THE ENERGY-SOURCES IN ONTOGENESIS II. THE URIC ACID CONTENT AND THE GENERAL PROTEIN METABOLISM OF THE DEVELOPING AVIAN EGG BY JOSEPH NEEDHAM, M.A., PH.D., Fellow of Gonville and Caius College, Cambridge. (From the Biochemical Laboratory, University of Cambridge.) (Received 2nd May 1926.) (With Fifteen Text-figures.) CONTENTS. PAGE Introduction 114 Technique 115 Experimental results . . . no Discussion: (a) The relationship of uric acid to urea during development . . 122 (b) The combustion of protein by the embryo (c) Calculation of Szneroffna's ratio (d) Calculation of the respiratory quotient . (e) The succession of energy-sources during development The controlling factor in embryonic metabolism 12s 129 136 141 Summary 143 INTRODUCTION. IN the preceding paper of this series (32) a report was given of experiments in which the urea present in the developing hen's egg was estimated as accurately as possible on each day of incubation. In the experiments now to be described the uric acid has also been estimated, and it has been found possible to draw definite conclusions from the resulting figures about the utilisation of protein by the developing embryo. The primary object of these researches has been to test the truth of the hypothesis that in the course of ontogenesis there is a succession of energy-sources, carbo- hydrate being first made use of, then protein, and finally, fat. It was shown in the preceding paper that a large amount of the previous work on the chemistry of embryonic development fell into line and formed a connected whole if that hypothesis was adopted. As regards the hen's egg, which was the material used in these investigations, there was a large gap in our knowledge, for data about the nitrogenous excretory products of the embryo were very few in number. Although we possessed a certain amount of moderately exact information about the movements of fat and of carbo- hydrate, we were still in the dark as far as protein was concerned. Systerr^^ estimations of the urea production had never been done, and the work of Fridericia(i3) was the only systematic examination of the uric acid production. His
Transcript
THE ENERGY-SOURCES IN ONTOGENESIS
II. THE URIC ACID CONTENT AND THE GENERAL PROTEINMETABOLISM OF THE DEVELOPING AVIAN EGG
BY JOSEPH NEEDHAM, M.A., PH.D.,
Fellow of Gonville and Caius College, Cambridge.
(From the Biochemical Laboratory, University of Cambridge.)
(Received 2nd May 1926.)
(With Fifteen Text-figures.)
CONTENTS.PAGE
Introduction 114Technique 115Experimental results . . . n oDiscussion:
(a) The relationship of uric acid to urea during development . . 122(b) The combustion of protein by the embryo(c) Calculation of Szneroffna's ratio(d) Calculation of the respiratory quotient .(e) The succession of energy-sources during development
The controlling factor in embryonic metabolism
12s129
136141
Summary 143
INTRODUCTION.
IN the preceding paper of this series (32) a report was given of experiments in whichthe urea present in the developing hen's egg was estimated as accurately as possibleon each day of incubation. In the experiments now to be described the uric acidhas also been estimated, and it has been found possible to draw definite conclusionsfrom the resulting figures about the utilisation of protein by the developing embryo.The primary object of these researches has been to test the truth of the hypothesisthat in the course of ontogenesis there is a succession of energy-sources, carbo-hydrate being first made use of, then protein, and finally, fat. It was shown in thepreceding paper that a large amount of the previous work on the chemistry ofembryonic development fell into line and formed a connected whole if that hypothesiswas adopted.
As regards the hen's egg, which was the material used in these investigations,there was a large gap in our knowledge, for data about the nitrogenous excretoryproducts of the embryo were very few in number. Although we possessed a certainamount of moderately exact information about the movements of fat and of carbo-hydrate, we were still in the dark as far as protein was concerned. Systerr^^estimations of the urea production had never been done, and the work ofFridericia(i3) was the only systematic examination of the uric acid production. His
The Energy-sources in Ontogenesis 115
:s were for two reasons unsatisfactory: in the first place, they did not beginthe eleventh day of development, by which time many important things
might have happened, and in the second place, they were obtained by the oldmethods of copper and silver precipitation. These necessitated a separation of theuric acid from all the purine bases and, as has been found by many workers, sucha technique is not reliable. An additional criticism is that he only used about200 eggs, while in the experiments now reported, more than double that numberhave been analysed.
TECHNIQUE.Two methods were made use of in estimating the uric acid in order to allow
for the fact that owing to the growth of the embryo the whole scale of uric acidproduction is outside the total range of one method. As a micro-method for theearly stages that of Benedict and Franked) was used, and for the later period theammonium chloride precipitation method of Hopkins (21).
The eggs were all laid by White Leghorn hens and incubated as nearly as•possible under the standard conditions suggested by Murray(30, that is to say,temperature constant at 38-8 ± 0-4° C , humidity constant at 67-5 ± 2-5 per cent.,and continuous ventilation by warm air. The eggs were aired every morning for15-20 minutes and rolled once a day.
It was found that for every stage in development from the fourth day onwards,excellent separation of white and yolk from the embryo and its membranes couldbe obtained by the following method. Before being opened the egg was held overa powerful electric lamp, and the area occupied by the white marked on the shellin pencil. The shell was then cracked inside the circle so formed, the crack enlargedwith forceps, and the albumen allowed to flow out through the opening. The yolkwas then freed from the allantois and allowed to escape in the same way. In ninecases out of ten the embryo and its membranes were left in the shell free from yolkand white. They were then tumbled out into a tared vessel and weighed when asufficient number had been collected. The whole material was ground up withwell-washed quartz sand in a mortar and extracted three times with distilled wateracidified with acetic acid. During the first extraction the flask was held in a boilingwater bath so that all the protein was coagulated. The separation from the proteincould be made exceedingly clean by adjusting the reaction of the liquid with aceticacid to give a greenish-yellow colour with brom-cresol-purple, and when theextractions were finished all the extracts were evaporated down on a water-bathtogether to a low bulk. As a rule a very small quantity of protein separated outduring this process but it was removed by centrifuging or filtration, and on theclear pale yellow liquid the estimation was done. In the case of the micro-methodthe arsenophosphotungstic acid reagent and the sodium cyanide were added to
amount of the solution, and the resulting blue colour compared with thatdard solution of uric acid similarly treated. The micro-method in question
had been evolved in the first instance for the purpose of estimating uric acid inurine, and there was a certain danger lest the maximum reading should not represent
n6 JOSEPH NEEDHAM
the amount of uric acid present but simply the maximum colour obtainablethe quantities of reagents added. It was easy to overcome this difficulty, hoby doing in each case a series of readings, diluting the original solution ioo per cent,every time. If the results came to just half each other, then the highest readingcould be accepted as correct, but if in any given instance the result was not halvedalthough the strength of the solution had been halved then, obviously, the methodwas not recording the full value of the uric acid present.
