THE ORIGIN AND FATE OF UREA IN THE DEVELOPING HEN'S EGG BY JOSEPH NEEDHAM, JEAN BRACHET · 321 THE...

321

THE ORIGIN AND FATE OF UREA IN THEDEVELOPING HEN'S EGG

BY JOSEPH NEEDHAM, JEAN BRACHETAND ROBERT K. BROWN.

(From the Biochemical Laboratory, Cambridge.)

(Received 20th February, 1935.)

(With Three Text-figures.)

CONTENTS.PAGE

I. The formation of urea by the chick embryo . . 321(1) Introduction . 3 2 1(2) Material and methods . . . . . . 323(3) Experimental results . . . . . . 323

(«) Production of urea from ammonia . . . 323(A) Production of urea by the ornithine cycle . 324(c) Production of urea from uric acid . . . 324(d) P r o d u c t i o n o f u r e a f r o m a r g i n i n e . . . 3 2 4

I I . U r e a a n d t h e f o r m a t i o n o f u r i c a c i d i n t h e e g g . . 3 2 0( 1 ) I n t r o d u c t i o n . . . . . . . 3 2 9( 2 ) M a t e r i a l a n d m e t h o d s . . . . . 3 3 0( 3 ) E x p e r i m e n t a l r e s u l t s . . . . . . 3 3 0

I I I . S u m m a r y . . . . . . . . . 3 3 4A c k n o w l e d g m e n t s . . . . . . . 3 3 5R e f e r e n c e s . . . . . . . . . 3 3 5

I. THE FORMATION OF UREA BY THE CHICK EMBRYO.

(1) INTRODUCTION.

SINCE the work of Needham (1926), reported in this Journal, the question of theexcretion of nitrogen by the chick embryo during its development has not receivedmuch further consideration. In this work it was shown that the embryo at anearly stage of its development (fourth to seventh day) may excrete more urea thanuric acid, but that later the elimination of uric acid greatly exceeds that of urea.On the tenth day of development, uric acid may form 95 per cent, of the excretednitrogen.

More recently, Dickens and Greville (1933) have studied the excretion of ureaand ammonia by the chick embryo in vitro by more sensitive methods. They foundonly a very small urea excretion on the part of 5th day embryos, and establishedthe fact that the addition of glucose or fructose did not diminish this excretion.This might be interpreted either as evidence of lack of protein-sparing action or asevidence that the urea of the chick embryo does not arise from the ammonia of

322 JOSEPH NEEDHAM, JEAN BRACHET and ROBERT K. BROWN

protein deamination. That the embryo at this age can deaminate amino acidsappeared from the work of Glover (1931), who observed small increases of ammoniaand urea when embryos were shaken in vitro with amino acids.1

Since up to the present time no definite information about the origin of theurea of the chick embryo has been forthcoming, the following hypotheses presentedthemselves for examination. (1) The urea might arise, as it would do according toclassical theory in a ureotelic animal, from the direct combination of carbondioxide and ammonia. (2) The urea might be formed, as it is in ureotelic organisms,by means of the ornithine cycle of Krebs and Henseleit (1932). Working withmammalian liver slices, these authors, as is well known, found that ornithine wasthe only amino acid which would give rise to urea, and that it would do so evenwhen present in minute quantity. Ornithine, in the presence of carbon dioxideand ammonia, forms citrulline, this with another molecule of ammonia formsarginine, and the arginine, acted upon by arginase, gives urea and ornithine.(3) The urea, however, might also be formed from uric acid. Przylecki and Rogalski(1928) showed that the chick embryo, during the first week of its development, iscapable of destroying uric acid by a uricase. Unfortunately they did not investigatethe products of its action (allantoin, allantoic acid, urea, etc.), so that it mightequally well be an enzyme similar to that discovered by Stransky (1933) in Amphibiaand fishes, which breaks uric acid down to urea; or the common uricase of mam-mals which only carries the process as far as allantoin. As Przylecki's chick uricasedisappears from the embryo at about the time when the excretion of uric acid beginsin earnest, the urea formed beforehand might well be derived from this source.(4) Finally, the urea might be derived simply from the action of arginase on arginine.

