Book - Chemical embryology 2-9 (1900)
Embryology - 19 Apr 2024    Expand to Translate
|
|---|
| Google Translate - select your language from the list shown below (this will open a new external page) |
|
العربية | català | 中文 | 中國傳統的 | français | Deutsche | עִברִית | हिंदी | bahasa Indonesia | italiano | 日本語 | 한국어 | မြန်မာ | Pilipino | Polskie | português | ਪੰਜਾਬੀ ਦੇ | Română | русский | Español | Swahili | Svensk | ไทย | Türkçe | اردو | ייִדיש | Tiếng Việt These external translations are automated and may not be accurate. (More? About Translations) |
Needham J. Chemical Embryology Vol. 2. (1900)
| Online Editor |
|---|
|
| Historic Disclaimer - information about historic embryology pages |
|---|
|
Chemical Embryology - Volume Two
Section 9 Protein Metabolism
9-1. The Structure of the Avian Egg-proteins before and after Development
The hen's egg contains at the beginning of development, as we have already seen, about six or seven different proteins and a number of smaller nitrogenous molecules. At the end it consists of another set of proteins, mostly the avian body and serum proteins, a further collection of simpler nitrogenous substances, differently distributed, and, in addition, a quantity of incombustible protein breakdown products, the outcome of the embryonic oxidations. How does the constitution (especially as to amino-acid distribution) of the original egg-protein molecule differ from that of the finished embryo-protein molecule? One answer is afforded by the work of Plimmer & Lowndes who massed together the proteins at o, 15 and 21 days of incubation, and made van Slyke estimations of the amino-acid distribution, employing Plimmer's modifications of this technique. In Table 130 are shown the results, expressed in percentages of the total nitrogen.
Table 130.
Plimmer and Lowndes' figures (massed proteins) : Experiment
I
Experiment
2
% of the total nitrogen
% of the total nitrogen
Stage
...
15 days
Hatched
15 days
Hatched
Amide nitrogen
8-2
8-7
8-2
9-8
9-5
8-7
Humin nitrogen
1-5
1-8
1-7
1-8
1-9
1-8
Di-amino fraction
Total nitrogen ...
25-8
27-1
27-4
24-5
24-2
26-4
Amino nitrogen ...
14-8
I5-0
14-5
13-2
II-3
12-2
Non-amino nitrogen
II-O
II-9
13-0 i6-8
II-3
12-9
I4-I
Arginine nitrogen
14-3
15-0
13-2
13-5
14-5
Histidine nitrogen
0-4
1-2
0-5
2-1
4-8
il
Lysine nitrogen
II-O
lo-g
10-2
9-2
6-4
6-6
Cystine nitrogen
1-8
1-7
2-6
1-2
1-2
0-6
Mono-amino fraction
Total nitrogen ...
64-0
6i-3
6o-2
63-7
62-9
62-8
Amino nitrogen ...
67-1
64-7
59-1
59-3
57-1
55-3
Non-amino nitrogen
i-i
4-3
5-8
7-4
Arginine nitrogen
4-4
3-6
3-2
4-0
3-1
3-3
Cystine nitrogen
3-5
4-9
5-7
3-9
—
3-3
Over the whole period the percentage of amide and humin nitrogen showed no change, except a very slight decrease in amide nitrogen in one experiment. The total di-amino nitrogen percentage, however, increased to the extent of 2 per cent., and, corresponding with this, the arginine nitrogen increased by i per cent. Histidine and lysine probably account for the difference. Conversely the monoamino-acids decreased — from 64-0 to 60-2 in one experiment, and from 63-7 to 62-8 in another. The non-amino nitrogen of this fraction, on the other hand, increased, and, as far as could be ascertained with these methods, the cystine nitrogen did so too. As the figures for bromine absorption decreased continually through development, Plimmer & Lowndes suggested that the cystine was increasing relatively at the expense of tyrosine, but, as Plimmer & Phillips had previously shown, the bromination method is not very quantitative, nor even very specific. The main result of the investigation was the finding that the mono-amino-acids decreased and the di-amino-acids increased, with the suggestion that the former were those principally used for furnishing energy by combustion. As will be mentioned later, an analogous process seems to go on in the eggs of the trout and the salamander, the only other material which has been treated from this point of view.
Very similar experiments to those of Plimmer & Lowndes were carried out by Russo, whose main interest lay in the origin of the purine bases. He had already found a diminution in the arginine and histidine content of the echinoderm testis corresponding to an increase in the purine bases, and, in accordance with the original suggestion of Hopkins & Ackroyd, he was inclined to regard that way of derivation as universal. Russo's results with avian eggs are shown in Table 131; they were obtained with the van Slyke method applied to the massed proteins of all the parts of the egg-contents. It is evident from the figures that in his experiments the amide nitrogen remained constant (agreeing thus with those of Plimmer & Lowndes) , as also did the cystine (differing from theirs) . Russo's treatment of the cystine question, however, cannot be considered final, for, as Plimmer & Lowndes have shown in another paper, some of the cystine passes into the filtrate from the phosphotungstates of the hexone bases in the van Slyke method, and must be looked for there. The question is also complicated by the presence of Mueller's sulphur-containing amino-acid in the monoamino-acid fraction. Perhaps more serious, therefore, is the divergence between the results of Russo and those of Plimmer & Lowndes over the acids arginine and histidine, for, whereas in the latter they definitely though slightly increased, in the former they equally definitely decreased. And the mono-amino-acids, which in Plimmer & Lowndes' work decreased, in Russo's work increased, an eflfect seen in both the amino and non-amino fractions. All the changes in Russo's experiments were greater relatively than those in Plimmer & Lowndes'.
Table 131.
Russo's figures (massed proteins) :
Days .
10
15
Amide nitrogen
5-12
5-17
5-15
Humin nitrogen (not determined)
Di-amino fraction
Total nitrogen
24-62
24-26
20-8o
Arginine nitrogen
11-84 3.68
10-25
7-89
Histidine nitrogen
4-04
3-13
Lysine nitrogen
g-io
9-97
9-78
Cystine nitrogen
0-49
0-47
0-47
Mono-amino fraction
Total nitrogen
48-25
51-90
52-91 42-61
Amino nitrogen
40-79
42-35
Non-amino nitrogen ...
7-46
9-55
10-30
Nitrogen unaccounted for
21-52
18-20
20-67
Calvery's figures (massed proteins, including shell) :
% of the total nitrogen
Days ...
5
10
15
20
Acid melanin nitrogen
0-88
0-81
1-04
0-94
0-99
Amide nitrogen
8-69
6-84
8-44
8-88
8-22
Humin nitrogen ...
0-90
0-89
I-I5
1-49
1-65
Di-amino fraction
Total nitrogen ...
26-30
27-75
27-05
25-93
28-25
Arginine nitrogen
14-53
14-72
13-25
13-32
15-82
Histidine nitrogen
1-82
Ml
4-74 8-53
3-01
1-69
Lysine nitrogen ...
8-93
9-60
10-12
Cystine nitrogen...
0-80
0-91
0-72
0-93
0-65
Mono-amino fraction
Total nitrogen ...
64-05
62-54
62-25
63-95
62-00
Amino nitrogen ...
61-40
6o-35
58-72
59-40
'1%
Non-amino nitrogen
2-65
2-19
3-53
4-55
The third investigation along these lines was that of Calvery, who reversed Russo's findings and agreed with Plimmer & Lowndes that the mono-amino-acids were combusted rather than the di-amino acids. As Table 131 shows, Calvery found no change in acid melanin, amide, or cystine fractions, a fall followed by a rise in the arginine fraction and a rise in the lysine fraction. His data, however, differed from those of previous workers by including the shells, so the subject is by no means closed and in spite of the large amount of laborious work that has been done on it we cannot as yet be said to know much about the origin of either the nucleins or the incombustible end products. The change over from amino to non-amino nitrogen in the filtrate from the bases, which appears in all three sets of figures may be due to the accumulation of proline and oxyproline.
Sznerovna's figures:
Days Proteins of
o White
Yolk
White and yolk massed 14 Embryo 18 Embryo
White and yolk massed
Table 132.
% of the total nitrogen
Ammonia Melanin
nitrogen
6-64 7-88
lit
5-98
7-14
nitrogen
0-73 i*6o 3-02 2-46 1-45
Di-amino Mono-amino
nitrogen
64-52 66*02 65-12 58-15 64-07 64-77
nitrogen 26-66 25-37 26-14 33-25 27-49 26-64
Calvery's figures for whole c^gg (including shell) :
Histidine nitrogen % of total nitrogen
'
van Slyke
Mercuric
"*
van Slyke
total nitrogen Bromination Colorimetric
sulphate
distribution in histidine Plimmer
Hanke &
Kossel &
Isolation
Days
method
fractio]
1 & Philli
ips
Koessler
Patten as flavianate
2-31
5-86
6-70
5-03
3-75
3-8i
5
1-36
6-07
5-83
4-93
3-76
3-68
10
4-74
6-14
5-90 6-00
4-42
3-79
3-51
15
3-01
6-25
4-00
3-13
3-13
20
1-69
7-98
6-01
3-41
1-35
1-38
Arginine and lysine nitrogen % of total
nitrogen
Arginine nitrogen
Lysine nitrogen
van Slyke
Total
van Slyke Total
distri
nitrogen in
Alkaline
Isola
distri
nitrogen in
Isola
bution
arginine
hydrolysis 1
tion as
bution
lysine
tion as
Days
method
fraction
van Slyke fli
avianate method
fraction
picrate
14-63
11-04
9-90
9-48
8-40
10-90
7-04
5
14-74
9-93
9-86
9-63
11-00
8-86
6-40
10
13-25
11-63 12-66
9-58
9-59
8-53
8-60
^•f
15
13-32
9-33
9-53
9-60
9-10
4-06
20
15-82
12-84
9-91
9-83
10-12
8-30
SECT. 9] PROTEIN METABOLISM 1059
Experiments of a somewhat similar kind were made by Sznerovna, who took the proteins of yolk, white and embryo at different stages, and hydrolysed them, effecting then a simple separation into diamine and mono-amino fractions. Her figures are given in Table 132. They do not give any answer to the problem attacked by Plimmer & Lowndes and by Russo, for she did not state the results for the massed embryo and yolk proteins at the end of incubation, and they cannot be calculated in the absence of information concerning the relative weights of her i8th day embryos. Nevertheless her data for the amino-acid distribution in the massed white and yolk proteins strongly support the view that there is no intrinsic change in them during development, for it is substantially the same on the i8th day as at zero hour. On the other hand, her figures show a big difference between the embryo protein of the 1 4th and that of the 1 8th day, trending in a direction favourable to Plimmer & Lowndes rather than to Russo. The mono-amino-acids in the embryo protein certainly seem to be diminishing, and the di-amino-acids to be increasing, although such a change in the composition of the body protein contradicts Cahn's suggestion of its constancy. The whole question is confused, and needs further analysis than it has received.
9-2. Metabolism of Individual Amino-acids
Individual amino-acids, also, have been estimated in the hydrolysates of the massed egg-proteins at different stages of development. Thus Abderhalden & Kempe in 1907 obtained the amounts of amino-acids in the egg at o, 10 and 21 days, tyrosine by simple crystallisation, glutamic acid by the hydrochloric acid method, and glycine by esterification. In grams per cent, of the egg, none of the three manifested any change, but the methods employed were not delicate enough to settle the question. Levene was another pioneer along these lines, but his data were few, and so erratic that no good purpose would be served by discussing them.
An important series of experiments on the metabolic behaviour of individual amino-acids during incubation was carried out by Sendju. Tryptophane in the embryo, the yolk and the white was estimated by the von Fiirth colorimetric technique. As Fig. 295 shows, there was a marked diminution of it in absolute amount throughout the period, and this diminution took place in two stages separated from each other by a plateau. Sendju identified the first of the two falls
io6o
PROTEIN METABOLISM
[PT. Ill
with the production of haemoglobin and the second with the formation of the bile pigment, which is so noticeable a constituent of the egg in the last few days before hatching, Sendju estimated tyrosine in the same way, using the method of von Fiirth & Fleischmann, and the results he obtained are plotted in Fig. 296. The general picture is very like that for tryptophane — the total amount of the amino-acid decreases, both in yolk and white, while the fraction of it contained in the embryonic body rises to meet the descending total curve at hatching. Sendju's values for the total amount of tyrosine agree to
Sendiu (Trypbophane) e Embryo I -f ■" <- o White
• Yolk e Whole egg
Days
400
^
X
Tyrosine ® Embryo
c350
I300
i^
\
e Wholeeggj Nm ® Whole egg, PiimmerSo \^ Lowndes
a.
0,250
~^^~^
^ ^ g
2
\
•
© /
^200
ns
^
\
0^150
^
^K. /
100
^^X^
50
, . , 1 , . , , 1 , . . , 1 .
Days -> 5
Fig. 295.
Fig. 296.
some extent with those given by Plimmer & Lowndes, better at the latter part of the curve than at the former. The fall was regarded by Sendju as indicating the utilisation of the tyrosine for hormones and other special purposes.
Sendju's estimations of the content of arginine, histidine and lysine are shown in Figs. 297 and 298. There is a general similarity between the graphs, for the amount of substance in the embryo regularly rises till it has absorbed all that outside, while the yolk and white curves show that the material of the white is used regularly before that of the yolk. The behaviour of the curves for total amino-acid, however, shows some variation, histidine distinctly rising, lysine less so, and
SECT. 9]
PROTEIN METABOLISM
1061
arginine distinctly falling. Sendju himself attached no special importance to these variations, which would agree with those found by Plimmer & Lowndes in the case of histidine and lysine, but not in that of arginine.
The best work on this subject is that of Calvery who made a comparative study of histidine, arginine and lysine, using a variety of different methods.^ His figures, which are given in Table 132, show, as regards the histidine (percentage of total nitrogen of whole egg.
Arginine
Days-* 5
Days-»-5 Fig. 297.
shell included), in one case no change, in another case a slight increase, and in four cases, a definite decrease. Calvery regards Sendju's increase as due to errors of technique, but thinks Russo's decrease was genuine. As regards arginine, all four methods showed no change. Sendju's diminution is again considered by Calvery as being due simply to analytical errors, but no explanation is available for the large fall found by Russo. Out of three methods used for lysine, one showed no change and two a decrease, which Calvery thought
1 Calvery's later work confirmed Sendju's fall in tyrosine and cystine but showed no fall in tryptophane.
io62
PROTEIN METABOLISM
[PT. in
probably real. This again is not in agreement with the work of Plimmer & Lowndes; Sendju, and Russo.
The fact of the matter is that the estimation methods available are insuflficiently delicate to show up clearly the slight changes which occur in these amino-acid distributions. One reflection, at any rate, may be made, namely that the raw material of the embryo is not very different from the embryo itself: half the di-amino-acids, for instance, do not have to be made into mono-amino-acids. Would
Lysine
500
© Embryo O White • YotW ® Whole egg
Nitrogen not preci pi table with
phosphotungstic acid
(monoamino acids)
Days -+5
5 20 Days ^5
Fig. 298.
this be equally true of an embryo which did not have to suppress its protein catabolism, as is the case with terrestrial animals? Parallel studies with aquatic eggs should certainly be undertaken. And one also wonders, if the hen's egg economises its amino-acids to such an extent, whether they ever go down to individual simplicity at all, or whether they do not rather enter the embryo in the form of proteose bundles.
