes.

Globulin. See coagulable proteids, cocoa
edestin.

Index.

739

Glucoproteids. See mucoids.

Glucosamin from mucoids, bb, 4.

Glucose. See sugar.

Glyc£emia, ee, 25 : adrenalin.

Glycerin devoid of antitoxic power, n, 29.

Glycogen : absent from fluid of pancreatic
fistula, 28 ; not affected by adrenalin, 23.

Glycosuria, 23 : adrenalin ; effect of ad-
renalin greater when introduced intra-
peritoneally than when injected sub-
cutaneously ; of pancreatic origin.

Glycuronic acid, 5. See tendomucoid.

Goodman's mucin-like substance from
muscle probably alkalialbuminate, aa.

Growth. See eggs, germination.

Guanin : from cerebro-nucleocompounds,

12 ; in ligament, 4.

I

I

H ion : in chromate-proteid reaction, jj ; in '

peptic proteolysis, kk, mm ; without an- ^

titoxic power, n, 29.

Heemocyanin, 14. I

Hair : cage for collection, II ; composition |
and importance of collection in metab-
olism experiments, 16, 21. j

HCl secretion affected by tellurium, j, 20,
21.

Heart : no " Rabuteau crystals ' ' in blood ■
after death from selenium, gg ; content of I
tellurium after subcutaneous injection, 21.

Heart beat : influence of section of cord, j
24 ; relative order and time of cessation '
in gradual anemia, m, 26.

Heat : influence of boiling on complete
precipitation of acidalbumin, 34 ; no
effect (boiling) on activity of adrenalin, |

23- j

Heat of combustion : chondromucoid, os- \
seomucoid and tendomucoid, 2, 3, 5 ;
elastin and ligament gelatin, 4.
Hemoglobin : absent from urine in a case
of simple anemia, 27 ; amount in blood
after perfusion, m, 26, in pernicious and
simple anemia, 27 ; value of its deter-
mination in anemia, 27.
Hemorrhage : into intestines after admin-
istration of adrenalin, 23, 25 and tel-
lurium, 20, 21 ; in various organs after
administration of adrenalin, 23.

' Hepatic vein, 25 : sugar in blood.
Hexone bases : from elastin, ii, f, 4 ; in
I growing plants, 39. See arginin, his-
I tidin, lysin.
! Histidin from elastin, 4.
Hydration products. See digestion, elas-
tin, mucoids, purin bases.
Hydrogen : artificial respiration with hydro-
gen, 24. Determined in most of the
substances under the head of ' ' com-
position." See H ion.
Hydrolysis. See hydration products.
Hypersesthesia, 24 : due to strychnin
and section of cord ; effect of artificial
respiration.
HyperglycEemia caused by adrenalin, ee,

25. See adrenalin.
Hypoxaothin in ligament, 4.

Inflammation of mucous membranes by
adrenalin, 23, tellurium, j, 20, 21, borax
and boric acid, 16.

Inhibition, 24 : influence of artificial res-
piration on strychnin spasms.

Inorganic matter. See ash.

Intes tinal putrefaction . See putrefaction.

Intestine : increased consumption of sugar
in intestine in experiments with adren-
alin, 25 ; increased secretion of mucus after
administration of borax and boric acid,
16, tellurium 20, 21 ; effect, on putrefac-
tion, of borax and boric acid, 16, and
tellurium, 20, 21 ; excretion of kynurenic
acid not dependent on putrefaction, 30 ;
influence of contents on detection of
minute amounts of strychnin, 17, 18 ;
lesions caused by adrenalin, 23, tel-
lurium, j, 20, 21. See cyanosis, disinte-
gration, hemorrhage.

Intraperitoneal injections : greater effect
in adrenalin glycosuria than subcutaneous
injections, 23 ; adrenalin glycemia, 25.