The first of these possibilities was the most unlikely, since there is now littlereason for supposing that direct synthesis ever occurs. The recapitulatory aspectof the phenomena would be satisfied by any of the other three possibilities, for theornithine cycle is present in Amphibia and reptiles, the uricase of Stransky ispresent in fishes and Amphibia,2 and the presence of arginase, even in high activity,is widespread throughout invertebrates and vertebrates (cf. Baldwin, 1935). Ifthe ornithine cycle were present in the chick embryo, it would be retaining for atransient period a mechanism which had been of value to the Amphibia andwhich has also been so to the mammals; if on the other hand the embryo derived itsurea solely from arginine, it would possess an adult characteristic. In 1932 Clementiclearly showed that neither ammonia nor purines, but only arginine, produces ureain the adult hen. Hens on a normal diet excrete some 70 mg. urea daily, and inhunger this is reduced to about 20 mg. If the animals are then given 350 mg.arginine parenterally, the output goes up to normal. No other substance will bringabout this effect.

1 The results of Kaieda (1930) were very confusing. Injecting amino-acids into the hen's eggat o days and estimating the urea at 16 days, he got small increases with some and decreases withothers. Ammonium carbonate gave no increase. His control value at 16 days was three time9 ashigh as that usually accepted.

1 The appearance of this uricase in developing Amphibia has recently been investigated byTruszkowaki and Czuperski (1933). It appears late, about the time of the limb-buds.

The Origin and Fate of Urea in the Developing Hen's Egg 323

In order to settle the question, we have added ammonia, ammonia and ornithine,uric acid, and arginine to chick embryos in vitro, following subsequently theformation of urea.

(2) MATERIAL AND METHODS.

The White Ixghorn eggs used were obtained from the pure-bred flocks of theCambridge University Farm, Messrs Chivers, and the Papworth Industries. Mo9tof the experiments were made on embryos of 4! or 5 days of development. Afterseparation from amnion, allantois and yolk-sac, the embryos were washed in Ringersolution of the same composition as that used by Benzinger and Krebs (1933) intheir work on uric acid formation, previously brought to 370 C. Yolk-sacs, whenused, were freed from yolk and white by being dragged for some time over cleandry glass surfaces, after having been washed in Ringer solution. Weighed amountsof tissue were then incubated in suitable vessels slowly agitated for a known periodin a thermostatic water-bath at 3 8° C. Buffering at pH 7-4 was accomplished byfilling the vessels with a gas mixture of 95 per cent. Oa and 5 per cent. CO2 inequilibrium with the bicarbonate in the Benzinger-Krebs solution.

After a sufficient time had elapsed (usually 4 hours) an aliquot portion of thesuspension liquid was pipetted off, and the urea contained in it estimated accordingto the delicate manometric method of Krebs and Henseleit (1932) in which thecarbon dioxide arising from the decomposition of urea by urease is measured. Weexpress our results in the same manner as these authors:

™s. _ cmm. urea CO2

^ " mg. dry weight x hours *

Dry weights were calculated according to the data in the literature (Byerly, 1932;Needham, 1931), since the material is known to be very constant.

As controls, numerous experiments were made in order to determine the Qn

of the embryos and the membranes without the addition of any substances. Theresults are given in Table I. Another control was regularly made on the solutionsadded, in the absence of any tissue; this was always very small, but was deductedfrom the experiments in which tissue was present.

(3) EXPERIMENTAL RESULTS.

(a) Production of urea from ammonia.

To the Ringer-bicarbonate solution was added an amount of ammoniumchloride equivalent to 31-8 mg. NH3 per cent. This concentration of ammonia wasthat which in the experiments of Krebs and Henseleit (1932, p. 57) gave the highestsyntheses of urea by means of the ornithine cycle in mammalian liver. Table Ishows the values found for embryos of 4̂ —5 days' incubation age. It must beconcluded that the urea production at this stage is extremely small (some 100 timesless than the starving mammalian liver), and that it is not raised appreciably by theaddition of ammonia.

Some experiments were also made on the membranes of 5-day embryos (yolk-


sacs). Here also the addition of ammonia caused not the slightest increase in theurea formation, which normally was even lower than that of the embryo (Qu 0-005).

Table I.

Embryosalone

O-O20 0 3O O I

0 0 3—

Average 002

38° C. Q™ .

Embryos withammonia

O-O20-03O'O2OO3O-O2

Average 002

(b) Production of urea by the ornithine cycle.