If the macro-method was being used, then the pale yellow tissue extract wasfully saturated with ammonium chloride and made alkaline with strong ammonia.This precipitated all the uric acid and also a certain amount of ammonium phosphate.After standing for three or four hours, the precipitate was filtered off, washed withsaturated amrrionium chloride solution, removed with hot distilled water into abeaker, strong hydrochloric acid added, and left for the uric acid to crystallise outover night. Next morning, the uric acid was filtered off, dissolved up again in dis-tilled water made alkaline with sodium carbonate, and titrated against standardpermanganate.
EXPERIMENTAL RESULTS.The results for the uric acid are tabulated in Table I. Column i gives the
number of days of incubation, column 2 the number of eggs used for the deter-mination in question, and column 3 the amounts of uric acid present every dayin mg. per embryo. Column 4 gives the uric acid present in mg. per cent, of wetweight of embryo, and column 5 does the same thing for dry weight of embryo.These calculations were made using the figures for wet and dry weight of WhiteLeghorn chick embryos given by Murray (3o. The data are graphically representedin Figs. 1-3. Fig. 1 shows the gradual increase in mg. per embryo of uric acid. Itwill be seen that the points fall on a very regular curve. In Fig. 2 is shown themg. per embryo wet weight and here it is very significant that a plateau appears.In the first seven days of development the uric acid mg. per cent, wet weight isexceedingly small in amount, but from the seventh to the eleventh day it risesrapidly until on the twelfth day it attains a constant level which it does not leave.There is thus a specially intensive production of uric acid between the seventhand the eleventh days of incubation. It will be seen that the points are fairly closetogether, and the only serious divergence occurs on the eleventh day. There canbe little doubt but that these differences are due to unequal rapidity of develop-ment, some embryos being more advanced than others at the beginning, as, forinstance, "body-heated eggs." The only way to avoid such errors is to take a verylarge number of eggs, and as far as possible that was what was done. The ascendingcurve is, of course, the place where the greatest divergences would be expected,if they were due to inequalities on the time-scale.
Fig. 3 gives the uric acid in mg. per cent, dry weight of embryo. The ctreaches a definite peak on the eleventh day after which it descends and seems t$reaching a steady level by the time of hatching, at about 460 mg. per cent. Therelation between the results now reported for uric acid in mg. per cent, of wet
The Energy-sources in Ontogenesis 117
?ht of embryo and those which can be calculated from the data given by Fride-I(i3) is shown in Table II. On the eleventh, twelfth and thirteenth days, his
figures, though some 20 mg. per cent, higher than mine, yet manifest a constancy,but after that they begin to mount until the time of hatching, with a slight dropon the twentieth day. This cannot possibly be due to differences in breed or race.
The explanation seems to be afforded by the work of Le Breton and Schaeffer (*6).In the course of a long and very careful research on the purine metabolism of thedeveloping embryo, they had occasion to examine the method used by Fridericia^Bthe separation of uric acid from purine bases and concluded that it was ratherunsatisfactory. It appeared that even under the best conditions, from 6 to 34 percent, of the total purine bases might be lost from the purine fraction and add
n 8 JOSEPH NEEDHAM
45
40
35
I 30
i 25
'§ 20*c3w 15 "tE
10
5
0
100-
90-
_ 80"
§ 70•c•5- 60
iI 50-
5 40-
i so-20-
10
Q Fridericia° Needham
O points• averages
-<?-<?—9-
10Days
Fig. I.
I15
o
10Days
Fig. 2.
I15
20
20
1200
1100
1000"
900
I 800o
3 700
•§> 600
£ 500-T3
OO400
300
200
100
The Energy-sources in Ontogenesis
o
119
O points•averages
5 15 20\
10Days
Fig. 3-themselves on to the uric acid fraction. Le Breton and Schaeffer accordingly madeuse themselves of the processes of Hopkins feo and Ronchese (4°). But it is somewhatsignificant that in Fridericia's estimations, there is found a plateau on the purinebase curve beginning just exactly at the point when the uric acid curve rises anddraws away from that described in this paper. It certainly seems as if the explana-tion of the difference in our results might lie in this direction.
Table II.
Day
89
1 0
111 2
1314IS161718192 0
Uric acid mg.
Needham
7 1 137-S°6800
820086-s86-s86-s86-s86-s86-s86-s86-s86-s
% wet weight
Fridericia
——
106-0110-5ioo-o1393175-52086243 225362525236-0
120 J O S E P H N E E D H A M
In Table III are shown the smoothed curve values for uric acid in absolutemg. per embryo, mg. per cent, wet weight, and mg. per cent, dry weight. B ^ f ethem, for purposes of ready comparison, are placed the smoothed curve valuesfor the data for urea in absolute mg. per embryo, mg. per cent, wet weight andmg. per cent, dry weight, contained in the preceding paper. Figs. 4-6 show graphi-cally the rather striking relationships between them. In Fig. 4 is shown the ureaand the uric acid in mg. per cent, wet weight of embryo plotted on the same scale.It shows the very interesting fact that on the third, fourth, fifth, sixth and seventh
Table III.Smoothed curve values for uric acid and urea.
days of incubation, the uric acid is rising distinctly more slowly than the urea,and, indeed, columns 3 and 7 of Table III show that in absolute quantities per eggthere is less uric acid than urea until the eighth day is reached. Between the seventhand the eighth day, as is seen in Fig. 4, the uric acid rises tremendously in amountand overtaking the urea almost attains its final constant value. These relationshipsare better seen in Fig. 5 which gives the mg. per cent, wet weight for both uricacid and urea, the abscissa being arranged so as to get them both on to the ^ f egraph. When this is done it is obvious that though both urea and uric acid riscourse of time to a constant level, the urea starts rising much earlier than the uric
acid and reaches its maximum level a day or so before. Inevitably this is reflectedon the mg. per cent, dry weight curve, shown in Fig. 6, only now in a still r ^ Astriking way, for peaks appear, and it is seen that the urea is in advance of the uricacid by two days. It is as yet too early to speculate on the possible biologicalsignificance of this precedence of uric acid by urea in the nitrogen excretion of thedeveloping embryo, but the fact itself seems certain.
A further point on Table III is worthy of mention. For the combined estima-tions of uric acid and urea, 776 eggs have been analysed, a figure which comparesvery favourably with the 192 eggs used by Fridericia, the 80 of Le Breton andSchaeffer, and the 476 of Murray.
(a) The relationship of uric acid to urea during development.