Ions : acid (H) and alkali (OH) show no
antitoxic effects with other ions, n, 29 ;
antagonistic effects of ions of same and
different valencies and types, n, 29, 37 ;
antitoxic action, n, 29, 37 ; digestion
studies, kk, mm ; effects on cells, n,
9, 10, 29, 37 ; influence on development

740

Index.

of seedlings, 37, ova, n, 9, 10, 29 ;
precipitation of acidalbumin, 34 ; pro-
teid chromate reaction, jj ; toxicity, n,
29, 37 ; toxicity not affected by non-
tlectrolytes, n, 29, 37 ; valency and anti-
toxic action, n, 29, 37- See anions,
cations, non-electrolytes.

Irritabilty. See brain.

Islands of Langerhans the seat of gran-
ular degeneration after injections of
adrenalin, 23.

Kidneys : congestion due to adrenalin,
23 ; pigmentation due to tellurium, 21 ;
tellurium content after poisoning, j,
21.

Knife-grinder's lung. See lung.

Kynurenic acid : does not replace uric
acid in urine of dogs, 16, 30 ; metabo-
lism and excretion, 30 ; not dependent
on intestinal putrefaction, 30.

Liver : adrenalin glycemia after extirpa-
tion, ee ; sugar formation in liver before
and after administration of adrenalin, 25 ;
tellurium content after subcutaneous in-
jection, j, 20, 21. See portal vein.

Lung : edema, m, 26 ; methyl telluride
from, j, 20, 21, 22 (and tellurium, 21,
in), after poisoning ; jiignu-nts and other
constituents of lung of knife-grinder, ff.

Lymph, k, 1, 19 : capillary pressure not
the only factor in increased flow after ad-
ministration of lymphagogues ; comjx)si-
tion ; influence of osmotic pressure in
tissue spaces and of protoplasmic poisons
(arsenic, quinin) on formation after ad-
ministration of lymphagogues (dextrose,
leech extract) ; formation after death ;
physical and physiological factors ; sugar
from l)lood to lymph.

Lymphagogues. See l)-mph.

Lysin from elastin, 4.

Lactic acid absent from urine in a case of

simple anemia, 27.
Lecithin. See ether-soluble matter.
Lepidotic acid, 14.
Leucin : absent from urine in a case of

simple anemia, 27 ; from fluid from

pancreatic fistula, 28.
Lid reflex, m, 26 : relative order and time

of cessation in gradual anemia.
Ligament : ash, d, 7 ; CI, 7 ; coagulable

proteids, d, e, 4, 7 ; collagen (gelatin),

d, 4, 7 ; composition, d, 7 ; creatin, d,

e, 4, 7 ; elastin, f, ii, 4, 7, 35 ; extrac-
tives, d, e, 4, 7 ; fat, 7 ; mucoid, d, f,
4 ; nucleoproteid, 4 ; proteids, d, e, f,
'•' 4, 7i 35 ; purin bases, e, 4, 7 ; PO^,
7 ; SO,, <1, 7 ; Hfi, d, 7.

Ligamentomucoid : composition, f, 4;
glucosamin, 4 ; hydration products, 4 ;
quantity, d, 7 ; reactions and properties,

f, 4 ; reducing substance, 4 ; relation to
other mucoids, e, 2, 3, 4, 5 ; SO^, 4.
See mucoids.

Lipase in fluid from pancreatic cyst and

fistula, 28.
Lipochrom, 14.
Lipolytic enzyme. See lipase.

Meat, I, 16 : improved method of prepar-
ing for metabolism experiments, nitrogen
content, constancy of composition by this
method.

Melanin: in knife-grinder's lung, ff;
melanins, 14.

Metabolism : chemical changes in develop-
ing eggs, i, 15 ; conduct of metabolism
experiments on dogs, 16, 21 ; during
anemia, 27 ; improved cage for experi-
ments, 11 ; in cocoanut, r, 36 ; influence
of adrenalin, ee, 23, 25, borax and boric
acid, 16, selenium, gg, tellurium, j, 20,
21 ; improved method of preparing and
preserving meat for experiments, i, 16 ;
in seedlings, r, 36, 37, 39 ; similarity
in plants and animals, 39 ; with pancre-
atic ^st and fistula. See blood, diges-
tion, feces, ions, lymph, pancreas, proteid,
sugar, urine.