To the Ringer-bicarbonate solution was added as before 32 mg. per cent, ofammonia in the form of ammonium chloride, and in addition amounts varyingfrom 4 to 28 mg. per cent, of ornithine. On mammalian liver, the maximum effectis reached at about 30 mg. per cent, ornithine.

The results showed clearly that at this stage the chick embryo does not possessthe ornithine cycle. No increase in urea formation was observed in six experimentswith different concentrations of ornithine. The Qu was always between o-oi and0-02 and never rose above that of embryos maintained in physiological solution only.

In two further experiments the yolk-sac was found also to lack the ornithinecycle, as no increase above the controls could be observed.

(f) Production of urea from uric acid.

The reasons which make it worth while to test the possibility that chick embryourea could arise from uric acid have already been mentioned.

The uric acid was added to the Ringer-bicarbonate solution in the form ofsodium urate 84-5 mg. per cent. In some experiments a higher concentration(132 mg. per cent.) was obtained by raising the pH to 9-5 by means of a standardglycine buffer. In some cases the embryos were ground to a brei instead of beingused intact. But whatever the conditions of the experiment might be, it was neverpossible to observe an augmentation of the urea formation when uric acid waspresent (seven experiments). Three experiments were made on the yolk-sac bothat/>H 7-5 and 9-5, but here also there was no increase over the controls.

These negative results clearly show that the uricase of the chick embryostudied by Przylecki and Rogalski (1928) does not resemble that of fishes andAmphibia, but probably like that of mammals only decomposes uric acid as far asallantoin.

(d) Production of urea from arginine.

By the process of exclusion described in the preceding sections, this method ofproduction became more and more probable, and in fact it was possible to showthat it could fully account for the urea normally produced.


The Ringer-bicarbonate solution was now made up to contain 393 mg. per cent,arginine. In these conditions a Qa some 15-20 times higher than the controls wasvery regularly found. Table II gives the results for 4J-5 day embryos:

Table II. Arginase in 4^-5 day embryos.

Control, averageArginine

Average

O : w >

O O 2

0-460-580-50O-430-330 2 10 2 00-19

0-32

This demonstrates the existence of an active arginase, giving amounts of ureaof only about four times less than the starving mammalian liver by the ornithinecycle.

The 5th day yolk-sac also appears to possess active arginase, though not insuch great amount as the embryo. The results are shown in Table III. All thesefindings suffice for the conclusion that the urea normally produced by the chickembryo is derived entirely from the breakdown of arginine.

Table III. Arginase in §th day yolk-sac.

Controls

AverageArginine

Average

0-0050-0050-005

0-005

0-030-040-015O-O2

O-O25

The case of the chick embryo therefore seems to be analogous to that of malignanttumours in mammals, which, according to Neber (cit. in Edlbacher, Roller andBecker, 1934, p. 101) possess active arginase but cannot synthesise urea by meansof the ornithine cycle. The chick embryo therefore behaves as it should on Edlbacher'stheory (1934). Edlbacher has attempted to establish a correlation between highactivity of arginase, nuclease and phosphatase, and high rate of cell division andgrowth. He considers that these enzymes play a fundamentally important part inthe synthesis of the protein moiety of the nucleoproteins, and that the role ofarginase in growing tissues is not in connection with urea formation but rather in


connection with the disintegration (or synthesis) of the arginine contained in theprotamines and histones. It may here be noted that according to Lieben and Lieber(1934) arginase has no effect upon combined arginine; the substrate must be free.This, however, would not affect the possibility that the significance of arginine ingrowing tissues was synthetic. The generalisations of Edlbacher rest almost whollyon work with tumour tissue (Edlbacher and Kutscher, 1931, 1932; Edlbacher,Roller and Becker, 1934; Edlbacher and Jung, 1934); only in the paper of Edlbacherand Merz (1927) is there any information about embryonic tissue, and here theresults, which concerned guinea-pig material, were few and lacked precision re-garding the age and dimensions of the embryos studied.

It therefore seemed indispensable to study the activity of arginase in relation tothe progress of development. We know that the growth rate of the embryo declinesprogressively, and if Edlbacher's view were correct, one would expect a progressivediminution of the Qn during incubation. Table IV shows that this is actually thecase. The technique described above was used, except that in the case of the larger

Table IV. Change of arginase activity with age in the chick embryo.