Sendju did not give his amino-acid results in terms of the weight of the embryo, but they have been calculated in this way, and are shown in Fig. 299. Sagara has also made some estimations of the arginine, histidine and lysine content of the embryo, but they
SECT, 9]
PROTEIN METABOLISM
[063
were few in number, and the values were extremely small, disagreeing with the results of all other workers. They have not therefore been included in these graphs. Fig. 299 illustrates, of course, the increasing dryness of the embryo. On the same graph are plotted the more recent results of Cahn, who has estimated the arginine contained in the embryo during incubation. These are seen to agree to some extent with those of the Japanese worker, the difference being probably due to the fact that Cahn only gives his dry weights of embryos after removal of fat. These curves, however, do not bring out any important relation. The value of Cahn's work Hes rather in the fact that, just as Plimmer & Lowndes examined the structure of the protein molecule of the whole egg throughout incubation, so Cahn examined that of the embryo. As Table 133 demonstrates, the percentage of nitrogen remains perfectly constant in the dry fatfree and ash-free protein of the embryonic body. A few figures were also given by Cahn showing a constant nitrogen-content of the proteins of the yolk and white. This would suggest that, although the proteins of the egg as a whole undergo a rearrangement in being converted from egg into embryo proteins, the latter do not themselves change through embryonic hfe, but remain of the same constitution when once they are formed. Such a conclusion is strongly supported by Cahn's figures for the arginine-content of the protein of the embryonic body, for, as Table 133 shows, this also is constant. The very slight variations in the nitrogen-content of the protein molecule were regarded by Cahn either as technical errors due to incomplete removal of fat and ash, or perhaps as being due to the presence in different organs of different proteins varying slightly in this respect, and appearing now in one predominance, now in another, according to the differential growth of the various parts of
N E II 68
Days
Fig. 299.
io64 PROTEIN METABOLISM [pt. iii
the body. The arginine referred to in Fig. 299 and Table 133 was exclusively that resulting from the hydrolysis of the proteins, and was estimated by decomposition with arginase and estimation of the urea so formed by the xanthydrol reagent. Cahn also investigated the amount of total arginine in the whole egg during incubation and found no change whatever. This was contrary to Sendju's findings, but agreed more or less with those of Plimmer & Lowndes. Cahn stated that the arginine-content of the proteins of the non-embryonic parts of the egg remained approximately constant (7 per cent.) throughout incubation. These observations certainly make it appear as if neither the egg proteins nor the embryo proteins change intrinsically during development, apart from the breakdown which has to go on in order that the one may be transformed into the other.
Table 133.
Arginine % of water-, fat- and
ash-free embryonic proteins
•16
Cahn's
figures :
Nitrogen »/
of proteins water
Arginine %
fat
and ash-free
of total pro
tein nitrogen
Days
Embryo
Yolk and white
in embryo
I
15-85
—
46-0
i6-i
—
—
8
15-55
—
47-0
9.
1575
15-00
46-2
II
15-67
—
47-3
13
15-55
—
46-3
15
15-50
14-70
48-0
^1
15-35
—
—
18
—
14-50
—
19
15-55
46-0
21
15-50
15-55
45-6
Ik
7-2
7-1
7-IO
Another amino-acid which has been closely studied is cystine. Sendju's experiments, in which all the cystine, both free and combined in proteins, was estimated by the Okuda iodimetric method, did not give very illuminating results, though the passage of this amino-acid from the yolk and the white into the embryo is easily seen in Fig. 300. The diminution in total cystine stands in contradiction with the findings of Plimmer & Lowndes. Expressed in milligrams per cent, wet weight, the cystine as measured by Sendju can be observed in Fig. 299. The analogous curve constructed from Cahn's data is shown on the same graph, but his more important finding was that the amount of cystine in the protein molecule was not constant. The figures in Table 134 illustrate this. For the middle part
SECT. 9]
PROTEIN METABOLISM
065
of development the percentage of cystine in the embryonic protein is fairly steady, but during the first and last days it rises. The rise at the end of the period might be due to the appearance of feathers and the cystine of their keratin, but, as Table 108 shows, their weight is small, and will probably not account for it. Cahn's explanation involved the postulate of a central fixed nucleus in the embryonic protein molecule, of which arginine would be a constituent member, while cystine would not. These are hypotheses on which much further work might be profitably carried on.
According to Calvery, the cystine/cysteine ratio of the hen's egg falls from 5-9 to o-8 during development.
150 140
Sendju
(Cystine)
© Embryo ® Whole egg • Yolk White
130
^^"~T»~^_
g120
^
■^110
iioo
1 90
1
-) — ■
1
^ 80
3
70
/
^ 60
i 50
E 40
30
»
•
X
20
^@^^^
10
1 1 r 1 1 1 1
. , . 1 , . , , 1
Days -^
Fig. 300.
Table 134.
Cahn's figures :
Cystine grams %
Cystine grams %
of protein
of protein in
nitrogen in
Days
embryonic body
embryonic body
5
3-6i
22-8
8
4-25
27-0
9
4-37
28-2
II 13
4-38 4-87
27-0 24-6
15
4-20
27-1
17
5-53
35-9
21
5-03
32-6
9-3. The Relations between Protein and Non-protein Nitrogen
Many investigators, wishing to unravel the processes by which the egg proteins are transformed into those of the embryonic body, ha\'e estimated the non-protein and free amino-acid nitrogen in various
68-2
[o66
PROTEIN METABOLISM
[PT. in
parts of the egg from time to time, and it will be convenient at this point to consider what knowledge their work has led to. Albumoses and peptones have been shown to be present in the egg-white by qualitative analysis on the 15th day by Fischel and on the 6th by Emrys-Roberts. One of the first of these investigators was Tomita, who removed the proteins from the white and the yolk by boiling with
Non-protein nitrogen ©Total non-protein nitrogen \ ©Total non-protein nitrogen |
© Non-protein nitrogen pre- I Non-protein nitrogen pre- iNaka cipibable with phosphotungsbic acid>Tomita cipi table with phosphotungstlc acid imufa O Non-protein nitrogen not yQ— D Non-protein nitrogen not precipitablej
precipitable ®Total non-protein nitrogen (Aggazzotti) ♦ " " » " (Vladimirov Sc
Sclimidt^gQ
13 Total non-protein nitrogen (Wright)
Days-* 5
Days
Fig. 301.
acetic and precipitating with tannic acid, after which he estimated the total non-protein nitrogen in the filtrates, the nitrogen precipitable by phosphotungstic acid (the di-amino-acids, peptides, and some of the cystine), and, finally, that not so precipitable (the mono-aminoacids, and some of the cystine and arginine) . His results are shown in Fig. 301, together with those of Nakamura, who later confirmed him. Unfortunately neither of them took into account the varying water-content of the white and the yolk, so that the rise found in
SECT. 9] PROTEIN METABOLISM 1067
their values cannot be assessed at its true value from their data alone. As the white is drying up, and the yolk becoming wetter during the first 10 days of incubation, Tomita's figures might be said to be unduly small for the yolk and rather too large for the white, a correction which would exaggerate the difference between them.
Later still, another Japanese worker, Takahashi, estimated the free bases in the whole egg during development. All were found to rise :
Milligrams per whole egg
Purine
Histidine
Arginine
Lysine
ays
nitrogen
nitrogen
nitrogen
nitrogen
0-04
0-13
1-02
6-55
5
—
0-I2
1-90
6-97
9
0-04
0-02
2-31
9-38
4
0-19
3-40
15-97
7
0-20
4-03
16-30
9
0-95
0-38
5-15
27-35
The general order of magnitude of these figures agrees well with that shown in Fig. 301.
Aggazzotti, who was working at much the same time as Tomita, took the changing water-content of the yolk and white into consideration. Aggazzotti precipitated the proteins in the yolk and white by the Costantino acid sublimate technique, estimated the total and the amino nitrogen in the filtrate, and then, hydrolysing the proteins, estimated their total nitrogen, their amino nitrogen and their ammonia. The results he obtained are shown in Figs. 302 and 303. As regards the white, there was no change at all in the total protein nitrogen, or in the bound amino-acid nitrogen, though the ammonia of the proteins diminished by more than 50 per cent. The significance of this fall in the protein ammonia is not at all clear. Matters took very much the same course in the yolk, where the protein nitrogen and the bound amino-acid nitrogen remained constant, while the bound ammonia rose a little before falling. The free amino nitrogen, plotted in Fig. 304, showed practically no change when related to dry weight in the yolk, but rose and fell in the white with a maximum at the 8th day. However, when the free amino nitrogen was compared with the total free nitrogen, so that a measure was obtained of the extent to which the free amino-acids accounted for the non-protein nitrogen at any moment, the curves of Fig. 305 were obtained. From them it appears that there is a maximum of free
[o68
PROTEIN METABOLISM
[PT. Ill
amino-acids (expressed in this way) in the yolk at about the 3rd day and in the white at about the 7th day. As will appear presently, this result fits in well enough with other data obtained on the embryo itself. As regards the wet weight of the white, Aggazzotti was in agreement with Tomita in finding an increase of free amino-acids so related, in the case of the yolk precisely the opposite held good.
The main conclusion which can be drawn from the work of Tomita and Aggazzotti is that the changes in amino-acid concentration in the yolk and white are rather minute. There was certainly no a priori reason for expecting big changes, for the transfer of amino-acid mole
Aggazzotbifyolk) e Total protein nitrogen e Bound amino-acid nitrogen © » ammonia nibroqen
K
Days
Days ■*• 5
Fig. 302.
Fig. 303
cules from egg to embryo might vary very greatly in intensity without involving a difference in absolute amount or concentration of the intermediary substances.
Later, Vladimirov & Schmidtt precipitated the proteins of the eggwhite during development by the Fischer-Bang uranium acetate method, and by doing Kjeldahl estimations on the precipitate and the filtrate obtained figures for the protein and non-protein nitrogen. The former increased steadily as the water-content of the white diminished; the latter also increased steadily, so that the ratio protein nitrogen/non-protein nitrogen was constant. Both are plotted beside Tomita's results in Fig. 301, with which they agree. Aggazzotti's results are much higher all through. Thus all observers agree
SECT. 9]
PROTEIN METABOLISM
1069
that the non-protein nitrogen increases per cent, wet weight in the white, but for the yolk Tomita's rising curve stands in contrast with Aggazzotti's falling one. A private communication from Wright states that recently results have been obtained confirming Aggazzotti on this point (see Fig. 301). Free amino-acids have been found by Fiske & Boyden in the allantoic liquid of the chick, to the extent of 13 mgm. per cent, on the 5th and 7 mgm. per cent, on the loth day of incubation. This is lower than the amino nitrogen of the embryonic blood which Vladimirov & Schmidtt found to vary somewhat erratically between the 13th day and hatching around an average of 54-2 mgm.
AggazzoCti
Days -»■ 5
Fig. 304.
per cent. After hatching it has an average value of 52-7 mgm. per cent. Such high values are due to the nucleated erythrocytes.
Certain Japanese workers filled an obvious gap in the data which have so far been reviewed by estimating the total nitrogen of the entire egg throughout, and the whole of the free or combined aminoacid nitrogen. The figures of Sakuragi and of Idzumi are shown in Table 135. As Liebermann had originally thought probable, and as Tangl & von Mituch had later made very likely, there is no change at all in the total nitrogen of the whole egg. Not more than an infinitesimal quantity of this element can escape, in agreement with Krogh's findings discussed in the Section on respiration. Sakuragi, as the table shows, estimated the nitrogen in the proteins coagulable with acetic acid, on the one hand, and the
1070
PROTEIN METABOLISM
[PT. Ill
l>ri
oXjquig
r oXjquia I 1 I I
i I 1
O
r U330JJIU
uiajoid-uoivj
I I I
uaSojjtu in
ua3 O
-OJJIU IBJOJL CTV
U33
-OJjtu proE I
-OUltUB 331 j
U930HIU 'P
uiajojd-uoivi 2^
'[
1
I I
-n8B03 0siou §^ I
U330JJIM
3U33BJ0m3 O
>- 3iqE[n3B03 «
U330JJIISI
r 3iqBl CTi . . .
-n3E03osaou ei I I I
U330JJlts[
PPB to
0U33E qjIM ^111
3iqBin3eo3 ™ ' ' '
L U330J1I]SI
1 I
i3Bin5iBg m I I I
I I
1 1
I I
I I
I 1
? I g 11 ^ I
I l^l«
oj o CO et
o -■
1 1
1 I I I I I I I
I 1 ll
1 I ^1
I I 1
O (N
in
I 1 I
""ill
I l?l
^1
I 1^1
I SI
ill!
I \^\
00 ^ r^ r (M
-. I en
(M "" ►
I I I I S'l
(N CO t}« in<^ 1^00 O) o >-i c* CO ■>*< into t^oo oi o
SECT. 9]
PROTEIN METABOLISM
1071
icoag. protein nitrogen (o) \ per 100 gma. luncoag. protein niCrogen(»)J wet egg white
Oay= -^
Fig. 306.
Vladimirov S(,Schmidtt
nitrogen in the proteins not so coagulable plus that of all other nitrogenous compounds, on the other. Owing to protein combustion, the former diminishes and the latter rises. Idzumi, on the contrary, obtained the non-protein nitrogen separately from the uncoagulable protein nitrogen, and the free amino nitrogen in addition to that. The free amino nitrogen remains almost constant during development, thus agreeing with the data already presented, but the total non-protein nitrogen rises owing to the accumulation of protein waste products.
The protein just referred to, which is not coagulable with acetic acid, is, of course, ovomucoid, and in the Section on carbohydrate metabolism an account has been given of our knowledge of the physiology of this compound. Trichloracetic acid was found by Hiller & van Slyke to make a sharp separation between proteins of all kinds and nitrogenous bodies of lower molecular weight. Sakuragi gave for zero hour of development the values of 1 846 mgm. per cent, of coagulable protein nitrogen (acetic acid) and 209 mgm. per cent, not so coagulable. Using trichloracetic acid, Needham found i960 mgm. per cent, for all protein nitrogen and 90 mgm. per cent, non-protein nitrogen. The increase in the protein nitrogen caught by the trichloracetic acid method over that caught by the simple boiling with acetic acid was thus 114 mgm. per cent. Komori found by alcohol precipitation 480 mgm. of ovomucoid per egg, i.e. 124 mgm. per cent, of ovomucoid nitrogen, which gives close agreement with the extra 1 14 found on using trichloracetic acid as the precipitant.
Jays
Fig. 307.
1 072
PROTEIN METABOLISM
[PT. Ill
© Protein in yolU '\
^ " M yolk-sacs I
O » „ liquid part >Rlddle
® » " solid central core j
X " " inbracellularyolk)
Days —I L— J \ \ L_J
15 16 17 18 19 20 21
Fig. 308.
The subsequent fate of the ovomucoid fraction and the part played by it in metaboHsm have been discussed in Section 8, Here, however, it may be noted that the ovomucoid is not absorbed by the embryonic vessels at a different rate from the ovoalbumen. This was demonstrated by By waters' experiments, a graph -g of which is given in Fig. 306. The S ratio of the coagulable to the "S uncoagulable protein nitrogen ^" remains the same throughout \ development, though in some •; cases Bywaters' points are rather » erratic, and there may well be 1 2° some interesting relations hidden under this approximation (see L...L also Fig. 307).
