rite! Pa Tape
THE JOURNAL
OF
BIOLOGICAL CHEMISTRY
FOUNDED BY CHRISTIAN A. HERTER AND SUSTAINED IN PART BY THE CHRISTIAN A. HERTER MEMORIAL FUND
EDITED BY
KIN, New York City. LAFAYETTE B. ME
J. J. ABEL, Baltimore, Md. R. H. CHITTENDEN, New Haven, Conn.
OTTO FOLIN, Boston, Mass. A. S. LOEVENHART, Madison, Wis. a
_ WILLIAM J. GIES, New York. GRAHAM LUSK, New York. os
L. J. HENDERSON, Cambridge, Mass. A. B. MACALLUM, Toronto, Canada. 3
REID HUNT, Boston, Mass. J. J. R. MACLEOD, Cleveland, Ohio. ‘ WALTER JONES, Baltimore, Md. JOHN A. MANDEL, New York. a
J. H. KASTLE, Lexington, Ky. A. P. MATHEWS, Chicago, Ill. a
‘J, B, LEATHES, Toronto, Canada. F. G. NOVY, Ann Arbor, Mich. “-
| } THOMAS B. OSBORNE, New Haven, Conn. Bs i
t T. BRAILSFORD ROBERTSON, Berkeley, Cal. *
: P. A. SHAFFER, St. Louis, Mo. ;
} 4
A. E. TAYLOR, Philadelphia, Pa. F. P. UNDERHILL, New Haven, Conn.
| : V. C. VAUGHAN, Ann Arbor, Mich. 4 ALFRED J. WAKEMAN, New Haven, Conn. r HENRY L. WHEELER, New Haven, Conn. 2, }Y Ja \ ‘ 524 VOLUME xvi V*%~{b | BALTIMORE afi Y
1913-14
COMPOSED AND PLINTED AT THE :" ~ WAVERLY PRESS hh / By rae Wietrame & Wiuwins Courant
Bacrimonn, U.8, A, x
CONTENTS OF VOLUME XVI.
GrorGe Peirce: The partial purification of the esterase in pig's liver GerorGce Petrce: The compound formed between esterase and sodium GTO ak bo cn es Ieee ed: Larayetre B. Menpet and Rosert C. Lawanl The rate of elimina- ‘ tion of nitrogen as influenced by diet factors. I. The influ- ence-of the:texture of the diet.... ..,.<) hues ee ee LarayetTre B. MenpDEL and Ropert C. Lewis: The rate of elimina- tion of nitrogen as influenced by diet facters. Il. The influ-
LarayetTe B. Menpev and Rosert C. Lewis: ‘The rate of eli tion of nitrogen as influenced by diet factors. IIT. ence of the character of the ingested protein. _ Baa
ee N and B. a
+h
P. A. Levene and Sona D. Vor LYKE: The separation of d-ala- ee el ee a Donatp D. Van Stryke: The gasometric determination of aliphatic amino nitrogen in minute quantities......................... Dona.tp D. Van Stryke: Improved methods in the gasometric deter- mination of free and conjugated amino-acid nitrogen in the
q Car. O. Jonuns and Emit J. BAuMANN: Researches on purines. XIII. i On 2,8-dioxy-1,6-dimethylpurine and 2,6-dioxy-3,4-dimethy]-5-
nitropyrimidine (a-dimethylnitrouracil)...................... f Ray E. Nerpie: Polyatomic alcohols as sources of carbon for lower
BG tema er, Mt i, ee ak Epwarp B. Meres and Howarp L. Marsu: The comparative compo- ‘ sition of human milk and of cow’s milk.......................
Victor C. Myers and Morris 8. Fine: The influence of the admin- istration of creatine and creatinine on the creatine content of
Donatp D. Van Stryke: The fate of protein digestion products in the body. II. Determination of amino nitrogen in the tissues Donatp D. Van StYKe and Gustave M. Meyer: The fate of protein digestion products in the body. III. The absorption of amino- acids from the blood by the tissues........................... Donaup D. Van SLYKE and Gustave M. Meyer: The fate of protein digestion products in the body. IV. The locus of chemical transformation of absorbed amino-acids. .....................
iii
ence of carbohydrates and fats in the diet......... #4 vaca
19
121
iv Contents
Donatp D. Van StyYKeE and Gustave M. Meyer: The fate of protein digestion products in the body. V. The effects of feeding
and fasting on the amino-acid content of the tissues........... 231 K. Mryaxe: The influence of salts common in alkali soils upon the
growtl of the rice plant...[ogiimee es... 5... eee 235 Paitie A. SHarrer and W. McKim Marriotr: The determination
OF Geymputyric acid......... Signe... devas 265 W. M. Marriotr: The determination of acetone.................... 281 W. M. Marriorr: Nephelometric determination of minute quantities
Gr acetone... ...... 2. RRR s Gus 289 W. M. Marriorr: The determination of 8-oxybutyric acid in blood
and tiases. :........: Ge ie a oe ad aN ae 293
E. V. McCottum and D. R. HoaGianp: Studies of the endogenous metabolism of the pig as modified by various factors. I. The effects of acid and basic salts, and of free mineral acids on the endogenous nitrogen metabolism.....................64.02005 299 otLuUM and D. R. HoaGianp: Studies of the endogenous ism of the pig as modified by various factors. II. ence of fat feeding on —— a metab
ee ee |
The influence of ie 10
(i a lia A) 321
tabolism.............. ¥ Jacos RoseNBLooM and 8. Rov isis: The non-interference of “‘ptomaines”’ with certain tests for morphine................. 327
M. E. Penninoton, J. 8. Hepsurn, E. Q. Sr. Joun, E. Witmer, M. O. Srarrorp and J. I. Burrett: Bacterial and enzymic changes Th milk QMG'CROAM AGM 6 dvs ic 6 eee stolen. so cee Das 331 Howarp B. Lewis and Bren H. Nicoter: The reaction of some pu- rine, pyrimidine, and hydantoin derivatives with the uric acid
and phenol reagents of Folin and Denis....................... 369 Istpor GREENWALD: The formation of glucose from propionic acid
in dismeses mellitus: dees ide cis cPibee ccc dey come es ts Cae 375° EB. K. Marswary, Jr. and L. G. Rowntree: The action of radium
emanation on lipases... ae.) eae de ee. . a 379 S. R. Beneprer and J. R. Muruin: Note on the determination of
amino-acid nitrogen im urine..................ceee eee ceeeeees 385 W. Denis: Metabolism studies on cold-blooded animals. II. The
blood and urine of fights... 05)... 5: dues... ss. SS. ee 389 W. Dents: Note on the tolerance shown by elasmobranch fish towards
Certain nephrotoxic agents..........sccesesvceawale-coumueeee 395
Cyrus H. Fiske and Howarp T. Karsner: Urea formation in the liver. A study of the urea-forming function by perfusion with fluids containing (@) ammonium carbonate and (b) glycocoll.,. 399
P. A. Levene and C, J, Weer; The saturated fatty acid of kephalin, 419
Contents Vv
Tsomas B. Ossporne and Larayetre B. MENDEL (with the codpera- tion of Epna L. Ferry and ALFRED J. WAKEMAN): The influ-
ence of butter-fat on growth...) .. jiavaeesasee tenes scacsee-e- 423 J. Du P. OosrHuizEN and O. M. Suepp: The effect of ferments and other substances on the growth of Burley tobacco.. awake 3 ee
' J. R. Greer, E. J. WirzemMann and R. T. Woopyartt: Stuilies on the theory of diabetes. II. Glycid and acetole in the normal and phlorhizinized animal........ 2... .cigepe ene cs die eeeseeis 455 | A. T. Campron: The iodine content of the thyroid and of some bran- Gnier Clete OFeONsS,.. 6. ............ Hie os wknd tole e 465 P. A. Levene and C. J. West: A general methied for the conversion of fatty acids into their lower homologues..................... 475 Artuur W. Dox: Autolysis of mold cultures. II. Influence of ex- haustion of the medium upon the rate of autolysis of Aspergil- TE te a ee aS 479 Suiro Tasutro: Carbon dioxide apparatus III. Another special ap paratus for the estimation of very minute quantities of carbon Gigxidey Ue ee bwawaaess......... P eee On the rate of absorption of chol
ae ee
eee eee eee
Snithistive of Fa ge qaelees upor eae i are Ot a Se 515 W.R. Bioor: On fat absorption. III. Changes in fat during absorp-
ee ee es a ae
Donautp D. Van Stryke: The hexone bases of casein................. 531 Donaup D. Van Stryke and Freperick J. Bircnarp: The nature of the free amino groups in proteins...................-......... 539 P. A. Levene and C. J. West: On sphingosine. II. The oxidation of sphingosine and dihydrosphingosine.....................-.. 549 _ P. A. Levens and Gustave M. Meyer: On the action of leucocytes ! and of kidney tissue on amino-acids.......................40. 555 _ R. Leprne: “Sucre virtuel’’ and blood glycolysis.................... 559 A. I. Rineer and E. M. Franxet: The chemistry of gluconeogenesis. . VI. The effects of acetaldehyde and propylaldehyde on sugar ‘ formation and acidosis in the diabetic organism............... 563 SY OFM: OO VR cis cca Wiaa'ss . s dein <MMMins ws asuskvanccesclaiy 581
THE PARTIAL PURIFICATION OF THE ESTERASE IN PIG’S LIVER. By GEORGE PEIRCE. (From the Physiological Laboratory of the University of Wisconsin.) (Received for publication, August 1, 1913.)
_ The preparation of a substance which can be regarded as an enzyme free from foreign material has never been accomplished. Invertases and proteases of considerable strength have been fp
a paced, but very little work has been done on the pe ification
a was prepared 1
4 ‘as in is investigations. One hundrec
fresh pig’s iver vouont with sand and water, strained through _ cloth and made up to 1 liter with distilled water. Toluene was added as a preservative. After incubation at 37° for one day and after several weeks’ standing at room temperature it was _ filtered through a folded filter until clear. The filtrate will be referred to as 10 per cent crude enzyme solution. 20 per cent solutions were also made.
The crude enzyme solution was dialyzed in collodion bags for five or six days and filtered. About 90 per cent of the solid sub- stance was removed by this process, and the solution lost about 20 per cent of its total activity so that the purification was con- siderable. This solution will be referred to as dialyzed enzyme solution.
In it was now dissolved one-half the amount of ammonium sulphate necessary for complete saturation, and the solution was poured repeatedly through the same folded filter until a clear filtrate was obtained. The precipitate was practically inactive and was rejected. The filtrate was then fully saturated with ammorium sulphate and filtered till clear. The filtrate was inactive. The precipitate was taken up in water and the solution
dialyzed till it no longer gave a turbidity with Ba@l. This
I
i : | THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. 1.
p ~» , & ¥ Fail
2 Purification of Esterase
represents the most highly purified solution obtained (Solumin Bs No attempt was made to get a solid active substance.
A small portion of one of these solutions was mixed with an ethyl butyrate solution about two-thirds saturated. Both solu- tions had been previously warmed to 37° and the reaction was carried on at this temperature. The time of.the beginning of the reaction (which was not more than three seconds in error) was taken at the time when one-half of the enzyme had flowed from a pipette into the ethyl butyrate solution. At suitable intervals 50 cc. were removed with a pipette and allowed to flow into an Erlenmeyer flask containing 5 ee. of 1 per cent neutralized NaF. The mixture was titrated with #4 NaOH free from carbonate to a moderately deep shade of pink, phenolphthalein being used as the indicator. Nal of the above strength inhibits the enzyme completély in acid solutions, but allows the hydrolysis to proceed slowly in neutral or faintly alkaline solutions. Conseanep the titrations need not be made i e final stag must be performed in a few } ne of the for the selection of a deeper shade of pink than aad for an end point. The time of completion of the reaction is taken when the first drop from the pipette flows into the NaF. This allows for the slowing of the hydrolysis by cooling during the pipetting and for progress of the reaction in that part of the reaction mixture that has not yet been mixed with the Nal’. The error in the measurement of the time was probably less than ten seconds in all. The titration error probably averaged about 0.1 cc. or a little less. Proper allowance was always made for any initial acidity. The temperature regulation was efficient and no error is to be attributed to this source.
The activities of the enzyme solutions are represented on the basis of their solid content. It has been previously shown! that in solutions of equal acidity, a given amount of enzyme hydrolyzes ethyl butyrate with the same absolute velocity over a wide range of enzyme and ester concentration. Consequently it will be convenient for the present purpose to compare the activities of the different solutions by giving the number of parts of ethyl butyrate that one part of solid substance hydrolyzes per hour. The ethy] butyrate concentration must always be above yj and
1 Peired® Journ. Amer. Chem, Soc., xxxii, pp. 1525 and 1530, 1910,
George Peirce 3
_ the acidity must be the same in the solutions to be compared. _ In this paper it will be understood that the initia! acidity is “0” _ and the final acidity ‘‘10,”’ 7.e., 10 cc. of % acid per 50 ce. of reac- tion mixture.
___ 15ce. of Solution B contained 4.8 mgms. solid substance (dried over water bath). ‘
20 ce. of this solution in a total volume of 560 cc. hydrolyzed 650 mgms. _ ethyl butyrate in 28.1 minutes (final acidity “10”).
The activities of the various solutions follow:
PARTS OF ETHYL BUTYRATE DESIGNATION OF ENZYME SOLUTION HYDROLYZED PER HOUR BY 1 PART SOLID SUBSTANCE
OE CRS Se Ga AS a 10" : ¥ : Dialyzed 10 per cent....................... = 00 Partially} purified 10 per cent.............. 14eeor*
+The all Sr, Sala was omitted.
_ Kastle, Johnston and Elvove? eared a clear esterase solution _ and estimated its activity on the basis of its solid content. With- - out any allowance for the different conditions of the hydrolysis, " the solid substance in solution B was 400 times as active as that in their solution. Making a liberal allowance for differences in _ the conditions it seems safe to say that the solid substance of solu- _ tion B was 50 and probably 100 times as active as theirs.
No detailed chemical investigation was made of the dried mate- rial from these enzyme solutions, owing to the small amount of _ material available. Ag; contained about 0.6 per cent phosphorus, _ but the figure is only approximate. An accurate determination _ of the tyrosine content of B was made. 4.8 mgms. of the dried _ substance were heated on a water bath for several hours with 20 _ per cent HCl, the HCl was evaporated and the residue taken up with a few drops of dilute HCl. A determination according _ to Folin, using one-quarter of the given quantity of reagents and
diluting to 25 cc., gave the following result:
4.8 mgms. dried substance gave 0.283 mgm. tyrosine. Tyrosine=5.9 per cent.
2 Amer. Chem. Journ., xxxi, p. 526, 1904.
_ THE COMPOUND FORMED BETWEEN ESTERASE AND SODIUM FLUORIDE.
By GEORGE PEIRCE. (From the Physiological Laboratory of the University of Wisconsin.)
(Received for publication, August 1, 1913.)
In previous papers by Kastle and Loevenhart! and and Peirce? it was shown that sodium fluoride has a remar inhibiting action on lipases and esterases. In the present pa it,is proposed to show: (1) that this inhibition is du to tl
‘the reaction based on he mass hig agrees with the vations. _ The particular case investigated was that of the action of the esterase from pig’s liver on ethyl butyrate. The choice was '- made on account of ease of experimentation and it is planned | later to extend the work to other simple esters, and, if possible, to the true fats. Four different enzyme solutions were used. For the methods of their preparation the previous paper in this number of the Journal must be consulted. Preparation A was | the crude 10 per cent enzyme there referred to, while B was the |. most highly purified enzyme obtained in that investigation. The ' crude 10 per cent enzyme solution used in Table III was about \ one-third as active as A. C was prepared in the same manner | as B but was not quite so active.
The technique was the same as that of the previous paper ' except in Table V, where alcohol instead of sodium fluoride was used to stop the reaction before titration. "When sodium fluoride - was used in a reaction mixture it was mixed with the ethyl buty- - rate before the enzyme was added.
1 Amer. Chem. Journ., xxiv, p. 491, 1900. 2 This Journal, ii, p. 397, 1907.
5
6 Esterase and Sodium Fluoride
It has been shown’ that the concentration of ethyl butyrate ©
makes practically no difference in the absolute velocity of acid production when no sodium fluoride is present. This has not been shown to be true in sodium fluoride mixtures, so to obviate any difficulty the initial concentration of ethyl butyrate is the same in any given set of experiments. It varied from #5 to #5 in different sets of experiments.
Using enzyme solution B several series were run, alike in all respects except that the concentration of the sodium fluoride varied. The numerical data for this series of experiments are given in Table I, and the results shown graphically in Figure 1. wis the
! | ! | i ] a8 |
0 + 4 G0 owen O° 100 120 140 160 180 200
Fia. 1. To Intusrrare Tastp I,
number of cc. of # butyric acid produced in 50 cc. of reaction
mixture in ¢ minutes. If the decimal point is moved two places —
to the left, the normality of the solution is obtained. The time
taken to produce 10 ce. of acid is calculated for each series and — reappears in the third column of Table II. The reaction mix- —
tures were made up as follows:
250 ce. ethyl butyrate solution.
5ec. enzyme solution B. 25 ce sodium fluoride solution of different strengths. * | water.
® Peirce: Journ, Amer. Chem, Soc., xxxii, p. 1525, 1910,
George Peirce
_ NaF = 0.0268 mem. PER LITER
TABLE I. NaF = 0.00000 Mem. PER LITER NaF = 0.00893 Mem. PER LITER Observed Average Cbserved Average 2 t ee x t z i 1.22 5.22 1.18 5.03 1.29 5.92 1.32 6.52 12.14 4.83 ; 1.34 6.92 8,62 16.92 3.73 17.02 3.48 18.17 3.40 18.00 3.84 17.12 3.31 17.83 4,84 | 23.47 4.97 23 .56 5.65 32.75 5.62 32.63 ~ 8.10 23 .95 5.58 32.50 e262 37 .42 7.64 37 .52 7.92 49 .30 7.96. 49 .69 7.65 | 37.62 7.99 . of Oa 9.64 50.55 9.69 51.438 9.73 52.30 10.10 (10.00) | (53.7) 10.27
79.78
145 .56
(190. ) 201 .42
18.77 2.09 9.45 1.92 - 22.97 3.34 24.12 3.26 d 39.85 §.21 80.88 40.70 5.10 78 .67 74.47 8.15 | 146.78 74.28 8.01 | 144.33 (10.00) | (92.8) 10.09 | 94.28 10.18 94.83 10.52 | 202.83 10.26 | 95.37 10.42 | 200 .00 NaF = 0.268 MGM. PER LITER Observed Average z t F t 1.14 29 .08 1 29 .04 1.20 29 .00 3.85 133 .58 4.00 133.21 4.15 132.83 5.65 212 .83 5.70 213 .23 5. 213 .62 7.96 338 .08 8.01 338 .29 8.06. | 338.50 (10.00) | (450. 10.39 479.42} 10.51 479 .77 10.62 479 .92
8 Esterase and Sodium Fluoride
The most obvious explanation for this inhibition is that a cer- tain amount of the enzyme is inactivated by the sodium fluoride, either by destruction, or by the formation of some sort of inactive compound. We shall see later that the view that the enzyme is destroyed is untenable.
Any compound of sodium fluoride and esterase should be formed in accordance with the mass law:
(Cone. free Enz.)™ < (conc. free NaF)" =k (conc. NaF. Enz.)?..... {1]
where m, n and p represent the number of molecules of the sub-
stances involved in the reaction. Transposing:
2 (cone. free Enz.)™
~ (cone. NaF. Enz.)?
* (conc. free NaF)"........... [2]
P; but ittle consideration will enable us to ext de se possibilities at the outset ang leave only a few ot
of a reaction, wherein or Pimblecule of active enzyme would be broken up by sodium fluoride into two or more molecules of an inactive compound, in such a manner that the reaction could be reversible. This would indicate that in equations [1] and [2] m is equal to or greater than p. For simplicity we can let p = 1 and m = 1, 2,3 or more. The higher values are, of course, in- creasingly improbable. No values can be assigned to n on such considerations as these. |
On the basis of the figures already obtained, the most simple equation possible will first. be tested:
= oe ee conc. free Nak...) 0... ae
That is, we assume that one molecule of sodium fluoride combines with one molecule of enzyme to form one molecule of the inac- = bo Ean *- is calculated as follows: cone. Nal’, Enz. It has been shown repeatedly* that the absolute velocity of acid
tive compound. The term
‘Cf. Peirce: Journ. Amer. Chem, Soc., xxxii, p. 1529, 1910,
no data at present for assigning values to m, n and :
George Peirce 9
- production is very nearly proportional to the amount of enzyme present. In any event, a solution whose activity is inhibited by sodium fluoride behaves as if only a certain percentage of the total enzyme present were really acting. Let x represent this percentage. Then since the total percentage present is 100, the per-
cone. free Enz. _
conc.NaF.Enz. —
centage of the NaF’. Enz. present is 100—z2 and
a oH * For example it took a solution containing no sodium fluoride 60 minutes to produce 10 cc. of #% acid, and a solution i, containing 1: 40,000,000 sodium fluoride 100 minutes to produce he - the same amount. The one containing sodium fluoride had there-
260.) 100
i a, A 4g ide. The - rR ace a |
x the reaction so t a 7 sat by the reciprocal of the time taken to attain a given stage of the | reaction represents the average activity during this period. Since the _ curves with differept strengths of sodium fluoride are not exactly similar in shape a slight inaccuracy is involved. Absolutely accurate results can
theoretically be obtained by plotting the curves with x and t as ordinate
: and abscissa respectively, and taking the values of the tangents = at the
same values of z on the different curves as proportional to the values of the activities at these 4 oe Since now the curves have a nearly con-
stant curvature, the value of = 7 for z = 10 will be very nearly equal to o
for «= 5. The simpler aud is used for two reasons. First, the measurements are not sufficiently accurate to justify the labor involved in making an extremely accurate graphical measurement and, secondly, the method employed is absolutely objective. Expressed as non-mathematically as possible the argument runs: We _ wish to obtain the ratio of the activities of two solutions, one containing _ NaF and the other containing none. The rate of hydrolysis diminishes as the reaction proceeds, but we can represent the average activity during | the production of the first 10 cc. of acid by the reciprocal of the time taken to produce that acidity. Considering the form of the curves, this average | activity will, with sufficient accuracy for our present purposes, be also the actual activity at the point where 5 cc. of acid has been developed.
IO Esterase and Sodium Fluoride
therefore > = 1.5. The concentration of the free sodium fluoride
cannot be obtained directly, but it will be taken as equal to the total sodium fluoride present. This is, of course, not absolutely true; but we shall see in Table IV that very little of the total sodium fluoride present is combined with the enzyme, especially in weak enzyme mixtures. No appreciable error is therefore in- volved.
The headings in the following table are all self-explanatory. The data are derived from Table I.