Day

2l

3

3i

8

12

Hatched chick

OftI O Ii - i 7i - i 7I - 3 2

0-87

°-53

o-io0-080-08

0-050-040-04

0-05

Average 1-16

Average 063

Average o-oy

Average 004

embryos (8 and 12 days) a mince made with a razor was substituted for the intactembryos. The last value in this table, that for the hatched chick, was not obtainedin the present investigation, but is calculated from the measurements made byChaudhuri (1927) with the method of Edlbacher and Rothler (1925). The agree-ment is good.

The relative decline of arginase activity may conveniently be represented upona heterogonic diagram, as in Fig. 1, where the cmm. urea CO2 liberated by oneembryo in one hour are plotted against the dry weight of the embryo at succeedingstages. As explained in discussions of chemical heterogony (Teissier, 1931;Needham, 1934) an entity "growing" more slowly than the total body weight willshow a slope of less than 450 on the double log grid, while an entity "growing"more rapidly than the total body weight gives a slope greater than that angle. It


will be seen that arginase belongs to the former group of substances, and falls offrelatively as growth proceeds.

It may however be asked whether the decline of activity during development isnot a property common to all the enzymes of the embryo. The older literature(summarised in Needham, 1931) contained so few accurate determinations ofenzymic activity referable to dry weight of tissue, that an answer to this questionwould have been impossible till recently. But the work of Ammon and Schiitte(1935) now provides a comparison, for in measuring the relative activities of tri-butyrinase, esterase, and choline esterase during development, it was found that

10 100 1000

M g m . dry wt. of embryo

Fig . 1. Negative Heterogony of Arginase.

the two first-named enzymes continuously increased (units/gm. dry weight) whilethe choline esterase increased to a maximum and thereafter decreased. This is inharmony with what is known about the greater importance of fat metabolism inthe later periods of embryonic life, and suffices to show that all enzymes do notexhibit the negative heterogony of arginase.

Now it is known that the glutathione or total —SH content (Murray, 1926, andmany other workers) and the total reducing power (—SH plus ascorbic acid plusunknown reducing substances) (Bierich and Rosenbohm, 1935) fall relativelyduring embryonic development, referred to the dry weight. Since glutathione andascorbic acid (vitamin C) are known to activate arginase under certain conditions(Waldschmidt-Leitz, 1931; Salaskin and Solowjew, 1931; Edlbacher and Leuthardt,1933; Leuthardt and Roller, 1934; Edlbacher, Kraus and Leuthardt, 1934; Klein


and Ziese, 1934; Purr and Weil, 1934; Karrer and Zehender, 1934), it might besupposed that the decreasing activity of arginase during development was due tothe decreasing quantity of activators present. In order to test this possibility,glutathione, cystein, and ascorbic acid were added, each in M/100 concentration,to the 8 day embryos, but in no case could any increased activity over the normals(Qn 0-09) be observed. The same experiments were made with 5th day membranes,which show a low Qu, but the same results were obtained, see Table V. From

Table V. Effect of activators on urea production.

8th

8th8th8th

5th

5 *5th5th

day

daydayday

day

daydayday

embryo,embryo,embryo,embryo,

yolk-sac,yolk-sac,yolk-sac,yolk-sac,

aloneplus GSHplus cysteinplus ascorbic

aloneplus GSHplus cysteinplus ascorbic

Average

AverageAverage

Average

0-09

0-08009o-ioO-OQ

O'O25

O-O250-0150-015O'O2

these results it seems fairly certain that the diminution of arginase activity duringdevelopment is not due to a diminution of activators. The results constitute animportant argument in favour of the theory of Edlbacher. It would however bedesirable to extend them to developing eggs in which the synthesis of nucleoproteinsis only partial (cf. Brachet, 1933), such as that of the sea-urchin.

Finally, an attempt was made to estimate the arginase activity of the embryofollowing more closely the method of Baldwin (1935). Arginase is not normallybound to the cell proteins and its estimation is possible in brei. Unfortunately theresults were rather unsatisfactory as the tissue extract after filtration on being madealkaline throws down a slimy mass of semi-denatured protein which makes samplingdifficult. It was nevertheless clear that in these tissue extracts the activity of thearginase was of just the same order as in the intact embryos (£)n 0-3-0-4), at \\ days'incubation age, at all />H's from 7-5 to 9-0. The lack of a marked >̂H optimumcorresponds with the findings of Hunter (1934) on arginase from other sources.The action of activators on the arginase of 4.V day embryos in brei was also studied,but found to be absent.