Other points also emerge from Table 135. Sznerovna's figures illustrate the transfer of nitrogen from yolk and white to embryo, the embryo having at the end about as much nitrogen as the yolk had at the beginning. Iljin's figures illustrate the large increase (perhaps due to ovomucoid) in the unprecipitable nitrogen of the yolk towards the end of development. But more illuminating are the figures of Riddle represented graphically in Fig. 308, for they show that, in per cent, of the dry solids in the yolk, the yolkproteins rise during the last 5 or 6 days of incubation. This is due to a preference on the part of the embryo for lipoids and fats, ^'^' 3°9'
the concentration of which correspondingly rapidly decreases, and the graph has only to be compared with that in Fig. 252, where the absorption intensity of the embryo at different times is given, to show how good the correlation is. For 5 or 6 days before hatching
Days->-5
SECT. 9]
PROTEIN METABOLISM
1073
O Total non-protei
O " » " " (Nakamura)
<5 Non-protein nitrogen precipibabie with phosphotungsbic acid (Nalomura)^
<^ Non-protein nitrogen not precipitable with phosphotungsbic acid(Nakar
the absorption intensity for protein is falling rapidly and that for fatty substances is rising rapidly. On the other hand, it is known that the contents of the albumen-sac tend to be included in the yolk at the end of development, about the time of the opening of the sero-amniotic duct, and this may partly explain Riddle's results. Riddle also investigated the protein content of the yolk-sac itself from the 12th day onwards, finding practically no change, and obtained various figures for the
solid central core of yolk, the O Total non-protein n.trogen(Needham)
more Hquid part, and the yolk enclosed in the walls of the sac. These are given on the graph, but, as they are somewhat erratic and depend on an arbitrary selection of the material, they are not so important as the rest. These viscous bodies in the central core, which differed very markedly in chemical composition, were explained by Riddle as being in one case unaltered yolk from the beginning of development, and in the other case a clump of egg-white driven into the yolk and imperfectly infiltrated with it. Riddle also found that, when yolk is being resorbed in the hen by the follicle which secreted it, there is a more rapid removal of Hpoids than of fats, and of fats than of proteins.
It will have been remarked that, so far, nothing has been said about the non-protein nitrogen within the embryo. The early work of Demant on human and guinea-pig foetuses, in which a high concentration of peptones and albumoses was found, was discredited by Neumeister. In 1927 Needham estimated the nitrogen in the trichloracetic filtrate by Rose's modification of the Kjeldahl method. The curve obtained was regular (Fig. 309), but more interesting points were brought out by the expression of the non-protein nitrogen of the embryo in terms of wet weight. Fig. 310 shows this, together
Days
Fig. 310.
1074
PROTEIN METABOLISM
[PT. Ill
Fig. 311.
with a few later figures of Nakamura, which are not concordant. Concentrating attention on Fig. 311 for the moment, it can be seen that the wet weight curve, after a peak on the 6th day and a depression on the gth, gains a plateau and remains there for the rest of development. The dry weight curve presents the same peak and the same depression, but, since the embryo is growing much drier as it increases in size, the dry weight curve drops away steadily after the loth day. It is significant that there is a very vigorous period of protein absorption^ before the 5th day, and another from the 14th to the 1 8th days. The depression in the curves of Fig. 311 comes just between the two maxima of protein absorption. The amount of non-protein nitrogen in 100 gm. of embryo is related, then, to the amount of protein nitrogen which 100 gm. of embryo is receiving from the rest of the egg. Reference to Fig. 250 illustrates this.
Another curve which is worth studying is the curve for nonprotein nitrogen in percentage of the total nitrogen (Table 136 and Fig. 312). Low at first, the value rises to attain a peak on the Gth day, and thereafter falls, except for a slight oscillation about the gth day, probably comparable in cause with those noted as occurring
at the same time in the curves for Fig, 312.
non-protein nitrogen as percentages of wet and dry weight. It may be said, then, that, though little change seems to take place in the non-protein nitrogen outside the embryo, yet, inside it, definite peaks are found, and that these correspond with the periods of greatest intensity of protein absorption, such
1 This does not, of course, mean absorption of intact protein; see pp. 920 ff.
® Needham
-l
Murray
e Sznerovna
-3
®
® Le Breton 8t
^ ro
Schaeffer
"^ ^
\
Fridericia
-^ I
H
-S /
\®
e
° /
\
®
'•^ i
V^
P
~n
-I f
>D ® e e
- 5
-S
\
'■'
e \®e® A>
-i i !
-^ \x§gM>-^
z f \ 1 — 1 . 1 .11.
_j_
. ,1 .... , . . ,
Days ^5
10
15 20
SECT. 9]
PROTEIN METABOLISM
1075
fc |=§ i I I 9^^r9
c "J « lOco "CO
S g
^ 2
^ I ^ I ^ I ,• I
am o ■*'iO(Nrt-coococo I |COO|r^|^^(NcoaD Tt'co o(
-Q I 1 I I I I I Icbr^'w^lf^lLOLnliiti
I I
■ (X) o CO ■* i^ r- o -^(ri tj< i-i "H « CO
- IN CO'>p C< O Tt< r^iT) lO O CTi O CO o
I " CTicb <y> oicb f^^b io io -^ io iotb
hJ- O 1-1 5
2 £
1-1 C/D
ClCO COtX) ■*
o lot^ co'*'i^m-*mo o coo w •- i^t^ O p ^ ^'P^ T 9^?>cop P) mcor^iO( 666666
CI -^(^ CO O CO«D
I 1
"«:> ^co I 6 6 « - '
I ^ I •' I
1^1
1 1 1 1 r 1 1 it I
r^ r^co CO ■* cntD r^ in ojyD M ■* 10 N CO
- (N (N CO'* 't^
^cp in CTi
6) in I -^(Xi I r^
I i> I CO 05 I coco
I I I I I
Tj-CO CO •-. 01 O I^ -^CO ^ O O " CO
" c« coini^Mco;^ Tt-« i^N " " c< co-*rj- in
a CO •* m(0 t~-co o^ o " o» co tJ- intr> r^co cr; o
1076 PROTEIN METABOLISM [pt. hi
as have already been described in the Section on general metabolism. It would appear, therefore, that within the embryo there is a distinctly greater population of amino-acid molecules in the free state at some times than at others.
9-4. The Accumulation of Nitrogenous Waste-products
It is now time to turn to the engine room of the embryo, and to deal with the breakdown of the protein molecules which by their combustion furnish a supply of energy. The amount and intensity of combustion can be far more easily gauged in the case of protein than in the case of carbohydrate and fat, because of the incombustible residues left behind.
Urea may be taken first among the end products of protein combustion, although, as Fourcroy & VauqueUn found as long ago as 1805, by far the preponderant nitrogenous end product in the fowl is uric acid. Goindet was the first to find urea in avian urine, and in 1825 Prevost & Le Royer isolated from the allantoic fluid of a chick on the 1 7th day of development a substance which gave an insoluble compound with nitric acid, and which they identified with urea. In 19 12 Fridericia carried out some experiments in which he estimated the quantity of urea in the allantoic fluid of the chick by the Schondorff method. Thus on the 17th day of development Fridericia obtained 4-5 mgm. urea per embryo, or 12 per cent, of the total excreted nitrogen (urea + uric acid). On the 20th day he got much less, only 1-7 mgm. urea per embryo, or 1-9 per cent, of the total excreted nitrogen. He concluded that only small and variable amounts of urea were present, and that the chick, like the adult hen, excreted all its nitrogen in the form of uric acid.
The subject merited re-examination however, and in 1925 I went into it anew. The method used was that of Folin & Wu, which, involving as it does the use of the specific enzyme urease, was more satisfactory than Schondorff's. The results obtained, applying the method to embryo + allantoic fluid + amniotic fluid, are shown graphically in Figs. 313 and 314. In Fig. 313 is seen the milligrams per cent, wet weight plotted against the time and also the milligrams per embryo. The latter rise steadily, as might be expected. The former rises steadily also, until the 9th day, at which point it becomes stationary for the rest of development. In other words, as far as the wet weight is concerned, the rate of production or excretion of urea
SECT. 9]
PROTEIN METABOLISM
1077
is very intense after the 4th day and before the 9th day. At later periods, although excretion of urea is still going on, it only just succeeds in keeping abreast of the wet weight. It was at once noteworthy that this intensive period of urea production occurred exactly between the carbohydrate period and the period associated with the predominance of fat metabolism. The effect may, of course, be due to other causes than to a specially intense combustion of protein during this period. For example, it may be due to a limiting factor such as the small size of the Hver, or rather to the incapacity of the embryonic Uver at this stage to turn urea into uric acid. That the developing liver can act in this way is probable from what we know of the desaturation process in embryonic metabolism (see p. 1 1 7 1 ) . If this were the case, however, an inflection in the curve of milligrams per cent, should appear at the time when the liver takes on this function. The activities of the enzyme arginase may also be involved (see p. 13 1 2). But Kaieda has brought forward convincing evidence
00
90
Points
©
80
-| •
Averages
/^
J \o
70
±SI
&/
e e\
GO
--C^^
Q
50
CPta
1J
/©
\
i
i/»
\
40
7
^
30
-■a
/
>-^
20
E
10
E
I
1 1 1
, , 1 , , ,
,,,,!
Days 5
Fig. 314.
that the urea which the chick embryo produces is due to the deamination of protein breakdown-products. He injected each amino-acid into hen eggs before incubation and estimated the urea formed by the 1 6th day, with the following average results:
Control, Allantois ,, Embryo
After injection, both Average increase
mgni. I -00 1-37 2-37 3 "So 1-23 (ranging from 0-4 to 207
according to the amino-acid)
1078 PROTEIN METABOLISM [pt. iii
The only amino-acids which failed to give an increase were cystein and serine: ammonium carbamate was effective but ammonium carbonate was not. Per mg. of injected substance the increase in urea was from o-og to 0-40 mgm.
In Fig, 314 the urea content is seen related to dry weight of embryo. Here we have a peak at the gth day instead of an inflection; the urea excretion fails to keep pace with the increase in dry matter, and drops to a possibly constant level of 30 mgm. per cent. As before, it is the gth day which is prominent.
Would the excretion of uric acid follow a similar course to a peak about half-way through development? Uric acid had been first reognised in the allantoic Hquid of the chick embryo in 18 ig when L. L. Jacobson of Copenhagen ^ described it as at first clear and ool-l pale yellow, containing uric acid in solution, but later depositing urates in masses, which, he thought, also contained protein. "Diese Fliissigkeit", said Jacobson," die in der ersten Tagen der Ausbriitung hell ist, wird nachher mehr zahe und schleimig, weisse Anhaufungen schwim- p men in derselben ; diese vermeh ren sich, worauf die wasserigten Telle verschwinden, so dass man in den letzten Tagen der Ausbriitung eine bedeutende Menge diese Anhaufungen in einem dicken und zahen Schleim gehallt, in der Allantois findet." Jacobson identified the uric acid by the murexide test. Jacobson's discovery was confirmed by Prevost & Le Royer in 1825, by Sacc in 1847 and by Stas in 1850.
Two methods were used 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 acid production is outside the best range of one method. As a micromethod for the early stages the colorimetric technique of Benedict & Franke was used, and for the later period the ammonium chloride precipitation method of Hopkins. The gradual increase in milligrams per embryo of uric acid made a very regular curve. In Fig. 315 is shown the milligrams per cent, wet weight, and here the significant plateau appears. In the first 7 days of develop
SECT. 9]
PROTEIN METABOLISM
o79
ment the uric acid is exceedingly small in amount, but from the 7th to the I ith day it rises rapidly, until on the 1 2 th day it attains a constant level which it does not leave. There is thus a specially intensive production of uric acid between the 7th and the nth days of incubation.
Fig. 316 gives the uric acid in milligrams per cent, dry weight of embryo. The curve reaches a peak on the nth day, after which it descends, and seems to be reaching a steady level by the time of hatching, at about 460 mgm. per cent. Its shape is the same as that found previously for urea. In Fig. 3 1 7 are shown the urea and the uric acid in milligrams per cent, wet weight of embryo plotted on the same scale. It shows the interesting fact that, on the 3rd, 4th, 5th, 6th and 7th days of incubation, the uric acid is rising distinctly more slowly than the urea, and, indeed, in absolute '2001quantities per egg there is less uric acid than urea until the 8th day is reached. Between the 7th and the 8th day, the uric acid rises tremendously in amount, and, overtaking the urea, almost attains its final constant value. These relationships are better seen in Fig. 3 1 8, which gives the miUigrams per cent, wet weight for both uric acid and urea, the abscissa being arranged so as to get them both on to the same graph. When this is done, it is obvious that, though both urea and uric acid rise in course 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. This is reflected on the milligrams per cent, dry weight curve, shown in Fig. 319, and it is seen that the urea is in advance of the uric acid by two days.
In the hen, as we have seen, the excreted nitrogen is mostly in the form of uric acid, and the urea takes only a very small share of it. At what stage in embryonic development is the adult relationship reached? In Table 137 and Fig. 320 the ratio uric acid/urea is seen. From the 14th day onwards it is constant at about 16, that being the adult level, but before the 7th day its value is less than unity, because more urea is present and more urea is daily excreted than uric
N E II 69
Days
Fig. 316.
O In absolute mgms. per embryo per day present • In absolute mgms.excreted per embryo per day
• ^ » ADULT
V» O *0-0-LEVEL
Fig. 319
Fig. 320.
Days
Fig. 321.
55
r^
50
\
■4 5
_
\
ra
Y
Wet
weight
'h
\
Dry
weight
-3.':.
- E
\v
30
-<
^ \l
"25
-5>
\\
%
?0
- E
§ K
^^
X
Os^
15 .'0
-E
\>^
^^
5
Days
Fig. 322.
Figs. 318, 319 and 323 are smoothed curves.
SECT. 9] PROTEIN METABOLISM 1081
acid. The adult ratio is therefore seen to be attained well before hatching.
Another nitrogenous excretory product remained to be estimated, namely, ammonia. It was improbable that this would have much effect on the picture of protein metabolism as a whole, but there was a possibility that it might have importance at some special time in embryonic life. The quantitative estimations were done by the Folin method, and led to the values shown graphically in Figs. 321 and 322.
Table 137. Relations of uric acid and urea.