TABLE Il.
5 ec. enzyme solution B in a total volume of 280 ce.
Ey NaF CONC. OF ENZYME og y aici PERCENTAGES Free Ens. M 5 actpiry ‘‘10”* ) NaF. Enz. X10 iter | Normality Free | NaF. Enz. ea .
minutes 0.00000 0.000
0.00893 0.213 x 10-* a 3
0.0268 0.638X10-* 92.8 42.1 | 1.38 0.0893 2.13 X10-* 190.0 71.7 | 0.305 0.268 |6.38 X10-* 450.0 88.06 | 0.136
* Acidity “10’’ = 10 ce. ay acid in 50 ce. mixture.
As an additional example a similar experiment with a crude 10 per cent extract is included. Only the final results are given. k is different in the two tables. This is due in part to slightly
TABLE III.
5 ce. enzyme solution used to 250 ce. ethyl butyrate solution.
NaF CONC. OF ENZYME
aE te a TIME TO REACH + alba ae = axe
.—— ) Normality | = Free Ens. | NaF. Ens. ae Ear | minutes mer a
0,0008 0.000 | 47.9 100 | 0 0.0008 |0.233K10-" 53.9 89 11) Sa | 0.0195 |0.464X10-" 58.5 82 18 | 4.56 | 2.12 0.0481 |1.15 X10-*) 77.1 62 eo eee 0.0043 2.25 X10-*) 105.4 46 | 54 | 0.852 | 1.92 0.189 |4.50 X10-* = 158.3" 30 | 7 | 0.420 | 1.98
EES
George Peirce _ II
different conditions in the two experiments, but mainly to the fact that the enzyme solutions used were entirely different. Other experiments, performed with purified enzyme solutions, gave con- stants nearly equal to the ones in Table II. In every series, moreover, the values of k are constant within the limits of error of the experiment, so that the data agree satisfactorily with equa- tion [3]. No other values for m, n and p in equations [1] and [2] are so consistent with the observations.
If the equations as given are true the ratio®
portional to the free sodium fluoride. If a large amount of enzyme is used we should expect so much of the sodium fluoride to be combined that the concentration of the free sodium fluoride
would be appreciably diminished. In this event, a given con- '
centration of sodium fluoride would have less inhibiting effect in ti strong enzyme solutions than in weak ones. On testing this view 3 inhibition was apparently less in very strong
- i the i
80. erence was so slight as to be within the limits of experimental error. Unfortunately no great quantity of uniform purified enzyme remained for experiments in dupli- cate, and as it was quite evident that the enzyme was far from pure, it. did not seem advisable to repeat the experiment until a much purer enzyme could be obtained. The experiment did, however, show that very little sodium fluoride was bound even in enzyme solutions of considerable strength (five times the con-
centration in Table II), so that the assumption made in that ~
experiment, that the free sodium fluoride was very nearly equal to the total sodium fluoride, is justified.
The following table gives tlhe data on which the preceding conclusion is based. Only the last column requires any explana- tion. This is obtained as follows: The top figures of the first
five columns are obtained by extrapolation, and thus a series of ©
figures is obtained in the fifth column giving an irregularly de- scending series. The total amount of sodium fluoride present is 0.030 mg. per liter, and, starting from this figure, the last column gives a regularly descending series almost directly proportional to the figures in the next to the last column.
6 Note inversion of this ratio. This is done for convenience of presen- tation.
ee
12 Esterase and Sodium Fluoride
TABLE IV.
20 per cent purified enzyme B. Total volume 280 cc.
0.714 X 10-5N. 0.030 mgm. per liter.
CONCENTRATION OF ENZYME CONC. OF ENZYME ABSOLUTE AMOUNTS PERCENTAGES CALCULATED* NaF. ene COMER ate cc-gol. Bin | Mem. dried sub | ging. |Naw.Ems.| | | tents Pmm ts) 280 cc. reaction mixture Amounts ap-| Amounts ap-| (52.6) | (47.4) | (0.90) | 0.030 (total proaching proaching amount zero zero present) 5 §.7 53. 47. 0.89 | 0.030 - 10 11.4 52. 48. 0.93 | 0.029 25 28 .6 55. 45. 0.82 | 0.027
* The last column gives a uniform series, although the figures in the next to the last column diminish regularly.
16 showed the sodium fluoride was free, but fai t.e., it did not show how
riment was successful in its primary purpose; 7.e., it | in mixtures of low enzyme concentration almost all,
with a given amount of e e. | hee ae
One of the most important Points Shout this reneuon in. thal it is reversible. Loevenhart and Peirce? mixed esterase and so- dium fluoride and dialyzed the mixture. After dialysis the solu- tion had regained its original activity.
The experiment was conclusive evidence for dissociation of the inactive compound, provided an inactive compound was formed under those conditions (¢.e., mixture of the enzyme with sodium fluoride). It is, however, possible, and indeed probable, that the presence of ethyl butyrate or aleohol or butyric acid or even two or three of these substances is necessary for the formation of the inactive compound.* The evidence for the exact nature of this inactive compound will be presented in a succeeding paper, but the question does not concern us here. The reversibility of its formation is, however, easily demonstrated.
For instance, in a 250 ec. mixture containing 153.4 ce. } ethyl butyrate, 10 ec. of enzyme and 1: 6,000,000 sodium fluoride the action proceeded as if only 27.6 per cent of the enzyme present
’ This Journal, ii, p. 406, 1907. * For comment on these points, see Conclusion 7 at the end of this paper.
George Peirce 13
were acting. Ata given time (fixed by a preliminary experiment) 5 ce. 75 butyric acid had been produced per 50 ce., so that 50 ce. of the mixture then contained 5 cc. % butyric acid, 5 cc. 7 alco- hol and 25.68 cc. 7 ethyl butyrate. Fifty ec. of this solution were now added to 200 cc. of a mixture containing the same amount of ethyl butyrate, alcohol and butyric acid, but free from enzyme and sodium fluoride. In so doing, the enzyme and sodium fluor- ide were diluted five times, leaving all other factors unchanged. Two possibilities were now open for the further course of the reaction. In the first place, it might have proceeded one-fifth as fast as it did before dilution (where only 27.6 per cent of the enzyme was acting) or it might have produced acid at the same _ rate as a solution originally made up with 2 ce. enzyme in 250 ce. containing sodium fluoride 1:30,000,000. A control soluti
made up in this way worked as if about 59 per cent of the enzyme were rie and alee to what was actually ob-
niga v out 41 per cent Fok : present in n4 inactive form. The difference was” great enough to be unmistakable, and gave good evidence ' for the fact that the reaction is reversible, whatever the nature _ of the inactive compound. The data in the following experiment were obtained in the usual way, with two exceptions. First: the 50 cc. of solution to be titrated were run into 25 ce. of neutralized 80 per cent alcohol. This stopped the action more effectively than strong sodium fluor- ide. Second: 25 cc. instead of 50 ec. were in several instances used for a titration on account of lack of material. This accounts to a certain degree for divergence of the controls, as the titration errors must be multiplied by two.
A partial discussion of the results in the following table has just been given and the full data will now be presented.
ae 2
14 Esterase and Sodium Fluoride
TABLE V a.
200 ec. Ethyl butyrate solution (50 ce. = 38.35 ec. 35 solution). 10 cc. Enzyme solution C. 25 cc. Sodium fluoride 1:600,000.
15 ec. Water. A (OBSERVED) A (AVERAGE) a (CALCULATED) a(cc.X-actd)| ¢ (min.) 2 a bt | 5t-118.7 2.01 13 .08 2.11 13.94 2:21 14.80 : 3.68 29 .70 3.74 30.29 3.80 30.88 (4.88) (43.7) 218.5 99.8 (5 .00) (45 .2) Fh, 5.32 49.12 5.41 50.48 252.4 138.7 5.49 50.83 ¢ 8.85 | 98 .43 8.94 100.01 500.0 381.3 9.03 | 100.58 (10.00) (114.7) 573.5 10.34 | 120.08 10.43 | 120,78 603.9 | 10.52 121 .67 5 Bo, Pal ie
* For explanation of columns 5 and 6, see description of Figure 2.
A second similar solution was made up (also in duplicate), and at the end of approximately 44 minutes, 50 ce. of it were added to the following solution:
134 cc. Ethyl butyrate solution 20 cc. § Butyric acid.
5 ce, ¥ Alcohol.
41 cc. Water.
The first titration was made within 45 seconds of mixing and the time taken as 0 at this point.
en
George Peirce “15
TABLE V Bs. OBSERVED AVERAGE x t ree t
4.88 | 0.00 | 4.88 | 0.00 4.87 0.00 5.39 | 14.62 | 5.44 | 14.85 5.49 | 15.08 6.42 | 41.37 6.52 | 43.59 6.61 | 45.80 7.36 | 74.75 | 7.50 | 75.65 7.64 | 76.55 ss 8.84 1118.72 | 8.87 | 119.90 8.90 | 119.68 | 10.42 | 163.80 | 10.28 | 164.63 10.14 | 165.45
Toone 9 VcAND V bp.
9 eee butyl fholition: *% 500 cc. Ethyl butyre 10 ec. Enzyme solution C. 5 cc. Enz - Occ. Sodium fluoride. 12.5 ec. Sodium fluoride 1: 600,000. 40 cc. Water 107.5 cc. Water. OBSERVED AVERAGE OBSERVED AVERAGE z t x t z t z t 4.79 | 12.25 4.91 12.76 2.50 42.28 2.46 41.41 5.03 | 13.27 2.41 41.53 a2. 7.55) Suoee 7.57 21 .87 3.93 73 62 3.87 73.31 | 7.58 | 22.37 3.80 | 73,00 : (4.88) | (99.8) 9.50 | 29.07 9.42 29 .43 5.21 | 106.87 5.13 | 106.73 - 9.34 | 29.78 5.04 | 106.58 (10.00) | (31.6) 10.92 | 35.20 10.69 34.70 6.28 | 135.47 6.17 | 135.61 10.45 | 34.21 ‘ 6.05 | 135.75 7.53 | 175.58 7.39 | 175.29 7.24 | 175.00 8.92 | 222.03 8.75 | 222.20 8.57 | 222.87 (10.00) |(267.7) 10.41 76 .28 10.24 | 276.33 10.06 | 276.38
16 Esterase and Sodium Fluoride
The results are also expressed graphically in the following dia- gram. The letters of the curves refer to the preceding table.
* represents a reaction going - one-fifth as fast as A. ‘A, C ey D start from the origin. On the curve D, 4.88 ce. acid were pro- duced in 99.8 minutes; but B, as observed, begins at « = 4.88 and t= 0. To make the points = 4.88 on the two curves coincide, 99.8 is added to the values of ¢ in plotting the curve B. In plotting + the values of ¢, for curve A, are multiplied by 5. For az = 4.88,t = 218.5. In order to make the point x = 4.88 on this curve coincide with the corresponding points on B and D, 118.7 is subtracted from the values of ¢.
In spite of the apparent complexity of this experiment, the point that it makes is very simple. It shows that a given mix- ture of enzyme, sodium fluoride, ethyl butyrate, alcohol and bu- tyric acid, if diluted five times, with the proper mixture of ethyl butyrate, alcohol and butyric acid, is more than one-fifth as active as it was before dilution. Since an enzyme solution that con- tained no fluoride would have been only one-fifth as active, the additional activity must have come from the partial dissociation of some sort of inactive compound present in the solution. This reversibility of the formation of the inactive compound absolutely excludes destruction of the enzyme by the sodium fluoride. In
George Peirce © 17
q addition, the fact that curves B and D so nearly coincide, shows _ that the point of equilibrium demanded by equation [3] is reached _ almost instantly from both directions.
CONCLUSIONS,
1. Sodium fluoride forms a compound with the esterase from
_ pig’s liver. This compound has little, if any, hydrolytic action on ethyl butyrate.
2. The formation of this compound is reversible.
_ 3. When the concentration of the sodium fluoride is varied from
- 0.00893 mgm. per liter to 0.268 mgm. per liter, the inhibition .
increases from 20.8 per cent to 88.06 per cent.
4, Although theoretically we should expect a given amount of _ sodium fluoride to have less inhibiting effect in mixtures contain-
ing a large amount of enzyme, than in weaker enzyme mixtures,
the eeeronce actually found was very slight. cates that in the weal , at least, very little of al sodium
s into e formation of the inactive com-
=
: 5. "The following gixtion, based on the supposition that one molecule of the inactive compound contains one molecule of en- " zyme and one molecule of sodium fluoride, agrees with the obser- vations:
Cone. free Enzyme X Conc. free NaF = k Cone. (NaF. Enz.)
6. The observations will not agree with an equation based on |. any other supposition as to the number of molecules of sodium
- fluoride or enzyme entering into the formation of the inactive ' compound. For this reason it is justifiable to conclude for the |. present that one molecule of the inactive compound contains only one molecule of enzyme and one molecule of sodium fluoride. | 7. It is possible that ethyl.butyrate, alcohol or butyric acid | are also constituents of the inactive compound. This does not affect the argument in any way: It is merely necessary to con- k sider that the “free enzyme’ ” of the above equation represents
® Journ. Amer. Chem. Soc., xxxi, p. 1528, 1910.
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. 1.
18 Esterase and Sodium Fluoride
“free enzyme” is present in the form of a compound with ethyl butyrate, so that only a part of the so-called “free enzyme” is actually free. Whether the sodium fluoride combines with some compound of the enzyme or with the enzyme actually free is immaterial, provided the experiments are so arranged that — the concentration of the substance with which the sodium fluoride combines is proportional to some quantity that we know. This is done by never comparing any two solutions unless the con- centrations of the ethyl butyrate, aleohol, butyric acid and hydro- gen ion are the same at the stage of the reaction where the two solutions are compared. Under such conditions the concentra- tions of all enzyme compounds, except the fluoride compound, are proportional'® to the “free enzyme”’ of the equation, so that the mathematical treatment is justified.
1° This statement must be modified if any enzyme compound that con- tains two molecules of enzyme is present in large amounts, Practically, there is no evidence that such compounds occur, so that for a preliminary — investigation such as the present, the possibility of their existence may — be neglected.
ONE LD SEY eee
lad
THE RATE OF ELIMINATION OF NITROGEN AS INFLUENCED BY DIET FACTORS.'
I. THE INFLUENCE OF THE TEXTURE OF THE DIET.
By LAFAYETTE B. MENDEL ann ROBERT C. LEWIS.
(From the Sheffield Laboratory of Physiological Chemistry, Yale University ,- New Haven, Connecticut.)
(Received for publication, August 4, 1913.) INTRODUCTION.
The changing view as to the extent of digestion before absorp- tion and the probability that proteins are split to amino-acids has
_ raised the question whether all amino-acids are utilized alike. Are not some more resistant in metabolism than others? Does not
deaminization take place with greater difficulty in some cases? If it does, one can easily conceive how different may be the behavior of the various proteins in nutrition. Bearing in mind the well- known fact that proteins vary widely in chemical composition, it is evident that the products of absorption after the ingestion of one protein may be much unlike those when another is fed. Thus, if the assumption that amino-acids are of variable resist- ance is correct, we may have an entirely different metabolic picture in the two cases.
A study of the rate of elimination of nitrogen? in’ the urine sug- gests itself as a means of ascertaining whether or not the amino- acids behave alike in metabolism. It is obvious that any variation in the ease of deaminization of the amino-acids may lead to a
! The experimental data embodied in the papers of this series are taken from the dissertation submitted by Robert C. Lewis for the degree of Doctor of Philosophy, Yale University, 1912.
2 For a review of the literature on the rate of elimination of nitrogen see: Graffenberger: Zeitschr. f. Biol., xxviii, p. 318, 1891; Hawk: Amer. Journ. of Physiol., x, p. 115, 1903; Stauber: Biochem. Zeitschr., xxv, p. 187, 1910; Wolf: zbid., xl, p. 193, 1912.
19
20 Rate of Nitrogen Elimination
change in the nitrogen-output curve after the ingestion of differ- ent amino-acids or of proteins of unlike composition. Other points, however, must be taken into consideration. Will a change in the rate of elimination of nitrogen necessarily be due to a differ- ence in the metabolic behavior of the amino-acids? Certainly several other factors may play a part in this connection. Varia- tions in the rate of the different processes of alimentation—gastric digestion, discharge of the food residues from the stomach and their passage along the digestive tract, pancreatic and intestinal — digestion, absorption—may have a decided influence on the rapidity with which nitrogen leaves the body after a protein meal. Furthermore, metabolic processes distinct from deaminization, such as the behavior of the non-nitrogenous foodstuffs in influenc- protein metabolism, are not without significance in this con- ection. All these factors must be considered as having a bearing | in a study of the rate of elimination of nitrogen.
It seerhs quite probable that the lack of concordance in the nitrogen-output curves found by pr ious inv tors may — si due to a variation in incidental factors of the diet, s as the form in which the protein was taken, the amount of car-— bohydrate and fat ingested along with the protein, the water — intake with the meal, and finally the proportion of indigestible material. Since the initiation of these investigations Benedickt — and Roth’ have suggested comparable explanations for the dis- © crepancies in the results of earlier workers.
That indigestible materials have an influence on alimentary processes is a familiar fact. Hedblom and Cannon‘ have observed — that coarse branny foods in the diet cause a more rapid discharge of the stomach contents. Recently Mendel and Fine’ have shown that indigestible substances added to the daily meal even in small quantities cause a poorer utilization of protein. It seems quite probable, then, that the rate of elimination of nitrogen in the urine — may be affected by the texture of the diet. .
* Benedickt and Roth: Zeittschr. f. klin. Med., Ixxiv, p. 74, 1911. *Hedblom and Cannon: Amer, Journ. Med. Sci., exxxviii, p. 1, 1909. * Mendel and Fine: this Journal, xi, p. 5, 1912. .
Lafayette B. Mendel and Robert C. Lewis 21
METHODS.
The method employed in the present series of investigations for studying the rate of elimination of nitrogen in the urine has been to collect the urine at definite intervals after the ingestion of pro- tein, to determine its content of nitrogen for the different periods, and to obtain from the results a curve of nitrogen output. Bitches were used as subjects of investigation, the urine being obtained by catheterization. The elimination of individuality was secured by the use of more than one animal for each type of experiment. In the present paper, however, it will be necessary to limit our- selves to the report of a single experiment illustrating each point. The dogs used are designated by a specific letter in the number of the experiment.
_ each morning in a single meal a definite ration—the ‘Standard _ Diet.” On experimental days the meal differed from this “Stand- ard Diet’’ either by having something added to it | or by having (or more) of its constituents replaced. The same amount of iz en was always given, however; and in the replacement of F non-nitrogenous constituents isodynamic quantities of some other foodstuff were substituted. Preceding an experiment there was ’ always one day with the “Standard Diet’’ and generally there were two or three. Three of these preliminary days were intro- duced at the beginning of each series so that the animal might have plenty of time for adjustment to the new régime. Thus _ day after day throughout the whole series the food contained the same amount of nitrogen and, except in a few cases, approximately the same calorie value. _ On an experimental day the animal was catheterized in the morning and fed fifteen minutes after the beginning of catheter- ization. To avoid possible secretory disturbances, the tempera- ture of the well mixed food was always the same—20°C.—at the time of ingestion. Usually the animal ate the entire meal greedily. At times, however, the food had to be forced. In all cases feeding was complete in ten minutes or less. Collections of urine were made three, six, nine, twelve, fifteen and twenty-four hours after the beginning of the experiment. In some experiments a control specimen of urine was collected for an hourly period before the
While a series of experiments was in progress the animal received
22 Rate of Nitrogen Elimination
commencement of the experiment; but in the majority of cases a collection for the twenty-fifth hour was made. Catheterization was planned so as to take exactly ten minutes, the expiration of that time being awaited where necessary, before the completion of the final washing. Nitrogen was determined by the Kjeldahl- Gunning method.
The “Standard Diet”’ was, of course, arbitrarily chosen. The only requirements in selecting such a ration were that it must furnish at least the minimal protein requisite and sufficient calo- ries for the needs of the body. ° It seemed desirable, however, to use more than the minimal protein requirement in order to have a liberal output of nitrogen in the urine. The ration adopted consisted of meat,® lard, sucrose, bone ash’ (5 grams), and water, with the addition in some cases of NaCl (2 grams). The water was calculated on a basis of the dry constituents, three times as much water being added as there was dry material. In other words the water was present in the whole ration in about the same pro- portion as it is found in meat. The sugar and lard were given in quantities approximately isodynamie to each other. The meal always contained 0.6 gram of nitrogen per kilo of body weight — and furnished about 70 calories per kilo. The exact calorific value of the diets is not known because the fat content of the meat was not determined. By employing a fixed “Standard Diet” which was easy to duplicate, the rates of elimination of nitrogen under the various experimental conditions with the same and different animals were readily comparable.
Inasmuch as the addition of indigestible materials to the ‘““Stand- ard Diet’? suggests itself as a means of studying the influence of the texture of the diet on the character of the nitrogen-output curve, mineral oil, vaseline, paraffin, filter paper, ground cork, agar-agar, bone ash, and sand were added in various experiments. There is little question that these materials pass through the intestine chemically unchanged. Bone ash may possibly be dis- solved to some extent in the hydrochloric acid of the gastric juice. With respect to the mineral oil Bradley and Gasser* have reported
* Preserved frozen, according to the method of Gies.
? For the use of bone ash see Steele and Gies, Amer. Journ. of Physiol., xx, p. 343, 1907.
* Bradley and Gasser: Proceedings of the American Society of Biological Chemists, December 1911, this Journal, xi, p. xx, 1912,
Lafayette B. Mendel and Robert C. Lewis 23
that an emulsified mixture of olive and petroleum oils fed by sound to a dog leads to absorption of both fat and hydrocarbon—a result not in accord with the later experience of Bloor.’ The indigesti- bility of the other materials used is well established.
An amount of water equivalent to three times the weight of the superimposed substance was also added with two-fold purpose in the cases of the water-absorbing materials: filter paper, cork, and agar-agar. In the first place, without this extra water the added material could not have been soaked up and well mixed with the food. Secondly, the amount of water carried out through the bowel when these substances were used was relatively very great; and, in order to insure against a large loss of water from the tissues, it was necessary to give an added amount of water with the meal.
CONTROL EXPERIMENTS WITH THE “STANDARD DIET.”
Before attempting to determine the relation of the different _ diet factors to the rate of nitrogen elimination in the urine, it was necessary to ascertain the nature of the nitrogen-output curve after the ingestion of the “Standard Diet” and to see whether a characteristic curve always followed. In the text each type of experiment is illustrated by a curve, plotted from the data obtained. Curve I, a typical graphic illustration,’ shows the agreement of the nitrogen-output curves of two experiments with the “Standard Diet.” The abscissae represent equal increments of time; the ordinates, grams of nitrogen. Thus at a glance the average hourly rate of elimination of nitrogen for a single period is shown by the value of the ordinate. It is readily seen that after the ingestion of the “Standard Diet” there is a rise in the nitrogen output dur- ing the first period, reaching a maximum in the second three hours, followed by a fall to the initial level early the next morning. In the present work it has always been possible with the same animal to get “standard’’ experiments which agree within reasonably close limits (Curve I). Furthermore “standard’’ curves of duplicate character have been obtained repeatedly with different animals.