One corollary of the present findings on the arginase of the chick embryo is ofinterest. The capacity of the embryo to carry out protective syntheses was studiedby Takahashi (1928), who found that when benzoic acid was injected into the eggat the beginning of development no conjugation of the benzoic acid with ornithineoccurred before the 9th day. The capacity to produce ornithine from arginineappears therefore to be present considerably earlier than the capacity to conjugateit to form ornithuric acid.


II. UREA AND THE FORMATION OF URIC ACID IN THE EGG.

(1) INTRODUCTION.

No satisfactory explanation exists at the present time of the mechanism of uricacid formation from catabolic nitrogen by the avian organism. All that can besaid is that a number of hypotheses have been shown to be untenable. These willbe referred to at the end of this section. The suggestion of Wiener (1902), whichrather undeservedly became classical, was that a molecule of urea condensed witha three-carbon chain to form dialuric acid, which then condensed with a furthermolecule of urea to give uric acid. The three-carbon chain was commonly supposedto be tartronic acid, mesoxalic acid, or malonic acid.

COOH COOH COOH

CHOH CO CH,

COOH COOH COOH

Tartronic acid Mesoxalic acid Malonic acid

Later Fearon (1932) succeeded in condensing biuret with sarcosine in vitro withthe production of small amounts of uric acid.

NH, COOH NH—CO

CO + CH,—NHX -* CO C—NHVI XCH, I Jl >CONH—CO—NH, NH—C—NH/

Biuret Sarcosine Uric acid

This condensation, if found to occur in the body, would have the advantage ofproviding a function for sarcosine, which has long been known to occur widely inthe tissues of the vertebrates, but for which no likely function has ever beenput forward. Although biuret has never been isolated from tissues, its existencethere as an intermediate product in this synthesis might be fleeting.

The following experiments were designed to test these hypotheses with regardto the fate of urea in the hen's egg. Does the urea accumulating during developmentrepresent the whole of that which is formed, or only an excess over some furtherprocess which removes it? The method of injection of precursor substances intothe egg, much used by the Japanese workers of the school of Tomita, was employed.It has the disadvantage in this case that the limits of its applicability are rathernarrow. If too small a dose is injected, no marked effect is produced, the normalvariation in uric acid formation being considerable, but if the dose is too large,death or severe injury results. Similarly, if the injection is made too soon too manyeggs are required for the process to be practicable, and if the injection is madetoo late the estimation has to be done on allantoic liquids containing a large amountof very insoluble uric acid deposits.

JBB'IIliv


(2) MATERIAL AND METHODS.

Eggs from the same sources as those mentioned in the previous part of thepaper were used. Serially numbered and dated, they were incubated at intervalsof three days to provide a continuous supply of embryos at the right age.

Injection of precursor substances were made on the 12th day of incubation,which is just before the maximum point of uric acid production and well after thepeak of urea production. A sterilised glass hypodermic syringe was used, with a4-cm. needle, large and stout enough to puncture the shell easily. The solution,usually 0-5 or 1 c.c, was injected into the air space and the hole closed with adrop of paraffin wax. Each egg was carefully sponged with a mercuric chloridesolution before injection. In this way the loss by infected eggs was kept down tovery low figures.

The eggs were opened for uric acid estimation after three days further in-cubation. This allows sufficient time for the assimilation of the injected materialand is early enough (15th day) to avoid the worst accumulation of uric acid depositsas well as any of the complications of hatching. The allantoic liquid was carefullycollected, and together with the allantoia themselves ground with sand and washedinto a large flask. Frequently small granular masses of yellowish solid uric acidwere found in the allantoic cavity. These, which are covered with a protective coatof mucin, require special treatment, according to the work of Fiske and Boyden(1926). They were separated out by hand and let stand for an hour in a few c.c. ofconcentrated HC1. This was then evaporated off on a steam bath and the uric aciddissolved in the smallest possible amount of Benedict-Hitchcock phosphate solution(Benedict and Hitchcock, 1915). It was then added to the total extract.

Deproteination was effected by boiling at a pH yellow-green to brom-cresol-purple. The precipitate was extracted twice. Uric acid was estimated according tothe method of Benedict in a Klett colorimeter.