Metallic taste caused by tellurium, 20, 21.

Metals : influence as ions. See cations.

Methods (new or improved) : cage for
metabolism experiments, 11 ; cannula for
collecting blood, 25, and for modified
Eck fistula, ee ; chromate reaction for
proteids, jj ; production of gradual anemia

Index.

741

Tjy perfusion, m, 26. Preparation of
alkaverdin, 40 ; chondroalbumoid, 6 ;
cocoa proteids, 36 ; elastin, f, 4 ; liga-
ment gelatin, 7 ; ligamentomucoid, 4 ;
meat for metabolism experiments, 1,16;
nucleocompounds from brain, 12, from
ligament, 4 ; osseoalbumoid, h, 6 ; osseo-
mucoid, a, b, 2, 3; tendomucoid, 5, 13.
Quantitative determination : acidalbumin
in digestive mixtures, 34 ; strychnin in
intestinal contents, 17, 18 ; reducing
substance in blood, 25. Separation of
constituents of ossein, b. Criticisms :
Freund's method of detecting pepton in
urine and feces, t,t, ; Moor's " ureine "
and its production, 31 ; Pollacci's method
of detecting albumin in urine, 32.

TWethyl compounds (selenide and tellur-
i<^^)> J) gg> 20, 21, 22: occurrence and
elimination. See "bismuth breath."

"Methyl group in synthetic changes in the
body, j, gg, 20, 21, 22.

Milk of the cocoanut : composition, prop-
erties, quantity, p, q, 36, 38 ; absorption
during germination, r, 36.

Mineral constituents. See ash.

Mucin:* does muscle contain it? aa ;
mucin-like substances prepared from
bone, a, b, c, cc, 2, 3, from ligament, e,

4-
IMuCoids:* acid to litmus, a, 3, 4, 13;
antialbumid, 3, 4, 13 ; chondroitin
sulphuric acid-like body on hydration,
bb ; combining power with acid and
simple proteid, cc ; comparative —
composition, 2, 3, 5, 13, nitrogen con-
tent, 3, 5, 13, properties, bb, 3, 5,
reactions, bb, 5; relationship, g, bb, 2,
3, 5, sulphur content, bb, 3, 5, 13 ; di-
gestibility in pepsin-HCl, bb, 3 ; does
muscle contain it? aa ; ether-soluble
matter, 13, 35 ; electrolytic decomposi-
tion of salts, bb ; glucosamin, bb, 4 ;

^ In this volume the terms mucin and
mucoid are used synonymously when con-
nective tissue glucoproteids are described.
See footnote No. 3 on the opening page
<of reprint No. 5.

glycuronic acid, 5 ; heat of combustion,
bb, 2, 3, 5 ; hydration products and re-
ducing substance, a, g, bb, 2, 3, 4, 5,
13 ; in connective tissues (bone, cartilage,
ligament, tendon); non-crystallizable,
bb ; non-coagulable, 3, 4, 13 ; not " fat-
proteid " compounds, o, 35 ; phenylosa-
zone products, g, bb, 2, 3, 4, 5, 13 ; pre-
cipitin reaction, cc ; proteose and pepton,
bb, 3, 4, 13 ; quantitative precipitation,
CC, quantity in tissues, 7, 8, 13 ; salts,
bb, cc, 3 ; variability in analytic data, 3,"
5, 13. See varieties : chondromucoid,
ligamentomucoid, osseomucoid, tendo-
mucoid.

Mucous membrane. See mucus.

Mucus : secretion in gastro-enteric tract
increased by adrenalin, 23, by borax and
boric acid, 16, by tellurium, j, 20, 21.