Among the various deductions which can be made from the data now at ourdisposal, the first one may be the relative behaviour of the urea and the uric acid.In the hen, the excreted nitrogen is mostly in the form of uric acid, and the urtakes only a very small share of it. At what stage in embryonic development is
adult relationship reached? In Table IV the ratio uric acidurea
O in absolute mgms. per embryo per day present• in absolute mgms. excreted per embryo per day
ADULTLEVEL
UNITY
20
I 2 4 JOSEPH NEEDHAM
column 2 it has been calculated for the absolute amounts of uric acid and ureapresent in the egg each day, in column 3 for the absolute mg. excreted eper embryo. This is assuming that the absolute increment found each day iuric acid and urea curves consists of material all of which has been excreted intothe allantoic liquid. In point of fact, a very small proportion of the nitrogenousend-products might be expected to remain behind in the embryo, but this correctionis probably exceedingly slight. The ratio is shown graphically in Fig. 7. It will beseen that from the fourteenth day onwards the ratio is constant at about 16, thatbeing the adult level. Before the seventh day the value of the ratio is less than unitybecause more urea is present and more urea is excreted daily than uric acid. The
adult ratio is therefore seen to be attained well before hatching. Another mannerof expressing the relationship, which leads to a result slightly different, is shownin columns 4 and 5 of Table IV and also in Fig. 8. Here, assuming that the ureaand the uric acid together make up the total nitrogen (which is certainly notabsolutely true, but within 98 per cent.) the partition between them has beencalculated as mg. excreted by the embryo each day of urea and uric acid nitrogenin per cent, of the total nitrogen in mg. excreted by the embryo each day. Betweenthe fourth and fifth days, the uric acid only accounts for 9-4 per cent, of the ^nitrogen, but so rapidly does the change occur that between the eighth and nudays it accounts for as much as 907 per cent. It has therefore practically reached
The Energy-sources in Ontogenesis 125
its adult level, as is shown by the comparative standards to the right of the-graph.r ^ A shaded parts at the bottom represent urea and the rest uric acid; they arefigures taken from H. Meyer (29), von Knierem(24), Schimansky(44), Meissner(28)and Steudel and Kriwuschaus).
It is clear that in the first week of development the relationship of urea to uricacid is altogether different from what holds in the adult, but that in the last week ofdevelopment the adult value is rigidly adhered to. These results throw light on thefinding of Aggazzotti (2) that the amniotic fluid in the chick passes from/>H 7-2to pH 4-4 in the last half of incubation, whereas before the ninth day, it has beenconstant at about pH 7-2.
(b) The combustion of protein by the embryo.
Since the amount of urea and uric acid produced by the embryo during itsdevelopment are now known, we can calculate the amount of protein combustedby the embryo.
Urea uric acid
'3"1
25 •
•2-
•15 •
• 1 -
05 "
6-
5-
4-
3 -
2 -
1 -
D urea• uric acid
mgms. excreted perday per embryo
15
• • • ( • •
10Days
F ig . 9 .
•" " I ' '15
1 ' I I20
In Table V the figures are given by means of which the calculation is made.fc absolute mg. of uric acid and of urea excreted per embryo per day are given
in columns 2, 3, 4 and 5, and shown also in Fig. 9. The points are seen to riseon a uniform curve, and show the same relationship to each other for the whole
!" ? ? * ? ? ?I?? Fop !"PI'!"" vl mW0U.W N m b W N W W - 0 0 0 a m m m * 0.*0 b F mw v,WW r H W r 0 OIFW e m N N N N N - r
w v ) w 0 0 0 0 0
8 0 " E g 8 8 % - m o o w m m 0 0 ? ? ? r( ? P e? !"?'I? '+ N*? ?
r r r N N m m * w
The Energy-sources in Ontogenesis 127
half of development. From them it is easy to calculate the mg. of urea and^ ^ d nitrogen excreted per day per embryo (columns 7 and 6 in Table V)
the mg. of total nitrogen excreted per day per embryo (column 8 in Table V) andso, multiplying by the 6-25 factor, the mg. of protein catabolised per day per embryo(column 9 in Table V). These two last columns naturally follow a simple ascendingcurve and so are not plotted, but their totals give figures of importance for calcula-tions. Then in columns 12 and 13 are given the mg. of protein broken down eachday per cent, wet weight and dry weight of embryo. In order to prepare the figuresof columns 12 and 13 it was necessary to have the weights for the intermediate
mgms. % coagulable proteindisappearing per day: wetweight: calcd. from Sakuragi
PERIOD OFFAT
COMBUSTION
PERIOD OFCARBOHYDRATE
COMBUSTION
1 110
DaysFig. 10.
115 20
periods between days, and these, calculated from the weight curves of Murray (3 oare given in columns 10 and 11. The mg. protein combusted daily per cent, of wetweight and dry weight of embryo are plotted with abscissae arranged so as to havethem in one graph, in Fig. 10. It will be seen that in both cases the peak occursat eight and a half days of development, the significance of which fact is sufficientlyindicated by the labels relating to the respiratory quotient. The curve for the dry\^Bht declines considerably more than that for the wet weight after the peak ispassed, and this is due to the well-known fact that the embryo gets drier as it growsolder.
128 JOSEPH NEEDHAM
From the total given at the bottom of column 8, Table V, it will be seenthe total number of mg. of nitrogen excreted by the embryo during itsment (or, more accurately, transmuted into the nitrogenous waste-products, ureaand uric acid), is n-oo. Sendjuus) in some interesting recent work, has estimatedthe total amounts of various amino-acids in the hen's egg during the course of itsdevelopment. He found a very slight increase in the histidine, and an absoluteconstancy in the arginine, lysine and monoamino-acids, while in the tryptophaneand tyrosine, on the other hand, there was a very considerable falling-off. He corre-lated this with the formation of haemoglobin and the bile-pigments, and did nottake into account losses by combustion for the production of energy. Accordingto Sendju, the tryptophane per egg descends from 134 mg. to 60, a loss of 74 mg.;and the tyrosine, beginning at 420 mg., falls to 260, this being a loss of 160 mg.
Supposing, then, that these two amino-acids are the only ones, or even theprincipal ones combusted during development, it would follow that about 50 percent, of them was burnt and 50 per cent, was available for the work of haemoglobinformation or other uses. It is certainly very difficult to see how tyrosine andtryptophane can be precursors of a purine, but it must be remembered that in thebird uric acid corresponds to the urea of the mammals and would seem to beproduced from the ammonia due to de-amination of combusted proteins.
Another interesting point which arises out of Table V is the following. Saku-ragi(4i), in a paper to which reference will again be made, found that the coagulableprotein diminished during development from 1-846 mg. nitrogen per cent, to1-698 mg. per cent.; 148 mg. are therefore lost per cent, of egg contents whichamounts to 67 mg. per average egg, a figure showing very close agreement indeedwith that seen at the bottom of column 9 in Table V, namely 68-74 mS- nitrogenexcreted. Thus 100 per cent, of the coagulable protein disappearing is accountedfor by nitrogenous waste-products found, and considering the variability of eggs,as well as the technical difficulties associated with work on them, this agreement isas near as can be expected. Apparently all the protein combusted is coagulableprotein, probably albumen, the ovomucoid not taking part in a breakdown forpurposes of energy supply. According to Bywaters(6) there is no preferentialabsorption of ovoalbumen and ovomucoid; they enter the embryo in the sameproportions throughout development. Experiments are being continued in thislaboratory to try to throw some light on the significance of ovomucoid.