The compounds injected were:(1) Ammonium carbonate (B.D.H., A.R.). 10 mg. are equivalent to 8-4 mg.

uric acid.(2) Tartronic acid (B.D.H. special preparation). 10 mg. are equivalent to

14 mg. uric acid.(3) Urea (B.D.H. recryst.). 10 mg. are equivalent to 8-4 mg. uric acid.(4) Biuret (two specimens, one prepared by W. R. Fearon, the other Kahlbaum).

10 mg. are equivalent to 9-6 mg. uric acid.(5) Sarcosine (Kahlbaum). 10 mg. are equivalent to 4 mg. uric acid. 10 mg.

biuret plus 8-8 mg. sarcosine are equivalent to 13-8 mg. uric acid.

(3) EXPERIMENTAL RESULTS.

The results of the injections, given in Table VI, show a number of points ofinterest.

In the first place the control experiments may be added to the data already inthe literature about the exact amount of uric acid in the egg, about which there has


Table VI. Injection experiments with uric acid precursors.

Injection

Controls

Ammonium carbonate

Urea

Tartronic acid

Urea and tartronic acid

Biuret (Fearon)

Biuret (Kahlbaum)

Uiuret and sarcosine

Mg.

per egg

——————————

0 9

34231 2

1 2

84

1 0

8

131313

6J13

2 0

1 0

1 2

12 139 89 8

13 " 11 0

1 0

1 0

1 0

1 0

1 0

1 0

71 71

No.

Injected

———-———————

666666566

1 2

1 2

666567

955569

66666

5

of eggs

Survived

666665555550

0

0

0

2

5555884540

2

2

0

0

0

0

3664644

4

Av. wet weightembryos on

15th day gm.(normal =

11 gm.)

————

9 49 48 9———

——————————9 98 99 9——9 3————

9'58-8——

IO'I10-31 0 6

9-4

Uric acidmg. perembryo

I5-518-317-817-014-0

18-71 3 921-8

ig-818-61 8 9

———

23-826-61 7 926-728-6

10-3*U S *166i6-717-0

16-81 6 5

———

23-218-1

8-8»15-81 3 61 6 3

1 6 6

• Probably some loss owing to insufficient treatment of solid masses.

been some divergence. Fig. 2, a semilog plot, shows that whereas the values ofFiske and Boyden (1926) are uniformly high, and those of Kamei (1928) andTomita and Takahashi (1929) are uniformly low, those of Fridericia (1912),Needham (1926), Targonski (1927) and Calvery (1930) are in good agreement in anintermediate position. The results now reported, which appear as black discs onthe graph, agree again with this intermediate position, although obtained with


100-0

10-0 -

o

t.fiV

io

Friderlda (191a! • Silver precipitation

Needh*m (1936) NH4C1 titmtion

FlBke and Boyden (1926) Benedict (col on metric)

Tanjonski (1927! Moms and Mcl.eod (rokinmetrn I

Kninei (19381 FnUn and Wu (eolorimctric)

Toraita and Tslcahaihi ngjql u olonmetncl

CaJvery (1930I • Folln and Marenczy

Present worti Benedict |bl.ick dot^l

Tom itand

TakahasI

Days

2. Uric acid of the chick embryo.


a colorimetric method instead of the ammonium chloride precipitation method asused in this laboratory in 1926.

From Fig. 3, in which the figures have been represented diagrammatically, itcan be seen that the range of normal variation at the 15th day is between 14^ and20 mg. This range is much exceeded by embryos of eggs into which ammoniumcarbonate has been injected (24-29 mg.), unless, as in one case, the dose was verysmall. The first two urea experiments were vitiated by the fact that insufficienttreatment was given to the uric acid masses, but later experiments showed a rangewithin the normal. Tartronic acid also gave no increase, and with urea and tartronic

32-

28-

" 2 " .

T3 20-

.o 16-3

i

Fig. 3. Precursors of uric acid.

acid together, it proved impossible to get living embryos. This would not havebeen expected from the work of Tomita and Takahashi (1929), who after injectingurea and tartronic acid together into the egg obtained increases of more thanthree times the normal value in their best experiments. As has already been pointedout, however, their normal levels were so low that we cannot have confidence inthe view that their increases were outside the limit of normal variation.