® Bloor: This Journal, xv, p. 105, 1913.
10 All curves show the rate of nitrogen output in two experiments, a “standard’”’ experiment (broken line) being plotted for purposes of com- parison.
24 Rate of Nitrogen Elimination
Curve I. To illustrate the agreement of duplicate experiments after the ingestion of the ‘‘Standard Diet.’
N gm. O6 ; ta 1 BS. ; "Standard Diet" 1 ss aienihiees 225 gm. meat (N= 7.72 gm.) a4 ; 35" lard 1 is fs 70" sucrose roo : - 4 Sai ee, 5" done ash ' 325 " water pr 0.2 ag | tl ale —_——_—9 f . L On ee
A =: pas hrs.
----- Experiment D VII, ‘‘Standard Diet.’’ ———— Experiment D XIII,4‘‘Standard Diet.’’
Lafayette B. Mendel and Robert C. Lewis 25
EXPERIMENTS WITH INDIGESTIBLE MATERIALS. Mineral oil (Curve II).
When mineral oil was added to the diet the nitrogen output in the second period was notably less than in the corresponding period after the ingestion of the ‘Standard Diet” alone. Evidently mineral oil causes a slower rate of elimination of nitrogen.
Curve II. To illustrate the effect of an addition of mineral oil to the “Standard Diet’’ on the rate of elimination of nitrogen.
N g™- o "Standard Diet” ~~ 256 gm. meat (H =. | gm.) ‘7 “ae : ; ' “a Pek, 740". lard go * sucrose iin NaCl hy bone as ---4 soc " water i q — - #8 —_— & = oa See eure. . ide US 2h25 hrs.
----- Experiment G VIII, ‘‘Standard Diet.”’ —— Experiment G XII, ‘‘Standard Diet’? + 75 grams mineral oil.
1 A colorless, purified product sold under the trade name of ‘“‘Alboline.’’
26 Rate of Nitrogen Elimination
Vaseline (Curve ITT).
The effect of vaseline on the curve of nitrogen elimination is similar to, but more marked than that of mineral oil. The nitro- gen output in each of the first three periods is smaller than in the “standard” experiment; afterwards the two curves are almost identical. There is a delay in the excretion of nitrogen.
Curve III. To illustrate the effect of an addition of vaseline to the “Standard Diet’’ on the rate of elimination of nitrogen.
N gm. 0.6 2:0 | -> ; i 0.5 : : oe Standard Diet” ‘ 225 gm. meat (N= 7.27 gm.) Rtentd i. 85" lara ; 1
70" sucrose | bone ash
325 " water
Ti 2 a aa: has hs.
w---- Experiment D IV, ‘‘Standard Diet.’’ -——— Experiment D VI, ‘Standard Diet’’ + 75 grams vaseline.
? Yellow petroleum jelly (M.P.=38°C.).
——
Lafayette B. Mendel and Robert C. Lewis 27
Paraffin (Curve IV).
The experiment with paraffin shows a much more decided flat- tening of the nitrogen-output curve, 7.e., a preliminary’ delay in excretion of nitrogen, than was the case with either of the softer | petroleum products. The rate of elimination of nitrogen is not i only lower in the earlier periods than in the “standard” experiment, but also higher during the latter part of the day.
| Curve IV. To illustrate the effect of an addition of paraffin to the | “Standard Diet’’ on the rate of elimination of nitrogen.
N gm. | 07 aaah "Standerd Diet" ' 2 ! 500 gm. meat (N = 10.27 gm.) 0.6 | Lae 50" lara ‘ BS i er : 100" — sucrose 705) - I 2" Wecl H 5" bone ash 04 450" water | i ! eae leks en 03 }---—- } t ! i] u i a ay | 0.2 |i ena t a a 0.1 ; 5 Wie Gag. (Raais 425 hrs.
----- Experiment F V, ‘‘Standard Diet.’’ —— Experiment F IX, “Standard Diet’’ + 75 grams of paraffin.“
18 Fine shavings, obtained by scraping a cake of paraffin (M.P.=51°C.) with a knife. 14 Large quantity of paraffin feces during the night (15-24 hour) period.
28 Rate of Nitrogen Elimination
Filter Paper® (Curve V).
The rate of elimination of nitrogen during the earlier periods after the ‘ingestion of the “Standard Diet’’ plus filter paper is lower than in the “standard” experiment; during the later hours it is higher than normal. Thus, as was the case with paraffin, there results a very decided flattening of the nitrogen-output curve.
Curve V. To illustrate the effect of an addition of filter paper to the ‘Standard Diet’’ on the rate of elimination of nitrogen.
N g™. a ='=5 ' ‘ . : "Standerd Diet" 1 ' \ 0.6 | { ous S00 gm. meat (l= 10.27 gm.) ' 50" lara i . 0.5 | 1 gag 100 he sucrose .. a 2" Yeci 0.4 Bi gad 5" done ash L 450 * weter ° | a -——_—— qi = am o2h os 1 i 1 | ieee et Ol ; u o 5 — = a ie 2425 hrs. ----- Experiment F V, ‘‘Standard Diet.’’
Experiment F VIII, “Standard Diet’’ + 75 grams filter paper'® and 225 grams water.
* Cut up in small pieces. * Large quantities of paper feces during the 4th and 5th three-hour periods and during the night (nine-hour) period.
Lafayette B. Mendel and Robert C. Lewis 29
Cork” (Curve VI).
With cork there is likewise a much slower elimination of nitrogen than with the “Standard Diet’’ alone. In this case the total nitrogen output for the entire day is lower than in the “‘standard”’ experiment. This, however, is not the only cause of the sub- normal elimination of nitrogen in the early periods of the day; _ for the character of the nitrogen-output curve is radically different _ from the “standard’’—lower during the early periods and higher during the later periods of the day.
Curve VI. To illustrate the effect of an addition of cork to the “‘Stan- dard Diet’’ on the rate of elimination of nitrogen.
oN gn
o.1
"Standerd Diet™ a
® os 250 gm- meat (N= 8.68 gm.) 40 " lard
a _J
rt
B™ wacl
wae ---4
pea om ae
' 1 ' i} ; >, * bone ash 1
<4 350 " water
eR 5 ont i l 1 Ss a6 eo) Ne als 2has hes. ----- Experiment G VIII, “Standard Diet.”’
Experiment G XV, “Standard Diet”? + 50 grams cork!8 and 150 grams water.
1 Finely ground in a coffee mill. 8 Large quantities of cork feces during the 4th and 5th three-hour periods and during the night (nine-hour) period.
30 Rate of Nitrogen Elimination
Agar-agar® (Curve VII).
Of all the indigestible materials used the agar-agar caused the most pronounced delay in the nitrogen output. In the experiment
here reported there was a rise in the second period over the value —
of the first three hours and then very little change for twelve hours; in other experiments there was a similar though slightly less marked effect.
Curve VII. To illustrate the effect of an addition of agar-agar to the “Standard Diet’’ on the rate of elimination of nitrogen.
gm "Standerd Diet" of 200 gm. meat (N= 5.58 gm.) rae 30" lara 0.3) ! 50 " © sucrose ee L 5" bone esh 0.2. ee 250" water 1 fe 0.! Pisin on call a eee Ss .s & te 2425 hrs.
w--- Experiment B I, ‘‘Standard Diet.”’ Experiment B III, ‘‘Standard Diet”’ + 75 grams agar-agar®® and 225 grams water.
Very finely chopped. 2 First agar-agar feces during first three-hour period. No note kept of subsequent defecations; feces in almost every period, however.
Lafayette B. Mendel and Robert C. Lewis 31
Bone Ash (Curve VIII).
With the addition of bone ash to the ‘Standard Diet” there is a flattening of the curve, but by no means to such an extent as with any of the previously mentioned indigestible materials except the softer petroleum products. There is a delayed excretion of nitrogen in the first two periods, followed by a slight compensatory rise during the next six hours, the curve afterward running parallel to that of the ‘‘standard’”’ experiment.
Curve VIII. To illustrate the effect of an addition of bone ash to the “Standard Diet’’ on the rate of elimination of nitrogen.
gm. ro-c-5 : "Standard Diet" { é oe 225 em. meat (X= 7.79 on.) oan $5" lard of ' 70" sucrose 1 - 5" bone ash ee | + O35 325" water ' o2h ee ; 22 @& Out } ! J Bs f 9 He as 2425 hrs.
----- Experiment D VII, “Standard Diet.”’ —— Experiment D IX, ‘Standard Diet’’ + 75 grams bone ash.
32 Rate of Nitrogen Elimination
Sand (Curve IX).
The effect on the nitrogen-output curve of an addition of very fine sand to the ‘‘Standard Diet” is entirely different from anything so far reported. The rate of elimination of nitrogen during the first two periods is notably higher than in the corresponding periods of the “standard” experiment; afterward the two curves run © parallel. . ee
Curve IX. To illustrate the effect of an addition of sand to the “Stan- dard Diet” on the rate of elimination of nitrogen.
| : N i 0.7) | 06 Pe “— “Standard Die niet a eo Os ae , ~ 225 em- meat (N= 7.66 en.) id mt ; 35" lera 0.4 Q St 70 "sucrose ! 5" tone ash * i =. 325." water 0.3) | | Stabe. " ! ' 02h | i] pi 0.1} | 1 ‘ ! : ! 5 6 Pp Kk aS hrs «s--- Experiment D XIII, “Standard Diet.”
Experiment D XVI, “Standard Diet’’ +°75 grams sand.
Lafayette B. Mendel and Robert C. Lewis 33
DISCUSSION.
The experiments with a variety of indigestible materials have shown a slower rate of elimination of nitrogen after the addition of these substances to the “Standard Diet” except in the case of sand. Obviously there has been some delay in the processes of alimentation; for, excepting differences in the amount of water absorbed,” the purely metabolic conditions are the same when an indigestible substance is included in the daily meal as when the “Standard Diet” alone is fed. The prime factor in this delay must have been a slower rate of absorption, whether induced by a retardation of the discharge of the gastric contents, a delay in digestion, an adsorption of digestive products by the indigestible material, or a loss of absorbable material by an early evacuation of the bowel. Let us consider the bearing of each of these con- tributory factors on the present work.
In all probability no delay in the discharge from the stomach occurred when indigestible materials were added to the diet. With mineral oil, vaseline, paraffin, and bone ash the passage of
_ . food onward must have been as rapid as under normal conditions;
for during the first periods with these materials there was no - marked decrease in the nitrogen output below that of the ‘“stand- _ ard” experiment. The early appearance of the added indigestible material in the feces following the ingestion of filter paper, cork, and agar-agar” suggests an acceleration rather than a retardation of the normal gastric discharge. This is in harmony with the report of Hedblom and Cannon” that branny foods cause a more rapid emptying of the stomach.
A delayed absorption on account of a sub-normal rate of diges- tion in the experiments with indigestible materials is quite possible.
*t With several of the indigestible substances—cork, filter paper, and agar-agar—a large amount of water was excreted through the bowel. As a result the volume of urine, and hence the water absorbed, was very small. That the lack of water was not an important factor in causing a retardation of the rate of elimination of nitrogen has been shown by an experiment with agar-agar in which a very large amount of water was given. In this case there was a normal flow of urine, but the same effect was obtained as when the secretion of urine was small.
*2 The influence of these substances on the emptying of the bowel is indicated in the reports of the experiments.
*8 Hedblom and Cannon: Amer. Journ. Med. Sci., exxxviii, p. 1, 1909.
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. l.
34 Rate of Nitrogen Elimination
It was pointed out in the preceding paragraph that some of the indigestible materials caused a more rapid discharge from the stomach. This might very well lead to a slower rate of digestion because the preliminary gastric proteolysis was inhibited before it had advanced very far, and the intestine thus received much more complex residues than under normal conditions. This pos- sibility of an unfavorable result from sparing the stomach at the expense of the intestine has been previously mentioned by Cohn- heim™ and Cannon.” It is interesting to note that it was in the — cases where there was an early emptying of the stomach that the greatest delay in elimination of nitrogen, and hence in absorption, 7 occurred. Furthermore, digestion may have been delayed be- — cause the food residues, mixed as they must have been with the — indigestible materials, are rendered more or less inaccessible to the action of the digestive enzymes.
Another possible explanation for the delay in absorption when indigestible substances are added to the diet is that the products — of digestion are adsorbed by the added indigestible material. It .— is quite conceivable that Agar, for example, might adsorb the. — soluble intestinal contents. An examination of the data shows — that the indigestible materials exerted a retarding influence c the rate of absorption, as measured by the rate of elimination nitrogen. This retardation rogressively greater in the follo ing order—mineral oil, vaseline, bone ash, paraffin, filter paper, a cork, agar-agar—corresponding with the comparative adsorptive — power of the same substances. This parallelism between delayed absorption and power of adsorption seems to be more than a coinci- dence. Adsorption of intestinal contents by the indigeStible adju- vants might cause a slower rate of absorption in two ways: in the first place, the partially digested protein residues might im part be rendered less accessible to enzyme action; secondly, a smaller — proportion of the completely digested protein residues would come in contact with the intestinal wall, and for this reason absorption would be hindered.
It is unlikely that the delayed absorption in the experiments with indigestible materials is attributable to an bod emptying
~ Pe ee) ee ee eee Bo
*Cohnheim: Quoted by Cannon. * Cannon: The Mechanical Factors of Digestion, International Medical — Monographs I (Longmans, Green and Company), 1911, p. 123.
Lafayette B. Mendel and Robert C. Lewis = 35
of the bowel and a consequent loss of available nitrogen. The more than compensatory rise in the urinary nitrogen output above the normal which always occurs in the later periods with the substances causing early evacuation makes such an explanation questionable.
There remains a consideration of the results obtained with sand. The data presented emphasize an increased elimination of nitrogen in the first periods after the meal when sand is added to the “Stand- ard Diet.’’ Can this rise be caused by a more rapid discharge of the gastric contents? No evidence has been found in the litera- ture to warrant such a conclusion. On the contrary the results of Hedblom and Cannon,” showing that hard irregular pieces of dried starch paste in the diet caused a slower discharge of the stomach, appear to speak against such an explanation. It is ' unlikely that sand has mechanically stimulated an increased _ secretion, the reabsorption”’ of which has raised the nitrogen out- put of these first two periods; for Pawlow®* has demonstrated conclusively that the blowing of sand with force against the walls of the inactive stomach does not stimulate gastric secretion. The - secretion is f utherae by the follow-
q ” Dann the middle of the fourth day of a fast the urine was col- lected for a three-hour control period. ‘ A quantity of sand (and a little water) was then given; and the urine was subsequently collected at three-hour intervals for nine hours. There was no increase in the nitrogen output of these later periods over that of the control period.”
°® Hedblom and Cannon: Amer. Journ. Med. Set., exxxviii, p. 1, 1909.
*7 Mosenthal (Journ. Exp. Med., xiii, p. 319, 1911) has attributed to the intestinal secretion a considerable source of absorbable nitrogen. From experiments on dogs with isolated loops of intestine he estimated that the nitrogen content of the succus entericus secreted in twenty-four hours was equivalent to about 35 per cent of the nitrogen intake. Inasmuch as the | feces contained nitrogen equivalent to only 10 per cent of the intake, the _ major part of the intestinal secretion must have been reabsorbed.
** Pawlow: The Work of the Digestive Glands, translated by W. H. Thomp- son (Charles Griffin and Company), 1902, pp. 86-90.
Ina previous experiment we had ascertained that the eget
36 Rate of Nitrogen Elimination
SUMMARY.
The typical curve of nitrogen elimination on a selected mixed diet shows a rise in the first period, reaching a maximum in the second three hours, followed by a fall to the initial level early the next day.
With a definite diet it has always been possible to duplicate experiments on the same animal. Different animals on the same type of diet have given parallel curves. -
A delay in the elimination of nitrogen is caused by the addition
to the diet of such indigestible materials as mineral oil, vaseline, —
bone ash, paraffin, filter paper, cork, and agar-agar—substances which act in a purely mechanical as contrasted with a chemical
manner. Invariably there is a subnormal rate of nitrogen output — in the first periods following ingestion of the meal; with paraffin, — filter paper, cork, and agar-agar this is followed by a higher rate — in the later periods. The effect of the indigestible materials is —
progressively greater in the order in which they are given above. A delayed absorption of the nitrogen intake is presumedly
responsible for the slower rate of elimination of nitrogen. As
possible causes of this retardation of absorption the following have ! been suggested: (1) a slower rate of digestion caused by an early
emptying of the stomach and a consequent early exclusion of 4
gastric proteolysis, with the possibility of a more prolonged in- testinal digestion; (2) a slower rate of digestion caused by an
adsorption of partially digested protein residues by the added —
indigestible material, making them less readily accessible to the action of the digestive enzymes; (8) an adsorption ‘of the final digestive products by the indigestible substance whereby their absorption from the intestine is hindered.
Sand gives an exception to the results obtained with the other
indigestible materials studied, as it causes an elimination of nitro- — gen above the normal in the first six hours. This rise is presumably — not caused by an increased secretion and subsequent reabsorption — of digestive juices, for the ingestion of sand during starvation has —
no effect on the nitrogen-output curve.
o> jae el
.
THE RATE OF ELIMINATION OF NITROGEN AS INFLUENCED BY DIET FACTORS.
II. THE INFLUENCE OF CARBOHYDRATES AND FATS IN THE DIET.
By LAFAYETTE B. MENDEL ann ROBERT CG. LEWIS.
(From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.)
(Received for publication, August 4, 1913.)
Although the general effect of carbohydrates in the diet on the rate of elimination of nitrogen in the urine seems to be well estab- lished, no study of the comparative behavior of different car- _ bohydrates has been attempted. In the past the method of in-
E vestigation employed has been to superimpose the carbohydrate to be studied on a “standard” diet, to determine the rate of elimination of nitrogen after this augmented meal, and to ascer-
tain the effect of the carbohydrate on the nitrogen-output curve
by contrast with the rate when the “standard” diet alone was fed. _ The objection to this method is that on the day when carbohy- | drate is given the diet has a greater calorific value than the ‘‘stand- ard” diet, so that the conditions on the two experimental days are not comparable from the standpoint of energy intake. In the present investigation' an isodynamic quantity of carbohydrate
) was substituted for the non-nitrogenous constituents of the
1 “Standard Diet,” and thus the calorie value of the food of all '. days remained the same. The following carbohydrates were _ chosen for study: the polysaccharides, starch and soluble starch;
"the disaccharide, sucrose; and the monosaccharide, dextrose.
1 The methods employed were those outlined in our first paper (cf. this Journal, xvi, p. 19, 1913).
37
38 Rate of Nitrogen Elimination
EXPERIMENTS WITH CARBOHYDRATES.
Starch? (Curve I).
When starch was substituted for the non-nitrogenous constit- uents of the “Standard Diet” there was a distinct delay in the elimination of nitrogen, the amount of nitrogen excreted being smaller than in the ‘“‘standard’”’ experiment in the earlier periods of the day and larger in the later periods. .
Curve I. To illustrate the effect on the rate of nitrogen elimination
ee. oe ee
of substituting s‘arch for the non-nitrogenous constituents of the “‘Standard
Diet.”’ N gm "Standard Diet” 0.4 ; | aa ea 200 gm. neat (N= 5.58 gm.) i | 30 " lerd Oo. 3 Beet, —— 50" sucrose ic f 5° bone ash a2 4 a =f shee 250 " water Ht 1 0.1 \ ! 1 5 6 lg 2 5 2|4 hrs,
---- Experiment A IX, “Standard Diet.’’ Experiment A XIV, lard and sucrose of ‘Standard Diet”’ replaced by starch (118 grams).
—o — — _ — _ ee
* Arrowroot starch was mixed with water and heated in an autoclave for
fifteen minutes in order to rupture the starch grains.
le ee a
Lafayette B. Mendel and Robert C. Lewis 39
Soluble starch (Curve II).
With the use of soluble starch in place of starch the retarding effect on the nitrogen excretion was even more marked.
{ Curve II. To illustrate the effect on the rate of nitrogen elimination of substituting soluble starch for the non-nitrogenous constituents of the “Standard Diet.”
N gm
"Standard Diet”
225 em. meat (N= , a 35 " lard a a
a
70 " guerose
rf a eo" Bete LL ——+ 5" bone ash 325." water a 1 ! ! Reem y Rie. ee Poe ee ee ae of se Be ap Rey is 2has hrs.
---- Experiment D XXIV, ‘‘Standard Diet.’ _ —— Experiment D XXVII, lard and sucrose of ‘Standard Diet’’ _ replaced by soluble starch (150 grams).
40 Rate of Nitrogen Elimination
Sucrose (Gurve IED:
When sucrose was substituted for the lard of the “Standard Diet” the maximum output of nitrogen did not occur until the third three-hour period, as contrasted with the second period in the ‘‘standard”’ experiment and in experiments with the carbohy- drates above reported. The nitrogen output was relatively much smaller in the earlier periods of the day and much larger in the later periods than was the case with either of the polysaccharides. In other words the flattening effect of sucrose on the nitrogen- output curve was much more pronounced than that of starch or of soluble starch.
Curve II’. To illustrate the effect on the rate of nitrogen elimination of substituting sucrose for the lard of the “‘Standard Diet.”’
N g™: a OL ' } i "Standard Diet" 225 gm meat (N= 7.61 gm.) ! 0.6 35“ lard : 70 " gsucrose os : 2" Wacl ers — 5" bone ash of $25 " water \ f 0.3) . 4 i t=, o.2! - =e : io ag oe Cf a = oi} Sau we cite “als daas hrs
~--+=- Experiment D XXIV, “Standard Diet.’’ Experiment D XXVI, lard of ‘Standard Diet” replaced by suc- rose (80 grams),
Lafayette B. Mendel and Robert C. Lewis = 41
Dextrose (Curve IV).
With dextrose replacing the non-nitrogenous constituents of the “Standard Diet”’ the preliminary delay in nitrogen excretion was much greater than with any of the other carbohydrates studied, the nitrogen-output curve having a much more flattened aspect.
Curve IV. To illustrate the effect on the rate of nitrogen elimination of substituting dextrose for the non-nitrogenous constituents of the “‘Stan- dard Diet.”
x "Standard Diet” 0.5 | reocf 170 gm. meat (DN = 6.41 gm.) : 25" lara 0.4 : 60 " sucrose } | 2" acl 0.3| — ae) 5" bone ash ie mal
a 250 " water 02 o ! t
— © 2. ees 2425 hrs. ---- Experiment H I, “Standard Diet.”