There are various figures already in the literature regarding the loss of proteinnitrogen during embryonic development. These are summarised in Table VI.Krogh(2s) and Tangl and von Mituch(sO found that the hen's egg lost nowhatever in gaseous form during incubation, and many workers,Idzumiba), Szneroffna (49) and Aggazzottio), observed that there was no alterationin the amount of total nitrogen present during development. But this was only to
The Energy-sources in Ontogenesis 129
spected, since the nitrogenous waste-products cannot escape, and the reallynt figures are those for coagulable nitrogen. From Table VI it appears
that even in widely different classes of animals the same percentage of the proteinnitrogen present at the beginning of development is utilised to provide energyduring that process.
Table VI.
Investigator
Bialascewicz and MincoffnaFaure'-Fremiet and DragoiuFaure'-Fremiet and Vivien du StreelGortnerPearseWetzelTichomiroffPigoriniDakin and DakinIdzumiSakuragiNeedham
Reference
4111 21636555 2
377
2 2
4 iPresent
investigation
Egg of
Frog
Brook-trout
CrabSilkworm
PlaiceChick
it
Protein nitrogen lostduring development in
percentage, of totalprotein nitrogen present
at the beginning
9-112-0
g-29-2
1 7 050-018627-818-310-58-o7-5
(c) Calculation of Szneroffna's ratio.Szneroffnauo) made estimations of the nitrogen contained in the body of the
embryo, and that contained in the allantoic fluid at different stages of incubation.She found that the ratio of these, i.e.
Nitrogen in embryoNitrogen in allantoic fluid '
was practically a constant, wavering round about 17. In other words, for every1 gm. of nitrogen in the allantoic fluid, there were to be found 17 gm. of nitrogenin the body of the embryo. Her estimations did not begin before the tenth day,so in view of the relations which we have already found to hold between the earlyand late periods of development, a re-calculation of her ratio was necessary. Itwould be a legitimate criticism of her results to say that the determination of totalnitrogen in the allantoic fluid would be gravely upset by the presence of evenminute traces of the protein-containing blood, and in view of the delicacy of theblood-vessels it is not easy to see how it would be possible to obtain allantoic fluidabsolutely free from blood.
Szneroffna's ratio was calculated. It was assumed that only minute errors wouldbe introduced by calling the urea N plus the uric acid N the nitrogen present in theallantoic fluid, and two different sets of figures for the total nitrogen in the embryo
made use of. Actually three sets were available, those of Fridericiad3), Lebn and Schaeffer «6) and Murray 00, but only the last two were used because
Fridericia's fell on a curve, the points of which were averaged from widely divergentindividual differences.
JOSEPH NEEDHAM
In Table VII the results are tabulated. Column 3 contains the figures of Mifor total nitrogen of embryo, column 2 those of Szneroffna herself, and cothose of Le Breton and Schaeffer. Column 6 contains the mg. of urea and uricacid nitrogen found by me, and column 5 the nitrogen in the allantoic fluid foundby Szneroffna. It is to be noted that Szneroffna's figures usually largely exceedmine, which would be the case had any blood escaped into the allantoic fluid duringits collection. The important difference between the nitrogen figures of Le Bretonand Schaeffer on the one hand, and Murray on the other, is that the former ex-cluded the membranes in their estimations, while the latter probably includedthem. We therefore have a way of determining what part the membrane is playing
Table VII.
I
Days
56789
1 0
1112
131415161718192 0
2 3 4
mg. N present in embryoeach day
(cumulative)
Szneroffna
—
————1 6 5—
75-2—89-4—
216-4—
242-6—
384-1
Murray
1-3252-6614-8508-323
13-322I-OI32-575O-7877-85
124-00i88-8o264-30340-20410504719052870
Le Bretonand
Schaeffer
1-46
2-14 06-58-o
12-518832-057'5
I02-OI54-O
2I2-O265-0322-O39OO475-O
5 6
mg. N excretedby the embryoup to each day(cumulative)
in the protein metabolism, if any. These relations are shown in the form of a graphin Fig. 11. The newly-calculated ratio does not quite become a constant duringthe last ten days of incubation although it approximates to one, and it never reachesthe low figure obtained by Szneroffna. The extreme smallness of the proteincatabolism during the first six or seven days is reflected on this curve in the extremeheight of the ratio, but just as Fridericia's figures only began after the steady levelhad been reached, so Szneroffna's missed the early descent. The more intense theprotein metabolism, the lower the ratio because the greater the amount of nitrogenexcreted, and so it looks as if the metabolism of protein was more intense i ^ ^ eabsence of the membranes. From this it is perhaps legitimate to conclude thatthe part they play in the combustion of proteins is very slight.
The Energy-sources in Ontogenesis
(d) Calculation of the respiratory quotient.
Since we are now in possession of information concerning the amount ofprotein catabolised during the various stages of development in the hen's egg, itwould be possible, uniting the protein figures with those already in the literaturefor fat and for carbohydrate, to compute the respiratory quotient from purelychemical evidence. It could then be compared with the experimental respiratoryquotients obtained by Bohr and Hasselbalch(s) and by Lussanna(27).
600-
500-
o 400'•3
sCO
300-
200-
100-
50-^
O Szneroffna's valueso H. A. Murray• LeBreton and Schaeffer
10Days
Fig. I I .
15 20
Unfortunately, in the present state of our knowledge, such a calculation canbe only a rather poor approximation, and from the resulting curve no strict con-clusions can be drawn. In the first place we have no accurate information as to thecarbohydrate combustion. However, it is significant that Sakuragiui) finds theloss in total carbohydrate during development to be approximately equal to theloss in free glucose during the first ten days. If a curve is constructed from the datagiven by Pavybs), Tomitafo), Sato (42), Bywaters(6), Idzumite) and Sakuragiuo,a^Bging out all their points for the disappearance of free glucose during the firstten days of development, it is found that there is a loss of 166 mg. per egg. Now thefigure for loss of total carbohydrate found by Sakuragi(4i) is 130 mg. per egg.