Ratio :
Uric acid/
urea milligrams
per embryo
Day 4 0-8
5 0-2
D 0-2
7 0-45
8 1-63
9 10-80
10 1 1 -80
11 i3'io
12 , 14-31
13 14-29
14 15-15
15 15-73
16 16-03
17 15-70
18 15-77
19 16-00
20 16-50
Amounts excreted per day
Ratio :
per embryo as % of the
Uric acid/
total nitrogen excreted
urea milli
per day per embryo*
grams excreted
1 ^ — ^
per day
Uric acid Urea
per embryo
nitrogen nitrogen
0-145
Day 4-5
9-4 90-6
0-2I0
5-6
13-21 86-79
0-604
6-7
26-39 73-6i
7-18
l-^
83-43 i6-57
13-52
8-^
90-70 9-30 91-44 8-56
14-89
9-10
14-92
lO-II
91-44 8-56
15-92
11-12
91-90 8-10
16-28
12-13
92-10 7-90
17-28
13-14
92-52 7-48
17-96
14-15
92-76 7-24 92-34 7-66 92-48 7-52
16-88
15-16
17-03
16-17
16-69
17-18
92-28 7-72
16-55
18-19
93-22 6-78
17-65
19-20
92-68 7-32
Assuming that no other nitrogen compounds are excreted. For the increase in ammonia per embryo, the points lie on a welldefined curve, which is practically the same as regards its slope as those previously found for urea and uric acid (Figs. 313 and 328), but it will be seen that the total amounts with which we are now dealing are very much smaller than those of the urea curve, and infinitesimal compared with the amounts of uric acid which the embryo produces. Fig. 322 shows the amounts of ammonia present related to wet and dry weight of embryo. Inspection of this graph shows that the ammonia present in and around 100 gm. of embryo, falls steadily from the beginning of development. Whether the point at the 4th day represents a peak, or a descent from a yet higher value, we do not know. In Fig. 323 the amounts of ammonia,
69-2
[082
PROTEIN METABOLISM
[PT. Ill
• Ammonia
O Urea
O Uric acid
80
HOC
^ A y^SjT'^
1000
" ? /\ p Pk \
70
■900
I \ / / \\
■60
,800
700
s- V/ / qV>
_50
'600
."° JK f ^"bN-i
40
• 500
^ / \ / >i
.30
•400
f Y ^»/
20
.300
r / r*^
200
/ } ^^~*-~»^
10
rr.
■100
-.-^^MU^-PrV^ , 1 1 , 1 . 1
urea and uric acid, expressed as milligrams per cent, of dry weight of embryo, have all been placed on the same graph. Here the comparison becomes very interesting, for, just as urea was previously found to rise to its maximum 2 days before uric acid, so now ammonia is at its maximum (or, more correctly, higher than at any other time so far determined) 5 days before urea. These time-relations between ammonia, urea and uric acid, during ontogenesis, are summarised in Table 138. The nitrogenous excretory product which has the smallest molecular weight and the highest nitrogen percentage reaches its maximum earliest in ontogenesis ; that which has the largest molecular weight and the smallest nitrogen percentage reaches its maximum latest. The simplest product of deamination is the first to appear 1, the most complicated is the last. Yet the latter accounts for 91-5 per cent, of the total nitrogen excreted by an embryo throughout its development, and the former for only I per cent., while the intermediate compound, urea, accounts for 7*6 per cent.
Table 138.
Time of Absolute % of
peak of maxi- milligrams the total mum pro- nitrogen ex- nitrogen ex
Days
Fig- 323
Molecular weight of compound
% of
nitrogen
in the
compound
duction in
the chick's
development
(in days)
creted during
the whole development of the chick
creted during
the whole development of the chick
Ammonia Urea ... Uric acid
Z 168
82-3 46-6 33-3
4 9 II
0-I20
0-843 io-i6i
1-07
7-58
91-35
Total
11-124
A very interesting way of expressing the relationships here involved is shown in Fig. 324, where the milligrams of ammonia, urea, and uric acid nitrogen excreted per embryo per day are plotted against the time of development on semilog, paper. These curves correspond
^ The ammonia may of course be derived from some substance other than protein, such as adenylic acid.
1-0
Differential increase of ammonia, urea and uric acid excretion by chick embryos (Needham's experiments)
O Ammonia
• Urea
® Uric acid
•001
•0001
U1719 141719
Control +0-1CC. +0-1cc. +0'1cc. +0-1cc.
normal 10% 20% 10% lO/ourca
urea lactic tartronic +0-lcc.
acid acid 10^ tartronic acid
I I I' I 'I I
3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 2021
Days of development
Fig. 324.
io84 PROTEIN METABOLISM [pt. m
to McDowell's log. weight/age graph described in Section 2-2 and illustrate well the differential growth-rates of the three functions. Ammonia nitrogen appears first but grows slowly and is soon overtaken by urea nitrogen, which in its turn is overhauled and passed by the uric acid nitrogen "growing" very rapidly, and finally reaching much larger proportions than any of the others. The graph also shows that finally the embryo has produced about ten times as much urea nitrogen as ammonia nitrogen and about ten times as much uric acid nitrogen as urea nitrogen.
There seems no reason to hesitate in classing this sequence among the most remarkable instances we possess of the occurrence of a recapitulation phenomenon in chemical embryology. To designate it thus does not, however, explain it and I shall return to the theory of recapitulation in the Epilegomena. The recent researches of Przylecki & Rogalski have thrown some light on the sequence of nitrogenous excretory compounds in the chick from another angle. In connection with Przylecki's wide investigations concerning the manner of excreting nitrogen which characterises different phyla and species, he directed his attention to the ontogenetic side, and enquired whether the development of the embryo involved the appearance or disappearance of such enzymes as uricase. For this purpose Przylecki & Rogalski used chick embryos to find out when the embryonic tissues can form uric acid, and when they can break it down. For the first series embryos were minced to an emulsion in water, glycerine and chloroform, and to some flasks xanthine was added, to others not. The results are shown in Table 139. Obviously if xanthine oxidase were present and active, the flasks to which the xanthine was added should show in all cases a higher uric acid content at the end of the experiment than those which had received no such addition. Here there is a turningpoint between the 4th and 7th days of development, for before that time the xanthine flasks have no advantage over the others, but after that time an advantage is found without exception. This changeover point occurs at exactly the same time as the sudden rise of the control, which up to the 7th day reveals no preformed uric acid in the experimental material. The conclusion to be drawn then is that at some time between the 4th and the 7th day the embryonic xanthine oxidase awakens into activity, and the embryo acquires the power of forming uric acid from xanthine. If these time relations are compared with the appearance of uric acid shown in Fig. 317, the
SECT. 9] PROTEIN METABOLISM 1085
correspondence is striking, but of course the greater part of the uric acid formed in normal life originates from ammonium lactate rather than from xanthine. Morgan's results agree with those of Przylecki & Rogalski, and were obtained by a different method (methylene blue). At the 7th day the yolk-sac and blastoderm gave a positive result, but yolk and white were negative. Xanthine oxidase was present in the kidney on the 15th day and rose extraordinarily sharply in the liver at the time of hatching.
Table 139. Przylecki & Rogalski's figures.
Milligrams
uric acid
'
Flasks to
Flasks to
which
15 mgm.
which 25 mgm.
uric acid was
added
Age in days
Flasks alone
xanthine was added
Heated at
100° Not heated
2
o-o
o-o
6-5
4-1
o-o
o-o
7-0
?3
o-o
o-o
2-8
4
o-o
0-0
6-7
60
00
0-0
6-0
5"2
7
^•5
1-8
6-0
51
1-4
1-9
5-3
4-4
2-0
2-5
5-5
5-1
10
3-0
%l
4-5
4-5
2-8
5-1
5-1
2-7
3-7
5-5
5-5
14
2-5
3-4
7-0
7-0
3-5
»
4-1
4-1
3-1
3-9
11
21
^■^
3-7
80
1-8
3-4
4-3
4-3
2-6
3-6
4-0
40
Przylecki & Rogalski not only found that before the time at which uric acid appears in the excreta the embryo has no power of forming uric acid, but also that it destroys it if provided. The right-hand part of Table 139 makes this clear, for in the early stages of development a heated emulsion of embryonic tissues with added uric acid always contains more of this substance at the end of the experiment than a parallel flask which has not been heated. Between the 7th and loth days, however, a change takes place, and, after that time, the presence of uricase can no longer be demonstrated. It is plain that the facts revealed by the Polish workers fit in well with those described above. But the fact that the uricase does not disappear until the i oth day may mean that before that time uric acid is formed from substances such as ammonium lactate and equally rapidly broken down to allantoin.
io86 PROTEIN METABOLISM [pt. hi
Allantoin should therefore be estimated quantitatively in the allantois during the first week of incubation. Przylecki & Rogalski's findings with regard to the appearance of xanthine oxidase are in accord with those of the American school described in Section 14-10. The only information as to the way in which the avian uric acid is formed in the egg is due to the researches of Tomita & Takahashi. Believing that the following chain of reactions took place :
CH3 COOH
CHOH + 3O - H^O-^CHOH
COOH COOH
lactic acid from tartronic
carbohydrate breakdown acid
COOH NH— CO
CHOHfCO<JJ&-^CO CHOH I - I I
COOH -- NH-d:q
tartronic -^ dialuric
^"^ breakdown ^<^>^ dialuric acid +urea-> uric acid,
they injected tartronic acid and urea into hen's eggs and succeeded in obtaining an increased formation of uric acid by the embryo, as shown in Fig. 324. But some doubt may be expressed concerning their control of the range of variation in normal eggs and Clementi considers that urea cannot be an intermediate step in the synthesis of sauropsid uric acid.
9'5. Protein Catabolism
We must now return to the question of protein combustion in the chick. Knowing the amounts of ammonia, urea and uric acid present each day during development, it is simple to calculate the amount of protein burned. This gives the graph shown in Fig. 325, in which the milligrams of protein combusted by 100 gm. of dry weight of embryo per day are plotted against the time in days. A marked peak appears at 8-5 days' development, the significance of which is sufficiently indicated by the labels summarising other evidence. This has been discussed in Section 7-7. One may say that a given weight of embryo combusts 6 or 7 times as much protein on the 8th day of development as it does on the 4th or the 1 6th day. The combustion of protein thus goes on during both the carbohydrate and fat periods,
SECT. 9]
PROTEIN METABOLISM
1087
but not to anything like the same extent as it does when the former is passing over into the latter.
Rapkine has pointed out that this peak in protein catabolism precisely coincides with the time of development at which chick embryos are most suitable for use in providing the plasma for tissue cultures. Whether the growth-producing substance is a proteose, a peptone, a dipeptide, or even an amino-acid, it does at any rate seem clear that it is a nitrogenous substance of protein origin, and it is therefore interesting to note that it seems to be at its greatest concentration in the embryo just at the time when there is a maximum of protein catabolism. Carrel gives the 7th to the loth day as the period most suitable for taking embryos for this purpose, and the peak, as we have seen, occurs at 8-5 days of development. It is also very interesting that Remotti's curve for the activity of the yolk proteases reaches its maximum on the 9th day of development (see Fig. 423 fl). This brings up the whole question of how the yolk and white proteins are transformed into the tissue proteins during development. It is possible that the former are not
1200 ? 1100
o
^ 1000
g 900
55
,700 600 500400
,300 200 100
PERIOD OF
CARBOHYDRATE
COMBUSTION
PERIOD OF
FAT COMBUSTION
Days -5
Fig. 325
reduced to their constituent amino-acids entirely, but only to a proteose or peptone stage. If this were so. Carrel's growth-promoting substance might simply be one of the normal intermediate products. There have not so far been any researches designed to test this interesting possibility.
From the data in Table 138, it will be seen that the total number of milligrams of nitrogen excreted by the embryo during its development (or, more accurately, transmuted into the nitrogenous waste products, urea and uric acid) is 11 -oo. Sendju, as we have seen, estimated the total amounts of various amino-acids in the hen's egg during the course of its development, finding a very slight increase in the histidine, and a constancy in the arginine, lysine, and monoamino-acids, while in the tryptophane and tryosine, on the other hand, there was a considerable falling off. He correlated this with the formation of pigments, and did not take into account losses by
io88 PROTEIN METABOLISM [pt. iii
combustion for the production of energy. According to Sendju, the tryptophane per egg descends from 134 mgm, to 60, a loss of 74 mgm. ; and the tyrosine, beginning at 420 mgm., falls to 260, this being a loss of 160 mgm.
Nitrogen (mgm.) 74 mgm. tryptophane contain ... 9-868
160 mgm. tyrosine contain ... ... 12-374
Total ... ... 22-242
Supposing, then, that these two amino-acids are the only ones, or even the principal ones combusted during development, it would follow that about 50 per cent, was burnt and 50 per cent, was available for the work of haemoglobin formation or other uses. It is certainly strange that tyrosine and tryptophane should be precursors of a purine, but it must be remembered that in the bird uric acid corresponds to the urea of mammals, and is produced from the ammonia due to deamination of combusted proteins.
It is interesting to compare the estimates of protein catabolism obtained by assessing the coagulable protein disappearing with those obtained by assessing the total nitrogen excreted. Sakuragi found that the coagulable protein diminished during development from 1846 mgm. of nitrogen per cent, to 1698 (see Table 135). 148 mgm. are therefore lost per cent, of the contents of an average egg, which amounts to 67 mgm. per individual egg. Yet none of the investigators who have estimated the excreted nitrogen have recovered a corresponding amount:
Milligrams nitrogen per embryo Coagulable nitrogen disappearing ... Sakuragi 67
Waste products appearing (in nitrogen) Fiske & Boyden 36
Fridericia 23
Needham 1 2
Targonski 8
Kamei i
There is thus some reason for believing, whichever of these sets of data turns out to be nearest the truth, that the protein combusted is probably not more than a third of the coagulable protein disappearing. The fate of the rest still remains doubtful. Whether the ovomucoid takes part in combustion is a question at present unanswerable; all that can be said is that there is no preferential absorption of ovomucoid as against ovoalbumen from the white.
SECT. 9]
PROTEIN METABOLISM
1089
We can now examine again the part played by ammonia, urea and uric acid as excretory products. Another way of expressing the relationship leads to the result shown in Fig. 326. Here, assuming that the ammonia, urea and uric acid together make up the total excreted nitrogen (which is not quite true), the partition between them has been calculated as milligrams excreted by the embryo each day in per cent, of the total nitrogen excreted by the embryo each day. Between the 4th and 5th days, the uric acid only accounts for 9 per cent, of the total nitrogen, but so rapidly does the change occur that, between the 8th and 9th days, it accounts for as
• c t " a > ■ o (Oi
Fig. 326.
much as 90-7 per cent. It has therefore practically reached its adult level, as is shown by the comparative standards to the right of the graph. The shaded parts at the bottom represent urea and the rest uric acid; they are figures taken from Meyer; von Knierem; Schimansky; Meissner, and Steudel & Kriwuscha.
It is clear that in the first week of development the relationship of urea and ammonia to uric acid is altogether different from what holds in the adult, but that in the last two weeks of development the adult value is rigidly adhered to. These results throw light on the fact that the allantoic fluid in the chick passes from pH 7-2 to pH 6-0 in the last half of incubation, whereas, before the 9th day, it has been constant at about pH 7-2 (see Fig. 211).
logo
PROTEIN METABOLISM
[PT. Ill
In 192 1 Sznerovna 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,
Table 140.
Day
Milligrams nitrogen present in embryo each day (cumulative)
Milligrams nitrogen excreted by the embryo up to LeBreton each day (cumulative)
Sznerovna's ratio
(Nitrogen in embryo/
Nitrogen excreted)
Sznerovna
Murray
SchaeiTe
I
2
3
4
'1
1-325
1-46
2-661
2-1
7
4-850
!-^
8
—
8-323
9
13-32
8-0
10
i6-5
21-01
12-5
II
32-57
18-8
12
75-2
50-78
32-0
13
77-85
57-5
14
89-4
124-00 188-80
I02-0
15
154-0
16
216-4
264-30
212-0
'2
—
340-20
265-0
18
242-6
410-50
322-0
19
—
471-90
390-0
20
384-1
528-70
475-0
SchaeiTer Sznerovna Needham Sznerovna Murray LeBreton
0-95 4-2
5-5
[4-1
23-t
6 0-00234 0-00500 0-0149 0-095 0-279 0-5344 0-8772 1-3161 I -8699 2-5546 3-3810 4-3376 5-5101 6-9155 8-8452 [1-0038
17-5
16-4 15-3
566-2 533-4 325-6 106-5 47-7 39-32 37-13 38-59 41-64
48-53 55-86 60-94 61-74 59-35 53-55 48-07
9
624-0
420-9
260-6
68-3
28-7
23-4
21-4
24-3 30-8
39-9
45-5 48-9 48-1 46-6 44-1 34-3
i.e. nitrogen in embryo/nitrogen in allantoic fluid, was practically a constant, wavering round about 17 (see Table 140). In other words, for every i gm. of nitrogen in the allantoic fluid, there were to be found 1 7 grams of nitrogen in the body of the embryo. Her estimations did not begin before the loth day, so in view of the relations which we have already found to hold between the early and late periods of development, a recalculation of her ratio was desirable.