Experiment H ITI, lard and sucrose of ‘Standard Diet”’ replaced by dextrose (117 grams).
Sucrose; Lard being present in the diet.
In the experiments above reported the diets were fat-free and in this respect not comparable with the “Standard Diet.’’ What effect would an increased amount of sucrose, for example, have on the rate of nitrogen elimination if lard were also present in the diet? In a further experiment the meal contained an amount of sucrose isodynamic to the non-nitrogenous constituents of the
42 Rate of Nitrogen Elimination
“Standard Diet’’ and enough fat (94 grams of lard) to make the ratio of sugar to fat approximately the same as in the “Standard Diet.””. With such a procedure an increased number of calories was given, but to accomplish the desired end this was necessary. There was the same preliminary delay in the nitrogen excretion in this case with sucrose as when no fat was present in the diet.
DISCUSSION.
The substitution of the different carbohydrates for the non- — nitrogenous constituents of the “Standard Diet’’ resulted in a slower rate of elimination of nitrogen, a flattening of the nitrogen- output curve. The carbohydrates studied were progressively — more effective in the following order: starch, soluble starch, sucrose, and dextrose.
With the method employed in the present study with carbohy- drates two important changes have been made in the diet: (a) fat — has been removed; (b) an added amount of carbohydrate has been given. Which of these two changes is of paramount importance in causing the results above reported? An experiment in which fat as well as sucrose was added to the “Standard Diet” shows that. the removal of the fat was not the causal factor; for here, as in the cases where sucrose replaced lard, the curve of nitrogen elimination — is considerably flattened despite the presence of an abundance of — fat. The added carbohydrate must have been directly respon- — sible, then, for the slower rate of nitrogen elimination. .
The results of the present investigation with carbohydrates are completely in harmony with those of previous investigators. Vogt — (1906) found that the addition of rice or rice flour to a meat diet — caused a slower rate of elimination of nitrogen than meat alone. Levene and Kober (1909) reported that with the addition of — starch to a “standard’’ diet containing plasmon, cracker meal, : and lard the course of nitrogen elimination did “not differ mate- 7 rially from that of the standard diet.’’ A careful examination of — the data presented by these authors discloses, however, a slight — flattening of the nitrogen-output curve when starch was added to
j
Eee
the diet. According to Van Slyke and White (1911) starch super- — imposed on a diet of fish, cracker meal, and lard caused a delayed — elimination of nitrogen of about the same magnitude as in the — starch experiments of the present work. Falta and Gigon (1908)
Lafayette B. Mendel and Robert C. Lewis 43
found a delayed excretion of nitrogen after the addition of either wheat flour or levulose to a meat diet, the effect being more marked in the case of the latter carbohydrate. This result with levulose agrees with that of Falta, Grote, and Staehelin (1907). Pari (1908) reported that with the addition of sucrose to a meat diet there was a retardation of the nitrogen excretion. Interesting in this _ connection are the experiments of Boettcher and Vogt (1909) in _ which subcutaneous injections of dextrose (5-10 grams) caused a flattening of the nitrogen-output curve. All of these investiga- tors worked with dogs.* Lusk (1912) fed dextrose alone to dogs _ 24 hours after the last meal and found a nitrogen output ‘ower than the fasting level during the hour following the dextrose in- take. Subsequently there was a compensatory rise in the nitrogen elimination.
Concerning the manner in which carbohydrate may be responsi-~ ble for a delay in nitrogen excretion several possibilities must be considered, viz: a subnormal rate of discharge of the stomach contents, a retardation of digestion, a delayed absorption, altered - metabolic processes. Van Slyke and White (1911) have given no _ experimental proof for their conclusions that the retardation of nitrogen elimination when starch is added to the diet is caused by a delay in digestion and absorption. The expefiments of Boett- " cher and Vogt (1909), showing a delay in absorption after sub- cutaneous or intravenous dextrose injections in five out of seven eases, are hardly comparable with the experiments of the present _ series where the carbohydrate was given per os. In fact, no con- _ clusive evidence has been found in the literature to the effect that - a subnormal rate of any of the alimentary processes is caused by an addition of carbohydrate to the diet. On the contrary, the report of Cannon‘ that a mixture of carbohydrate and protein foods leaves the stomach more rapidly than protein alone, whereas f fat has a retarding action on the emptying of the stomach, makes _ it probable that the addition of carbohydrate to, and the removal of fat from the diet in the present experiments is, if anything, | followed by a more rapid discharge of the gastric contents than in the “standard” experiment.
3 Wolf (1912) studied the rate of elimination of nitrogen after the inges- ' tion of starch by a fasting man. The results have no bearing on the ex- periments here reported.
4Cannon: Amer. Journ. of Physiol., xii, p. 387, 1904.
44 Rate of Nitrogen Elimination
There is some evidence in the literature that variations in meta- bolic processes are responsible for the slower rate of nitrogen elimination under the influence of carbohydrates. Falta and Gigon (1908) and Par. (1908) have attributed: this delay to the protein-sparing action of carbohydrates; for after a fast, when the
glycogen depots are almost depleted, the carbohydrates no longer —
exert a retarding action on the nitrogen-output curve. The reason for this, according to these authors, is that the carbohy-
drates now go to make up the depleted glycogen supply in preference — to being immediately burned. Boettcher and Vogt (1909) think — that a disturbance of intermediary metabolism is in part responsi- — ble for the delay in nitrogen excretion obtained after subcutaneous — dextrose injections, although they offer no experimental proof for — their contention. The consensus of opinion, then, seems to favor — a disturbance of metabolic processes, rather than a delay in ali- mentation, as the causal factor in the retardation of nitrogen ex- —
cretion when carbohydrate is present in the diet.
Although the experimental data obtained in this study do not —
warrant the adoption of a final theory as to how the carbohy-
drates act to retard nitrogen excretion, the writer is inclined to the — belief that the protein-sparing action of carbohydrate causes this — delay. When carbohydrate is present in the diet, it is digested, —
absorbed, and burned; while the protein residues, which are simul-
taneously absorbed, are temporarily spared to some extent and — are only completely metabolized when carbohydrate is no longer
available, a preliminary delay in nitrogen excretion thus occurring.
If such a theory holds, the physiological, six-carbon sugar dex- —
trose should be more efficient than the polysaccharide starch in
causing a retardation of nitrogen excretion; for the more nearly —
the carbohydrate is prepared for absorption when ingested, the
sooner should its sparing action come to expression, and so the — greater should be the delay in nitrogen elimination. As a matter — of fact the carbohydrates studied did show a progressively greater
retarding effect in the order: starch, soluble starch, sucrose, dex- trose. Thus the theory accounts for all the results obtained.
In conclusion reference should be made to the effect of indigesti- i ble carbohydrates in the diet on the rate of elimination of nitrogen. — In the previous paper® it was shown that cellulose—filter paper —
* Mendel and Lewis: This Journal, xvi, p. 19, 1918.
ee ee ee
Lafayette B. Mendel and Robert C. Lewis 45
and cork—and agar-agar caused a delay in nitrogen excretion. It is obvious, however, that the explanations of the similar effect on the rate of nitrogen elimination after the ingestion of digestible and indigestible carbohydrates, respectively, are radically different.
SUMMARY OF RESULTS WITH CARBOHYDRATES.
Carbohydrates in the diet cause a slower rate of elimination of nitrogen after a protein meal, the various carbohydrates studied having a progressively greater effect in the following order: starch, soluble starch, sucrose, dextrose.
The experimental data do not warrant the adoption of more than a tentative theory as to the explanation of the retardation of nitro- gen excretion when carbohydrates are present in the diet. It seems quite probable, however, that the protein-sparing action of carbohydrate is responsible for this delay. At any rate all the results obtained in the experiments with carbohydrates may be explained by such a theory.
Tue RELATION oF Fats IN THE DIET TO THE RATE OF ELIMINATION OF NITROGEN.
| Nocomparative study of fats of different texture—soft or hard— has been attempted in the few previous investigations of the influ- ence of fat in the diet on the rate of nitrogen elimination. Most of the work in the past has been done by superimposing the fat on a “standard” diet, and determining its effect on the nitrogen-out- put curve. Inasmuch as this procedure is open to the objection that with the addition of fat to the diet an increased number of calories is given, a method similar to that employed in the study of carbohydrates® was adopted for fats, the non-nitrogenous con- ‘stituents of the “Standard Diet” being replaced by the fat to be
_ studied. The following fats of widely different textures were used:
cotton-seed oil, lard, and “Oleo-stearin’’? (M.P.=53°C.).
® See the first part of this paper. 7 Armour and Company kindly furnished this product.
46 Rate of Nitrogen Elimination
EXPERIMENTS WITH FATS.
Cotton-seed oil (Curve V).
The substitution of cotton-seed oil for the non-nitrogenous constituents of the “Standard Diet” had very little effect on the course of the nitrogen-output curve, the only variation from the “standard” occurring in the second three-hour period where the nitrogen output was decreased.
Curve V. To illustrate the effect on the rate of nitrogen elimination of substituting cotton-seed oil for the non-nitrogenous constituents of the ‘Standard Diet.’’
N gm. 0.6 a "Standard Diet” i 250 gm meat (N= 6.68 gm ) 1 40" lard ibe. 0.5 fl on on all /. l 80 " sucrose ‘ae ! ‘ 0.4) 4 2 NaCl = 5 " bone ash UJ ” 0.3 a 350 water t U U 0.2] <a U 1 ot} ! 3 6 9 wt Is 4425 hrs.
-~--- Experiment G VIII, ‘Standard Diet.”’
by cotton-seed oil (75 grams).
ee
Experiment G IX, lard and sucrose of ‘Standard Diet’’ replaced |
Lafayette B. Mendel and Robert €. Lewis 47
Lard (Curve VI).
_ When lard was substituted for the sucrose of the ‘Standard Diet,” the nitrogen excretion during the first two three-hour periods was considerably larger than in the “standard” experiment; afterwards the nitrogen-output curve was identical with that i ' after the ingestion of the “Standard Diet.”
| Curve VI. To illustrate the effect on the rate of nitrogen elimination _ of substituting /ard for the sucrose of the ‘Standard Diet.”
N g™.
"Standard Diet"
240 gm. meat (N= 8.95 gm.)
re —
40" lard
2° iad sucrose
Bee Bad} ” bone ash
350 * water
ee ee ae ee i]
= 6 Ve B2 SS 425) hrs.
---- Experiment G IV, ‘Standard Diet.” Experiment G VI, sucrose of ‘Standard Diet’’ replaced by lard (35 grams). .
48 Rate of Nitrogen Elimination
*Oleo-stearin”’ (Substitution) (Curve VII).
The result of substituting “‘Oleo-stearin” for the non-nitrogenous parts of the “Standard Diet’ was similar to that with lard, a nitro-— gen output above the normal occurring in the first three three-hour
periods.
Curve VII. To illustrate the effect on the rate of nitrogen elimination of substituting ‘‘Oleo-stearin’’ for the non-nitrogenous constituents of the
“Standard Diet.”’
ee a a
N gm. O71 "Standard Diet" 0.6 — 240 gm. meat (!1= 8.95 gu.) 40" ilerd %» 0.5) . 80" sucrose j ae = NaCl OA 5" bone ash as ' 350 " water 0.3}! —_ ep 4 ' 0.2} --29 rr 0.1 ‘ 3 6 we oie cas 2425 hrs, ---- Experiment GIV, “Standard Diet.’’
~——— Experiment G III, lard and sucrose of ‘Standard Diet’’ replaced — |
by “‘Oleo-stearin’’ (75 grams),
Lafayette B. Mendel and Robert C. Lewis 49
“Oleo-stearin” (Addition) (Curve VIII).
In the above experiments with fats the sugar was completely removed from the diet. When fat was superimposed on the “‘Stand- ard Diet,” the sucrose thus being retained, a nitrogen-output curve _ of the same character as that occurring after the ingestion of the “Standard Diet” followed.
: Curve VIII. To illustrate the effect of an addition of “Oleo-stearin”’ _ to the “Standard Diet’’ on the rate of nitrogen elimination.
oN _ gm. — O71) sa "Standard Diet" { 300 gm. meat (N= 10.27 gm.) 06 f — er 60 ” lard H 100 " sucrose 0.5 : Nall 2" Waci 4 1 5" done ash : 1 04 | : 450 ” water == : j 0.2. f i RR ai oe A | a ashe On u
ae e ae oS 2425 hrs.
---- Experiment F V, “Standard Diet.” —— Experiment F VI, ‘Standard Diet”? + 75 grams “Oleo-stearin.”
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. 1.
50 Rate of Nitrogen Elimination
EXPERIMENT WITHOUT FAT OR CARBOHYDRATE IN THE DIET.
(Curve IX.)
In the discussion which’ follows reference will be made to an experiment where meat alone was given, the “Standard Diet” minus its content of sucrose and lard being fed. In the first two periods the nitrogen output under these conditions was larger than when the “Standard Diet” was fed intact; in the later periods, practically identical with the ‘‘standard.”
Curve IX. Comparison of the rate of nitrogen elimination after the ingestion of a mixed diet with that when meat alone is fed.
N gm. om - 0.6 | ae i "Standard Diet" i 225 gm. meat (N= 7.27 gn.) ea. oO t =i oa 35" lard 7 t , 70" sucrose 0.4 i -- aq ' | 5" bone ash a icr---4 " 03! 325 water \ ) ie o2} 7 t ‘ Loos sae OL CO ee ee 1 , 1 4 i ’ l Bae ae FR als 2h25 hrs.
-~--- Experiment DIV, “Standard Diet.”
: | |
—— Experiment D V, “Standard Diet’ minus its lard and sucrose, —
ee
Lafayette B. Mendel and Robert C. Lewis 51
DISCUSSION.
The substitution of the different fats for the non-nitrogenous - constituents of the ‘Standard Diet’ did not yield concordant results. With cotton-seed oil the nitrogen-output curve was _ practically identical with the “standard; with lard and “Oleo- stearin,”’ higher in the first periods and afterwards the same as _ when the “Standard Diet” was fed. In these substitution experi- _ ments with fats two important changes in the diet were made: _ (a) a larger amount of fat was given; (b) carbohydrate was removed. _ One or both of these two changes must be responsible for the _ results obtained. The experimental data show that the typical _ “standard” curve was obtained when a large quantity of ‘“Oleo- stearin’’ was superimposed on the ‘Standard Diet.” Therefore, | the larger amount of fat was not the important factor in causing _ the initial rise of the nitrogen-output curve above the normal ' when the non-nitrogenous constituents of the diet were replaced - by “Oleo-stearin” (and a priori by lard). Furthermore, the ” nitrogen-output curve following the ingestion of the “Standard Diet’ minus its content of sucrose and lard shows a close similarity to those obtained when lard and “Oleo-stearin,” respectively, re- _ placed the non-nitrogenous constituents of the “Standard Diet.” ’ In both types of experiment the sucrose of the “Standard Diet” ' has been removed. From the results obtained with carbohy- drates® it is evident that sucrose in the diet exerts a retarding in- | fluence on nitrogen excretion. It is not surprising, then, that _ with removal of the sucrose there was an increased output of nitro- _ gen in the early periods. Obviously lard and “Oleo-stearin” per se have no influence on the rate of elimination of nitrogen. With cotton-seed oil there was a slight preliminary delay in the ' nitrogen excretion. In the light of the foregoing discussion the | effect of this fat must have been much more pronounced than is ' evident from the data. With the removal of the sucrose of the ’ “Standard Diet”? there would be a tendency for a more rapid elimination of nitrogen in the early periods. The fact that there is, on the contrary, a slower rate can only be explained by assuming | that the cotton-seed oil has caused a marked fetardation of the nitrogen excretion. This is made the more evident by contrasting
8 See the first part of this paper.
52 Rate of Nitrogen Elimination
the nitrogen-output curve obtained when cotton-seed oil was pres- ent in the diet with that when meat alone was fed (cf. Curves V and IX).
The results with cotton-seed oil are in accord with those of earlier investigators who studied the influence of fat on the nitrogen- output curve. Panum (1874) and Feder (1881) found that lard caused a delay in the nitrogen excretion. Pari (1908) and Levene and Kober (1909) confirmed these findings; and Vogt (1906) like- wise reported that fat (no mention of its character is made) caused a flattening of the nitrogen-output curve. All of these investi- gators used dogs as subjects of experimentation.? The discrepancy between the results of the experiments with lard here reported and those of former workers awaits an explanation.
As causes of the retardation of the rate of nitrogen elimination on the part of cotton-seed oil there are several possibilities, viz: a delay in gastric discharge, a subnormal rate of absorption, altered metabolic conditions. The reports of Cannon" that fat causes a retardation of gastric discharge, and of Vogt (1906) and Boettcher and Vogt (1909) that fat in the diet leads to a delayed absorption, make it quite probable that a sub-normal rate of alimentation is the prime cause of the results with cotton-seed oil; for no evidence has been found that altered metabolic conditions play a part in this connection.
The lack of concordance in the results obtained with the differs ent fats may be explained by the fact that cotton-seed oil becomes more thoroughly incorporated in the diet and thus has a greater effect on the processes of alimentation, and hence on the nitrogen- output curve, than do the solid fats. An example of how varied the behavior of fats of different textures in the diet may be is af- forded by experiments of Tangl and Erdélyi," showing that the - rate of discharge of fat from the stomach is dependent to a great. extent upon its melting point and viscosity. :
® Although the results have no bearing on the experiments here reported, it should be noted that Wolf (1912) studied the effect on the nitrogen-output — curve of feeding fat to a fasting man. a
1° Cannon: Amer. Journ. of Physiol., xii, p. 387, 1904. ' Tangl and Erdélyi: Biochem. Zeitschr., xxxiv, p. 94, 1911.
Lafayette B. Mendel and Robert C. Lewis 53
i SUMMARY OF RESULTS WITH FATS.
The effect on the nitrogen-output curve of replacing the non- _ nitrogenous constituents of a mixed diet by fat varied with the _ character of the fat as follows: (a) with the fluid cotton-seed oil there was a slower rate of nitrogen elimination; (b) with lard and _ “Oleo-stearin’’ the nitrogen excretion in the early periods following _ the meal was above the normal. The apparent action of these latter fats was shown to be in reality the result of removing the sucrose from the diet. _ The action of cotton-seed oil per se was to cause a marked delay in the rate of elimination of nitrogen. Neither lard nor ‘‘Oleo- _ stearin’’ by themselves had any effect on the nitrogen-output curve.
BIBLIOGRAPHY. |
BoertcHer and Voat: 1909, Arch. f. exper. Path. u. Pharm., xi, p. 7.
Fara and Gigon: 1908, Biochem. Zeiischr., xiii, p. 267.
Fauta, Grote and STaAEHELIN: 1907, Beitr. z. chem. Physiol. u. Path.,
) ix, p. 333.
if Fever: 1881, Zeitschr. f. Biol., xvii, p. 531.
LEVENE and Koper: 1909, Amer. Journ. of Physiol., xxiii, p. 324.
Lusk: 1912, Journ. Biol. Chem., xiii, p. 27.
» Panum: 1874, Nord. med. Ark., vi, p. 12; Jahresber. w. d. Fortschr. d. _ Tierchem., iv, p. 361.
’ Part: 1908, Biochem. Zeitschr., xiii, p. 274.
Van StYKE and Wuire: 1911, Journ. Biol. Chem., ix, p. 219.
Voat: 1906, Beitr. z. chem. Physiol. u. Path., viii, p. 409.
Wotr: 1912, Biochem. Zeitschr., xl, p. 234.
ENCED BY DIET FACTORS.
Ill. THE INFLUENCE OF THE CHARACTER OF THE INGESTED : PROTEIN.
By LAFAYETTE B. MENDEL anp ROBERT C. LEWIS.
_ (From the Sheffield Laboratory of Physiological Chemistry, Yale University, New Haven, Connecticut.)
(Received for publication, August 4, 1913.)
The influence which the character of the protein intake has on _ the rate of nitrogen elimination may he considered to better ad- _ vantage now that the effect of various other diet factors has been determined.! The fact that there has been considerable discus- sion in recent years as to whether or not all proteins are catabo-
_ lized with equal rapidity gives stimulus to further research on _ this subject.
In comparison with previous experiments the “Standard Diet”’ ‘used in the present study contained a small amount of water;? and the relative amounts of fat and carbohydrate were changed.* _ With this low water intake two of the animals invariably gave
| of in the second.' The fact that there was a delay in nitrogen _ excretion when these animals were given the modified “Standard
1 Cf. Mendel and Lewis: this Journal, xvi, pp. 19 and 37, 1913.
ie 2 The proteins were all in the form of dry powders and, if an amount of | water as large as had been given were now used, it was thought that there would be difficulty in getting the animal to eat the entire ration.
3’ Inasmuch as carbohydrates have been shown to exert a retarding in- _ fluence on the rate of nitrogen elimination (cf. Mendel and Lewis: this Journal, xvi, p. 37, 1913), it seemed advisable to reduce the amount of car- . bohydrate in the diet. Except for the changes in the “Standard Diet’’ we ' employed the methods of our first paper (this Journal, xvi, p. 19, 1913). 4 Cf. Mendel and Lewis: this Journal, xvi, p. 23, 1913.
55
56 Rate of Nitrogen Elimination
Diet” could not be explained by the change in the relative amounts of fat and sugar in this diet.5 The only other explanation, then, was that the variation was due to the decrease in the water in- take. This possibility was suggested by the comparatively low volume of urine per kilo of body weight of these atypical animals. In order to determine whether this hypothesis was correct, an experiment was conducted on one of these animals in which the water content of the diet was made proportional to that of meat.® This time the typical “standard” curve with the maximum out- put of nitrogen in the second three-hour period was obtained. Thus it is conclusive that too great a diminution in the intake of water will cause a marked slowing of the rate of elimination of nitrogen in the urine. In the experiments to be reported the results are in all cases compared with those obtained on the same animal after the ingestion of this modified diet, a fact which the reader should bear in mind.
PRELIMINARY EXPERIMENTS. Dried meai (Curve I).
In the present study the rate of elimination of nitrogen was determined after the ingestion of several different proteins, the majority of which were in the form of dry powders. The differ- ence in texture between such dry preparations and moist meat suggested itself as a possible cause for a variation in the nitrogen-— output curve and so demanded a preliminary investigation. For . this purpose a day’s portion of meat was dried at about 55°C., then finely ground in a coffee mill, and fed. The drying of meat had practically no effect on the rate of nitrogen elimination.
* In a previous paper (this Journal, xvi, p. 37, 1913) the authors have — shown that carbohydrate in the diet causes a delay in nitrogen excretion, — The reduction of the amount of carbohydrate in this case would tend . to have an opposite effect. “f
6 The water addition in our original “Standard Diet” was calculated on this basis (cf. this Journal, xvi, p. 19, 1918).
Lafayette B. Mendel and Robert C. Lewis 57
Curve I. To illustrate the absence of effect on the rate of elimination of nitrogen of previously drying the meat of the “‘Standard Diet.”’