Muscle : does it contain mucoid ? aa ; tel-
lurium content after subcutaneous injec-
tion, j, 21.

Myosin, aa : Goodman's mucin-like sub-
stance probably myosin alkalialbumi-
nate. See acidalbumin.

Nausea produced by adrenalin, 23, borax

and boric acid, 16, tellurium, j, 20, 21.

Necrosis following injections of adrenalin,

23-
Nephrectomy, 17 : elimination of strychnin

after nephrectomy.
Nervousness caused increased flow from

pancreatic fistula, 28.
Nitrogen content. See mucoids and other

substances under head of ''composition."
Nitrogenous equilibrium : influence of

borax and boric acid, 16, chinic acid,

hh, tellurium, j, 20, 21.
Non-electrolytes : alcohol, cane sugar,

glycerin and urea devoid of "antitoxic

action," n, 29, 37 ; toxicity, 29, 37.
Nose reflex, m, 26 : relative order and

time of cessation during gradual anemia.
Nuclein bases. See purin bases.
Nucleocompounds. See nucleoproteid,

purin bases.
Nucleoproteid : in bone, b ; in brain (see

cerebronuclein, cerebronucleoproteid) ,

743

Index.

12 ; in ligament, 4 — separation, P-con-
tent, purin bases on hydration.
Nutrition. See metabolism.

OH ion : influence on proteid-chromate re-
action, jj ; without antitoxic power, n, 29.

Operation for removal of pancreatic cy.st, 28.

Organs. Sec toxicology.

Osmotic pressure a factor in lymph forma-
tion, k, ig.

Ossein : digestible in gastric juice, 6 ; in-
digestible in pancreatic juice, b ; separa-
tion of constituents, b. See bone.

Osseoalbumoid ( a new constituent of bone ) :
preparation, b, h, 6 ; composition, diffi-
culty of removing ash, digestibility, no P,
neither elastin nor keratin, properties, h,
6 ; derived albuminate, location and
quantity in bone, 6.

Osseomucoid (a new constituent of bone):
alkali salt, digestibility, varieties, 3 ; com-
position, ethereal sulphuric acid, a, c, 2,
3 ; distribution, cc ; preparation and fac-
tors modifying it, a, b, 2, 3 ; heat of
combustion, 2, 3, 5 ; hydration products,
reducing substances, a, 2, 3 ; no P, c
3; not " fat-proteid " compound, 35;
reactions and properties, a, c, bb, 2, 3 ;
relation to other mucoids, cc, 2, 3, 5.
See mucoids.

Ossification, a, 2, 3 : connective tissue
matri.x not completely absorbed.

Oxybutyric acid absent from urine in a
case of simjile anemia, 27.

Oxygen. See strychnin spasms. De-
termined in most of the substances under
the head of "composition."

Ova. See eggs.

I

Pancreas : changes in blood, ee, 25, and
in urine, 23, after direct treatment with
adrenalin ; cyst, with qualities of fluid, |
etiology, operation, etc., 28 ; effect on j
pancreas of adrenalin (see islands of Lan-
gerhans), 23 ; fistula, with composition j
of fluid, character of excretions, etc., j
28 ; functions maintained with fistula |
for over three years, 28 ; influence of
pancreas in glycosuria, 23, in glycemia, i

25 ; lesions caused by adrenalin, 23,
25 ; sugar in blood from (see pan-
creatico-duodenal vein), 25 ; tellur-
ium content after subcutaneous injec-
tion, 21.

Pancreatico-duodenal vein, 25 : sugar
conttiit of blood.

Paralysis cauxil ]>\ tellurium, 20, 21.

Paramucin-like substance in bone, b.

Paraxanthin, m, 26 : influence on reflexes,,
etc., (luring perfusicjn.

Parthenogeuesis, artificial, caused by
ions, 9, 10.