I 3 2 JOSEPH NEEDHAM
Accordingly we may conclude that of the 166 mg. of free glucose disappeauigduring the first ten days of development all but about 35 mg. are combusted.^Pledifficulty arises when it is necessary to distribute this difference of 35 mg. overthe first ten days. It is possible to gain some idea as to how this distribution shouldbe done by using the lactic acid figures of Tomita(53). He found a peak of lacticacid on the fifth day, and it is significant that at its highest point it reaches 34 mg.per egg, thus almost exactly accounting for the 35 mg. glucose disappearing butnot combusted. In Table VIII the average values of the six workers mentionedabove have been brought together in column 2, and the daily differences betweenthem in columns 5 and 6. In column 3 are placed the figures for the lactic acidpresent each day according to Tomita, and in column 7 the lactic acid accumulatingeach day. If this is subtracted from the glucose disappearing the corrected curvefor sugar combusted is obtained and is shown in the last column. We may supposethat the lactic acid so formed is converted after the fifth day into alanine for somesynthetic purpose. During this early period, however, the protein burnt is so smallin amount that this correction does not affect the R.Q., so in Table X the uncorrectedcurve will be found.
Table VIII.
I
Day
012345
6789
10
1112131415
2
mg. freeglucose
present inwhole egg
175-51687157-51440130-1114-7
9678 1 064-349533-71894-5000
3
mg. lacticacid
present inwhole egg
6-618-124-0280320342
26-018013-589
5-75
t'l2*52-2O
4
Intervalsof
days
0- 11- 22- 33- 44- 55-6
6-77-88-99-10
IO-II11-1212-1313-1414-15
5 6
mg. glucosedisappearing
Experim.
6-811-213513915-4180
15-716-714-8158148
14-44-500
Smoothed
7-5IO-O129i|-o16-5173170167157149140
n-55000
7
mg. lacticacid
accumu-lating
II-55940402-2
•
8
mg. glu-cose com-busted, i.e.cols. 0-7
o-o
UII-O14-317317016715-7149140II-55000
* After this point the lactic acid begins to disappear.
Our knowledge of the combustion of fat is unsatisfactory. The position isexperimentally a difficult one, for we have to deal with a system containing enormousamounts of fat, and yet in which the tiniest changes are of great theoretical im-portance. It is not surprising that the methods in use up to the present ^have not succeeded in solving the problem. In Table IX are collectedthe figures obtained by averaging out all the points for total fat in the egg obtainedby Eaves (9), Idzumi (22), Murray (31) and Sakuragi uo. Column 2 shows the amounts
The Energy-sources in Ontogenesis 133of fat actually present in the egg each day during incubation, and column 4 the fatld^^ach day, the smoothed curve figures of which are shown in column 5. Incolumn 6 are the figures calculated by Murray (31) from his estimations of CO2-production, assuming that all the CO2 produced each day was derived from thecombustion of fat. These reveal a considerable divergence, and it is to be notedthat from the seventh to the fourteenth day the fat lost as determined by theaveraged chemical analyses, is considerably in excess of that lost as determinedfrom the CO2-output, even supposing that all the CO2 was derived from fat, whichis not true. The figures of Bohr and Hasselbalch (5) for CO2-output would givean even worse divergence, for during this particular period they were lower than
Table IX.
I
Days
012
3456789
1 0
111 2
1314I S
1617181 92 0
2
gm. fat inwhole GSS
S-65-6S-6S-6S-6S-6S-65-65-585-535-455-355-255-135-oi4-864674 S O4-253 9 13-45
3
Intervalsof days
0- 11- 22- 33- 44- sS - 6
6 - 77 - 88- 99-10
10—11
11-1212-13
13-1414-1515-16
16-1717-1818-1919-20
—
4 S
mg. fat utilised per day
Experim.
000000
02 0
£1 0 0
1 0 01 2 01 2 01 5 01 9 0
1 7 02 5 0
34°460—
Smoothed
000000
02 0
5°8094
1 0 51 1 61 2 7
145168
2 0 52 5 0
3314 6 0
6
mg. fatutilised per day
calc. by Murrayfrom CO2-output
0000
36
112 0
33456 0
80i°5132164198
2362533S9——
Total 2150
those of Murray. The explanation for this missing fat must be either that duringthat period it is used for other purposes than combustion, or perhaps more prob-ably, that the estimations of fat are wrong. However, since we are purposing tocalculate the respiratory quotient, we cannot make use of Murray's computed fatloss curve, because it was itself derived from respiratory data", and must, therefore,adopting the chemical curve, neglect the error in question.
The fact that there do exist these considerable errors in the curves for fat and^ ^ d r a t e loss, however, make it impossible to lay any stress on the figures for
percentage utilisation shown in Table X. Columns 2, 3 and 4 of that table give thecombustion of foodstuffs in absolute mg. per embryo per day, and their sum the
JOSEPH NEEDHAM
total amount of foodstuff catabolised each day in column 5. At the bottom 1of these columns there will be found its total. In columns 6, 7 and 8 the pfat and carbohydrate combusted each day in percentages of the total foodstuffcombusted each day, are shown. For the reasons given above no significance canbe attached to the exact shape of these curves. Finally, the calculated respiratoryquotient is given in column 9.
Fig. 12 shows the way in which the respiratory quotient calculated from chemicalanalyses only, assuming carbohydrate as i-oooo, protein as o-8oi and fat as 0707,agrees with the respiratory quotient determined experimentally by Hasselbalch (19),
by Bohr and Hasselbalch (5) and by Lussannato). The approximation is inexactbut suggestive. It is interesting to note that of the high respiratory quotients of thefirst five days, the only point actually given by Bohr and Hasselbalch was that at0-890 for the fourth day. In their tables, however, there were several blank spaces,and when the respiratory quotients were calculated for these with the aid of theoxygen utilisation curve of Hasselbalch (19) five other high points came to light.These are all shown on the graph.
At the beginning of development the albumen contains a certainalkali reserve, which might be expected to bind CO2 and so to depress the apparentR.Q. This has been measured by Aggazzotti (2) and by Healy and Peter(20), who
The Energy-sources in Ontogenesis 135
^ very divergent results in titrating to different indicator end-points. Ih^^calculated what the correction for the R.Q. should be, on the basis of thehighest figures of Healy and Peter: the resulting points are seen in Fig. 12 and showthe effect of the alkali reserve to be quite small.
Since at no time does the protein reach a level of more than 3-5 per cent, ofthe total foodstuff catabolised, it hardly affects the respiratory quotient curve. Asfar as the observable respiration is concerned, the burden is borne by the fat and,to a lesser degree, by the carbohydrate.
The totals at the bottoms of columns 2, 3, 4 and 5 in Table X are of someinterest. To make them comparable with other data in the literature the followingcorrections may be made:
Total mg. offoodstuff combusted Correction for
during the whole foodstuff disappearingof incubation but not combusted
ProteinCarbohydrateFat
6834166-50
2I7I-0O
O40
IO5
6834126-50
2066-002260-84
From these figures two comparisons may be made. In the first place, they
136 JOSEPH NEEDHAM
agree well with the results of some previous workers in the absolute loss oweight by the egg during its development, this corresponding more or lessthe protein is not burnt altogether away) to the foodstuff catabolised. Table XIshows the results brought together.