N gm. "Standard Diet" 0.6 | ' Ae fodiresa | t 300 gm. meat (N= 10.73 gm.) i ! 65 " lerd O53) 1 a I " 45 sucrose J Rabat ong 2 " 04 ; i NeCl bone ash te 03 | water ae ee ee ee oe we ae ny 0.2 ] out} Ss |\6mee la tis 24 hrs. ----- Experiment G XVIII, ‘‘Standard Diet.’’
Exper ment G XX, “‘Standard Diet,’’ except that meat had been previously dried (dry weight=75 grams) and finely ground. Extra water= 225 cc.
Extracted meat? (Curve II).
Inasmuch as a notable proportion of the nitrogen of meat is non-protein in character, it seemed desirable to study meat de- void of extractives and in such a form as to be directly compar- able with the extractive-free isolated proteins. The nitrogen- output curves of the latter are compared with that occurring after the ingestion of an extracted meat powder. The use of such an extractive-free meat is made the more necessary because of the recognized influence of extractives on secretory processes in the stomach, and because the nitrogen of extractives like crea- tine and the purines is in a different chemical structure from that
7 A light brown, impalpable powder obtained from Armour and Company,
58 Rate of Nitrogen Elimination
Curve II. Comparison of the rates of nitrogen elimination on diets containing meat, extracted meat, and extracted meat+ extractives, respectively.
N gm.
"Standard Diet" 225 gm. meat (N= 7.61 gm.) 35" lard
70" sucrose aa. NaCl
5" bone ash
water
3. |é Beye ons ----- Experiment D XXIV, ‘Standard Diet.’’
grams extracted meat (N=7.65 grams) and 170 grams water. —-— Experiment D XXXI, meat of “Standard Diet’’ replaced by 51
grams extracted meat (N=6.84 grams), beef extract (N=0.77 grams) and 170 grams water.
of the familiar amino-acids. The nitrogen-output curve with the extracted meat was considerably more flattened than that after the ingestion of fresh meat—-a fact which may be explained in part by the absence of extractives in the former product, the fol- _ lowing experiments showing that extractive-nitrogen is very rapidly eliminated.
Experiment D XXX, meat of ‘Standard Diet’’ replaced by 57 —
Lafayette B. Mendel and Robert C. Lewis 59
Extracted meat + Extractives (Curve II).
When extracted meat furnished 90 per cent, and Liebig’s beef extract 10 per cent of the nitrogen of the diet, there was a larger ~ nitrogen output in the first periods than after the ingestion of extracted meat alone, showing that extractive-nitrogen is elimi- nated with comparative rapidity.
Meat + Urea (Curve III).
When the nitrogen of the diet was furnished in equal portions by meat and urea, the nitrogen excretion in the first two periods was enormously larger than when meat alone was fed. It is evident that the urea-nitrogen is rapidly eliminated.’
In the first of these two experiments (Curve II) extractive- nitrogen was present in approximately the same proportion as it occurs in meat; the nitrogen-output curve, however, was con- siderably more flattened than when fresh meat was fed. It is quite apparent, therefore, that the absence of extractives can only account in part for the slower rate of nitrogen elimination when extracted meat replaces the meat of the “Standard Diet.” The cause of this difference between the nitrogen-output curve of
fresh meat and that of extracted meat may be that the latter product is richer in connective tissue and so less readily digestible. _ The finding of Mendel and Fine® that this same extracted meat was not as well utilized as fresh meat bears out such an assumption.
i we :
PROTEIN MATERIALS EMPLOYED BESIDES MEAT.
In several cases the materials used in the present study were isolated proteins; in others, products relatively rich in protein. A description of each of the substances used follows.
Casein!*—a purified preparation in the form of an impalpable powder containing 13.36 per cent nitrogen.
8 Wolf (1912b) and Cathcart and Green (1913) have reported a rapid elimination of nitrogen after feeding urea to man.
® Mendel and Fine: this Journal, xi, p. 5, 1912.
10 The casein and a portion of the edestin were contributed by Dr. T. B. Osborne, New Haven, Conn.
60
Rate of Nitrogen Elimination
CurveEIII. To illustrate the effect on the rate of elimination of nitrogen of replacing one-half of the meat of the “Standard Diet”’ by urea.
N LS pie 1.0) 0.9 0.8 "Standard Diet" 300 gm. meat (= 10.73 en.) 0.7) 65" lard 45" sucrose 06. ee 2" Nec1 Pd bone ash 0.5) I 150" water ! base 04 ‘ 1 TE ae ! t---4 = -—— — — = = == “—-——— = : a2 lt t It 0.! ; t f 1 f Poe PR ® oe ----- Experiment G XVIII ‘Standard Diet.’’
replaced by 11.5 grams of urea (N =5.37 grams) and 115 grams of water.
, _ — ae ee
Experiment G XXII, one-half of meat of “Standard Diet”
Lafayette B. Mendel and Robert C. Lewis 61
Ovovitellin'—a purified product; an impalpable powder con- taining 13.78 per cent nitrogen.
Edestin—a purified preparation. Two lots of this material were used, both of which were impalpable powders containing 16.23 per cent and 16.95 per cent of nitrogen, respectively.
“Glidine’”’!2—a commercial preparation from wheat;!* an impal- pable powder giving no starch reaction and containing 14.8 per cent nitrogen.
Gelatin—a commercial preparation in the form of a fine powder containing 15.1 per cent nitrogen. :
Soy Bean“—an impalpable powder containing 7.25 per cent nitrogen, thus being poor in protein as compared with the other dry materials used. Besides protein the soy bean contains a large amount of fat and considerable cane sugar.”
Liquid Egg-White—the whites of eggs thoroughly strained and mixed (nitrogen content=1.95 per cent).
Dried Egg-White—the whites of eggs dried at 50°C. and then ground to a fine powder in a mortar (nitrogen content=13.6 per cent). ;
Coagulated Egg-White—the whites of hard boiled eggs passed through a sieve (nitrogen content= 1.92 per cent).
- Ovalbumin'*—a purified product in the form of an impalpable powder containing 15.4 per cent nitrogen.
Water was added to the powdered proteins (with the excep- tion of gelatin, dried egg-white, and ovalbumin) the night pre- vious to feeding, for the purpose of allowing ample time for “hydration” of the material. The following morning a thoroughly hydrated mush was always found.
4 Prepared by Mr. R. L. Kahn in our laboratory.
12 Obtained from Menley and James, New York City.
13 This material, ‘‘according to Bergell, and Thiemar, is prepared from wheat flour by a process of washing and centrifuging.”
4 Mr. M. F. Deming of the Cereo Company, Tappan, New York, con- tributed this material.
15 For a complete analysis of soy bean, see Ruhrih: Journ. Amer. Med. Assn., liv, p. 1664, 1910; also quoted by Mendel and Fine: this Journal, x, p. 435, 1911.
16 This material was prepared in our laboratory by Dr. Martha Tracy.
62 Rate of Nitrogen Elimination
EXPERIMENTS WITH PROTEINS.
Casein (Curve IV).
When casein was substituted for the meat of the “Standard Diet,” the nitrogen-output curve was practically the same as that with extracted meat.
Curve IV. Comparison of the rates of nitrogen elimination on diets containing extracted meat and casein, respectively.
N 9", "Standard Diet” os 200 gm. meat (N = 7.15 gu.) r---74 50" lara 0.4 30." sucrose ! wm - NaCl ash] 5" bone ash t---4 100" water 02 === ! aaa - ae au 3 f t 1
3 6 pit 5 24.
~---- Experiment H VII, meat of ‘Standard Diet’’ replaced by 53 grams extracted meat (N=7.11 grams) and 150 grams water. ' Experiment H VIII, meat of “Standard Diet’’ replaced by st grams casein (N=7.21 grams) and 150 grams water.
Lafayette B. Mendel and Robert C. Lewis 63
Ovovitellin (Curve V).
The rate of nitrogen elimination after the ingestion of ovovi- tellin was identical within the limits of experimental error with that when extracted meat was fed.
Curve V. Comparison of the rates of nitrogen elimination on diets containing extracted meat and ovovitellin, respectively.
N- g™ : "Standard Diet” ' 0.6 : # : 225 gm. meat (N= 8.04 gn.) ; 55" lard . 35." sucrose * ! " ' fe 2 NaCl > 5" bone ash 125" water U Se 1 9 Ie is 214 ----- Experiment D XXXIII, meat of ‘Standard Diet”’ replaced by
’ 60 grams extracted meat (N=8.05 grams) and 170 grams water. 4 Experiment D XXXVIII, meat of ‘‘Standard Diet’”’ replaced by 58 grams ovovitellin (N=7.99 grams) and 170 grams water.
64 Rate of Nitrogen Elimination
Edestin (Curve VI).
The nitrogen-output curve with edestin was not of such a flat- tened aspect as that with extracted meat. The nitrogen excre- tion in the earlier periods was larger; in the later periods, smaller than with extracted meat. The edestin curve, however, was very much the same as that with fresh meat. - |
Curve VI. Comparison of the rates of nitrogen elimination on diets containing meat, extracted meat, and edestin, respectively.
N g™ 0.1 "Standard Diet” 0.6) 200 gm. meet (Ne@ 6.84 gm.) 50 " lerd 0.5 30" sucrose = §6Racl 04 5" bone ash . 250" water a 4 h t a t———., 0.2 od i oo en oo i eo A 4 Ol aso a ee ; 1S. 6 ape Sle -ouls 24 hrs —--— Experiment H XIV, “Standard Diet,’ --+-- Experiment H VII, meat of “Standard Diet’’ replaced by 58
grams extracted meat (N=7,11 grams). Experiment H XI, meat of ‘Standard Diet” replaced by 44 grams _
edestin (N=7,14 grams),
Lafayette B. Mendel and Robert C. Lewis 65
“Glidine” (Curve VII).
When “Glidine” constituted the protein intake, the nitrogen excretion was larger during the earlier periods of the day than with extracted meat. The character of the nitrogen-output curve was practically the same as that with fresh meat; the two curves ran parallel, that with ‘Glidine” being at a higher level.
ea ae eee ™ aT)
Curve VII. Comparison of the rates of nitrogen elimination on diets containing meat, extracted meat, and ‘‘Glidine,’’ respectively.
N gm 0.1) ss "Standard Diet” 0.6 | 200 gm. meat (N= 6.84 gm.) ‘ —_ ' a, | | 50" lerd — OS! | | "sucrose ¢.| , NeCl . ca} " bone ash a i Bsa : " water iJ ames 02
“™s bP we wis 214 hrs.
—-— Experiment H XIV, “Standard Diet.’
weeee Experiment H VII, meat of “Standard Diet’’ replaced by 53 grams extracted meat (N=7.11 grams).
Experiment H IX, meat of “Standard Diet’’ replaced by 48 grams “‘Glidine’’ (N=7.10 grams).
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL, XVI, NO. l.
66 Rate of Nitrogen Elimination
e
Gelatin (Curve VIII).
Again with gelatin the nitrogen-output curve was not as flat- tened as that with extracted meat, but identical in character with the “standard” (fresh meat) curve. There was a negative balance with gelatin and the nitrogen excretion for all the periods was higher than with meat.
Curve VIII.- Comparison of the rates of nitrogen elimination on diets containing meat, extracted meat, and gelatin, respectively.
gm. 0.7 "Standard Diet" ee 200 gm. meat (N= 6.84 gm.) 06 | | 50" ara : l 30" sucrose 0.5 | | 2" NaCl oant n b tr Ii 5 one ash | 04 Ii HT 250" water —" 1 Biiee i} ‘hes 4 andslened Lu 0. 0. 0.! 3 | 9 le as 4 hrs —--— Experiment H XIV, “Standard Diet.’ w<-0- Experiment H VII, meat of “Standard Diet” replaced by 53
grams extracted meat (N=7,11 grams).
gelatin (N=7,08 grams),
Experiment H X, meat of ‘Standard Diet” replaced by 47 grams”
Lafayette B. Mendel and Robert C. Lewis 67
Soy bean (Curve IX).
On account of the comparatively poor utilization of soy bean!’ _ its ingestion was followed by a smaller nitrogen output in all the _ periods of the day than when extracted meat was fed. Further- more, this lower curve was not parallel to that with extracted meat; its character was quite different. In the earlier periods of _ the experiment here reported a smaller percentage of the 24-hour nitrogen output was excreted than when meat was fed; in the later periods, a larger percentage. In a second experiment the maximum nitrogen excretion did not occur until the third three- hour period, instead of the second. In both cases, then, there was a delay in the elimination of nitrogen independent of the poorer utilization of the soy bean.
Curve IX. Comparison of the rates of nitrogen elimination on diets containing extracted meat and soy bean, respectively.
. N ie 7 -~ J gm. "Standard Diet" 200 gm. meat (N= 7.15 gm.) co lard sucrose bone ash Nacl water SS; Se Se U ! t l i a fl CS zz. 214 hrs,
_ ----- Experiment H VII, meat of ‘Standard Diet’”’ replaced by 53 rams extracted meat (N=7.11 grams) and 150 grams water.
Experiment H XII, meat of ‘‘Standard Diet’”’ replaced by 99 rams soy bean (N=7.18 grams) and 150 grams water.
17 Mendel and Fine (this Journal, x, p. 345, 1911) found soy bean nitrogen ‘0 be poorly utilized.
68 Rate of Nitrogen Elimination
Uncoagulated egg-white (Curve X).
On the days when native egg-white was fed the nitrogen out- put was only about half as large as the intake. It is probable, as the discussion to follow will show, that this material was poorly utilized. The character of the nitrogen-output curve with un- coagulated egg-white was somewhat different from that with extracted meat, the maximum excretion occurring during an earlier period—in two experiments during the first three-hour period,
Curve X. Comparison of the rates of nitrogen elimination on diets containing extracted meat and liquid egg-white, respectively.
N gm. 0.6 oo. "Standard Diet” a i 225 em. meat (N= 8.04 gm.) OS) : 55" lara : ; 35" ousreae . 0.4 <a 2" Ral : iat ee 5" bone esh . 0.54 a oe 0.2 | et {eo 2 : O.1} je bomt ia, IS 24 hrs “<--> Experiment D XXXIII, meat of ‘Standard Diet’’ replaced by
60 grams extracted meat (N=8.05 grams) and 170 grams water. Experiment D XXXII, meat and water of ‘Standard Diet’ replaced by 412 cc. liquid egg-white (N =8,03 grams).'*
ania
* Diarrhea during 5th three-hour period; urine contaminated, Thi ingestion of dried native egg-white was also followed by diarrhea,
a
ad
y
Lafayette B. Mendel and Robert C. Lewis 69
instead of during the second; in a third experiment during the second period, instead of during the third. The results with liquid egg-white and dried egg-white, respectively, were concor- dant.
Coagulated egg-white (Curve XI). Coagulated egg-white was evidently well utilized; but the nitro-
gen-output curve after its ingestion was more flattened than that
when extracted meat was fed. In other words there was a com- parative delay in the elimination of nitrogen when coagulated egg-white constituted the protein intake.
Curve XI. Comparison of the rates of nitrogen elimination on diets
| containing extracted meat and coagulated egg-white, respectively.
gm. "Standard Diet" Pt on 225 gn. meat (N= GimeneeD 0.6 | 55" lard 35" sucrose a =~ 6 NaCl
" bone ash
water .
a b6 BP RR Ws 24 hrs.
----- Experiment D XXXIII, meat of ‘Standard Diet’’ replaced by
60 grams extracted meat (N=8.05 grams) and 170 grams water.
Experiment D XXXVII, meat of ‘‘Standard Diet’’ replaced by
419 grams coagulated egg-white (N=8.04 grams).
70 Rate of Nitrogen Elimination Ovalbumin® (Curve XII).
When ovalbumin was fed the urinary nitrogen-output was much smaller than the intake, the marked diarrhea about nine hours after the meal suggesting a poor utilization of the ovalbumin as the cause of the smaller nitrogen excretion. , Furthermore, the rate of nitrogen elimination with this material was quite different from that with extracted meat. There was a rise to a maximum in the second three-hour period, then a slight fall during the third three hours, followed by a second rise to a maximum in the fifth
Curve XII. Comparison of the rates of nitrogen elimination on diets containing extracted meat and ovalbumin, respectively:
-
> N "Standard Diet" gm. 200 gm. meat (N = 6.84 gm. 50 " lard
Z0-* sucrose nue eam Neal Nacl .
hy bone ash
— --4 260 be water
3 6 9 te As 24 hrs
----- Experiment H XV, meat of ‘Standard Diet” replaced by 51 grams extracted meat (N =6.84 grams ). Experiment H XVII, meat of “Standard Diet’’ replaced by 50 grams ovalbumin (N=6,73 grams).?°
1” We are greatly indebted to Mr. R. L. Kahn for performing this and several other experiments of the present series.
2° Voluminous diarrhea during third three-hour period; animal ate some feces,
Lafayette B. Mendel and Robert C. Lewis 71
period. The fact that the animal ate a portion of the diarrheal feces at the beginning of the third period may account in part for the second rise.
DISCUSSION.
The nitrogen-output curves following the ingestion of unaltered meat and extracted meat powder, respectively, were quite differ- ent. The slower rate of elimination of nitrogen when extracted meat was fed cannot be explained by the dry condition of this material; for the nitrogen-output curve was unchanged by a pre- vious drying of the meat of the “Standard Diet.” The absence of extractives in the extracted meat can only account in part for the delayed nitrogen excretion. This product presumably contains proportionately more connective tissue than the fresh meat used and thus is digested more slowly. The nitrogen-output curves following the ingestion of casein and ovovitellin are practically identical with that of extracted meat; the character of the curves with edestin, “‘Glidine,” and gelatin is the same as that with fresh meat. .
Soy bean, egg-white, and isolated ovalbumin gave nitrogen- output curves radically different from those of either of the meat products studied. The comparative delay in nitrogen elimination independent of the poor utilization of soy bean may be explained
_in part by a greater difficulty of digestion of this product, and in part by the presence of sucrose in soy bean, it having been shown in a previous paper that the presence of carbohydrate in the diet delays nitrogen excretion. The comparatively small excretion of nitrogen after the ingestion of native egg-white and ovalbumin is caused in all probability by a poor utilization of these materials, the early diarrhea following their ingestion making such an ex- planation quite likely. That uncoagulated ovalbumin is poorly __ utilized was reported by Falta (1906), who found that the coefficient of utilization of this material in man was only about 70 per cent. Wolf (1912b) fed a large quantity of native egg-white to man and reported that only about half was utilized. When liquid egg-white” was fed it is probable that very little gastric proteolysis occurred;
21 Mendel and Lewis: this Journal, xvi, p. 37, 1913. 2 The dried egg-white mixed with water would be essentially the same as the natural product.
72 Rate of Nitrogen Elimination
for Cannon," and London and Sulima,” working with cats and dogs, respectively, have reported that this material begins to leave the stomach almost immediately after ingestion. The early dis- charge of the stomach, the comparatively early emptying of the bowel, and the unfavorable character of liquid egg-white for the action of digestive enzymes, together with a possible resistance of native protein to digestion, may all contribute to a poor utiliza- tion of this material. Coagulated egg-white was well utilized; but following its ingestion there was a comparative delay in nitro- gen excretion. The slower rate of elimination of nitrogen with this source of protein cannot be accounted for by a delay of gastric discharge; for Cannon, and London and Sulima have demon- strated that egg-white coagulated by heat leaves the stomach more rapidly than most proteins. It is probable that the delayed excretion of nitrogen may be caused in part, at least, by a com- _ difficulty of digestion of coagulated egg-white on account of the compact and impermeable character of fine particles of coaguium.
A few reports in the literature are in harmiaal with this view that such changes as do occur in the rate of elimination of nitrogen after the ingestion of different protein materials may be explained by variations in alimentary, rather than metabolic processes.
’Van Slyke and White (1911), using the method of the present — work, demonstrated that different nitrogen-output curves were obtained after feeding various boiled fish meats to a dog; and attributed this result to a variation in the rate of digestion. The validity of such an explanation is made very clear by a comparison of the results obtained by these authors with fresh and salt cod, there being as much difference in the nitrogen-output curves of the two preparations of this one fish as in those of any of the different fish. Vogt (1906) investigated the effect on the rate of elimination of nitrogen of superimposing various proteins in con- siderable quantities on a standard diet, finding that both coagu- lated and uncoagulated ovalbumin caused a delay in nitrogen excretion whereas edestin and a casein preparation (Nutrose) gave a nitrogen-output curve of approximately the same character
#1 Cannon: Amer. Journ. of Physiol., xii, p. 387, 1904. *? London and Sulima: Zeitschr. f. physiol. Chem., xlvi, p. 282, 1905,
‘
Lafayette B. Mendel and Robert C. Lewis 73
as meat. This author believed that the delay with ovalbumin was caused by a comparatively slow rate of digestion of this material. Loeb (1911) studied the rate of elimination of nitrogen after replacing about one-half of the meat of a standard diet by another form of protein; and demonstrated that there was only a very slight change when meat and casein, respectively, were fed, although considerable difference existed between the curves of these proteins on the one hand and those of their hydrolyzed prod- ucts on the other. In experiments where the urine was collected only for twelve-hour periods Falta, Grote, and Staehelin (1907) found approximately the same rate of nitrogen excretion with casein as with meat. All of these investigators worked on dogs. Wolf (1912a, 1912b) added various proteins and non-proteins to a “standard” diet in man and collected the urine in hourly periods, studying among other things the rate of elimination of nitrogen... He found little difference in the nitrogen-output curves following — the ingestion of gelatin and plasmon, respectively. With veal, however, there was a somewhat slower rate of nitrogen output. Native egg-white and coagulated egg-white gave results quite similar to those reported in the present paper. Wolf (1912c) also studied the rate of nitrogen elimination in dogs after feeding cooked and raw meat, respectively, obtaining practically identical results in the two cases.