It was assumed that only small errors would be introduced by calling the urea nitrogen plus the uric acid nitrogen the nitrogen present in the allantoic fluid, and two different sets of figures for the total nitrogen in the embryo were available. The important difference between the nitrogen figures
600
[ 1
® Sznerovna
500
\\
H. A. Murray
■5 V
\ • Le Brecon ScSchaeffer
400
■t \
4
300
2
w
200
01
en
\
100
Vv
"lO
\Wo-2=J:=3=»=«^*=«=*^
■ ' . — 1 — 1 — .
. . ^^^ts:^-^^_^ ,
Days ^
Fig. 327.
SECT. 9] PROTEIN METABOLISM 1091
of LeBreton & Schaeffer, on the one hand, and Murray, on the other, is that the former excluded the membranes in their estimations while the latter probably included them. We therefore have a way
Table 141.
Uric acid milligrams per embryo
< — — ^ ^ ^
Tomita & Needham Fridericia Fiske & Boyden Kamei Targonski Takahashi
" l^g^ 'll I ^ Jl s „ I
2 S S 2 era . i « j; "" Ji 'c -%-, E
e-S.s li^s-""?
Day i5+£8l? <^ag ^£b " ^fe8 ^^8 l+SBfel
o
Embryo +amr + allantois 1 (Benedict &Fr ' colorimetricmi Hopkins' amm chloride meth(
c:3
>.-5 111
m
111 ■ 111
0-00029 0-00095 0-0020
0-007 0-030 0-055 0-33
o-oii6
—
0-72
—
o-oii6
0-0845 0-0845 0-950
1-77 2~87
0-266
0-503
9
0-969 — — — — —
9-10 I -301 — — — — —
10 1-873 — 4-33 — 2-o6 — 1-344 _____
11 3-410 3-5 7-1 _ _ _ 3-733 _____ 1-917 _____
12 4-276 5-2 10-61 — 3-51 — 4-077 _____
13 5-314 5-5 15-06 _ _ _ 6-330 _____
14 6-930 11-7 _ 0-507 4-75 0-55
15 10-760 18-6 — — — —
9-920 — — — — —
16 1 3*650 28-8 — — 9-30 —
17 13-940 42-8 — 3-u — 3-50
17-18 17-640 _ _ _ 13. y^ _
18 — 49-7 _ _ _ _
19 - - _ _ _ 7.50
19-5 26-090 57-4 lOO-O — — —
20 32-700 — — — — — 25-600 64-8 — — — —
of determining what part the membranes are playing in the protein metabolism. These relations are shown in the form of a graph in Fig. 327. The newly calculated ratio does not quite become a constant during the last 10 days of incubation, although it approximates
I092
PROTEIN METABOLISM
[PT. Ill
to one, and it never reaches the low figure obtained by Sznerovna. The extreme smallness of the protein catabolism during the first 6 or 7 days is reflected on this curve in the extreme height of the ratio,
50 r—
Days -^ 5
but Sznerovna missed the early descent. The metaboHsm of protein seems to be more intense in the absence of the membranes, but it is perhaps legitimate to conclude that the part they play in the combustion of proteins is sHght.
SECT. 9]
PROTEIN METABOLISM
1093
Among the investigators who have estimated the uric acid produced by the chick embryo there is some divergence in the absolute magnitude of their values. Table 141 illustrates this, and ,: shows that Fridericia's results, for instance, though at first ^ of exactly the same order as J mine, draw off about the S 15th day, and rise much o'^ higher during the remaining ^ time. Fiske & Boyden's, on "fthe contrary, are always 5 higher than either Fride- ^ ^ ricia's or mine, while Kamei's ^ are the lowest of all. Tar- |, gonski embodied his results ^ in singularly confusing and obscure tables, but the uric acid production of his embryos can be calculated from
O Fiske &;,Boyden © Needham
Days
Fig. 329.
them, with the result that his data confirm mine, none of his points
being very far away from the curve of Fig. 328. These are all the sets of
figures for uric acid production
which we have at present, and,
until a thorough comparative
assessment is made of the effect
of breed, etc., on uric acid pro
duction, the differences will
remain difficult to account
for^. But it is important that
the peak in nitrogen cata bolism emerges from at least
two of them, and it may be
supposed that this is due to a
relative rather than a uni
Fig. 330.
versal validity inhering in each series. This is illustrated by Fig. 329.
1 Differences of technique may account for much divergence; thus some methods estimate ergothioneine as well as uric acid. Calvery's later figures for uric acid agree exactly with those of Needham.
1094 PROTEIN METABOLISM [pt. iii
The data of Fiske & Boyden and of Needham, when plotted in terms of milligrams of protein combusted each day by lOO gm. dry weight of embryo, show the peak in both cases. Fridericia's points, according to Cahn, confirm the downward trend from the 1 1 th day onwards.
It will be noticed from Table 141 that in my set of figures the uric acid in the embryonic body is included in the estimations. I did no experiments to determine the degree of uric acid retention, but Fiske & Boyden reported that, between the 5th and nth days, the uric acid content of the embryonic body never exceeds 2 mgm. per cent. Between the 7th and 8th days, then, when the embryo weighs about a gram (wet weight), 0-02 mgm. uric acid would be contained in it, although during this time 0-5 mgm. would be excreted.
The data for the daily excretion of uric acid — a necessary step in these calculations — are shown in Fig. 330. Fiske & Boyden attributed significance to the irregularities in the points of their curve, but for this there is no warrant.
Table 142. Allantoic liquid [milligrams per egg).
Fiske & Boyden Targonski
Nitrogen
Nitrogen
other than
other than
uric acid
Sznerovna.
Kamei.
Uric
uric acid
Total
Uric acid (residual
Total
Total
Total
acid
(residual
Day
I 2
nitrogen
nitrogen
nitrogen)
nitrogen
nitrogen
nitrogen nitrogen nitrogen)
3
4
0-034
0-002
0032
I
4-8
0-068
o-oi
0-058
—
—
—
—
—
o-io
0-02
o-o8
—
—
—
—
—
6
0-92
o-ii
0-18
—
—
—
—
—
7
0-45
0-24
0-21
—
—
—
—
—
8
0-89
0-59
0-30
—
—
0-71
0-17
0-54
9
1-52
0-96
0-56
—
4-8i
10
2-29
1-44
0-85
0-95
ii4
0-67
1-17
1 1
3-38
2-37
I-OI
—
—
12
4-8i
2-54
1-27
2-8
—
2-77
i-o8
1^9
13
7-35
5-02
2-33
—
—
—
—
14
—
—
—
5-5
52-74
3-19
1-82
2-37
—
—
—
14-1
—
3-75
3-65
o-io
17
—
—
—
—
i3'44
—
—
—
18 19
—
—
—
17-9
9-65
5-65
4-00
20
SECT. 9]
PROTEIN METABOLISM
1095
® Uric acid nitrogen) Risked O Residual .. / Boyden "P Uric acid n 1^ A Residual - Targonsk.
9-6. Nitrogen-excretion; Mesonephros, Allantois and Amnios
The total nitrogen in the allantoic fluid has been measured quantitatively by Fiske & Boyden; Sznerovna; Kamei, and Targonski. Kamei's figures are obviously aberrant, but the rest correspond more or less closely, as an inspection of Table 142 indicates well enough. The principal interest of it is the fact, emerging from Fiske & Boyden's work, that at the earlier stages of development uric acid is by no means the predominating constituent of the nitrogen excretion. The uric acid rises above the rest, however, at the 7th day of development. This confirmed very strikingly my original finding, and the harmony of the
70 -b 60-^ 50-1' 40 -i
Days ->■ 5
t be remembered that Tarffonski '. for 58% of the tt>taJ nitrogen of the ajlojitoic Hqtud
Fig- 331
results is shown in the comparison of Figs. 326 and 331. Targonski's more erratic figures also show the typical cross-over in the importance of uric acid and other nitrogenous molecules as excretory products. What is the residual nitrogen of the early stages ? A balance sheet can be drawn up as follows :
Table 143. Allantoic liquid {milligrams per embryo).
Fiske & Boyden
NTpf-rlV^o^
Amino
iN eer " ""^
a^iciii
Unac
%un
Total
Uric acid
Creatine
acid
Urea
Ammonia
counted
coun
Day
nitrogen
nitrogen
nitrogen
nitrogen
nitrogen
nitrogen
for
for
4-0
—
—
—
—
0-0002
0-0033
—
—
4-8
0-082
001 1
o-ooi
—
—
—
—
5-0
0-107
0*017
0-007
0-047
0-0025
0-004
0-03
28
%^
0-I20
0'025
0-008
0-058
—
—
0-02
17
—
—
—
—
0-005
0-006
—
8-0
8-2
1-27
0-86
0-039
—
0-026
0-014^
— j
0-33
26
9-9
2-32
1-82
—
0-28
—
— 1
0-13
6
lO-O
—
—
—
—
0-068
0-025 1
12-9
13-0
6-74
5-21
—
0-47
0-192
0-048 /
0-82
12
From this it is clear that an amount of nitrogen varying from 6 to 28 per cent, of that contained in the allantoic liquid is as yet unaccounted for. Examination of the table shows the constant presence
1096
PROTEIN METABOLISM
[PT. Ill
150
13 140
vertical lines-fiske 8c
Boyden'a limits - O Targonski's points for uric acid nitrogen • Targonski total nitrogen A Kamel total nitrogen
of creatine and amino-acid nitrogen, which latter in the early stages accounts for as much as 40 per cent, of the total nitrogen ^
Although the absolute amounts of the excreted nitrogenous waste products are the values which it is most important to know, the concentration of them in the allantoic Hquid is of some interest. Here the most complete series of estimations is that of Fiske & Boyden, plotted in Fig. 332. The vertical lines show the range of variation in their figures, and the white and black circles refer to Targonski's points. At the beginning the composition of the allantoic fluid, as regards its nitrogenous crystalloids, quite closely resembles the concentrations
found in the plasma of adults.
. y 120
Fiske & Boyden regard it as a o
mere filtrate till the end of the | 5th day. After that time the con- ^ centration of uric acid steadily ^ rises, at least until the end of the ^ 13th day. The concentration of c the residual nitrogen, on the '^ contrary, falls markedly until | the gth or loth day, as would § be the case if the formation of o any quantity of urea and ammonia was suspended when the formation of uric acid began, and afterwards rises again, as would be the case if other substances, such as amino-acids or creatinine, began to enter into it. About the end of the 2nd week of incubation the allantoic fluid acquires more urates than it can dissolve— thus it is sometimes milky as early as the 7th day, and sometimes clear as late as the 13th. Besides this turbidity, the urates are deposited as slimy stringy masses, which, though at first soft, eventually become hard and almost brittle. Fiske & Boyden observed that as much as 87 per cent, of the total uric acid present may be contained in these deposits. Boyden has drawn attention to the blister-shaped vesicles which often, if not always, occur within the inner wall of the chick's allantois in the region of the amnio 1 See the foot-note on p. 977 and Table 163.
Days
Fig. 332
SECT. 9] PROTEIN METABOLISM 1097
allantoic fusion. These first appear about the time that the lymphatic circulation is established in the allantois (7th day), and increase in number and size till the end of development. Gentle heating coagulates the liquid inside the blisters. They may either be concerned with the absorption of protein, or they may be caused by the sharp edges of the uric acid crystals, or, again, they may secrete mucin. As regards the excretion of urates, Fiske & Boyden pointed out that the anatomical "evidence" that the mesonephros is functionally inert is no more than an expression of opinion, and that all we know about the presence of nitrogenous waste products in the allantois goes to show that it is functional. That water passes through the mesonephros even against pressure as early as 2*5 days of incubation was proved by the experiments of Boyden, who obstructed the Wolffian duct, and afterwards observed hydronephrosis. Indeed, without the mechanical distention produced by the excretion of the mesonephros the allantois fails to grow normally in size, and consequently the proper respiratory apparatus of the allantoic vessels does not develop. And the mesonephros is alone in a position to dispose of waste products up till the nth day. Aggazzotti, in his work on the pH of the allantoic liquid (see Fig. 211), concluded that the allantoic liquid was not a true excretion before the i ith day, because it was not acid, although the yolk was, but, as Fiske & Boyden point out, no one could have predicted a priori what the reaction of the embryonic excreta would be.
Fridericia was much interested in the problem of relating morphological knowledge about such organs as the liver and the mesonephros to the results obtained on uric acid excretion. For the former organ he could find no hint in the literature, but he made an attack on the mesonephros by measuring its dimensions in a large number of embryos. In the chick embryo the mesonephros is a large yellowish-red organ on the 1 6th day, but as it hands over its functions to the metanephros at a later stage it atrophies, and by the 20th day is small, thin and pale. Fridericia's measurements of its size are shown in Fig. 333, from which it appears that the mesonephros reaches its greatest size upon the i6th day. Fridericia related this to the peak which his figures for daily uric acid excretion show at that point of development, but, as the only other set of data covering it do not show such a falHng-off (Needham), it is doubtful if any emphasis can be laid upon it.
1098
PROTEIN METABOLISM
[PT. Ill
A good deal of light has been thrown on the functioning of the embryonic excretory organs in the chick by the injection of vital dyes into the egg during development. The pioneer in this line of work was Bakounine, who in 1895 injected indigo-carmine intravenously into chick embryos varying from 3 to 1 5 days' development, and in all cases observed the dye in the cells of the proximal portion of the mesonephric tubules. Undoubtedly excretion of the stain was occurring. Zaretzki was the first to inject dyes into the air-space. He used trypan blue, trypan red, neutral red, methylene blue, fluorescein, eosin and ethyl green. With trypan blue there was in the late stages of incubation a slight colouring of the amniotic liquid, but not of the embryo. When the dye was injected into the outer | wall of the allantois, he observed a diffuse staining of the embryo, amniotic liquid and foetal membranes. These results were rather difficult to interpret, as were those later obtained by Graper, who unsuccessfully tried to use an in vitro technique for keeping stained whole blastoderms alive outside the egg. Hanan has shown more recently that trypan blue, once within the circulation of the embryo, is excreted through the kidneys, and appears in the allantoic but not in the amniotic liquid, strongly suggesting that, in the case of birds, foetal urinary water does not contribute to the formation of the latter. The excretory activity of the mesonephros, he found, began on the 4th day of incubation, which agrees very well with the earlier results of Bakounine and of Wislocki, and with the fact established by Boyden that hydronephrosis cannot easily be produced before the 4th day. The more recent researches of Atwell & Hanan and of Hurd with trypan blue have established (if the behaviour of the dye in the cells is any criterion) that the excretory activity of the mesonephros begins on the 4th day, and goes on alone till the 1 1 th day, at which time the metanephros comes into operation. Both function together till the
bZQ.