Pathological. See adrenalin, albumin,
anemia, anthracosis, arsenic, " Bence
Jones' body," borax, boric acid, chinic
acid, chloralbacid, cyst, fistula, glycemia,
glycosuria, ions, paraxanthin, proteosuria,
quinin, selenium, strychnin, tellurium,
" ureine. "

Pepsin : action in various acids under dif-
ferent conditions of strength and disso-
ciation, kk, mm; influence of tellurium,
j, 20, 21. See digestion, enzymes.
I Pepton : absent from cocoanut, q, 36^
from feces in a case of simple anemia,
' 27, from fluid from pancreatic fistula,.
' 28 ; criticism of J"reund's new method of
detecting in urine and feces, 33 ; formed
from elastin, 4, and mucoid, bb, 3, 13 ;
in growing plants, 39 ; no influence on
the quantitative precipitation of acidal-
bumin in digestive mixtures, 34 ; not
"fat-proteid" compound, 35.

Perfusion experiments, m, 26 : anemia
of the brain. See anemia, reflexes.

Peritoneal cavity, 23, 25 : effect of adre-
nalin after injection into. See pancreas,
subcutaneous injection.

Phenylosazone products. See mucoids.

Phlorhizin, 23 : effect of adrenalin after
phlorliizin treatment.

Phosphorus content. See mucoids, osseo-
all^umoid and other substances under
head of "composition."

Physico-chemical. See ions, non-electro-
lytes, osmotic pressure.

Physiological action. See toxicology.

Pigmentation of intestinal contents, of or-

Index.

743

gans and urine by tellurium, j, 20, 21 ;
bile pigment in urine after tellurium
poisoning, 20, 21 ; urobilin and uroery-
thrin in the urine during anemia, 27.
See coloring matters.

Pigment metabolism disordered in ane-
mia, 27.

Pigments, respiratory and miscellaneous,
14. See coloring matters.

Pitcher plant [SarracenJa p^irpurea), 40 :
alkaverdin, constituents, digestion.

Plants. See "botanical studies."

PoUacci's method of detecting albumin in
urine, 32 : criticism.

Portal vein, 25 : sugar in blood.

Postmortem lymph formation, k, 1, 19.
See autopsy.

Precipitin reaction with mucoid, cc.

Pressure. See capillary, osmotic.

Properties. See substances under head
of " composition. ' '

Prostration after administration of adren-
alin, 23, 25, tellurium, 20, 21 ; in per-
nicious anemia, 27.

Protagon, 11 : cerebron from it, composi-
tion, does not contain the bulk of the P
of the brain, fractional products, not a
chemical individual, a mixture of sub-
stances.

Proteid metabolism. See metabolism.

Protei S ; abnormal absent from urine in a
case of simple anemia, 27, and after treat-
ment with borax and boric acid, 16 ;
carbohydrate in proteid synthesis, 39 ;
chromate reaction, jj ; coagulation inter-
fered with by chondroitin sulphuric
acid and chondromucoid, 4 ; combining
power of simple proteid with mucoid,
cc ; Freund's "pepton" method, 23 '^
heat of combustion, bb, 2, 3, 5 ; meat
for metabolism experiments, i, 16 ; mol-
ecule, 39 ; origin, 39 ; PoUacci's method
of detecting albumin, 32. See cyst,
fistula, lung, metabolism, methods. Also
the following substances : acidalbumin,
albumin, albuminate, albuminoids, alka-
lialbuminate, " Bence Jones' body,"
cerebronuclein, cerebronucleoproteid,
chloralbacid, chondroalbumoid, chondro-

mucoid, coagulable proteids, cocoa pro-
teids, collagen, compound proteids, edes-
tin, elastin, " fat-proteid " compounds,
gelatin, globulin, glucoproteids, hemo-
globin, ligamentomucoid, mucoids, mus-
cle mucin, myosin, nucleoproteid, osseo-
albumoid, osseomucoid, paramucin-like
substance, pepton, proteose, simple pro-
teids, tendomucoid.

Proteolysis. See digestion.