Table XI.
Investigator
DrogeHasselbalchMurrayNeedham
Tangl and von Mituch
Reference
81931
Presentinvestigation
S i
Total loss during development of
mg. dry weight
2230—
16642260
22102180254°225024602420
mg. fat
195°226015772066
180021502180214022102230
mg. (othersubstances
by difference)
280—87
194
4 1 030
360n o250190
225 (Av.)
Secondly, it may be enquired what part of the total energy of development isprovided by fat, protein and carbohydrate. In Table XII the available figures areshown, though, like those in Table XI, they are only rough. It is evident that thereis no agreement among different classes of animals as to the substance from whichthey shall principally derive their energy during their development. And it maywell turn out that some of them do not utilise a fat, a protein, or a carbohydrate,but a sterol instead.
Table XII.
Investigator
FarkasTichomiroffGortnerDakin and DakinFaur6- Fremiet and
DragoiuWetzelNeedhamPearse
Reference
10
s7
n
55
11
Egg of
Silkworm
Brook-troutPlaiceFrog
CrabChickBrook-trout
Foodstuff combusted-in percentage of totalfoodstuff combusted during development
Protein
3330-1463080043 9
250302
75-o
Carbohydrate
1506« 37"< 2O-194
O
5-570
Fat
67-054-2
° 36775-0914250
(e) The succession of energy-sources during development.
Since the preceding paper of this series was written the hypothesis of a successionof energy-sources in embryogenesis, carbohydrate preceding protein and p ^ ^ i npreceding fat, has received further confirmation from recent work. The pointswhich have arisen will now be dealt with.
The Energy-sources in Ontogenesis 137
i) The undoubted utilisation of carbohydrate by the hen embryo in the early^ A of its development naturally leads to the question of whether embryonic
tissues vary in glycolytic power with their age. Negelein (34) has recently gone intothis problem and gives a curve relating the anaerobic glycolysis to the age of ratembryos. There is a high peak at 0-47 mg. dry weight, after which time the activitysteadily declines till at birth (1 i-o mg. dry weight) the glycolytic power is less thana third of its former value. "Der anaerobe Glycolyse," says Negelein, "istum sokleiner je alter der Embryo ist." The aerobic glycolytic power also manifests apeak at 0-47 mg. dry weight, but Negelein considers that this is not a physiologicalphenomenon.
(2) The silkworm embryo seems to come into line with other embryos asregards early carbohydrate utilisation, according to the recent work of Pigorini(38),who estimated the glycogen in the embryos of Bombyx mori throughout theirdevelopment. Tallarico (50) has brought forward evidence showing that the lipaseof the hen's egg increases very markedly in activity towards the end of incubation,especially after the ninth day.
(3) Attention was drawn in the previous paper to the fact that the calorificquotients obtained by Meyerhof (30) on the eggs of Arbacia pustulosa and Aplysialimacina did not agree with the view that carbohydrate was being utilised as anenergy-source in the very earliest stages of development. His calorific quotientsvaried about 2-6 and he concluded that there was combustion of fat, though thetheoretical figures are 3-2 for protein, 3-3 for fat and 3-5 for carbohydrate.
Very recently, Rogers and Cole (39) have made more accurate estimations ofthe heat-production of Arbacia eggs and have obtained higher values than anypreviously recorded. Their technique was modelled on the micro-calorimetricmethods applied to muscle by A. V. Hill, and as the chief trouble in such work isleakage of heat, it is probable that their values are the most accurate we have. Theratio of heat-production between fertilised and unfertilised eggs they found tobe the same as that of Meyerhof and Shearer (47). They themselves drew no con-clusions about the calorific quotient, but clearly higher values for heat-productionwill bring the C.Q. more into line with the theoretical values, so it was worth whileto calculate what the c.Q. would be on the new figures of Rogers and Cole. Referenceto Fig. 13 will show that the new calorific quotients are higher than the theoreticalrange, but not so far from it as the old ones, and what is significant is that theypoint to a combustion of carbohydrate both before and after fertilisation.
(4) Indirect evidence about the utilisation of protein by the embryo can begained by the work of Scheminski(43). Scheminski determined the resistance ofthe trout egg to electric currents during its development. The whole period was55 days, and for the first 30 days there was practically no change in the resistance,but after that time it rose tremendously, the strength of current required to produceprecipitation of the egg globulin in 1 minute increasing six times in the last 25 daysof^»elopment. The effect of the current was to render the egg-membrane per-meable to kations, which would diffuse out and cause the globulin to be precipitated(cf. Gray(18)). Jarischta) showed that lipoids and fats in systems poor in salt
-ivii
138 JOSEPH NEEDHAM
favour the precipitation of globulin, so if the current dismisses the kations fromJheegg»t n e precipitation of globulin will be more favoured the more fatty subsii^Pesthere are present. Scheminski's curve becomes, then, in some measure, an indexof the amount of fat absorbed by the embryo, and the fact that it is of so graduala slope during the first two-thirds of development may be interpreted as showinga greater intensity of fat absorption (and combustion?) towards the end of develop-ment than towards the beginning. These findings may be compared with those ofGage and Gage (14) on the hen embryo, discussed in the previous paper.
Theoretical values for:Carbohydrate
Fat
Protein
AFTER FERTILISATION4
|Meyerhof^I^Shearer- Warburg1 IRogers and Cole
Fig. 13-
Tomita(s3) and von Grafe(i7) were not the only workers who drew attentionin the past to the evidence showing that fat was not the only energy-source of thechick embryo. Drogew considered that protein must take a share in the work,and Sakuragi(4O specifically went into the question of the other energy-s<^Besof the embryo. In the German summary to his Japanese paper, he says, " Obwohlbisherige Autoren, welche sich mit Stoff- und Energiewechsel von bebriitenden
The Energy-sources in Ontogenesis 139
Hiihnereiern beschaftigen, die Bedeutung des Kohlehydrates fur Energiewechselg^^vernachlassigten, glaubt der Verfasser, dass der schon vorhandene Trauben-zucker in den ersten Bebriitungstadien besondere Wichtigkeit und grosse Bedeu-tung dafiir hat, und dass der erste chemische Vorgang in den bebriitenden Eiernin der Zersetzung von Traubenzucker besteht." Sakuragi estimated the free andcombined sugar, the fat, the various fractions of nitrogen, and the glycogen at thedifferent stages of development and interpreted his figures as showing that through-out development carbohydrate was combusted, the fat at the late stages beingturned into carbohydrate before being burnt. His arguments for this processwere not very convincing but his experimental results were very valuable indeed.