With biuret the limits of normal variation were not exceeded except in onecase, and the single experiment in which biuret and sarcosine were injected togethergave a result about the centre of the normal range. It would be perhaps desirableto continue these experiments if another opportunity presented itself, but so faras they go, they do not give support to the idea that biuret can condense with


sarcosine to give uric acid in the avian organism. More certain is the conclusionthat urea itself will not form uric acid.

This is in agreement with the whole trend of work on this subject during thepast ten years. The only evidence in favour of the view that urea is a precursor ofuric acid in the bird derives from the old work of Meyer and Jaffe (1877) and ofWiener (1902), the technique of which cannot be accepted now as adequate. Afterthe classical liver perfusions of Kovalevski and Salaskin (1901), who showed thaturic acid could be formed from ammonium lactate, Izar (1911) reported a similarsynthesis by perfusion methods from urea, but this claim was not as convincing asthe negative results of Friedmann and Mandel in 1908. In recent times the subjecthas been exhaustively studied by workers of the Italian school. Urea has beengiven to birds (hens, geese, pigeons) by injection, at the same time as the adminis-tration of the following substances by the mouth, and in no case was there anysignificant increase in the quantity of uric acid excreted:

Malonic, tartronic and lactic acids (Clementi, 1929).Pyruvic and propionic acids (Torrisi and Torrisi, 1931).Dialuric acid (Torrisi, 1932).Glycerol and glyceric acid (Biondi, 1932).Tartronic, lactic, barbituric and dialuric acids (Russo, 1934).Glycerol, malonic, tartronic and lactic acids (Schuler and Reindel, 1933).In some of these experiments the urea was quantitatively recovered. Entirely

similar results were obtained by Benzinger and Krebs (1933) using tissue-slices ofavian liver, and by Russo and Cuscuna (1931) and Cuscuna (1934) using the methodof liver perfusion.

On the other hand, the formation of uric acid from ammonium carbonate, firstdemonstrated by Minkovski in 1886, has been repeatedly confirmed, e.g. byClementi (1930) and Russo (1933), and is seen again in the present experiments.

III. SUMMARY.

1. The urea formed by the chick embryo during its development is not derivedfrom the ammonia of general protein catabolism, either directly or by way of theornithine cycle.

2. Nor is it derived from uric acid by a complex uricase similar to that offishes and Amphibia (Stransky's complex: uricase, allantoinase and allantoicase).The uricase discovered in the chick embryo by Przylecki probably breaks downuric acid only as far as allantoin.

3. The urea of the chick embryo is entirely derived from an active arginine-arginase system present at all stages examined from the second day of incubationonward.

4. The arginase of the embryo, quantitatively estimated, falls off very regularlyas development proceeds, reaching a minimum about the 12th day. This negativeheterogony is experimentally shown not to be due to the parallel diminution ofactivators such as glutathione and ascorbic acid, but must be regarded as a real


diminution in the relative amount of the enzyme. It forms an important supportfor the view of Edlbacher, hitherto mainly based on observations of tumour tissues,that arginase is an enzyme associated with high growth rate and probably nucleinsynthesis, as well as with the production of urea as a vehicle of waste nitrogen.

5. Arginase is also contained in the yolk-sac, but in much smaller quantitythan in the embryo. Here also the low activity is not due to the absence of activators.

6. Experiments made to test the possibility that the urea, once formed, might,as would be the case on Wiener's theory, participate in the formation of uric acid,took the form of injection of possible precursors into the egg on the 12th day ofdevelopment and estimation of the uric acid in the egg on the 15th. In this wayit is shown that urea cannot be converted into uric acid by the chick embryo. Thisconclusion is in agreement with much recent work on the adult bird.

7. A preliminary test was also made of the possibility that biuret and sarcosinecould be condensed to uric acid by the chick embryo. The results were negative.

ACKNOWLEDGMENTS.

The authors wish to express their thanks to the Government Grant Committeeof the Royal Society for a grant which partially defrayed the cost of these researches.One of us (J. B.) is indebted to the Administrative Council of the University ofBrussels for a Stipend during the tenure of which the present work was carried out.Another (R. K. B.) is indebted to Harvard University for a travelling Fellowship.

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THE ORIGIN AND FATE OF UREA IN THE DEVELOPING HEN'S EGG BY JOSEPH NEEDHAM, JEAN BRACHET · 321 THE...

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