Fig. 333.
SECT. 9] PROTEIN METABOLISM 1099
1 8th day, after which the metanephros continues by itself. For experiments on the excretory powers of the mesonephros in the salamander, Necturus maculosus, see the papers of Dawson and of White & Schmitt.
Table 1 44. Concentration of constituents in the allantoic liquid of the chick. [Milligrams °/^.)
Kamei Targonski
S| II li li I i U li Ih HI
Day feh h2 ZcJ Z'c < P E- Z S Z E J 5^ g.^ x^ ^ c
2 — — — — — — — — — — —
3— _____— — — — —
4 28 _____ _____
5 26 __________
6 27 ____- _____
7 29 _____ _____
8 34 _____ _____
9 43 4-6 3-6 i-i 0-8 5-6 — — — — —
10 55 — — — — — — — — — —
11 68 _____ 46 18 — 40-2 59-8
12 85 _____ _____
13 106 — — — — — — — — — —
14 151 41-6 0-6 4-7 3-6 21-2 85 43 42 51-0 49-0
15 — — — — — — — — — — —
16 — _____ 92 60 32 65-1 34-9
17 — 87-1 20-7 8-5 12-5 56-7 — — — — —
18 — _____ 116 50 — 43-2 56-8 19— _____—___ — 20 — — — — — — — — — — —
In the previous discussion, nothing has been said about the other estimates of the urea present in the allantoic fluid. Kamei made a few determinations of ammonia and urea, and the figures are shown in Table 144. On the 14th day he found 21-2 mgm. per cent, of urea in the allantoic fluid, or, taking its volume at 6-5 c.c, 1-28 mgm. absolute, and on the same day 3-4 mgm. per cent, urea in the amniotic fluid, or, its volume being roughly 2 c.c, o-o68 mgm. absolute. The total amount present per embryo was therefore I '35 mgm., a value somewhat in excess of my 14th day determination, namely, 0-5 mgm. Kamei did not state what method he used for his urea determinations. In the same way his estimate of 0-276 mgm. of ammonia in the amniotic and allantoic liquids on the 14th day
iioo PROTEIN METABOLISM [pt. m
exceeds my value of o-o6 mgm. for amniotic and allantoic liquids and embryo. Such discrepancies seem to be inevitable in the first approximations, and can only be abolished by further work. Kamei's figures for the non-protein nitrogen fractions are not easy to interpret.
Table 145. Concentration of constituents in the amniotic liquid of the chick. {Milligrams °l^.)
Fiske & Boyden Kamei Targonski
1 ^ c
c o • Ji "sc -s^a o i; -5
1 ^ -I is 2^ g'c I I c gc
I -Sc % Is g2| §2 I S I I §2
Day h D& h hS 2c^ Zc < D h £ Zc
2 — — — — — — — — — — —
3— ___ — —_ — ___
4— __ _ _______
5— __________
6 — o-io — — — — — — — — —
7 2-9 — — — — —— — — — —
8— __________
9 — o-io 1 1-9 33-1 1-5 0-6 0-8 3-4 — — —
10 — 0-13 — — — — — — 17-0 — —
11 7-5 o-ii — — — — — — — — —
12 156 _______ 40-5 — —
13 2450 — — — ^ — — — — — — —
14 — — 3184 37-1 1-3 3-5 1-9 3-4 58-0 — —
15 _ __________
16 1500 0-17 _ _ _ _ _ _ 5450 5200 250
17 — — 2003 30-3 4-4 2-2 1-7 14-3 _ _ _ 18— _______ 5300 _ —
19 _ __________
20 — — — — — — — — — — —
The effect of the nitrogen metabolism on the amniotic fluid of the chick has been investigated by Fiske & Boyden; Targonski, and Kamei. As Table 145 shows, the total nitrogen concentration of the amniotic liquid rises very slowly until the 12th day, at which time it increases in prodigious strides, but, as Fiske & Boyden put it, "this merely serves to give a quantitative aspect" to the fact, discovered by Hirota and Fiilleborn, that in the last half of the 2nd week of incubation a communication is established between the albumen-sac and the amnion at the site of the sero-amniotic junction. As the data of Targonski and of Kamei in Table 145 show, this large increase of nitrogen is almost entirely due to protein. As for the uric acid, its concentration does not increase proportionately to the growth of the
SECT. 9]
PROTEIN METABOLISM
embryo, as is the case in the allantoic liquid, but it remains constant throughout. No communication between cloaca and amniotic fluid exists till later — after the 17th day, according to Gasser. Kamei's figures for total non-protein nitrogen basic and non-basic by phosphotungstic acid remain difficult to interpret, partly owing to their small number, but it is interesting that he found a definite increase in the ammonia and urea concentration of the amniotic fluid. Such easily soluble and diffusible molecules would be expected to penetrate the walls of the allantois, and to turn up elsewhere.
Targonski has calculated the ratios Nitrogen in embryo/Nitrogen in amniotic liquid and Nitrogen in amniotic liquid/Nitrogen in allantoic liquid. Both these are plotted in Fig. 334.
The first ratio is high and rising, for the denominator is remaining constant and the numerator is steadily increasing, but a maximum occurs on the 12th day, owing to the sudden inrush of protein from the albumen-sac into the amnios through the seroamniotic connection, and afterwards a fall takes place. The same relations hold inversely for the amniotic and allantoic liquids, for the nitrogen of the latter is at first abundant compared to that of the former, but, after the events of the 12th day, this is no longer the case. Targonski 's curves illustrate the effects of the opening of the seroamniotic junction.
9-7. The Origin of Protective Syntheses
The only other matter which must be considered under the heading of the protein metabolism of the bird's egg is the synthesis of ornithuric acid by the embryo. It is obviously a matter of great interest to determine the time in ontogenesis at which the embryo becomes able to carry out those chemical protective syntheses which are so interesting a feature of the metabolism of the adult, Takahashi, who
Days 5
Fig. 334
II02 PROTEIN METABOLISM [pt. iii
first attacked this problem, estimated the sulphates in the allantoic liquid, and found, as will be described in Section 12-7, that at least as early as the gth day ethereal sulphates were accumulating in the embryonic excreta, showing a detoxication of phenols by synthesis with sulphuric acid. But his work on ornithuric acid was more remarkable. As is well known, the metabolism of the bird reacts to the presence of benzoic acid by combining it with ornithine (diaminovalerianic acid) and excreting it in that form — a mechanism doubtless developed because of the vegetarian diet, and its accompanying abundance of unbreakable benzene rings. In order, therefore, to discover at what stage in development the chick could first bring about this synthesis, Takahashi injected small amounts of sodium
Table 146.
Amount of benzoic acid
Amount of omi
ithuric acid
found afterwards
found afterwards
Day
"111
&1U
it
ill III
.2
1 <
1 t
1
is
Allantois Yolk and white
1
ii
All injections made
9
1014
842
3-537
o-o6i
14 0-1372
0-0
0-0 O-O
0-0
'A
2094
1411
5-928
00
O-O
0-0
0-4541 0-0
0-0
1292
774
3-251
o-o
o-o
0-0
0-5964 O-O
o-o
The ornithuric acid was identified by solubilities, melting-point and complete elementary analysis. In control normal embryos no ornithuric acid was ever found in any part of the egg.
benzoate into the egg-white before incubation, and then worked up the various component parts of the egg for benzoic and ornithuric acid. His results, which are given in Table 146, were remarkable, partly because of the large number of eggs used, amounting to some thousands, each one of which received by injection 5 mgm. of sodium benzoate in 10 per cent, solution. The results showed definitely that no ornithuric acid was formed up to the gth day, and that the benzoic acid was then being excreted unchanged, but that on the 14th day, on the contrary, ornithuric acid was undoubtedly being manufactured. It would probably be legitimate to conclude from this that the power to conjoin benzoic acid and ornithine is not always present in the chick embryo, but arises at a definite stage in its development.
SECT. 9] PROTEIN METABOLISM 1103
9*8. Protein Metabolism of Reptilian Eggs
A group of Japanese workers has studied the development of the marine turtle, Thalassochelys corticata, which lays eggs the size and appearance of ping-pong balls in the warm littoral sand. Dealing first with the end products of protein metabolism, Tomita reported a preponderance of urea over uric acid. His figures were as follows, both urea and uric acid being given as absolute amounts in milligrams
per egg: Days Urea Uric acid
2-0
00
15
3-7
0-r
30
lO-O
o-i(
45 24-5 0-15
They lead to a typically aquatic nitrogen utilisation (see Table 163). The preponderance of urea is curious in view of the uricotelic metabolism of the sauropsida, but the nitrogen partition of chelonian urine almost certainly does not follow the usual course in reptiHa, and there is abundant evidence that the turtle ^gg is not a cleidoic system (see Section 6-6 and the Epilegomena).
The more general aspects of nitrogen metabolism were investigated by Nakamura, who observed a diminution in the total nitrogen of the eggs from 592 to 506 mgm. per egg. This is very interesting in view of Tomita's findings, for the egg of this turtle thus not only absorbs water from its environment but also gives off end-products of nitrogen catabolism to it. Transition from ureotelic to uricotelic habit occurs, then, not between amphibia and reptiles but between Chelonia and Sauria. The following table shows the movements of the nitrogen within the €:gg according to Nakamura's analyses :
Table 147.
Milligrams nitrogen
^
Amniotic and
Day
Whole egg
Egg-white Egg-yolk allantoic liquids Embryo
592
41-2 548 3
16
586
17-8 544 24
30
549
12-2 501 25 10
45
— 119 64 323 Grams weight of the fractions
'
Amniotic and
Day Egg-w
hite Egg-yolk allantoic liquids Embryo
i8
7 1 1-4 3-3 —
16 10
5 IO-8 12-5 —
30 5-i
B 104 15-1 II
45 —
2-6 12-7 17-5
II04 PROTEIN METABOLISM [pt. iii
Table 147 shows clearly the passage of nitrogen from raw materials into embryo and also the fact that, just as with the bird, the eggwhite is used up before the yolk. Nakamura estimated the non-protein nitrogen in the various parts of the egg during development but it did not vary much, remaining always at from 14 to 25 mgm. per cent., and showing only a slight rising tendency.
Another member of the group, Sendju, studied the behaviour of the amino-acids with the following interesting results :
Table
148.
Total amino-acids in
milligrams per egg
Trypto
Tyro
Histi
'
Purine
Days
phane
sine
Cystine
Arginine
dine
Lysine
bases
55
173
59
208
33
212
0-4
15
46
156
52
195
35
201
0-7
37
39
135
41
45
187
2-7
45
38
"5
31
176
47
201
33 3-6
Hatched
36
"5
27
181
48
196
it is clear that the turtle's egg has only half as much tryptophane, tyrosine and lysine as the hen's egg, and still less cystine and histidine. The loss during development seems to bear mainly on the tryptophane, tyrosine, cystine and arginine, i.e. precisely those amino-acids in which definite diminutions had been found by Sendju to occur during avian ontogenesis. In both the turtle and the hen, the lysine remains constant, and the purine bases are synthesised although it may be questioned whether Sendju's figures for the latter are not much too low. Tomita found the following distribution as between white and yolk in the turtle's egg:
Milligrams % (fresh egg) wet weight
White
Yolk
Tryptophane
Tyrosine
Cystine
Arginine
Histidine
103 123 32
9
55
153
201
1089
201
Lysme
9
1354
Purine bases
3(?)
9-9. Protein Metabolism of Amphibian Eggs
Faure-Fremiet & Dragoiu in their analysis of the frog's egg and the hatched tadpole, observed a definite diminution of protein. The figures were as follows:
SECT. 9]
PROTEIN METABOLISM
1 105
Table 149.
Terroine & Barthelemy Zero hour
Faure-Fremiet & Dragoiu Zero hour
Faure-Fremiet & Dragoiu Hatching
Therefore lost during this time Faure-Fremiet & Dragoiu Loss of yolk-sac
Therefore lost during this time
Lost during the whole time
Protein
% wet
% dry
Milligrams
weight
weight
absolute
27*9
—
—
26-5
610
1-1696
23-2
—
I -0459
3-3
—
0-1237
c
—
0-9000
—
—
0-146
0-270 (Le.23-i %of protein originally present)
Consumption of dry solid by amphibian embryos 10 20 30 40
Evidently a good deal of the protein material with which the egg begins is transformed into the protein of the larva, while a smaller amount is combusted. Unfortunately Faure-Fremiet & Dragoiu did not estimate the protein present at the last stage of all, and relied on approximative assessment, but still their data give a general idea of the process as a whole.
The other investigators who have studied the chemistry of the developing tadpole have for the most part paid no attention to its protein metabolism. But a notable exception is afforded by the interesting paper of | Bialascewicz & Mincovna which appeared in 1921. These workers estimated both the nitrogen contained in the embryos at different stages and the nitrogen excreted into the circumambient water on each day. Bialascewicz & Mincovna first established the fact that some dry weight is lost by the frog embryo — an important point, for earlier workers had confused the issue by always expressing results in percentages. The data are shown in Fig. 335, in addition to some others collected by Bialascewicz himself in another paper, and by Galloway. Using different temperatures, and consequently different hatching times (68, 78, 90, 96, 100, 120 and 172 hours), Bialascewicz & Mincovna found that the loss of dry solid was always practically identical at
Days 5 10 15 20 25 30
O Rana fu&ca (Bialascewicz) ©Rang sylvatical
® " " (Bialascewicz S(Mcncovna) •Amblystoma flGalloway) ® " •' (Barthelemy ^Bonnet) tigrinum )
Fig- 335
iio6 PROTEIN METABOLISM [pt. iii
0-076 mgm., or 5-6 per cent, of the initial dry weight. They next estimated the nitrogen in the egg at the beginning and in the hatched tadpole at the end, using the micromethods of Pilch and Bang, and obtaining the following averages :
Milligrams nitrogen per 100 embryos
Bialascewicz
Barthelemy & Bonnet
& Mincovna
(for comparison)
Just after fertilisation
13-14
i8-7 24-1 (without mucin) (with mucm)
At hatching
"•95
—
At time of disappearance of external gills
13-9
Loss
I-I9
4-8
One hundred embryos during their pre-natal life, then, lost i • 1 9 mgm. of nitrogen, or g-i per cent, of the initial quantity provided. After hatching this decomposition of protein still goes on, as Fig. 336, taken from Bialascewicz & Mincovna's figures, shows. Turning now to the determination of the end products, the total nitrogen excreted by the embryos into the surrounding water was estimated, and related to a standard number of embryos in 24-hour periods. This led to the striking curve in Fig. 337, which shows the relative intensity of nitrogen excretion from frog embryos, and as it is an intensity graph it can be compared directly with that for the chick as given in Fig. 325. To express the amphibian production of nitrogen in milligrams per cent, dry weight of embryo is not possible, as the embryo cannot be dissected away from the yolk. We can only tell what 100 mgm. dry weight of frog embryo-plus-yolk excretes per day, but, as the total change of dry weight throughout development is not more than 6 per cent., there is no necessity to turn the excretion curve into terms of dry weight, and it is sufficient to have it in terms of a constant number of embryos. It is obvious that, if we could express it in terms of "live" dry weight, the peak would be much more in evidence than it is, for the "live" dry weight is continually increasing, and the "dead" or yolk dry weight is continually decreasing — therefore the descent after the 125th hour would be emphasised considerably. It may be said, then, that in the frog, as well as in the chick, there exists a period towards the middle of embryonic development at which more protein is combusted than at any other time. The observation of this peak in a bird and an amphibian would suggest that the phenomenon is a general one.