In considering the significance-of the results of the present study attention should be given to Falta’s conclusions from his work on the rate of metabolism of proteins. The method of in- vestigation employed by this author was to determine the average daily nitrogen-output of a dog in nitrogen equilibrium for a period of several days, then to superimpose the protein to be studied on the “standard” diet, and to ascertain how long a time was required for a reappearance of an excess of nitrogen in the urine equivalent to the nitrogen of the superimposed material. Falta (1904 and 1906) studied different proteins on man and found that with most of these more than half of the excess nitrogen reappeared on the first day, about three days being required for the entire amount to show up. With casein, for example, about two-thirds of the excess nitrogen reappeared during the first day, and most of the remainder on the second day. A few exceptions occurred, however,
74 Rate of Nitrogen Elimination
the most striking being with ovalbumin and ovovitellin. In these cases only about 27 per cent of the excess nitrogen appeared the first day; and five days were required for all to reappear, although all but a very small amount had come out in three days. When coagulated ovalbumin was the added protein no longer time was required for the reappearance of the excess nitrogen than was the case with casein. With dogs the results with ovalbumin and casein were the same as for casein with man. These observa- tions on man were confirmed by Himiiliinen and Helme (1907), who demonstrated that a longer time was required for the reappear- ance of the excess nitrogen with egg-white than with a casein prep- aration (Proton) or roast veal. Cathcart and Green (1913), employing the Falta method of superimposition on man, reported that with egg-white, both coagulated and uncoagulated, only a small part of the extra nitrogen appeared in the urine even after several days. There was little difference in the rate of elimina- Aion of the extra nitrogen after adding veal and gelatin, respec- tively; to the diet, greater differences being obtained with the same sample of gelatin in two experiments where the basal rations varied considerably. Vogt (1906) used Falta’s method of study on dogs and found that a longer time was required for the reappearance of the excess nitrogen when egg-white, both uncoagulated and coagulated, was superimposed on the standard diet than when edestin or a casein preparation, Nutrose, was added. All of these communications are in harmony with that of Graffen- berger (1891), who showed by a somewhat different method that when gelatin or fibrin was superimposed on a standard diet the excess nitrogen reappeared more rapidly than when peptone con- stituted the increased nitrogen intake.
From the results of his experiments Falta (1906) has concluded that the longer time required for the reappearance of the excess nitrogen after superimposing ovalbumin on the standard diet was the result of the absorption of comparatively large cleavage prod- ucts of this protein, a longer time being required for the catab-
olism of these higher protein residues, Hiimiiliiinen and Helme —
(1907) held a similar view; and Levene (1909a, 1909b, 1909¢, 1910) and his co-workers from a series of studies of an entirely different
*” Only one experiment with ovovitellin is reported and the author says that no definite conclusion should be drawn from a single experiment.
ee
—, ———————————=————EEE== cere errr erm ree
LT PTS
Lafayette B. Mendel and Robert C. Lewis 75
nature likewise came to the conclusion that the higher protein cleavage products are catabolized more slowly than the simple amino-acids. Vogt (1906) was not inclined to favor such an ex- planation, maintaining that a slower rate of digestion and absorp- tion might account in part for the results obtained by him with egg-white, and that certain unknown factors of intermediary metabolism might play a part.
Although Falta’s experiments are not directly comparable with those of the present study, yet if one recalls how readily texture of the diet influences the rate of digestion and absorption inde- pendently of the character of the protein, it seems quite likely that Falta’s results were caused in part by a difference in the rate of digestion of the various materials studied. Let us consider what would be the effect of a markedly delayed digestion and absorption in studies of the type that Falta made. In the ex- periments of this author on man the superimposed protein as well as the standard diet was fed in four portions distributed over the day. Under such conditions it is quite likely that absorption of the digestion products of a difficultly digestible protein would not be complete until after the beginning of the second day. It is not surprising, then, that the excess nitrogen eliminated on the day when the protein was added to the diet should be smaller than when the superimposed protein was readily digestible; nor that it should be greater on the following day, the amount of nitro- gen absorbed on this second day being greater than that usual on a normal day. The fact that, when Falta fed a single meal to dogs at the beginning of the day, he obtained a result with oval- bumin similar to that with casein makes such an explanation more probable; for in this case digestion and absorption would certainly be complete during the first day.
SUMMARY OF RESULTS WITH PROTEINS.
The nitrogen-output curves following the ingestion of meat and extracted meat, respectively, differ considerably, that with the latter product being more flattened. This slower rate of elimina- tion of nitrogen cannot be explained by the dried condition of the extracted meat; and only in part by the absence of extractives in this material. It is suggested that the extracted meat may
76 Rate of Nitrogen Elimination
have contained proportionately more connective tissue than the fresh meat used and thus have been less readily digestible.
The nitrogen-output curves following the ingestion of most of the proteins studied—casein, ovovitellin, edestin, ‘“Glidine,” gela- tin—differ in character to no greater extent than those obtained by feeding the two meat products employed. With egg-white, ovalbumin, and soy bean, however, curves of a character radically different from that of either of the meats were obtained. These results may be explained to a great extent by a difference in the rate and completeness of digestion and absorption of these mate- rials; while the sucrose in the soy bean will also.account in part for the delay in nitrogen elimination with this product. When these alimentary differences are duly taken into account, the conclu- sion seems justified that proteins do not differ materially in their rate of metabolism.
Falta’s conclusions from his work on the rate of protein metabo- lism and the similar conclusions of Hiimiiliinen and Helme, and of Levene and his co-workers were discussed. From the results of the present study it seems quite probable that the findings of these authors may be explained by other factors than an assumed difference in the rate of metabolism of proteins caused by an ab- sorption of larger or smaller cleavage products.
The results of the experiments reported in the papers of this series show that apart from the character of the protein ingested a large number of diet factors—the water intake, the presence and nature of indigestible materials in the diet, the amount and character of the carbohydrate fed, and to some extent the pres- ence of fat in the diet—play a réle in modifying the rate of elimi- nation of nitrogen after a meal containing protein. With most of the proteins studied the nitrogen-output curves differed to only a slight extent from one another; and in no case did the nature of the protein have a greater effect on the rate of nitrogen elimina- tion than some of the non-protein diet factors mentioned above.
Lafayette B. Mendel and Robert C. Lewis 77
BIBLIOGRAPHY.
Catucart and GREEN: 1913, Biochem. Journ., vii, p. 1. Faura: 1904, Deutsch. Arch. f. klin. Med., |xxxi, p. 231;1906, ibid., Ixxxvi, p. 517. Fara, Grote, and STAEHELIN: 1907, Beitr. z. chem. Physiol. u. Path., ix, p. 333. : GRAFFENBERGER: 1891, Zeitschr. f. Biol., xxviii, p. 318. HAMALAINEN and Heume: 1907, Skand. Arch. f. Physiol., xix, p. 182. LEVENE: 1909a (Levene and Kober), Amer. Journ. of Physiol., xxiii, p. 324; 1909b (Levene and Meyer), ibid., xxv, p. 214; 1909¢ (Levin, Manson, and Levene), ibid., xxv, p. 231; 1910 (Carrel, Meyer, and Levene), ibid., xxv, p. 439. Logs: 1911, Zeitschr. f. Biol., lv, p. 167. Van SLYKE and Waite: 1911, Journ. Biol. Chem., ix, p. 219. Voar: 1906, Beitr. z. chem. Physiol. u. Path., viii, p. 409. _ Wor: 1912a, Biochem. Zeitschr., xl, p. 193; 1912b, ibid., xl, p. 234;1912¢c, ibid., xli, p. 111. ae *
5 al
THE CARBON DIOXIDE AND OXYGEN CONTENT OF THE BLOOD AFTER CLAMPING THE ABDOMINAL AORTA AND INFERIOR VENA CAVA BELOW THE DIAPHRAGM.
By J. R. MURLIN, LEO EDELMANN anp B. KRAMER.
(From the Physiological Laboratory of Cornell University ‘Medical College, New York City.)
(Received for publication, August 22, 1913.) *
In search of experimental support for the over-production theory of diabetes mellitus Porges' and Porges and Salomon® have found that ligation of the abdominal aorta and inferior vena cava just below the diaphragm, both in normal rabbits and in depanereatized dogs, causes a rise in the external*® respiratory quotient. They interpret this result as proof (1) that the organism is dependent upon the liver for its power to oxidize protein and fat, carbohy- drate only or carbohydrate chiefly being oxidized when the liver is excluded; and (2) that the depancreatized animal retains its power to oxidize sugar.
A rise in respiratory quotient after this radical interference with the circulation has been confirmed by Rolly,* who, however, finds the rise not at all constant and gives an altogether different explanation. Verzdr® likewise witnessed a sudden change in the R. Q., but a change in the same direction, when the liver was partially excluded by anastomosis of the portal vein with the lower
1 Porges: Biochem. Zeitschr., xxvii, p. 131, 1910.
* Porges and Salomon: Jbid, p. 148.
’ The term external R.Q. is used here in order to emphasize the fact that the exchange of gases between blood and outside air does not under all cir- cumstances take place at the same rate as the exchange between blood and tissue. The volumetric relations between the CO, gain of venous blood and O, loss from arterial may be called the internal R.Q.
‘Rolly: Deutsch. Arch. f. klin. Med., ev, p. 494, 1912; Miinch. med. Woch- enschr., 1912, Nos. 22 and 23.
5 Verzdr: Biochem. Zeitschr., xxxiv, p. 52, 1912.
79
80 ° Metabolism after Clamping Abdominal Vessels
part of the inferior vena’ cava (Queirolo operation). Fischler and Grafe® ligated the hepatic artery of dogs which, some weeks before, had been successfully operated for the Eck fistula, and found in two out of six cases a distinct rise in the R.Q. Béhm’ reports but a very slight rise “after exclusion of the abdominal organs even in depancreatized dogs.”
In ‘all these experiments showing a higher R. Q.° there is, as would be expected, a reduction in the total respiratory exchange, depending in amount upon the kind and amount of tissue excluded from the circulation. The reduction in the absorption of oxygen is greater than that for the elimination of carbon dioxide. Hence the higher R. Q. In other words, after the crucial operation there is, relatively, a greater output of COs,
There are the following possible ways of explaining this result: (1) A greater production of CO, with no change in the rate of @limi- nation. If carbohydrate or carbohydrate-like bodies should be oxidized instead of protein or fat, moré CO: would be produced. This is the explanation adopted by the von Noorden school. : (2) Greater elimination of CO, from the blood with no essential change in the rate of production. The influences which may be con- ceived of as driving out more CO. may be (a) chemical or (b) mechanical. If more acids were produced, or if acids produced as usual were not neutralized, after exclusion of the liver, more CO, would be liberated from its combination in the tissues and the blood, and would eseape. This is the explanation adopted by Rolly, and approved of by Fischler and Grafe. Rolly has actually found in the serum of his operated animals a lower de- gree of alkalescence than in that of normal animals and Porges in a recent paper’ has himself shown that acidification of the blood by intravenous infusion of sodium dihydrogen phosphate will raise the respiratory quotient, though not so much as occurred in his earlier experiments.
The mechanical factors have not been sufficiently emphasized.
*Fischler and Grafe: Deutsch. Arch. f. klin. Med., eviii, p. 516, 1912,
7 Bohm: Zentralbl. f. Physiol., xxvii, p. 120, 19138,
* Except one animal which had convulsions in Fischler and Grafe's series. Béhm’s complete paper is not accessible and it is possible that his series may contain other exceptions,
* Porges: Biochem. Zeitschr., xivi, p. 1, 1912.
: : :
J. R. Murlin, Leo Edelmann and B. Kramer 81
Porges, in his original article assumes that any change due to over- ventilation which might result would be equalized in fifteen min- utes. Presumably he means by over-ventilation only exaggerated breathing for he cites in support of his view, the work of Born- stein and Gartzen’® on the effects of over-ventilation by voluntary effort in human subjects, showing that after fifty minutes no more CO. can be pumped out in this manner! He also cites one of his own experiments in which the R. Q. in the second period was slightly higher than in the first period after ligation of the vessels. According to Porges’ view, the quotient in the second period should be smaller if any factor of over-ventilation were operative.
Neither of these citations offers any convincing evidence which would exclude the mechanical factors; for in the experiments of Bornstein and Gartzen the circulation was in no way disturbed, while the forced breathing was maximal, and Porges’ own experi- ment proves only that, whatever was the controlling cause of the higher quotient after ligation of the vessels, the conditions were the same in the second period as in the first. Moreover it should be borne in mind that over-ventilation may mean something more than exaggerated breathing: there may be over-aération due solely to a disturbance to the pulmonary circulation.
Fischler and Grafe appreciated the possible effect of the dis- turbance to the circulation resulting from the Porges procedure, saying, ‘‘One does not know to what extent the results may be due to the direct consequences of this alteration.’
In the writers’ opinion the work of Fischler and Grafe is suffi- cient refutation of the position taken by the von Noorden school as to the réle of the liver in the metabolism of the food-stuffs. Excluding the liver by ligation of the hepatic artery after Eck fistula did not cause a permanent rise in the respiratory quotient in dogs which survived from six to twenty hours. On the other hand, there is no doubt, in certain cases at least, about the rise of quotient after ligation of -the abdominal aorta and inferior vena cava just below the diaphragm. It remains to give a satisfactory explanation of this phenomenon.
It seems almost incredible that the purely mechanical effects of so radical a procedure as one which excludes at a stroke fully
10 Bornstein and Gartzen: Pfliger’s Archiv, cix, p. 628, 1905. 1 Fischler and Grafe: loc. cit., p. 519.
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. 1
82. Metabolism after Clamping Abdominal Vessels.
one-half of the blood and considerably more than one-half of the animal’s weight from the circulation should not have been more seriously considered. What effect would it have on the heart rate, on the blood pressure, on the rate of blood flow through the lungs? Neither Porges nor Porges and Salomon gives any data as to the pulse, blood pressure, rate of respiration or volume of respiration, to say nothing of the gaseous content of the blood before and after ligation of the vessels, and yet the results are presented as proof that, by turning a valve, so to speak, the meta- bolic processes are suddenly changed so that one fuel and one only can now supply the body’s energies!
The first and most obvious control which one would think of in connection with so radical a change in the R. Q. would be the gaseous content of the blood. There are numerous experiments in the work of Zuntz,!* Krogh," Barcroft,'* Henderson® and others showing that the O2 and CO: contents of the blood are subject to considerable variations, particularly under operative conditions.
It is a commonplace laboratory exercise to clamp off the abdom- inal aorta below the diaphragm and witness the enormous rise in systemic pressure (carotid) which results. Simultaneous clamping of the inferior vena cava with the aorta will likewise produce the rise in systemic pressure. But if the two vessels are not clamped simultaneously, the mechanical effect will depend on the order in which the two are clamped. Clamping the inferior vena cava without clamping the aorta will produce a great fall in blood pres- sure for very obvious reasons. Hence, if the vena cava be clamped even as much as fifteen seconds before the aorta, the rise in carotid pressure is not so great as when the two are clamped together. Vice versa should the aorta be clamped first and even a small interval of time intervene before the vena cava is clamped, the blood from the abdominal viscera will continue to flow into the thorax until the vis a tergo is exhausted and the pressure on clamp- ing the aorta will mount even higher, ofttimes so high as to cause heart failure.
These mechanical effects must, of necessity, affect the circula-
'* Zuntz, N.: Deutsch. med, Wochenschr., 1892, p. 109.
' Krogh: Skand. Arch. f. Physiol., xxiii, p. 179 et 8eq., 1910,
“ Bareroft: Lrgeb. d. Physiol., vii, p. 699, 1908.
'* Henderson: Amer. Journ. of Physiol., xxi, p. 126, 1908; xxiv, p. 66, 1909.
J. R. Murlin, Leo Edelmann and B. Kramer 83
tion through the lungs, the aération of the blood in other words, and in turn the exchange of oxygen and carbon dioxide between blood and outside air.
Suppose the two vessels be clamped exactly at the same moment. If the heart remains competent to empty itself against the increased pressure, the blood will of necessity circulate more rapidly through the lungs, unless the heart compensates by beating more slowly. On the other hand, if the heart be not competent, it may go into fibrillations or beat imperfectly, in which case blood will accumu- late in the lungs, producing passive congestion.
The former set of conditions should result in a decrease in the carbon dioxide in the blood because, the blood being exposed more often to the alveolar air, the carbon dioxide has more oppor- tunity to escape. The latter set of conditions should result in an interference with oxygen absorption with or without a decrease in the carbon dioxide.
EXPERIMENTAL PART.
,
Reasoning along these lines, the writers have undertaken to determine to what extent clamping of the abdominal aorta and inferior vena cava would alter the carbon dioxide content of the blood as it leaves the heart (carotid artery).
Method.
The method of procedure in the earlier, orienting experiments was as follows. Normal dogs were anaesthetized with chloretone. Urethane, which Porges and Porges and Salomon employed, has been avoided because it has been the experience in this laboratory that this drug excites the respiratory center of dogs much more than does chloretone. Morphine has likewise been avoided, except in one experiment, because it tends to increase the CO, in the blood.'®
When anaesthesia was fully established, the abdominal incision was made, the abdominal aorta exposed just above the origin of _ the coeliac axis, and hemostatic clamps were adjusted all ready to be closed at a signal. Before clamping, the pulse and respira- tion were usually counted and the control sample of blood was
16 Cushny: Textbook of Pharmacology, 1910, p. 221.
84 Metabolism after Clamping Abdominal Vessels
drawn. By these precautions one had knowledge of the condition of the animal just before the crucial operation. Then at a signal the two clamps were closed simultaneously, the closure of the vessels being immediately verified by examination. After the lapse of a varying interval of time, during which the pulse and respiration were counted frequently, the second sample of blood for analysis was drawn in the same manner as the first.!”
The blood analyses were made by the chemical method of Hal- dane'® using the apparatus devised by Brodie.’® All determina- tions were made in duplicate.
The results of the preliminary experiments on five animals are presented in Table I.?° It is evident, from an examination of these results, that a very great change in the aération of the blood is brought about by the occlusion of these vessels. How serious a matter this change is for the life of the animal is seen in the fact that Dog III survived for only twenty minutes after the obstruc- tion was accomplished. In all probability death was due to failure of the left ventricle. ;
Out of the four experiments in which a second analysis of blood was made, three exhibit a marked fall in the carbon dioxide of the ‘arterial blood. Two show, in addition, a material fall in the oxygen content.
It is not to be supposed that the blood alone loses carbon dioxide. Arterial blood, containing less than the usual percentage of COs, will carry away CO, by diffusion and this will continue the more rapidly the more the tension in the tissues exceeds that of the
17 The clamps which have been used for obstructing the vena cava are those known to surgeons as gastero-enterostomy clamps, fitted with rubber. Much difficulty has been experienced in placing a clamp on the aorta above the origin of the coeliac axis without serious rupture of the diaphragm and a number of animals were killed prematurely in this way. After this experi- ence, however, it was found that by exposing the coeliac axis itself and just above it applying the clamp in such a way as to include the arcuate fibres about the aorta within the clamp it was possible to effect a complete ob- struction without injury to other structures, Later a heavy wrapping cord was passed about the aorta at this point by means of a ligature carrier.
'* Haldane: Journ. of Physiol., xxii, p. 465, 1897-8; Haldane and Bar- croft: Ibid, xxxii, p. 232, 1902.
1° Brodie: Tbid, xxxix, p. 391, 1910.
10 Of, Proe, Soc. Exp. Biol. and Med., x, p. 174, 1918.
J. R. Murlin, Leo Edelmann and B. Kramer 85
arterial blood. The total amount of extra carbon dioxide appear- ing in the respiration after the clamps are applied, therefore, will depend upon the amount of the gas stored in the tissues. There is no perfectly satisfactory method of estimating the total carbon dioxide stored in the body at one time; hence it is impossible, from the percentages in the blood, to say just how much would escape in a given time. There is scarcely any doubt, however, that sufficient CO, has escaped from the animal’s body in each of the three experiments, to cause a considerable rise in the respira- tory quotient had it been determined for the period during which the vessels were clamped.
Respiration experiments.
Proof of the correctness of this view could only be had by repeti- tion of the experiments of Porges accompanied by blood-gas analy- ses. Should the carbon dioxide of the arterial blood fall as in the foregoing experiments, during a respiration period occurring imme- diately after clamping of the vessels, and showing a higher R. Q., the conclusion would be irresistible that the higher quotient was due simply to an alteration in the rate of discharge and not an alteration in the rate of production of this gas. Again if the factor of over-ventilation, in the sense of increased breathing, were a controlling one, a period of exaggerated breathing preceding _ the clamping off of the vessels should nullify the effect of clamping on the respiratory quotient. It was desired also to make blood- pressure determinations before and after clamping to ascertain if possible what change is produced in the rate of flow through the lungs.
Method of respiration experiments.
The apparatus used was a respiration incubator constructed for the special purpose of studying the respiratory metabolism in new-born infants. It consists of a copper chamber (303276 em.) placed inside a Freas electric incubator," by means of which the chamber can be kept at a constant temperature, and connected
*1 The Freas incubator can be purchased from Eimer and Amend, New York. The assemblage of apparatus as used in these experiments will be described in detail soon.
86 Metabolism after Clamping Abdominal Vessels
to a small Benedict” respiration machine, by means of which it is ventilated.
The chamber will accommodate a dog of 6-10 kgm. The entire cubic contents of the air circuit is about 80 liters and, with the subject inside, the air space is correspondingly less. This fact permits of the determination of the R. Q. by the well-known method of Benedict”* with an unusual degree of accuracy; for the reason that a small variation of temperature or of barometric pressure makes but a slight error in the oxygen determination. By making residual analyses at the beginning and end of each Jespiration period, even these errors may be eliminated. The apparatus has been thoroughly tested by burning alcohol inside it, with results, for the R. Q., very close to the theoretical value (0.666); namely, 0.661, 0.654, 0.662, 0.667.
For the blood-pressure readings the pulse-pressure instrument of Dr. C. J. Wiggers,** who very kindly instructed us in its use, was employed. Instead of the usual levers for graphic records, the maximal and minimal pressure tubes were connected directly to long mercury manometers and the maximum and minimum pressures were read off the millimeter scale directly. It was .* necessary .to have unusually long manometers on account of the very great rise in pressure which often, though not always, takes place on obstructing the vessels. In one of the earlier experiments of this serie’ the mercury was blown out by the excessive pressure and some 50 cc. of blood escaped from the carotid artery in the confusion which followed. The pressure readings were satisfac- torily obtained in only two experiments. In order to obtain the true pulse pressure and not the maximo-minimal pressures®* the readings were made while the respirations of the animal were inhibited temporarily by central stimulation of the vagus nerve.
All of the animals were anaesthetized with chloretone (usually given by stomach) and were tied lightly on an ordinary laboratory dog-board which had been sawed off to fit the respiration chamber, Once the dog is in the chamber and the latter sealed air-tight
* Benedict, F.G.: Amer. Journ. of Physiol., xxiv, p. 345, 1909,
* U.S. Dept. of Agriculture, Office of Experiment Stations, Bulletin 175, 1907. “ Wiggers, C.J.: Amer. Journ. of Physiol., xxx, p. 233, 1912. *% Wiggers, C.J.: loc. cit.
J. R. Murlin, Leo Edelmann and B. Kramer 87
the respiration experiment begins (after a short preliminary period of 10 to 25 minutes during which the chamber is being ventilated by the Benedict machine) on the second of the minute, by throwing a switch and turning a valve excluding the absorbers. Oxygen is admitted automatically throughout the period, but at the end the pressure is brought to the starting-point pressure by hand. The period was usually one hour in length.