In the present paper, further evidence has been brought forward which seemsto show that there is in the developing embryo a succession of energy-sources,carbohydrate being the first one to be used, then protein and then fat. The intensityof production of urea, and of uric acid, the intensity of combustion of protein, haveall been shown to be greatest from the seventh to the eleventh day of development,in other words in the second quarter of incubation. The position can best beexpressed by saying that the points of maximum intensity of combustion of thethree classes of foodstuff probably follow each other in the order, carbohydrate,protein, fat.
It is interesting to note that in a closed system such as the hen's egg the com-bustion of protein is much less than that of the other two foods. Fat and carbo-hydrate can be burnt completely away while the products of protein metabolismremain as nitrogenous waste which cannot be got rid of. In an egg such as thatof the trout, however, where the nitrogenous end-products can escape into thesurrounding medium, we find that as much as 63 per cent, of the total energy usedduring development may come from protein.
It is also interesting that in agreement with Gayda's findings with the egg ofthe toad (is) the period when it is most expensive to double the weight of the chickembryo seems to coincide with the period of maximum intensity of protein com-bustion. In Fig. 14 is shown the mg. per cent, of wet weight of embryo proteincombusted per day, and as a background, the number of gram-calories evolvedper day per gm. wet weight of embryo during periods in which the weight ofembryo is doubled. The agreement is suggestive, and, if further criticism shouldleave this relationship established, it would seem to be due to the specific dynamicaction of the protein combusted. If we accept the findings of Sendjuus) that theamino-acids principally combusted are tyrosine and tryptophane, it may in thefuture be possible to calculate what the extent of this specific dynamic action shouldbe. According to Seth and Luck(46) the S.D.A. of an amino-acid is proportional toits power of raising the blood amino nitrogen, and hence to the rapidity of itsabsorption from the gastro-intestinal tract. In the case of the egg, where the amino-acids presumably pass directly from the albumen into the blood-stream, there is^feesent no means of calculating what the S.D.A. ought to be. Possibly in the egg,where the influence of the intestinal wall is eliminated, all amino-acids may havethe same S.D.A,
140 J O S E P H N E E D H A M
The ultimate nature of the succession of energy-sources presents a problemofgreat interest. It is possible that carbohydrate is first combusted because it r e q ^ Kno preparation. Proteins must be deaminated, fats must be desaturated, andperhaps the embryo in its early stages cannot do either of these things: but, on theother hand, glucose lies ready for use, and it is significant that what is then com-busted is free, not combined, carbohydrate. There is already strong evidence thatthe power of desaturation of fats only arises at a comparatively late stage of develop-ment, e.g. the tenth to the fifteenth day in the chick (Needham (33), p. 21). Andwe may look on the unsaturated fatty acids present in egg-yolk as a preparation forthese conditions.
Or it may be that some conception of " ease of combustion " will prove helpful.Quastel and Whetham (56) studying the action of B. coli on various organic sub-stances, found that carbohydrates were much better hydrogen-donators thansubstances of protein or fatty type. The following figures, taken from their paper,are very striking:
Reduction coefficient(The reciprocal of the molar concentration
required to reduce i c.c. of 1/5000methylene blue in presence of standard
amount of organism in J hour)"Carbohydrate"
Glucose 5000Fructose 5000Lactic acid 583
"Protein"Alanine 1Glycine o-8Glutaminic acid 25
"Fat"Nonylic acid o-4Heptylic acid 0-4
The succession of energy-sources might in some such way be related to achanging oxidation-reduction potential of the embryonic cells, and a micro-injection study of such cells in tissue-culture, using rH indicators, would now bein order.
An ontogenetic succession of carbohydrate, protein and fat, could appear inthree ways:
(1) Combustion of foodstuffs by the embryo.(2) Absorption of foodstuffs by the embryo.(3) Storage of foodstuffs by the embryo, i.e. its constitution.The following papers in this series will be devoted to investigations dealing
with the relations between combustion and absorption on the one hand and com-bustion and constitution on the other. For a discussion of this subject from asomewhat different angle, reference should be made to the second paper ofMurray (30.
The Energy-sources in Ontogenesis
THE CONTROLLING FACTOR IN EMBRYONIC METABOLISM.
Assuming, for the time being, that an ontogenetic succession of energy-sourcesexists, we may enquire whether it is due to the influence of the embryo or to theinfluence of its food supply. In other words, whether it is embryogenic or ovo-genic, whether the embryo combusts protein because it cannot obtain carbohydrate,or because it must by its functional constitution, do so.
115-
90
A certain amount of evidence already exists which contributes to a solution ofthis problem. It will be seen from Table VIII that the free sugar—for reasons givenabove probably the only carbohydrate fraction burnt by the embryo—does notentirely disappear until the twelfth day of development. Yet, as Fig. 10 showsclearly, it is between the eighth and the ninth day that the peak of utilisation ofprotein occurs1. The embryo then by no means awaits the exhaustion of its carbo-hydrate supplies before beginning to combust protein. This fact is strong evidencein favour of the view that the embryo and not the supply of food at its disposal isin command of the situation.
In order, however, to make more certain of this, some injection experimentswere carried out. Eggs were injected in the manner described in a foregoing^ (Needhamfo)) with a solution of glucose containing 500 mg. per c.c.
1 Moreover at that moment the egg also contains about 140 mg. per cent, of glucose in tne uuunuform of ovomucoid.
142 JOSEPH NEEDHAM
The only modification in the method of injection was that the glucose was injectedinto the air-space, from which it was quickly absorbed; this process consider^freduced the mortality of embryos. As will be seen from Table XIII and Fig. 15no significant effect was produced upon the uric acid curve. If the embryo had
Table XIII.
Day
99999
1 0IOIO
Whethernormal orinjected
NNNIINNI
On whatday
injected
——44
——8
mg. glucoseinjected
——
250500
——
500
mg. perembryo
09500-9691-3011-2341-1821-8731-3441-526
mg. %wet weight
52-253-471-667965-0
70-3550-5257-80
100-
90 -
:igh
•0
aci
0
•c3M
s
80 -
70 -
60 -
50—
40 -
30 •
20 -
10 -
INJECTION INJECTION
SIGNIFICANT^LIMITS
110
DaysFig. 15.
1 '15
120
been burning protein because carbohydrate was absent or not easily obtained, thenthe uric acid curve should have been depressed after the injection of glucose, hutthis was never the case.
We may conclude provisionally that the succession of energy-sources in onto-genesis is embryogenic and not ovogenic, that it is in some way intimately bound
The Energy-sources in Ontogenesis 143
the metabolic potentialities of the growing embryo. The embryo does notve as regards its nourishment as does the bacterial cell.
SUMMARY.1. The uric acid content of the hen's egg has been investigated from the fourth
to the twentieth day of incubation. There is a period of intensive uric acid pro-duction from the seventh to the eleventh day. After that point the excretion of uricacid fails to keep pace with the growth and differentiation of the embryo.