Proteolytic enzyme in cocoanut, r, 36 ;
in fluid from pancreatic cyst and fistula,
28. See digestion.

Proteolytic products. See hydration pro-
ducts.

Proteose : absent from urine and feces in
a case of simple anemia, 27, from fluid
from pancreatic fistula, 28 ; criticism of
Freund's method of detecting "pepton ' '
in urine and feces, t,2, ; from elastin, 4,
mucoid, bb, 3, 13 ; in cocoanut, p, q,
36, seedlings, 39 ; no influence on the
quantitative precipitation of acidal-
bumin in digestive mixtures, 34 ; not
"fat-proteid" compound, 35; peculiar
reaction of elastin proteose, 4 ; precipi-
tated by acid and chromate, jj ; pro-
teosuria, 2)?)-

Proteosuria, ;^2) '■ criticism of Freund's
method of detecting "pepton" (pro-
teose).

Protoplasmic poisons on lymph formation,
k, 1, 19 : arsenic, quinin. See lymph.

Ptomains absent from urine and feces of
cases of simple and pernicious anemia,
27.

Ptyalin affected by tellurium compounds,
20, 21.

Punicin, 14.

Purin bases : absent from fluid from pan-
creatic fistula, 28 ; in developing eggs,
i, 15, ligament, e, 4, 7, tendon, e, 4,
8 ; from brain nucleocompounds, 12,
ligament nucleoproteid, 4. See adenin,
guanin, hypoxanthin, xanthin.

Putrefaction, intestinal : influence of
borax and boric acid, 16, tellurium, 20,
21 ; excretion of kynurenic acid inde-
pendent of it, 30.

744

Index.

Putrescin alisent from urine and feces in
three cases of pernicious anemia, 27.

Quinin on lymph formation, k, 1, ig.

ReactiODS : proteid-chromate, jj, mucoid
liricipitin, cc. See substances under head
of "composition."

Reducing substances. See blood, cocoa-
nut, cerebron, mucoids, phenylosazone
]>n)(hicts, pitcher plant, urine.

Reduction of tellurium compounds in cells
and organs, j, 20, 21.

Reflexes : effect of adrenalin, 23, tellurium,

20, 21, artificial respiration, 24 ; rela-
tive order and time of cessation during
experimental anemia, m, 26.

Respiration, m, 26 : relative order and
time of cessation during gradual anemia.
Sec artificial respiration.

Respiratory movements : influence of
artificial respiration and section of cord
and vagi, 24, tellurium, 20, 21 ; chemi-
cal factors, 24.

Respiratory pigments, 14.

Restlessoess caused by adrenalin, 23, tel-
lurium, 20, 21.

Salts of Mucoids : formation, bb, 3 ; dis-
sociated by the electric current, cc. See
mucoids.

Salt solutions, 34 : solvent action on acid-
albumin. See ions.

Secretion: influence of adrenalin, 23, borax
and boric acid, 16, and tellurium, 20,

21, on the secretion of mucus in the gas-
trointestinal canal ; influence of tellurium
on secretion of acid in the stomach, j,
20, 21. See epidermal secretion.

Seedlings. See germination.

Selenium, toxicology, gg ; effects, distri-
bution, elimination, methylation, no pro-
duction of " Rabuteau's crystals " in the
blood of the heart at death.

Simple proteids not " fat-proteid " com-
pounds, 35.

Skin reflex, m, 26 : order and time of
cessation during gradual anemia.

Solutions : in determinations of toxic and

antitoxic action, n, 29, 37 ; of enzymes,
kk, mm ; for digestive studies, kk,
mm, and perfusion experiments, m, 26.
See extracts.

Somnolence caused by adrenalin, 23, tel-
lurium, 20, 21.

Spasms. See convulsions.

Spectrum, 40 : alkaverdin without influ-
ence.

Spermatozoa, 9, 10 : characters of ex-
tracts ; extracts are without fecundative
effect ; neither proliferative en/yme nor
zyniogen could be detected. See eggs.