I, Experiment on depancreatized dog showing higher R. Q. The first subject of this series was a depancreatized dog (Table II). That the animal was thoroughly diabetic is seen by the R. Q. obtained in two successive hours at the beginning of the experi- ments. The blood sample drawn at 3.12 p.m., just three minutes before clamping the vessels, showed a rather low percentage of both gases. It is well known that in diabetic subjects the CO, tension falls at times to a very low point. Another sample drawn fifteen minutes after clamping however shows both gases still farther reduceed—the carbon dioxide more than 10 per cent. Twenty-five minutes after taking this sample of klood the second respiration experiment began and continued for nearly two hours. The R. Q.s are decidedly increased, although the total respiratory exchange is very much reduced. Accompanying the higher R. Q. is a very great depression in the carbon-dioxide content of the blood. In view of the elevated blood pressure, which continued for twelve minutes at least after clamping the vessels, and the increased percentage of oxygen it seems likely that the true explanation of the lower percentage of CO. and hence of the higher R. Q. in this case is an increased aération of the blood by more rapid cir- culation through the lungs.
II. Experiment in which the R. Q. remained the same after clamp- ing the vessels. This was a normal dog, anaesthetized as usual. Blood pressures were determined before the respiration experi- ment. The dog had been fed the day before on dog biscuit, con- taining a high percentage of carbohydrate, and may have eaten some of it left over from the previous day, on the morning of the experiment. The R. Q. is rather high (probably for this reason) the first hour, but fell the second hour to a point more nearly within the range of a true niichtern value.
*6 Beddard, Pembrey and Spriggs: Lancet, 1903, I, p. 1366.
88 Metabolism after Clamping Abdominal Vessels
Upon clamping, the blood gases fell rapidly within the next fifteen minutes and the blood pressures which were high at first fell rather suddenly to very low levels. The R. Q. did not rise in the second respiration period and the CO: did not fall as in the previous experi- ment. The explanation of the low blood pressure was found at autopsy in the fact that the clamp on the aorta had caught a bit of the stomach and for this reason was not quite competent to hold the arterial pressure. It was possible, after sectioning the aorta below the clamp, to squeeze blood through. The animal therefore must have bled into his abdominal vessels until the arterial pressure reached a level which could no longer pass the obstruction.
This experiment proves the relatively greater importance of CO, than of O, in the blood in determining the external R. Q. Oxygen is not stored in the tissues in any quantity as is carbon dioxide; consequently a considerable change in ‘the O, content of the circulating medium does not affect the R. Q. materially.
III, Experiments in which the R. Q. fell after clamping the vessels. In the next experiment (Table IV) the dog exhibited a high R. Q. in the first period but instead of falling as is usual the further the time from feeding, it rose. Unfortunately the respiration rate was not recorded during these preliminary periods. It must be supposed however that there had been a considerable over- ventilation of the lungs and a consequent Auspumpung of COs; for upon clamping the vessels there was no fall, to speak of, in the CO; content of the blood within the first twenty minutes, and during the subsequent respiration period the CO, rose to 54.8 per cent while the oxygen fell. Again the CO, proves to be the determining factor; for its rise in the blood denotes a very considerable storage in the tissues and it is the holding back of this CO, which causes the R. Q. to fall to the extremely low level of 0.61 in the second period. The respiration apparatus was thoroughly tested imme- diately after this experiment and proved to be absolutely correct, giving a R. Q. with the alcohol flame of 0.667.
The results of the preceding experiments are fully confirmed in the following one (Table V) which was more complete. The dog had had no food since the previous day. The respiration rate was recorded during the preliminary respiration periods and established the cause of the high R. Q.s unquestionably to be the
J. R. Murlin, Leo Edelmann and B. Kramer 89
Auspumpung of CO:. The temperature of the respiration chamber during these periods was very close to the critical temperature at which dogs begin to pant. This fact together with a rather light state of anaesthesia probably accounts for the high rate of breathing. The pumping out of CO, in this case was so complete that upon clamping the vessels there was no reduction of the CO, in the blood, but instead a slight rise. In all probability this rise started from a still lower level the moment the dog was removed from the respiration chamber, for the respiratory rate fell at once to normal. In the respiration experiment which followed clamping of the vessels the R. Q., instead of rising, fell to an abnormally low point. The clamps were absolutely competent.
Severe congestion of the lungs with oedema was found at autopsy, a circumstance which explains the very low percentage of oxygen found at the end. The carbon dioxide was not so high, however, as in the previous expe iment. The rate of respiration declined rapidly and the dog was near death when removed from the chamber. a.
One other experiment, not reported in detail, was performed on a normal fasting dog, in which the R. Q.s in the preliminary periods were 0.72 and 0.85, while after clamping it was 0.67. The same explanations probably apply.
Two other experiments on depancreatized dogs were attempted _ but both died upon clamping of the vessels. Porges and Salomon succeeded in obtaining respiratory periods after ligation of the vessels in only four depancreatized dogs out of fifteen. There are obvious reasons why the animals do not survive longer. The strain upon the heart is tremendous. In several dogs, both normal and depancreatized, of this series, the heart failed at once and could not be revived. Aside from this the very rapid fall in the CO, percentage, which cannot be entirely compensated for by reduced rate (see Dog V, Table I) must produce a profound effect on all the higher brain centers. When, added to this, we consider 4 that the congestion of the lungs is such as to interfere with the _ absorption of oxygen, the wonder becomes that so many animals survive as long as they do.
90 ©Metabolism after Clamping Abdominal Vessels
Alkalinity of the blood.
Rolly?’ has established, by a new and much improved method, the fact that in dogs operated after the Porges procedure, the H-ion concentration of the blood is increased and the OH-ion concentration is diminished. This observation has been confirmed in a single examination of the blood reaction made in these experi- ments. From Dog IX, 20 ce. of carotid blood were drawn (10 ce. into each of two centrifuge tubes containing 0.5 cc. each of 0.1 per cent hirudin solution) before clamping the vessels and again just after drawing the last sample of blood for gas analysis. Ten ec. of the hirudin plasma titrated to the first pink color of phenol- phthalein with 4; NaOH required for the first sample 4.2 ec. and for the second 7 ce. The acidity, in other words, had nearly doubled, and yet in spite of this change the CO, was held back coincident- ly so as to reduce the R. Q. to 0.633! From this single observa- tion the indications are that this greater acidity (H-ion concen- tration) cannot be the only cause of the extra elimination of COs.
DISCUSSION OF THE FACTOR OF EXAGGERATED BREATHING.
This series of experiments was undertaken in the full expecta- tion of finding mechanical factors adequate to explain any altera- tion in the R. Q. which could result from sudden obstruction of the main vessels leading to and from the abdominal organs One such factor, exaggerated breathing, unquestionably is; for in these experiments it has been shown (Dogs VIII and IX) that increased respiratory activity may keep the quotient far above normal for at least two hours. That over-ventilation (exaggerated breathing) was present in the experiments of Porges and of Porges and Salomon may be inferred, in the absence of direct data, from the expressed assumption of Porges that after fifteen minutes of exaggerated breathing no more CO, could be pumped out. Fur- thermore it is the experience of this laboratory that urethane, which Porges and Salomon used, always excites the respiratory center (in dogs) and that it cannot always be controlled with moderate doses of morphine. In a former series of experiments in which the respiration apparatus was attached directly to the
*7 Rolly; Minch. med. Wochenschr., 1912, Nos, 22 and 23,
J. R. Murlin, Leo Edelmann and B. Kramer 1
trachea* urethane was tried and was given up for this very reason. In the original experiments of Porges and of Porges and Salomon, the higher quotients are doubtless due in part to this form of over- ventilation.
DISCUSSION OF THE FACTOR OF BLOOD FLOW THROUGH THE LUNGS.
That some other factor than exaggerated breathing may account for a great reduction in the CO, of the arterial blood and there- fore for a rise in the respiratory quotient after clamping of the ves- sels, is seen from the experiments with Dogs I, II and V (Table I) and Dog VI (Table II). In none of these experiments was any in- creased breathing observed. The blood-pressure determination with Dog VI gave a clue which it was hoped would lead to definite conclusions on the matter of blood-flow when the pulse pressures were more accurately determined in the experiments with Dogs VII and IX. Unluckily the leak in Experiment VII invalidated the blood-pressure findings, as a criterion of blood-flow in that experiment; for the mean pressure changed. In Experiment IX however it may be seen that the minute volume of blood-flow through the heart, and therefore through*the lungs has changed greatly after clamping and that this change is consistent with the change in blood gases.
According to the law of von Recklinghausen®® the amplitude of the pulse wave (pulse-pressure) at any given mean pressure is a measure of the systolic output, provided the distensibility of the arterial wall is constant. The product of the pulse-pressure | by the pulse-frequency is then a measure of the minute-volume.
There is no reason to suppose that the distensibility coefficient per se of the arterial system is in any way altered by the clamping of the aorta and vena cava. Therefore if the mean pressure remains about the same the product of pulse-pressure into pulse- frequency would afford a criterion of the effect of the operation on the blood-flow.
Referring to Table V it is seen that the pulse pressure just before clamping is three and one-half times as great as just after clamping. The mean pressure hasrisen slightly but not sufficiently
28 Murlin and Greer: Amer. Journ. of Physiol., xxvii, p. xviii, 1911. 29v. Recklinghausen: Arch. f. exp. Path. u. Pharm., lvi, p. 1, 1906.
92 Metabolism after Clamping Abdominal Vessels
to offset the difference in pulse pressure.*° The pulse frequency is considerably higher before the operation than after it. Hence the blood-flow through the lungs has been greatly reduced by the operation. The surprising thing is that such a change in the blood-flow should not have produced a greater effect on the ex- change of gases.
Two facts then stand out with some significance in the matter of blood-flow. In Experiment VI where the CO, in the blood fell rapidly after clamping of the vessels (while the O, rose), and the R. Q. as a consequence rose, the pulse pressure was maintained. Since there is no reason to believe that the pulse rate suffered any diminution (see Experiments I-V), the minute volume after clamp- ing was at least as great as before. In Experiment IX where the CO, in the blood rose slightly (while the O, fell) and the R. Q. as a consequence was falling (after the previous over-ventilation) the minute volume was distinctly less. These two facts are offered not as final proof but as evidence, consistent as far as it goes, that
R
8° y. Recklinghausen’s formula is A = ——_. X 1/k where A is ampli- . er) an dp/ p tude or pulse pressure, R is pulse volume, the expression (2), denotes
distensibility of the arterial wall, at the mean pressure and k is a constant determined by viscosity, diameter of vessels, etc. The pulse A (distensibility) I/k ; Making substitutions from Table V the pulse volume before clamping 22 x distensibility at 54 I/k
volume R then would be expressed by the formula
would be
; after clamping it would be
6 X distensibility at 72 “it Vk ’ Supposing the distensibility and the value of k to be the same the pulse
volume before clamping is more than three times the value after clamping. The minute volume would be found by multiplying the value of the pulse- volume, or systolic output, by the pulse-frequency. Taking 210 as pulse- frequency just before clamping and 180 just after, the minute volume proves to be less than one-third its former value. In all probability this differ- ence is too great; the point is to show that distensibility or the value of k would have to change a great deal to offset the difference in pulse pres- sure observed.
J. R. Murlin, Leo Edelmann and B. Kramer 93
the altered rate of blood-flow through the lungs is an important factor in determining the CO, (and O2) content of the blood and therefore in explaining the altered respiratory exchange.
CONCLUSION.
Whether one or both of the factors discussed above are control- ‘ing, there can be no doubt as to the significance of the blood-gas analyses. In each instance the blood-gas changes are consistent with the mechanical explanation of the altered respiratory quo- tients after clamping the vessels. Where the R. Q. rose (Experi- ment VI) the CO, of the blood fell; where the quotient remained stationary (Experiment VII), the CO, did not change; and where the R. Q. fell (Experiments VIII and IX), the CO, in the arterial blood rose. Clamping off the blood from the abdominal organs therefore does not alter the character of the metabolism, and the © experiments of Porges and of Porges and Salomon have no bear- ing on the problem of the oxidation of sugar. .
94 Metabolism after Clamping Abdominal Vessels
TABLE I. Dog. I. 8Skgm. March 22, 1913. Chloretone per rectum.
BLOOD
TIME EVENT PULSE bagi Se
Oz CO: p.m. per cent| per cent 3.20 | 4.3 cc. carotid blood drawn 15.48 438.6 3.25 | Vessels clamped simultaneously 3.30 96 36 3.40 144 30 3.45 138 30 3.54 | 4.2 cc. carotid blood drawn ' 15.52) 22.53 3.55 | Clamps removed 4.00 120 35
Dog IIT. 12kgm. April 10,1913. Chloretone intraperitoneally.
2.15 | 4.4 cc. carotid blood drawn 17.10) 38.35 2.17 | Vessels clamped simultaneously 66 | 35 2.20 120 24 2.35 % 102 |, 12 2.40 ue 102 30 2.41 | 3.2 ce. carotid blood drawn 17.16) 37.47 2.47 | Clamps removed ht
Dog III. 7.5kgm. April 12, 1913. Chloretone anaesthesia.
2.08 108 | 30 2.20 114 24 2.30 114 24 2.35 120 | 15 2.36 | 4.5 cc. carotid blood drawn 18.85, 42.01 2.40 120 | 24 2.46 | Vessels clamped simultaneously; heart stopped 2.58 24 2.59 | Artificial respiration ' 3.00 96 | 30 3.06 120 | 54 3.10 | Dog died; clamps on only 20 minutes; cause of death not apparent
ane - — en —
J. R. Murlin, Leo Edelmann and B. Kramer 95
TABLE I.—Continued. Dog IV. 9kgm. April 19, 1913. Chloretone by stomach.
BLOOD TIME i EVENT PULSE * ante 6 vara Oz COz
p.m. per cent) per cent 1.50 138 35
2.25 — | 120 72 ‘2.32 | 4.4 cc. carotid blood drawn 138 66 | 19.52) 39.42 2.38 132 60
2.40 | Vessels clamped simultaneously
2.43 104 96
2.52 126 64
3.02 120 78
8.15 120 80
3.25 120 72
3.35 126 72
3.43 | 4.35 cc. carotid blood drawn 17.09) 24.28 3.44 | Clamps removed
3.45 120 60 |
Dog V. 10 kgm. May 10, 1913. Morphine subcutaneously. Chloretone by stomach.
2.238 96 34
3.15 120 32
3.17 | 4.15 ce. carotid blood drawn 13.32 51.06 3.20 | Vessels clamped simultaneously
3.22 102 16
3.27 120 16
3.33 120 14
3.48 120 14
4.03 120 14
4.13 120 18
4.18 - 120 16
4.22 | 4.3 ec. carotid blood drawn 11.60, 34.16 4.23 | Clamps removed 120 | 24
4.29 108 24
4.39 108 24
Metabolism after Clamping Abdominal Vessels
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THE SEPARATION OF ¢d-ALANINE AND d-VALINE.
By P. A. LEVENE anp DONALD D. VAN SLYKE,
(From the Laboratories of the Rockefeller Institute for Medical Research, New York.)
(Received for publication, August 23, 1913.)
In the ester method of protein hydrolysis the esterifiable amino- acids are separated by distillation into two fractions, a higher boiling containing aspartic and glutaminic acids, phenyl alanine, and serine, and a lower boiling fraction containing proline, /-leu- cine, d-isoleucine, d-valine, d-alanine, and glycocoll. For several years we have been trying to devise methods to approximate as nearly as possible a quantitative separation or determination of the six amino-acids composing the latter mixture.
Proline, unlike the other members of this fraction, is very sol- uble in alcohol,’ and is partially separated from them by alco- holic extraction. The extract,,however, usually consists of about two-thirds proline and one-third of a mixture of other amino- acids which have gone with the proline into solution in the alcohol.
- Proline, however, contains no primary amino nitrogen, while all
the nitrogen of the other acids of this ester fraction is in the form of primary amino groups. Therefore, a determination of the total and the primary amino nitrogen,’ respectively, in the extract per- mit one to calculate accurately the amount of proline, which is indicated by that of the non-amino nitrogen.
The other five amino-acids can be distributed by fractional crys- tallization among subfractions the composition of which varies
, greatly according to the proportions in which the different acids
are present. As glycocoll and alanine dissolve at room tempera- ture in only four parts of water, while the other three, particularly
1 Fischer: Ber. d..deutsch. chem. Gesellsch., xxxix, p. 530, 1906. *Van Slyke: Quantitative Determination of Proline obtained by the
4! Ester Method in Protein Hydrolysis, this Journal, ix, p. 205, 1911; Quan-
titative Determination of Aliphatic Amino Groups, this Journal, ix, p. 185,
1911 and xii, p. 275, 1912.
103
104 Separation of d-Alanine and d-Valine
the leucine and isoleucine, are much less soluble, one can usually obtain by crystallization the greater part of the mixture in two fractions, a comparatively insoluble one consisting of the leucine and isoleucine, together with much of the valine, and a very solu- ble fraction containing glycocoll and alanine. For the quantitative determination of the proportions in which leucine, isoleucine, and valine are present in the less soluble fraction we have already published methods which have been utilized with “satisfactory results.’ More recently we have described the separation of gly- cocoll from alanine in the more soluble subfraction by means of glycocoll picrate, which is difficultly soluble in cold water.‘ Besides the leucine-isoleucine-valine and the glycocoll-alanine crystallized fractions, however, one usually obtains another, in- termediate between these two, containing alanine and valine in such proportions that they cannot be separated by crystalliza- tion. This paper presents a method for the separation of the alanine and valine of this intermediate fraction. One can now determine all the six amino-acids from the lower boiling ester fraction with a fair degree of accuracy. This does not mean that they are completely regained in the amounts in which they are present in the proteins. Losses which prevent this still occur in the esterification and distillation “of the esters. The uncertain- ties, however, which were formerly connected with the separation — of these amino-acids after the distillation, are now reduced comparatively small proportions. We have determined the following data, on which is based the method for separating valine from alanine, and from glyeocoll in — case this also should occur in the intermediate fraction.
Data on which the separation is based,
d-Alanine in the presence of 10 per cent sulphurie acid is pre- cipitated by phosphotungstic acid as a crystalline salt which contains approximately 14 parts of phosphotungstic acid to 1 of |
* Levene and Van Slyke: this Journal, vi, p. 391, 1909. Abderhalden and — Weil have recently isolated from nerve tissue a third leucine isomer. We — did not find evidence of it in casein or edestin; but if it proves to be a general constituent of the proteins still further development of special methods for this fraction will be necessary, Zeitschr. f. ec rat Chem., Ixxxiv, p. 39, 1913.
‘Levene and Van Slyke: this Journal, xii, p, 285, 1912.
P. A. Levene and D. D. Van Slyke 105
alanine. At 0° about twenty-four hours are required for precipi- tation of the maximum amount of alanine. The presence in solu- tion of about 20 grams of phosphotungstie acid (in excess of the amount precipitated with the alanine) per 100 cc. of solution is required to insure most complete precipitation. Under these conditions the amount of alanine left in solution at 0° in 100 cc. of mother liquor is 0.15 gram. The concentration of free phos- photungstic acid can be increased up to at least 70 grams per 100 ec. of solution without either increasing or diminishing to a sig- nificant extent the solubility of alanine phosphotungstate.
d-Valine has under the same conditions the much greater solu- bility of 1.2 grams per 100 cc. Valine phosphotungstate shows, under proper conditions, very little tendency to form mixed crys- tals with alanine phosphotungstate. In case a mixture of the two is obtained, one can readily separate them by reerystallization from a solution containing 10 per cent of sulphurie and 20 per cent of phosphotungstie acid.
The solubilities of the phosphotungstates of both alanine and valine are very dependent upon the concentration of sulphuric acid present.
Glycocoll is precipitated under the same conditions as d-alanine, only 0.2 gram of glycocoll remaining in 100 ce. of mother liquor.
Lead acetate, recently recommended by Benedict and Murlin® for the removal of phosphotungstic acid from solutions containing amino-acids, is the most satisfactory reagent which we have found for freeing both alanine and valine from sulphuric and phospho- tungstic acids. The precipitation of phosphotungstic acid is quan- titative, and the small amount of lead sulphate remaining dis- solved in the filtrate is reatlily removed by addition of an equal volume of alcohol. Five per cent, and sometimes even more, of the amino-acid present are usually adsorbed by the heavy pre- cipitate, but the loss is less than when barium hydrate is used, and the amino-acid regained after removing the excess lead as sulphide and concentrating the solution to dryness contains less than 1 per cent of ash.
Natural leucine is precipitated by concentrated solutions of phosphotungstic acid, the precipitate being redissolved by suffi- cient excess of the acid, as found by Levene and Beatty. Leucine
5 Proc. Soc. Exp. Biol. and Med., 1912.
106 Separation of d-Alanine and d-Valine
may interfere with the purification of alanine as the phospho- tungstate, however, and should be removed, either by crystalliza- tion or by precipitation as the lead salt® before the separation described below is begun.
Dilute methyl] and ethyl alcohol are unsuitable solvents for the recrystallization of valine when even a small proportion of alanine is present; because the relative solubilities of the two amino-acids in water are reversed in both alcohols, in which alanine is much less soluble than valine. This is the case to a less marked extent with acetone, and it is, therefore, better suited to throw valine out of water solution in the presence of alanine. If to 100 ec. of water at 20° one adds 200 ce. of 80 per cent acetone, the resulting solution will dissolve 3.2 grams of alanine and 3.4 of valine. The solu- bility relations are such that one can add 3, 4, 5, 6, or 7 volumes of 80 per cent acetone with nearly the same effect. A mixture of 100 cc. of water and 700 ce. of 80 per cent acetone dissolves 2.5 grams of alanirle and 3.4 of valine. Consequently, as the results are within a wide range independent of the volume of solu- tion added, 80 per cent acetone affords a convenient means for throwing valine out of water solution in the presence of small amounts of alanine.
Because of the fact that alanine is much less soluble than valine in ethyl and methyl] alcohol, especially the latter, it was thought
that valine could, perhaps, be extracted from a mixture of the two
amino-acids by means of methyl alcohol. It was found, however, —
that it was impossible to extract all the valine without also dis- *
solving a large proportion of the alanine.
Precipitation and purification of alanine as phosphotungstate.
The mixture of valine and alanine should preferably contain not over 50 per cent of valine. If more is present, part can readily be removed by recrystallizing from water, in which valine is much less soluble than alanine.
It is advisable, because of the appreciable solubility of alanine phosphotungstate, to precipitate it from as small a volume of 10 per cent sulphuric acid as will hold the valine in solution. In order to obtain ut once alanine phosphotungstate free from valingy
* Levene and Van Slyke: this Journal, vi, p. 391, 1909.
bi eral.