2. The point of maximum intensity of uric acid production occurs two dayslater than the point of maximum intensity in the production of urea.
3. From the fourth to the seventh day more urea is present than uric acid, andmore is excreted, but by the tenth day the adult relationship is attained, in which95 per cent, of the total nitrogen excreted is uric acid.
4. The maximum intensity of protein combustion is attained between the eighthand the ninth days. It is pointed out that this occurs midway between the periodswhen carbohydrate and fat are respectively the predominant energy-sources.
5. The protein used as a source of energy belongs entirely to the coagulablefraction; ovomucoid is not employed for this purpose.
6. The protein nitrogen lost by combustion during development amounts to7-5 per cent, of the total protein nitrogen present at the beginning, and to 3-0 percent, of the total foodstuff burnt.
7. The R.Q. for each day of incubation has been calculated on the basis ofchemical analyses of fat, protein, and carbohydrate, and agrees as well as can beexpected at present with those experimentally determined by Bohr and Hassel-balch, and by Lussanna.
8. Further evidence has been collected from the literature indicating that inembryogenesis there is a succession of sources of energy, carbohydrate precedingprotein, and protein preceding fat.
9. Injection experiments and other considerations lead to the conclusion thatfactors located in the embryo decide what the embryo shall make use of as a sourceof energy. It does not, for instance, combust protein because its supply of availablecarbohydrate has been exhausted.
My thanks are due to Prof. Sir Frederick Hopkins for his constant interest,to my wife for much valuable help, and to the Government Grant Committee ofthe Royal Society for a grant towards the cost of these researches.
144 JOSEPH NEEDHAM
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(Polish.)(5) BOHR, C. and HASSELBALCH, G. (1903). Skandinav. Archivf. Physiol. 14, 398.(6) BYWATERS, H. W. (1913). Joum. Physiol. 45, xl.(7) DAKIN, H. and DAKIN, S. (1925). Brit. Journ. Exp. Biol. 2, 310.(8) DROGE, A. (1914). Arb. a. d. Path. Inst. Tubingen, 9, 289.(9) EAVES, E." V. (1910). Journ. Physiol. 40, 451.
(10) FARKAS, K. (1908). Pflilger's Archiv, 98, 490.(11) FAURE-FREMIET, E. and DRAGOIU, J. (1923). Archives Internal, de Physiol. 21, 403.(12) FAURE-FREMIET, E. and DU STREEL, V. (1921). Bull. Soc. Chim. Biol. 3, 480.(13) FRIDERICJA, L. S. (1912). Skandinav. Archivf. Physiol. 26, 1.(14) GAGE, S. and GAGE, S. (1908). Set. N.S. 28, 494.(15) GAYDA G. (1921). Archivio di Fisiologia, 19, 211.(16) GORTNERJ R. A. (1913). Journ. Amer. Chem. Soc. 35, 632.(17) v. GR.XFBJ E. (1910). Bioch. Centralblatt, 6, 441.(18) GRAY, .1. (1920). Journ. Physiol. 53, 308.(19) HASSELBALCH, G. (1900). Skandinav. Archivf. Physiol. 10, 353.(20) HEALY, P. and PETER, J. (1925). Amer. Journ. Physiol. 74, 363.(21) HOPKINS, F. G. (1908). Chem. News (1893), 66, 106, and in COLE, Pract. Phys. Chem. 2nd ed.(22) IDZUMI, I. (1924). Mitt. Med. Fak. Univ. Tokyo, 32, 197.(23) JARISCH, A. (1922). Pfliiger's Archiv, 194, 2.(24) y. KNIEREM, P. (1877). Zeitsch.f. Biol. 13, 36.(25) KROGH, A. (1906). Skandinav. Archivf. Physiol. 18, 364.(26) LE BRETON, E. and SCHAEFFER, G. (1923). Trav. Inst. Physiol. Fac. Mid. Strasbourg.(27) LuSSANNA, E. (1905). Archivio di Fisiol. 3, 113.(28) MEISSNER, B. (1868). Zeit.f. rationelle Medizin, 31, 144.(29) MEYER, H. (1877). Diss. Konigsberg.(30) MEYERHOF, O. (1911). Biochem. Zeitschr. 35, Z46, 280, 316.(31) MURRAY, H. A. (1925). Journ. Gen. Physiol. 9, 1; (1926), 9.(32) NEEDHAM, J. (1926). Brit. Journ. Exp. Biol. 3, 189.(33) NEEDHAM, J. (1924). Biochem. Journ. 18, 1371; Physiol. Rev. (1925), 5, 1.(34) NEGELEIN, E. (1925). Biochem. Zeitschr. 165, 122.(35) PAVY, P. (1894). Physiol. of the Carbohydrates, London, p. 206.(36) PEARSE, E. C. (1925). Ecology, 6, 7.(37) PIGORINI, J. (1924). Ann. Royal. Staz. Bac. Padova, 44.(38) (1922). Ann. Rend, di 1st. Ven. Sci. Lett. 82, 351.(39) ROGERS, C. E. and COLE, J. (1925). Biol. Bull. 49, 338.(40) RoNCHESE, F. " Guide pratique d'analyse des urines." Bailliere, Paris.(41) SAKURAGI, G. (1917). Journ. Tokyo Med. Soc. (Japanese), "Tokio Iggakkwai," 31, 1.(42) SAT6, K. (1916). Ada Schol. Med. Kyoto, 1,375.(43) SCHEMINSKI, F. (1922). Biochem. Zeitschr. 132, 154.(44) SCHIMANSKY, C. J. (1879). Zeitschr. Physiol. Chem. 3, 396.(45) SENDJU, M. (1925). Japanese Journ. Biochem. 5, 391.(46) SETH, T. N. and LUCK, J. M. (1925). Biochem. Journ. 19, 366.(47) SHEARER, C. (1922). Proc. Roy. Soc. B, 93, 410.(48) STEUDEL, O. and KRIWUSCHA, L. (1914). Biochem. Zeitschr. 66, 126.(49) SZNEROFFNA, L. (1921). Trav. du lab. de phys. de Vlnst. Nencki, Varsovie, 1, No. 3. (Polish.)(50) TALLARICO, D. (1908). Arch. Farm. Sper. e Sci. Aff. 7, 535.(51) TANGL, F. and VON MITUCH, P. (1908). Pfliiger's Archiv, 121, 437.(52) TICHOMIROFF, H. (1882). Zeitschr. Physiol. Chem. 9, 526.(53) TOMITA, T. (1921). Biochem. Zeitschr. 116, 22.(54) WARBURG, O. (1915). Pfliiger's Archiv, 160, 324.(55) WETZEL, W. (1907). Archivf. Anat. und Physiol. 527.(56) QUASTEL, J. H. and WHETHAM, M. D. (1925). Biochem. Journ. 19, 522, 651.