SECT. 9]
PROTEIN METABOLISM
1 107
It was further found that the excretion was almost equally divided between urea and ammonia, as can be seen by the diagram at the top of Fig. 337. Bialascewicz & Mincovna noted that, while the loss in dry weight up to hatching was 0-076 mgm. per embryo, the loss in protein nitrogen was 0-012 mgm. per embryo or of protein 0-075 mgm. ; — they were inclined to hold, therefore, that before hatching only minimal amounts of fat and carbohydrate could be combusted. Other investigators, however, have not agreed with this conclusion (see p. 1 175). The 0-075 mgm. of protein disappearing would roughly
O Bialascewicz fie Mincovna
Barth^lcmy &i. Bonnet Hatching time between
these limits according
temperature
r
>iCRETtD
1
1^ 1
^/AMM'S'NTA'rN^TO^X'TS
ITRC&EN
_ Bialascewicz
S^Mincovna
Adult level
®
A
®
L \
1® \
j 1
Ve,®®
®
/
© ®
■^
\
1
.\
® /©
S^ Hatching
1 n
1
1 1
Fig. 336.
150 200 250 300
Fig. 337.
correspond with the 0-0039 c.c. of oxygen which was found by Bialascewicz & Bledovski to be taken up by one from embryo between fertilisation and hatching.
Barthelemy & Bonnet found, as has been stated above, that one egg of Rana temporaria contained 0-187 mgm. nitrogen without its mucilaginous envelope, and 0-241 with it, while at hatching one embryo contained 0-139 mgm. nitrogen. This was a loss of 25-7 per cent, analogous to the loss of 23-1 per cent, found by Faure-Fremiet & Dragoiu up to the end of the free-swimming yolk-provided stage. But the point on which Barthelemy & Bonnet laid stress was that, no matter what the temperature and therefore the rapidity of
iio8 PROTEIN METABOLISM [pt. in
development was, this value remained unaltered. In other words, it is useless to attempt to alter the total amount of nitrogen wastage associated with the making of one frog embryo by varying the temperature, for all that will change will be the rate of the whole process^. The following figures illustrate this:
Temperature 8
10
Nitrogen excreted in % Days taken till disapof the initial store pearance of external gills 26-5 30 28-9 22
14 21
25-1 20 25-7 8
Hibbard has described in the anuran, Discoglossuspidus, the accumulation and excretion of little superficial vacuoles in the epithelial cells of the pre-hatching stages. These she regards as possibly the mechanism of excretion of end-products prior to the formation of the kidneys.
With the work of Gortner we pass to another aspect of the attack on the protein metabolism of the amphibian embryo, although he was concerned with an icthyoid urodele, Cryptobranchus allegheniensis (the American giant salamander or "hell-bender"), and not with the anura. In many respects his findings diflfer considerably from those of the workers who have dealt with the latter material, and, as Cryptobranchus eggs are very difficult to obtain, it is not likely that he will be contradicted or confirmed very soon. Nevertheless, further work would be desirable on this or a related animal, for Gortner could find very little nitrogenous waste, and it is difficult to believe that his figures for this are correct. He approached the subject from the same angle as Plimmer & Lowndes, and, extracting the eggs and hatched larvae first with ether and then with alcohol, he divided them into what was soluble in ether and alcohol and what was not, which latter residue he called the "protein" fraction. Then he determined the different kinds of nitrogen in each. Table 150 shows his results.
Taking the protein first, Gortner found that 100 eggs had 575-4 mgm. of protein nitrogen and 100 hatched embryos had 568-8, a loss of 6-6 mgm., which he did not regard as significant. The character of the protein in the eggs certainly differed from that in the embryos at hatching, the main change being that, in the latter, the monoamino fraction had decreased and the di-amino fraction had slightly increased. This finding agrees with the conclusions of Plimmer & Lowndes on the hen's egg, and not with those of Russo. Slight rises
^ Cf. Section 6- 10.
SECT. 9]
PROTEIN METABOLISM
1 109
were noticeable in the ammonia nitrogen, the humin nitrogen, and in the non-amino nitrogen in the phosphotungstic filtrate. These are unexplained. Within the di-amino fraction the arginine, lysine and cystine rose, while the histidine fell, some of which changes agree with those found for the chick, and some of which do not.
Table 150. Gortnef s figures for Cryptobranchus allegheniensis.
% of the total nitrogen
Ether-soluble fraction
Undeveloped 1 Ammonia nitrogen ... ... 0-048
Humin nitrogen ... ... ... 0191
Basic (di-amino) nitrogen ... 0-048
Non-basic (mono-amino) nitrogen 0-114
Total ...
...
0-401
Alcohol-soluble fraction (ether
insoluble)
Ammonia nitrogen
0-024
Humin nitrogen
0-287
Basic (di-amino) nitrogen
0-654
Non-basic (mono-amino) nitrogen
0-231
Total ...
1-196
Protein fraction
Ammonia nitrogen
9-956
Humin nitrogen
2-250 19-56
Di-amino nitrogen total ...
Arginine nitrogen
13-56
Histidine nitrogen
5-63
Lysine nitrogen ...
10-16
Part of cystine nitrogen
0-213
Mono-amino nitrogen total
53-73
Non-amino nitrogen in filtration ...
2-76
Hatching larvae
o-io8 Rise
Rise
0-048
No change
0-239
Rise
0-778
Rise
0-024
No change
0-407
Rise
0838
Rise
0-635
Rise
1-904
Rise
IO-2I
Rise
2-324
Slight rise
19-38
Slight fall
13-90
Slight rise
4-93
Fall
10-27
Slight rise
0-282
Slight rise
51-92
Fall
2-686
Rise
5-256
86-52
Gortner unfortunately did not make analyses for urea and uric acid by direct methods, and their presence or absence has therefore to be discussed on the basis of their probable solubilities under his special conditions. Although it is always stated that uric acid is insoluble in ether and alcohol, and that urea is insoluble in ether, Gortner affirmed that in continuous extraction this was not the case, and that appreciable quantities of these substances would pass out into the extracting liquid. Although he did not fully prove this point, his main conclusion rests upon it. If the urea and uric acid were not passing out into the ether and alcohol, they were remaining behind masked in the "protein" fraction, so that it would not be surprising if by these methods no urea production could be demonstrated. As Gortner hydrolysed all his fractions, the urea and uric acid nitrogen
mo PROTEIN METABOLISM [pt. m
would, he thought, appear as ammonia nitrogen. This, as Table 150 shows, rises in two fractions, from 0-048 to o-io8 per cent, of the total nitrogen in the ether-soluble fraction, and from 9-956 to 10-21 per cent, in the protein fraction, and remains constant in one, the alcoholsoluble fraction. The total gain in ammonia nitrogen, namely, 0-314 per cent., would certainly not counterbalance the loss in monoamino-acids minus the gain in di-amino-acids, i.e. i -63 per cent. However, Gortner's conclusion that "no appreciable amount of urea or uric acid is formed during embryonic growth in Crypto branchus" does not follow, for the urea nitrogen may well have got mislaid among the numerous other fractions, e.g. the non-basic nitrogen of the alcoholsoluble fraction, which, in fact, does increase by 0-304 per cent. The question cannot be regarded as settled until the same material is reinvestigated, using direct and modern methods for ammonia, urea and uric acid. The gain in nitrogen of the ether-soluble fraction Gortner interpreted as being due to a synthesis of lecithin such as Tichomirov observed in the eggs of the silkworm. The gain in nitrogen of the alcohol-soluble fraction he interpreted as being due to the synthesis of purine and pyrimidine bases.
As already mentioned, the loss of protein nitrogen was, per 100 eggs, 6-6 mgm., or 1-15 per cent, of the initial value, but the protein itself, measured directly, fell from 4026 to 3828 mgm., i.e. 198 mgm., or 4-92 per cent. Thus the protein at the end of development had a distinctly higher nitrogen content than that at the beginning, being 14-86 instead of 14-3 per cent., and it is possible, as will be shown later (see p. 1 178), that the missing material contributed to the production of some synthesised fat. It must be remembered that it is not correct to regard the hatching stage in amphibia (for Cryptobranchus see B. Smith) as the end of embryonic development, for a considerable period subsequently elapses before the yolk-sac disappears, and before the complete larval form is attained. As regards the question whether any nitrogenous waste products are excreted into the water while the embryo is yet in the egg, Gortner found the total nitrogen to be practically identical at the beginning and at the end, being 584-9 mgm. and 584-5 mgm. per 100 embryos. It is probable, then, that in Cryptobranchus little or no waste nitrogen is excreted into the surrounding water before hatching, when what has accumulated is discharged all at once, after which the animal can get rid of its endproducts without hindrance. The loss in dry weight amounted to
SECT. 9] PROTEIN METABOLISM mi
96-9 mgm. (5825 to 5728) per 100 eggs, and reason will be given later for supposing that this is largely, perhaps preponderantly, due to the combustion of carbohydrate.
9-10. Protein Metabolism in Teleostean Ontogeny
Gortner applied the same methods to the study of the protein metabolism of the brook-trout's egg {Savelinus fontinalis) . This was more satisfactory than his study of the salamander's tgg, among other reasons because he pursued its development to completion, i.e. until the larvae had lost their yolk-sacs, and were ready to take food. The figures for total nitrogen were as follows:
Total nitrogen
Dry weight of
in 400 eggs
400 eggs
Days (mgm.)
(gm.)
873-5 21 890-8 35 860-3
51 (hatching) 823-8
7-5404
7-4920
7-5287
7-0668
72 (end of yolk-sac period) 68 1 -8
5-6295
Total weight lost
Total nitrogen lost 191 "7
1-9109
(or 21-9% of
(or 25-3 % of
initial value)
initial value)
Gortner attached no importance to the gain of 20 mgm. recorded for the second sample, or to the loss of 30 mgm. recorded for the third. He preferred to average the first three readings, and to say that, before hatching, 400 eggs of the trout have a constant figure for total nitrogen at 874-8 mgm. The difference between this figure and that first obtained after hatching, namely, 51 mgm., he put down to the loss of the egg-membranes, but it is more than probable that the greater part of it consisted of nitrogenous end products which had been retained in the eggs, and were now liberated. Certainly the consumption of protein after hatching during the last third of development was considerable. We can make a rough calculation to see how much of the 51 mgm. was egg-membrane protein, for Kronfeld & Scheminzki noted that the membranes of the trout egg were 8-25 per cent, of the total dry weight, i.e. 582 mgm. in this case at hatching, or, taking its nitrogen content at 14-5 per cent, and assuming that the membrane is at least 50 per cent, keratin, 42 mgm. of nitrogen. But this is a high estimate, for it is known that the egg-membranes of fishes become diaphanous and
III2 PROTEIN METABOLISM [pt. iii
thin towards hatching, and the operation of the hatching enzyme (see Section 24-2) will not be without a powerful effect. The amount of nitrogen accounted for by the membrane might therefore leave some 20 mgm. for the urea nitrogen, which would thus be 2-3 per cent, of the initial nitrogen content. But such a calculation is beset with the difficulties due to our ignorance, and no great emphasis can be laid upon it.
Table 151. Gortner' s figures for Savelinus fontinalis.
% of total nitrogen
Days ...
21
35
51*
72t
Ammonia nitrogen ... Humin nitrogen
\t
8-8o 2-26
7-86 2-32
8-78 2-70
9-19
2-73
Di-amino fraction
Total
Arginine nitrogen ... Cystine nitrogen ... Histidine nitrogen ... Lysine nitrogen
28-60 11-09
8-33
0-40 8-79 8-57
32-63 13-32 0-52 8-04
10-66
29-78
12-26
0-40
9-20
7-91
34-o6 12-52 0-50
I2-OI
9-02
Mono-amino fraction
Total
Non-amino nitrogen
61-55 3-90
59-00 4-27
Loss
56-87 4-09
or gain
(%)
55-85 3-85
54-33 3-90
To To end of hatching yolk-sac period
Total period
Ammonia nitrogen ... Humin nitrogen Di-amino nitrogen Mono-amino nitrogen
+ 1-22 + 0-98 + i-i8 -5-70
+ +
+4 -I
•41 •03 -28 •52
+ 1 + 1 + 5 -7
-631
•oil- +8-10
•46 1 •22
Hatching.
t End of
yolk-sac
peri
iod.
Gortner's data for the distribution of nitrogen are seen in Table 151. As he took no trouble to prepare the pure protein of the eggs and embryos, it is not easy to criticise the results, but there is an un Table 152. Gortner' s figures for Savelinus fontinahs.
% of total nitrogen
Protein at Protein in com zero hour busted fraction
Ammonia nitrogen 7-56 1-77
Di-amino nitrogen ... ... 28-60 9-17
Mono-amino nitrogen ... ... 61-55 87-14
mistakable trend from the mono-amino to the di-amino nitrogen, exactly as would be the case if there was a preferential utilisation of the former for combustion purposes. This appears well in Table 152
SECT. 9] PROTEIN METABOLISM 1113
where the composition of the lost nitrogen is shown. Here again is a correspondence with Plimmer & Lowndes. The ammonia and humin nitrogen also rise.
In this connection reference must be made to the paper of Tangl & Farkas on the trout egg, which has been already discussed on p. 955. In the course of their calorimetric study they obtained the following figures :
Milligrams per lOO individuals
Undeveloped eggs Hatched fry Gain or loss Wet weight ... ... 8850 8330 —520
Dry weight ... ... 2990 2915 —75
Fatty acids ... ... 639 663 +24
Nitrogen 359 357 -2
Carbon ... ... 1672 1635 -37
Tangl & Farkas did not regard the loss of nitrogen as significant, but there was certainly a gain in fatty acids. This phenomenon will receive some consideration in Section ii-8; all that need be said here is that this demonstration of the fixity of the total nitrogen does not imply that no protein had been catabolised, as the urea or ammonia would be included in the total nitrogen.
Gortner determined the phenol content of the phosphotungstic filtrate, and observed a loss of 29 per cent, throughout the whole period, principally after hatching, which may or may not have been tyrosine. As stated above, the 400 embryos lose throughout the whole period 191 1 mgm., of which 1190 mgm. is protein, so Gortner concluded that 62-7 per cent, of the total food-stuflf" catabolised must come from the proteins, and 37-2 per cent, from other substances. (See Table 126.)
Pearse has also studied the eggs of fresh- water fishes, particularly the brook-trout. In his paper of 1925 he stated that the nitrogen diminishes from the ist day of development till after the end of the yolk-sac period, but the published details are meagre. Hayes states that there is no diminution of the total nitrogen before hatching in the egg of the Atlantic salmon {Salmo salar) but that there is in that of the lumpsucker {Cyclopterus lumpus). Levene's experiments on the cod's egg [Gadus morrhua) were not unlike those of Gortner on the trout. The following table shows that the eggs as a whole lose water as they develop, and that there is a distinct loss of nitrogen, although the figures are erratic. The data for basic and non-basic nitrogen and protein nitrogen are so uneven, rising and falling in leaps between
III4
PROTEIN METABOLISM
[PT. Ill
each set of readings, that it is impossible to interpret them, and useless to present them.
Table 153.
Days after fertilisation
Water (%)
Ash (% dry weight)
Nitrogen (% dry weight)
I
II
20
94-66 94-80 92-02 93-59
10-09 17-17
Loss ... .