Spinal cord, 24 : influence of section on
movement, heart beat and on production
of apncea by artificial respiration ; strych-
nin spasms and respiratory movements
after section. See hyperesthesia.

Spleen, 21 : tellurium content after sub-
cutaneous injection.

Stomach r secretion of acid and mucus,
vomiting, etc., after tellurium poi.soning,
j, 20, 21 ; tellurium content after sub-
cutaneous injection, j, 21. See digestion,
gastro-enteric tract, gastric fistula.

Strychnin : detection and ([uantitative de-
termination in intestinal contents, 17, 18;
elimination into gastro-enteric tract after
nephrectomy, 17 ; toxic action of strych-
nin admixed with intestinal contents,
18 ; toxicological, 24. See strychnin
spasms.

Strychnin spasms, 24 : influence of arti-
ficial respiration, of section of the spinal
cord and vagi, of lack of oxygen in in-
spired air. See artificial respiration.

Subcutaneous injection : adrenalin sub-
cutaneously is less effective in producing
glycosuria than when injected intraperi-
toneally, 23 ; effects of selenium, gg,
tellurium, j, 20, 21, "ureine," 31.

Sugar : cane sugar in cocoanut milk, 36.
Dextrose : absent from urine in cases of
simple anemia, 27, of pancreatic cyst
and fistula, 28, and after treatment with
borax, boric acid, 16, and tellurium, 21 ;
in blood from various parts after ad-
ministration of adrenalin, ee, 25, fluid
from pancreatic fistula, 28, lymph after

Index.

745

injection of dextrose into blood, k, 1, 19,
urine after administration of adrenalin ;
normal content in blood, 25, lymph, 19 ;
source of excess in adrenalin glycemia,
ee, 25. Dextrin-like substance in cocoa-
nut endosperm, 36. Saccharates with
cations, n, 29, 37. Sugar in parts of
the cocoanut, p, q, 36, 38, and in pitcher
plant, 40. No. antitoxic action, n, 29.
See adrenalin, carbohydrate, reducing
substance, urine.

Sulphur content of elastin, mucoids and
other substances under the head of
" composition."

Sulphuric acid, ethereal. See mucoids.

Summaries. Conclusions are summarized
at the ends of the following reprints :
I, 2, 3, 4, 5, 6, 7, 8, 9, 10, II, IS, 16,
19, 21, 23, 25, 26, 28, 29, 31, 32, 33,
34, 35, 39. Contents are indicated on
the opening page of each of the follow-
ing papers : 3, 4, S, 6, 9, 16, 21, 34,
36. Analytic and experimental data are
presented in special tabular form in the
following publications : r, i, 3, 4, 5, 6,
7, 8, II, 15, 16, 19, 21, 25, 26, 27, 28,

30, 3i> 34, 35, 36, 37-

Suprarenal glycosuria probably not con-
nected with diastatic ferment, 23. See
adrenalin.

Suprarenals congested after injection of
adrenalin, 23. See adrenalin.

Sympto 1 s. See anemia, pancreatic cyst,
pancreatic fistula, toxicology.

Tables. See summaries.

Taste. See metallic taste.

Tellurate, j, 20, 21 ; therapeutic value.

Tellurium: cause of "bismuth breath,"
elimination, methylation, j, 20, 21, 22 ;
digestion, absorption, assimilation, dis-
tribution after subcutaneous injection,
influence on metabolism and gastro-en-
teric tract, soluble in body juices, thera-
peutic value of tellurates, toxicology,
j, 20, 21 ; personal experiences, 20,
21.

Temperature. See coagulation tempera-
ture, heat.