P. A. Levene and D. D. Van Slyke 107
the volume of solution must be as great as 100 cc. for each gram of valine present. If the alanine phosphotungstate is recrystallized, however, one need use but 30 to 40 ec. for each gram of valine, recrystallizing once from a similar volume of fresh solution. One thus completes the separation, using in all only 60 to 80 per cent of the volume of solution required when one does not recrystallize, and one is also somewhat more certain of the absolute purity of the alanine. The process which gives the most satisfactory separa- tion is the following:
The mixture of alanine and valine is dissolved in a hot solution which contains 10 grams of sulphuric acid per 100 cc. The volume of this 10 per cent sulphuric acid used should be 30-40 ec. for each gram of valine which analysis of the mixture indicates can, as a maximum, be present. In the hot solution one further dissolves enough purified phosphotungstic acid to combine in the ratio of 14:1 with the maximum amount of alanine which previous anal- ysis has indicated can be present in the mixture, and in addition leave 1 gram of excess phosphotungstic acid for every 5 ee. of the 10 per cent sulphuric acid used. The use of a greater excess of phosphotungstic acid does not interfere with the separation, but leaves one an unnecessarily large amount to remove at the end of the operation. The solution prepared as above directed is placed in a refrigerator at 0° and allowed to remain there for at least
___ twenty-four hours.’ In case the volume of the solution is large,
time must be allowed for it to cool before beginning to count the period allowed for crystallization. The precipitate separates in large, transparent crystals, which form a solid layer about the walls and bottom of the flask. When sufficient time has been allowed for the separation, the supernatant solution is decanted off as completely as possiblé. The crystals are then redissolved by heating with a volume of 10 per cent sulphuric acid equal to that originally used. Phosphotungstic acid, in the ratio of 1 gram to each 4 or 5 cc. of 10 per cent sulphuric acid used, is then dissolved in the hot solution, and the alanine phosphotungstate is again allowed twenty-four hours at 0° to crystallize. The supernatant solution is again decanted, and the crystals are washed with suction with a small volume of an ice-cold solution containing
10 per cent of sulphuric and 20 per cent of phosphotungstic acid.
7 If only an ordinary ice box, which usually gives a temperature of 8°, is available, the flask should be immersed in ice water.
108 Separation of d-Alanine and d-Valine
Determination and isolation of the precipitated alanine.
The alanine phosphotungstate is at once dissolved in hot water, where it forms a solution that is usually somewhat turbid. It is diluted in a measuring flask to such a volume that 10 cc. contain from 50 to 100 mgms. of alanine, and aliquot parts are used for determination of the nitrogen present. The determination is most conveniently performed by the nitrous acid method for determina- tion of amino nitrogen.’ If the micro-apparatus (cf. p. 121) is used 2 ce. of solution are sufficient; with the larger apparatus one uses 10 cc. The determination can also be done according to Kjeldahl, — although in this case it is necessary to draw air through the mix- _ ture, while it is digesting with sulphuric acid, in order to prevent the violent bumping which the precipitated tungstic acid causes.° It is preferable to base the calculation of the amount of alanine present on the nitrogen determination rather than on the sub- stance actually isolated, because, when the phosphotungstic acid is removed with lead, the bulky precipitate of lead phosphotung- state adsorbs several per centof the alanine present, and the amount actually recovered is only 90-95 per cent ofthat in solution before the removal of the mineral acids. To the amount of alanine calculated from the nitrogen determination one may add a solu- bility correction for the amount dissolved in the total volume of solution from which the alanine was precipitated and recrystal- — lized. This amount is calculated on the basis of a solubility of — 0.15 gram of alanine per 100 ce.
The remainder of the solution, after the portion for the analysis has been removed, is washed into a Jena beaker and heated to boiling. A 20 per cent solution of neutral lead acetate is added in portions until an excess is present, and can be detected, by means of the sulphuric acid test, in a drop removed from the sur- face of the solution in the beaker. The heavy precipitate of lead sulphate and phosphotungstate is filtered with suction and washed thoroughly with water. The filtrate is concentrated to a volume
* This Journal, ix, p. 185, 1911, and xii, p. 275, 1912. As there is so much mineral acid present, it is advisable to add, to the nitrous acid solution in the apparatus, enough 4 or 5 Nn NaOH to nearly neutralize the sulphuric acid before the solution containing the latter is run in.
* Denis: this Journal, viii, p. 427.
bir a
P. A. Levene and D. D. Van Slyke 109
of about 50 ec. for each gram of alanine present, and mixed with an equal volume of 95 per cent alcohol. This precipitates a small amount of lead sulphate which had remained, owing to its slight but appreciable solubility in water. The solution is allowed to stand on the water bath for an hour or more to complete the pre- cipitation, the sulphate is filtered off, and the excess of lead in the filtrate is removed with hydrogen sulphide. The lead sulphide is washed with water through which H.S has been bubbled, and the filtrate is concentrated, preferably in vacuum, to a small volume. It is then transferred to a Jena glass evaporating dish and the concentration continued on the water bath until all the visible liquid has been evaporated. The drying is completed in a vacuum desiccator over sulphuric acid and potassium hydrate. It is not advisable to try to drive off with heat the last traces of water and acetic acid, for this is likely to somewhat discolor the substance. The product, dried in vacuum, is perfectly colorless, nearly ash- free (if pure reagents have been used), and free from valine. Besides the alanine isolated as above described, a small amount, left in solution when the alanine phosphotungstate was precipitated, is later obtained from the mother liquors of the
valine.
In case the original valine-alanine mixture contained glycocoll, the latter will now be found with the alanine, from which it can be separated as the picrate, according to the method described by us.??
Determination and isolation of the valine.
The decanted filtrates and the washings from the alanine phos- photungstate are diluted to a definite volume and the amino nitro- gen determined in an aliquot part, in the manner described for the alanine solution. A special blank determination to ascertain the correction for the reagents should be made, using as the con- trol solution 10 per cent sulphuric acid instead of water, as the presence of so much mineral acid increases the correction. The phosphotungstic and sulphuric acids are removed with lead acetate, as in the isolation of alanine, and the valine solution, free from mineral acids and bases, is concentrated on the water bath until the valine begins to erystallize at the surface. Two or three
10 Levene and Van Slyke: this Journal, xii, p. 285, 1912.
110 Separation of d-Alanine and d-Valine
volumes of 80 per cent acetone are then added, and the mixture is rinsed, using more 80 per cent acetone, into a flask. This is stoppered to prevent evaporation of the acetone, and allowed to stand over night while the valine crystallizes. The latter is filtered, washed with 80 per cent acetone, and thus obtained free from alanine in a yield of 80 to 85 per cent of the amount present.
The filtrate from the valine contains the small amount of ala- nine which escaped precipitation by phosphotungstic acid, and an amount, usually about equal, of valine, which remained in solution in the dilute acetone. The filtrate is concentrated to dryness, weighed, and the alanine and valine separated with phosphotung- stic acid as before. This second crystallization makes the separa- tion practically quantitative.
When refrigeration facilities do not enable one to keep the solu- tions at 0° during the entire period while the alanine is being pre- cipitated, one can let the solutions stand over night at room temperature, and then place them in ice water for several hours, stirring them occasionally to complete the crystallization at 0°. The precipitation is nearly, though not quite, so complete as when the solution is kept at 0° for the entire period.
Working at room temperature entirely, one can precipitate at, least 75 per cent of the alanine in purity, using one-half the vol- ume of solutions given in the above directions.
Purity of reagents. a
Because of the large amounts of lead acetate and phospho- tungstic acid used, both reagents must be pure or the amino- acids obtained after their use will be accompanied by ash. The lead acetate should leave no residue after precipitation of a solu- tion with hydrogen sulphide and evaporation of the filtrate to dryness. We have had no difficulty in obtaining good lead acetate from the manufacturers. The phosphotungstic acid should leave no residue after precipitation with pure lead acetate and evapora- tion of the filtrate. We,purify the commercial phosphotungstic acid by Winterstein’s method. ‘The acid is dissolved in a small amount of water, from which itis shaken out with ether. With the latter it forms an oily solution much heavier than water. The ether solution is washed several times with water, and the ether is driven off on the water bath. The produet is not hydroscopie, and forms a colorless solution,
P. A. Levene and D. D. Van Slyke Ii
EXPERIMENTAL. Analysis of materials.
d-Alanine was obtained from hydrolyzed silk by the ester method. The glycocoll accompanying the alanine in the amino-acids ob- tained from the low boiling fraction of esters was removed with picric acid," and the d-alanine was purified by recrystallization from dilute alcohol. It gave the following figures on analysis.
Substance, 0.1195 gram; CO:, 0.1764 gram; H2O, 0.0825 gram.
Substance, 0.0909 gram; nitrogen gas at 21°, 763 mm. (nitrous acid method), 25.20 cc.
Substance, 0.1817 gram; solution (containing 1.3 mols. HCl), 2.4910 grams; concentration, 7.29 per cent; sp.gr., 1.03; rotation in 2 dm. tube with yellow light from a spectroscope, +2.07° +0.01°.
Substance, 0.1422 gram; solution in 20 per cent HCl, 2.5810 grams; con-
centration, 5.51 per cent; sp. gr., 1.087 at 25°; rotation in 2 dm. tube, +1.64°
*(0.01°. Calculated for Found CesH70.:N: Se RUS 40.25 40.41 Be... a ae es 7.42 7.92 TS See 15.74 15.73
[a], with 1.3 mols. HCl.. .. +9.77° +10.30° (Calculated for HCl salt. )!? [a], with 1.3 mols. HCl.. ..+13.78° Calculated for amino- acid. [a] with 20 per cent HCl. .. +9.72° Calculated for HCl salt. [a]> with 20 per cent HCl. . +13.69° Calculated for amino- acid.
From the above figures it is apparent that the d-alanine was analytically pure and as free from dl-alanine as one can usually prepare it from hydrolyzed protein. The rotation is, as stated by Emil Fischer, practically unaffected by the amount of excess hydro- chloric acid present.
d-Valine was prepared from casein by esterification and the use of our lead method. The preparation gave the following figures on analysis: _ .
Substance, 0.1203 gram; COs, 0.2260 gram; HO:, 0.1013 gram.
Substance, 0.1081 gram; nitrogen, 22.9 cc. at 25°, 762 mm. (nitrous acid method).
1 Levene and Van Slyke: this Journal, xii, p. 285, 1912. 2 E. Fischer: Ber. d. deutsch. chem. Gesellsch., xxxix, p. 464. 8 Levene and Van Slyke: this Journal, vi, p. 391, 1909.
112 Separation of d-Alanine and d-Valine
Substance, 0.1510 gram; solution in 20 per cent HCl, 2.6240 grams; sp. gr., 1.10; rotation in 2 dm. tube with yellow light, +3.28° +0.01°.
Calculated for Found: CsHi102N: ora ecko esc cnc Once 0 bcs eee pi 21 51.24 lt Lp II 9.43 9 .47 |. ee a eee eh 11.97 11.96 MR +25 .93° +28 .80°
The valine was analytically pure. The rotation was lower than that obtained by Fischer for synthetic d-valine,“ but is as high as one usually obtains in the natural product after acid hydrolysis. As the valine obtained by acid hydrolysis of proteins usually has a rotation of +24° to +26°, the use of the above material gives one more nearly the conditions actually met in hydrolysis work
than would employment of the optically pure synthetic substance.
Composition of alanine phosphotungstate.
Levene and Beatty found that alanine combines.with phospho- tungstic acid to form a crystalline salt." We have prepared the salt as nearly pure as possible in order to determine its composi- tion. Preliminary preparations showed that the ratio of alanine to phosphotungstic acid was approximately 1:14. We dissolved the two constituents in this ratio (0.5 gram of alanine and 7
grams of phosphotungstie acid) in 15 cc. of normal hydrochloric _
acid, and let the solution stand over night while the salt crystal- lized. The crystals were filtered on a clay plate and dried over solid potassium hydrate until the chloride reaction disappeared. The product was further dried in a vacuum at 100°. The pro- portion of alanine was then determined by estimation of the amino nitrogen with nitrous acid. The results were:
a RRS. SS “D 1.036 per cent. PEED, +. cots 5 MEMES + 6 SMEs ss MME ov aEWe 6.57 per cent. RRR SSSR OB SRR SA 93.33 per cent. pemeumlaning: PT Ags... .uss+-cctuesscddaes =1:14,1
The salt forms with water of crystallization, The air-dried substance loses 3.8 per cent of its weight when dried in vacuum at
100°, and the anhydrous salt when exposed to air takes up a sim-
4 Ber, d. deutach. chem, Gesellach., xxxix, p. 2320. * Levene and Beatty: Zeilschr. f. physiol, Chem., xlvii, p. 149, 1906,
"
P. A. Levene and D. D. Van Slyke 113
ilar weight of moisture. This corresponds to approximately 3 molecules of water for 1 of alanine, the ratio, 1 alanine: 3 H,O, requiring 3.99 per cent water.
Solubility of d-alanine phosphotungstate in varying concentrations of sulphuric acid.
Solutions each containing 0.250 gram of d-alanine, 5 grams of phosphotungstic acid, and varying amounts of sulphuric acid were made up to 10 cc. volume and left at 0° for forty-eight hours. The solutions were then decanted through dry filter papers into the 10 cc. burette of the aminometer (apparatus for determination of amino nitrogen) described in this Journal, xii, p. 275. The nitro- gen in the measured volume of filtrate was determined by the nitrous acid method, and from the result the amount of alanine present in 100 cc. of filtrate was calculated. The percentages of sulphuric acid indicate grams per 100 ce. of solution.
TABLE I. CONCENTRATION He2SO« | ALANINE IN 100 cc. OF FILTRATE
per cent grams 3 0.56
4 0.38
5 0.36
6 0.30
8 0.19
10 0.14 10 0.15 12 0.16 14 0.18 16 0.18
As 0.250 gram of alanine combines with 3.5 grams of phospho- tungstic acid, the excess of the latter in solution was 1.5 grams, or 15 grams per 100 ec. ‘The above table indicates that, in the - presence of this excess of phosphotungstic acid, sulphuric acid decreases the solubility of alanine phosphotungstate, the maximum
effect of the sulphuric acid being exerted in 10 per cent concen- tration. Under these conditions the solubility of alanine at 0° is only 1 gram per 700 ec. of solution.
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVI, NO. 1.
114 | Separation of d-Alanine and d-Valine
Effect of the concentration of free phosphotungstic acid on the solu- bility of d-alanine phosphotungstate in 10 per cent sulphuric acid at 0°.
Portions of 50 mgm. of d-alanine were dissolved in 5 ce. each of 20 per cent sulphuric acid in test tubes, and varying amounts of a solution containing 2 grams of phosphotungstic acid per cubic — centimeter were added to the different solutions, all of which were then made up to 10 cc. with water allowed to stand thirty hours at 0°. The amounts of alanine remaining in solution were then determined as described in the preceding section. The excess phosphotungstic acid was estimated by subtracting from the amount added the 0.7 gram combining with 0.05 gram of alanine,
TABLE II.
P PTA ADDED PER 100 cc. ee ee mii ae sindhond = grams . grams
15 8 0.22
20 13 0.21
25 18 0.18
30 >. 2 0.15
40 Bh 33 0.14
60 53 0.15
80 73 0.13
It is evident that about 20 per cent of free, excess phospho- tungstic acid in solution insures a maximum precipitation of the alanine at 0°. At 20°, in the presence of 20 per cent phospho- tungstic acid solution, the solubility is 0.38 gram per 100 ce.
Time required for the precipitation of d-alanine phosphotungstate at 0°.
Portions of 0.05 gram of d-alanine were dissolved with 3 grams of phosphotungstie acid in 10 ce. of 10 per cent sulphuric acid, — The solutions were left at 0° for varying periods, at the end of — which they were decanted through dry filters, as in the solubility determinations described in the preceding sections, and the nitrogen remaining in solution was determined, .
—
: : | / r
P. A. Levene and D. D. Van Slyke 115
TABLE III. TIME ALLOWED FOR PRECIPITATION ALANINE IN 100 cc. FILTRATE
hours grams
3 0.21
6 : 0.20
15 0.17
22 0.16
40 0.14
While the greater part of the alanine is precipitated in three hours, over twenty are required for the complete attainment of solubility equilibrium.
Solubility of dl-alanine in 10 per cent sulphuric acid containing varying concentrations of phosphotungstie acid.
The results in the following table show that the phosphotung- — state of dl-alanine is more than twice as soluble at 0° that of
d-alanine. The conditions of the solubility tests were t e same as those of the foregoing experiment. | TABLE IV. PTA apogp vem 100 cc, | Bxoms PTA wuesr7 ome ie grams grams 10 3 0.43 20 13 0.35 30 23 0.35 60 53 0.37 80 73 0.37
Solubility of d-valine phosphotungstate in varying concentrations of sulphuric acid at 0°.
Portions of 0.4 gram of valine were dissolved with 6 grams of phosphotungstic acid each in 5 cc. of 2, 4, 6, 8, 10, and 12 per cent sulphuric acid respectively. The amount of phosphotungstic acid was found by a separate experiment to be a sufficient excess to depress the solubility of the valine to its minimum. The solu- tions were cooled to 0° and kept at that temperature for three days. The solubilities of the valine were then determined as in
116 Separation of d-Alanine and d-Valine
the similar experiments with alanine. The solution with only 2 per cent of sulphuric acid showed no precipitate. The others showed crystalline precipitates varying in bulk with the concen- tration of the sulphuric acid. The percentages of sulphuric acid indicate grams per 100 ce.
TABLE V. HeSO. VALINE IN 100 cc. OF FILTRATE per cent ; grams 4 we 4 4.95 6 2.78 8 1.87 10 1.21 12 0.88
At 20° the solubility in 10 per cent sulphuric acid in the presence of an excess of phosphotungstic acid is 3.4 grams per 100 ce.
Solubility of valine and alanine in varying concentrations of acetone.
As stated before, acetone was found a better agent than methyl or ethyl aleohol for throwing valine out of solution in the presence of the small proportions of alanine that escape precipitation with the main crop of alanine phosphotungstate. To ascertain the optimum proportion of acetone to add to the water solution of valine in order to cause it to crystallize most completely with- out carrying down alanine also, the solubilities of the two amino- acids in varying concentrations of acetone were determined at 20°. Fifteen cubic centimeters of the solvent were in each case shaken two hours with an excess of amino-acid, and 10 ee. of the filtered solution evaporated in a weighed dish.
TABLE VI. ACETONE | satr a IN ee ie ant IN a taal cent | y grams grams
100 0,008 0.002 90 0,028 0.012 80 0.164 0.097 : 66.7 0,560 0.402 50 1,290 1.315
we ee
P. A. Levene and D. D. Van Slyke 117
The following table shows that when 80 per cent acetone, in the ratio of from 2 to 7 volumes, is added to 1 volume of water, the solvent power of the water for alanine and valine is reduced to a point which remains nearly the same, whether 2, 3, 4, 5, 6, or 7 volumes of the 80 per cent acetone are added. The decrease in solubility caused by increasing the percentage of acetone is ap- proximately compensated by the increase in volume.
TABLE VII. AMINO-ACID DISSOLVED IN MaceronE ACETONE IN MUON MIXTURE. q ag a ro 100 cc... | THE MIXTURE ; OF WATER Alanine Valine Alanine Valine cc. per cent grams grams | grams grams 200 53.3 1.08 1.16 3.24 3.48 300 60.0 | 0.71 0.85 2 84 3.40 400 64.0 052 | 0.67 2.60 3.35 500 66.7 0.40 0.56 | 3.40 3.36 600 68.6 0.35 0.48 | 2.45 3.36 700 70.0 01 | 04 | 2.48 3.44
The solubilities in the third column were graphically inter- polated from those given in the preceding table.
Separation of a mixture of d-valine and d-alanine.
The following separation serves as an example of the applica- tion of the method.
One gram each of d-valine and d-alanine was dissolved in 35 ce. of hot 10 per cent sulphuric acid (prepared by diluting 10 grams of acid to 100 cc.) with 23 grams of purified phosphotungstie acid. The solution was allowed to stand till it had cooled to room tem-
perature, and was then placed in a refrigerator at 0° for twenty- four hours. The crystals which had separated formed a solid
layer about the walls and bottom of the flask. The supernatant liquid was decanted off, and the crystals were redissolved on the
_ water bath with 35 cc. of fresh 10 per cent sulphuric acid. Eight
grams of phosphotungstiec acid were then dissolved in the hot solu-
_ tion, which was cooled and placed in the refrigerator for twenty-
four hours as before. The mother liquors were again decanted
off, and the crystals were quickly washed on a suction funnel with
118 Separation of d-Alanine and d-Valine
several small portions of a solution containing 10 grams of sul- phurie acid and 20 grams of phosphotungstic per 100 cc., the washing solution being at a temperature of 0°.
Alanine. The crystals were transferred as completely as pos- sible with a spatula from the funnel to a Jena beaker. A small residue adhering to the funnel and filter paper was washed into the beaker with hot water, and the flask in which the crystals had formed was also washed out with hot water, in order to obtain a few crystals of alanine phosphotungstate which the previous washing had not removed to the funnel. Enough water was added to the alanine phosphotungstate to bring the volume to 75-100 ec., and the beaker was covered and heated on the water bath until the crystals were dissolved to a slightly turbid solution. The latter was transferred to a 150 ec. measuring flask and diluted to the mark. Two cubic centimeters of the solution used for deter- mination of amino nitrogen in the micro-apparatus gave 3.37 cc. of nitrogen gas at 25°, 758 mm., indicating 0.1398 gram of nitrogen, or 0.889 gram of alanine in the entire solution. The remaining 148 cc. of solution were treated as described on pp. 108 and 109 to remove phosphotungstic and sulphuric acids. Thealanine regained weighed 0.83 gram, and gave the following figures on analysis.
Substance, 0.1222 gram; ash, 0.0013 gram; substance, ash-free, 0. 1209 gram; CO:, 0.1777 gram; H,O, 0.0855 gram.
Rotation in 20 per cent HCl: Substance, 0.1029 gram=0.1016 aslo solution, 1.9060 grams; concentration, 5.33 per cent; sp. gr., 1.1; rotation in 1 dm. tube, +0.80°.
Calculated for Found: d-alanine ee ae OR 40.10 40.40 ae Sa COU Oe Oe 7.92 7.92 NE RR a +13.7° +0,2° +13.7°
It is evident that the precipitate consisted of pure alanine phos- photungstate. The correction for the solubility of alanine‘as phos- photungstate in 70.ce. of solution under the conditions of precipi- tation and recrystallization is 0.70 * 0.15 = 0.105 gram of alanine. Adding this to the 0.889 gram precipitated gives 0:994 gram of alanine found to be present out of the 1 gram originally added,
Valine. The filtrate and washings from the alanine phospho- tungstate were diluted to 150 ee. and 2 ce. of the solution taken
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P, A. Levene and D.