Tendomucoid : associated impurities, pre-
paration, 5, 13 ; composition, nitrogen
content, varieties, sulphur content, d, g,
5, 13 ; difficulty of removing ether-
soluble matter, 13, 35 ; fractional pro-
ducts, Loebisch's data for sulphur con-
tent not verified, reducing substance on
hydration, g, 5, 13 ; glucosamin, g, 5 ;
glycuronic acid, lability, 5 ; heat of com-
bustion, 2, 3, 5 ; not " fat-proteid "
compound, o, 35 ; proteid hydration
products, 5, 13 ; quantity in tendon, 8,
13 ; reactions, bb, cc, 5, 13 ; relation to
other connective tissue glucoproteids, g,
bb, 5 ; mucoid from parts of t endon, d,
g, 5. See mucoids.

Tendon: ash, collagen (gelatin), compo-
sition, elastin, total SO^ and ethereal SO^,
water, d, 8 ; CI, fat, PO4, 8 ; coagulable
proteids, extractives, d, e, 4, 8 ; creatin,
d, 4, 8 ; mucoid, proteids, d, g, bb, CC,
o, 2, 3, 5, 8, 13, 35 ; purin bases, e, 4,8.

" Test papers." See alkaverdin.

Tetanus. See strychnin spasms.

Tetronerythrin, 14.

Therapeutic value: tellurates, j, 20, 21 ;
parts and constituents of cocoanut, 36, 38.

Thermochemical. See heat of combus-
tion.

Tissue fluids, j, 20, 21 : tellurium soluble.
See osmotic pressure.

Tissues, 14 : pigments. See connective
tissues, organs, toxicology.

Toxicology. See adrenalin, borax, boric
acid, chloralbacid, ions, paraxanthin,
selenium, strychnin, tellurium, "ureine. "

Transfusion experiments. See perfusion.

Transudate (pancreatic), 28: quahties,
composition.

Treatment for anemia, 27 ; for pancreatic
cyst and fistula, 28.

Tremor caused by strychnin, 24, tellurium,
20, 21.

Trypsin : in fluid from pancreatic cyst and
fistula, 28 ; influence of tellurium, j, 20,
21.

Tryptophan absent from fluid from pan-
creatic fistula, 28.

Tumor. See cyst.

746

Index.

Turacin, 14. I Urobilin increased in urine in a case of

Tyrosin absent from fluid from pancreatic simple anemia, 27.

cvst and fistula, 28 ; from urine in a case ' Uroerythrin increased in urine in a case of

of simple anemia, 27. I simple anemia, 27.

Unconsciousness in tellurium poisoning,
20, 21.

Unsteady gait after administration of ad-
renalin, 23, tellurium, 20, 21.

Urea : absent from fluid from pancreatic-
fistula, 28 ; no antitoxic power, n, 29, 37;
related compounds in seedlings, 39; tox-
icity, 29, 37.

" Ureine," 31 : preparation, composition,
a mixture of many substances, not the
cause of uremia, toxicity.

Uremia not caused by "ureine," 31.

Uric acid : influence of chinic acid on elim-
ination, hh; not replaced by kynurenic
acid in dog's urine, 16, 30.

Urine affected by adrenalin, anemia, borax,
boric acid, chinic acid, pancreatic cyst and
fistula, selenium, tellurium, "ureine."
See also albumin, " Bence Jones' body,"
cage for collection, Freund's peptone
method, glycosuria, kynurenic acid, met-
abolism, Moor's "ureine," Pollacci's
albumin method, proteosuria.

Vagi, 24 : efi"ect of section on apncea by
artificial respiration ; influence of section
on respiratory movements and strychnin
spasms.

Valency, n, 29, 37 : relation to action of
inns.

Vomiting caused by adrenalin, 23, borax
and boric acid, 16, tellurium, j, 20, 21,
30.

Water. See materials under head of
" composition."

Xanthin : in developing eggs, i, from
brain nucleocompounds, 12 ; from liga-
ment nucleoproteid, 4. See purin bases.

Xanthin bases. See purin bases.

Zoonerythrin, 14.

Zymogen not detectable in sperm extract

(fecundative), 9, 10, nor in pitcher

plant (digestive), 40.
Zymolysis. See digestion.