10-90
9-96
11-22
9-52
1-38
A related form which has been investigated is the egg of the plaice, Pleuronectes platessa, the nitrogen content of which was determined by Dakin & Dakin in 1925. Their table,
Table 154.
2000 eggs
(weights in
milligrams)
fertilisation
hatching
Wet weight
6720
6638
Dry weight
479
418
Water
6251
6220
Fatty acids
6
21
Protein ...
426
348
Protein nitrogen
68
56
shows that, during the period preceding hatching, 78 mgm. of protein are lost by this quantity of eggs, or 18-3 per cent, of the original amount. In this tgg, therefore, the membranes must clearly resemble those of the eggs of the anura rather than those of the eggs of the salmonidae, in letting the products of protein combustion pass out to the exterior. It is also obvious from Table 154 that the loss of protein almost wholly accounts for the loss in dry weight, so that between 80 and 95 per cent, of the total material catabolised must be, in the case of the plaice, protein. The oxygen consumption (90 mgm. per 2000 eggs), as found by Dakin & Dakin, nearly, but not quite, equals that calculated from the lost protein, assuming that it was all combusted. At the same time there is the 15 mgm. increase in fat to be remembered.
9-11. Protein Metabolism in Selachian Ontogeny
With this we pass to the elasmobranch fishes. Little has been done on the protein metabolism of their embryos, but it could have been predicted beforehand that in this they would differ from other fishes.
SECT. 9] PROTEIN METABOLISM 1115
In the adult elasmobranch, the urea produced by protein breakdown is retained within the circulation, causing an extraordinarily high blood urea and counterbalancing the osmotic pressure of the saline external medium. This retention of urea as an osmotic device has already been discussed in Section 1-13. As early as 1834 John Davy noted the presence of urea in the uterine cavity of Squalus squatina, a selachian, but did not succeed in finding any in the uterus of Torpedo marmorata. He never detected uric acid in these fishes. Parker and Parker & Liversidge also found abundance of urea, but no protein or uric acid, in the " pseudamniotic Hquid" (i.e. the periviteHine liquid within the egg-cases of the ovoviviparous Mustelus antarcticus, after the disappearance of most of the yolk) .
Whence comes all this urea? In 1890 von Schroder extirpated the livers of a number of fishes [Scyllium canicula) and observed only a small reduction of the urea content of the muscles (1950 mgm. per cent, before and i860 mgm. per cent, afterwards). The Hver can therefore not be the main source, and probably all the tissues have the power of forming urea from the amino-groups in the food. Arginase appears to be found in great quantities in elasmobranchs; thus Hunter & Dauphinee found the following typical figures:
Arginase units
Liver Kidney SclachidL-a {Squalus sucklii) ... ... 319 31
Teleostean {Sebastodes maliger) ... 29 4
The arginase of the dogfish liver was twice as active as that from the most active teleostean liver (in the herring, Clupea pallasii) and forty times as active as that from the feeblest (in the tommy-cod, Hexagrammos stelleri) . It is probable, however, that the contribution of urea made by arginase to the total urea content of the elasmobranch cells would not be large. Hunter & Dauphinee made the interesting observation that the undeveloped eggs of Squalus sucklii contained notable amounts of urea, but no arginase, while both were present in an embryo of 20-5 cm. (see also pp. 1077, 1142 and 1312).
It may be added that Baglioni found that the selachian heart could not beat properly unless a certain amount of urea was contained in the perfusion fluid.
The excretion of urea in selachians does not appear to take place wholly through the kidneys. Denis found that only 20 to 50 mgm
iii6 PROTEIN METABOLISM [pt. iii
were excreted through the kidneys of an adult dogfish per kilo per day. The gills have been found by Duval & Portier to be absolutely impermeable to urea. But van Slyke & White found that large amounts of urea were contained in the bile (up to 72-3 per cent, of the total biliary nitrogen), so that the intestinal tract is probably concerned together with the liver cells in regulating the urea-content. The kidney certainly does not seem to do much regulation, as Denis found the blood urea to be uninfluenced by experimental nephritis induced by uranium nitrate or potassium chromate. Another mode of elimination of urea from the elasmobranch body is through the peritoneal pores, which Smith believes to have an excretory function, for the peritoneal fluid of a dogfish contains 680 mgm. per cent.
More recently, Needham & Needham made an investigation of urea production in the embryos of Scyllium canicula and Pristiurus melanostoma. We first of all carried out a series of experiments to ascertain whether the dogfish tgg was a closed system, and, by allowing eggs to remain in comparatively restricted amounts of sea water, we found that not more than traces of urea are lost to the exterior, no matter whether the embryo be less than i cm. long, or more than 7, i.e. nearly ready to hatch. Rather complicated controls were here necessary as the diatoms in sea water destroy urea, a fact insufficiently taken into account by earlier workers. By constructing osmometers of the thick horny egg-shells, we further found that, although they themselves were permeable to urea in the outward direction yet the sHmy coat on the inside effectually made them impermeable. But the fact that the egg was a closed system as regards nitrogenous end products led to a paradox, for, as is well known, the egg-cases of the dogfish possess from the beginning four slits at one end, which, at first plugged with albumen, open to the sea water about two-thirds of the way through development. It is even probable that a current of sea water may pass through them, introduced by the waving of the embryo's external gills. Why, then, does not the urea escape? We found the answer when we estimated the urea in yolk, egg-white, and embryo separately:
Urea (mgm.)
0'5 cm. embryo and yolk 8-32
Corresponding white ... ... o- 1 65
3-0 cm. embryo and yolk ... ... ii"59
Corresponding white ... ... 0-03
SECT. 9]
PROTEIN METABOLISM
1117
Evidently the dogfish embryo excretes its urea into its yolk, and so retains it for its osmotic purposes. This peculiarity, then, made it possible to regard the egg as a closed system, and to estimate the amounts of urea produced by the embryo at different ages, the results of which are shown in Fig. 338. By the end of development some 15 mgm, of urea are present, but, as we do not yet know the exact amount of protein nitrogen present at the beginning, we cannot say what percentage this is.
Unfortunately, no good series of weighings exists for Scyllium embryos, but we know from the work of Fulton and Kearney that the length of fish embryos increases at first far more rapidly than the weight. Now this evidently implies that when the points are plotted against weight, the slight concavity towards the abscissa, shown in Fig. 338, will be greatly |' accentuated, because equal increments of length mean much greater increments of weight in the later stages than in the earlier. Plotted against weight, then, the urea-content curve would rise sharply to a certain point and then very slowly. Accordingly when the urea produced by the embryo is referred to unit weight of embryo, a peaked curve would result.
This expectation we found to be fulfilled as far as possible when we used as weight data the figures of Kearney for Mustelus canis. We laid no emphasis on the result though it will be admitted that the shape of the ascending weight/age curve for Mustelus probably does not differ much from that of the related Scyllium. As the last column of the following figures demonstrates, a descending curve is obtained over the range covered by Kearney.
The excretion of urea into its yolk by the elasmobranch embryo may perhaps throw light on some of the morphological results of Borcea, who studied in detail the development of the urinogenital system in these fishes.
Summing up what we know of the protein catabolism of the fish
iii8 PROTEIN METABOLISM [pt. iii
embryo, it may be said that it certainly uses more protein as an energy source than terrestrial embryos do, whether this be expressed in terms of the initial store of protein or of the total amount of material catabolised. Such a rule would be expected from the composition of the fish egg, which, as Greene said, has on an average 27 per cent, of protein in its yolk as against the 1 6 per cent, of protein present in the yolk of the chick.
Urea-nitrogen
Kearney's
Milligrams urea
milligrams
corresponding
nitrogen pro
per embryo
weights in
duced per 100
ngth in cm.
(embryo's con
milligrams
milligrams
{Scyllium)
tribution only)
{Mustelus)
wet weight
—
—
0-5
1-35
—
—
i-o
2-4
—
—
1-5
3-4
—
—
2-0
4-3
—
—
2-5
^'l
—
—
3-0
e:^
no
5-250
3-5
200
3-200
4-0
7-1
300
2-370
4-5
V
400
1-920
5-0
8-4
550
1-550
li
9-0
730
1-230
9-6
920
1-042
6-5
10-3
1 150
0-899
7-0
ii-o
1450
0-760
7-5
II-6
1720
0-668
8-0 12-2 2050 0-595
9-12. Protein Metabolism of Insect, Worm and Echinoderm Eggs
Among the insects, the egg which has received the most examination from this point of view is that of the silkworm, Bombyx mori. Tichomirov, who studied its metabolism in 1882, observed a diminution of protein (estimated roughly by solubility) from 11-3 to 9-2 per cent, of the dry weight, i.e. i8-6 per cent, of the original material, although, as only 14-9 per cent, of the original dry weight was lost, all the protein cannot have been combusted.
In more recent times, the proteins of the silkworm egg have been investigated anew by Pigorini. He divided them into several fractions : (^4) proteins soluble in distilled water, albumens, (J5) proteins soluble in 7-5 per cent, sodium chloride solution, globulins, (C) proteins soluble in 0-5 per cent, sodium hydroxide solution, vitellin and nucleoprotein, and finally (Z)) proteins soluble in water, but incoagulable by heat, and having the property of liberating
9]
PROTEIN METABOLISM
1119
1400-1100
Pigorini Proteins of silkworm eggs
ffl o
reducing substances on acid hydrolysis, ovomucoid. Pigorini subjected the eggs of the silkworm to three successive extractions, which gave him the proteins of groups A, B, and C, and then by coagulation of the extracts he was able to evaluate the amount of D. Fig. 339 gives the results.
Monzini has estimated the free amino-nitrogen in the silkworm egg at different stages of development. Expressed in terms of wet weight of egg, it shows at first a not very welldefined peak, and then declines till the larvae emerge (see Fig. 340). Tirelli continued Monzini's work, but his figures are so irregular that it is impossible to plot them, Russo extended the van Slyke method to the silkworm egg, with the results shown in Table 155. x\ccording to his data the total nitrogen per cent, of the dry weight is variable, falling by about 12 per cent., and then rising by about 7 per cent. There is every reason for supposing a />non that no nitrogen escapes from a terrestrial egg such as that of the silkworm, for the only form in which it could do so would be gaseous ammonia, and this would be so perceptible by its odour in the silkworm establishments that it would be well known. It is more probable that Russo's estimations were not sufficiently statistical or accurate to demonstrate a constancy in the total nitrogen of the egg. Ashbel, it is true, has stated that ammonia is lost by silkworm eggs, but only on the basis of erratic positive pressures in manometric experiments.
E 5
r
Monzini
S
Q)
°-^4
_
° ^
l?<
)
||3
^
1 ^
qo^^
i^
g-.
V5
£^2
a^
^
^
<^ ^
? 5
01
Coj 1
_
c
a
3
D>
a
£
... 1 .... 1 ,
. , 1"^
Days^5 10
15
Fig. 340.
II20 PROTEIN METABOLISM [pt. m
Farkas, although mainly concerned with the respiration and the calorific value of the silkworm egg at different stages, also estimated the total nitrogen in the eggs. In his first experiment a batch of 33*0 gm. of eggs (about 280 individuals) contained 1-27 gm. of total nitrogen before development, and 1-26 gm. at hatching — a constancy probably within the limits of error. His second experiment gave a different result, however, the total nitrogen falling from 1-84 gm. to I '54 gm. for a batch of 45-87 gm. of eggs. Farkas' explanation for this was that during the last few days of development the larvae were hatching irregularly, and some were dying, so that a mass of excreta, egg-shells, and dead larvae, all saturated with condensation water, remained on the floor of the incubation chamber. In this mass, micro-organisms were doubtless decomposing the uric acid. This explanation might go some way towards explaining Russo's decline in total nitrogen. Furthermore, it was shown by Peligot for the silkworm and by Henneberg for the bee that no ammonia was given off by the eggs as they develop. Farkas consequently affirmed that there was no nitrogen loss from the eggs, and that the missing 0-27 gm. of nitrogen in his second experiment was all uric acid. Further and more accurate observations would be very desirable in this confused subject. Farkas calculated from his calorimetric measurements that 63-4 per cent, of the total material catabolised in the silkworm egg was fat, and 36-6 per cent, not fat; and for this latter fraction he calculated a specific energy of 7-31 Cals. On this basis alone (the specific energy of protein being 7-9 Cals.) he concluded that most of the 36-6 per cent, was protein. It is not sufficient evidence.
Table 155. Russo's figures for the silkworm.
% of the total nitrogen
'~'
Total non
Total
Non
Free protein
nitrogen °l^
Protein
protein
amino amino Ammonia
Undeter
dry weight
nitrogen
nitrogen
nitrogen nitrogen nitrogen
mined
Just fertilised
IO-88
95-95
4-05
2-20 2-47 0-l8
1-4
After the diapause...
9-20
95-95
4-05
2-20 2-47 0-l8
1-4
At hatching
9-91
92-24
776
o-oo 3-93 0-30
3-53
More reliance, perhaps, can be placed upon Russo's figures for the distribution of nitrogen. These are interesting, for they show practically no change till the end of the diapause (as might be expected, the morphological work done till the end of that period being so
SECT. 9] PROTEIN METABOLISM 1121
slight), but definite changes during the three weeks of active growth and differentiation in the spring. The protein nitrogen decreases by 3-71 or 3-87 per cent, of the original value, and this loss, probably all uric acid, plus the undetermined and ammonia nitrogen, almost exactly makes up the 7-76 per cent, of non-protein nitrogen present in the egg-contents at hatching. It is interesting to see the complete disappearance of the free amino-nitrogen, a result which by no means agrees with Monzini. Nor does the small loss in protein nitrogen agree with the large loss found by Pigorini, but Russo's figures are the later ones, and are certainly to be preferred.
Another insect egg which has been investigated to some extent is that of the lackey-moth or tent-caterpillar, Malacosoma americana. Rudolfs determined the total nitrogen in it during embryonic life, and reported simply that it rose from 11-5 per cent, of the dry weight to 14-4 per cent, at hatching. As this figure was for the whole eggmasses, including the cases, it can be only an apparent rise, due to the diminution of something else, and, as Rudolfs did not publish his data for absolute weights, his results are dilBcult to interpret. The fat is known to diminish in this egg, and consequently the nitrogen/fat ratio rises during embryonic life. Rudolfs also gave one analysis of the egg-contents and egg-case separately towards the end of development, but there is nothing to compare it with. Free amino-nitrogen, free ammonia, urea, tryptophane, tyrosine, etc., were all tested for and found to be present. The presence of free ammonia (3-54 mgm. per cent.) in the egg-case and of free amino-acids (0-83 mgm. per cent.) suggested to him that these were decomposing, and furnishing amino-acids to the eggs inside. Such a process had previously been thought to take place in the silkworm egg.
We know nothing about the nitrogen metabolism of nematode eggs, except that Kozmina found as much nitrogen present at the end of Ascaris development as at the beginning.
Our knowledge of the protein metabolism of the echinoderm egg is also in a very backward state, but Ephrussi & Rapkine have done a good deal to fill the gap. Working on the eggs of Strongylocentrotus lividus, they found the following figures :
% of the dry weight
Hours from fertilisation
12 (blastulae)
40 (plutei)
Nitrogen IO-7
IO-2
9-7
Protein 66-88
60-62
II22 PROTEIN METABOLISM [pt. iii
It would thus appear that an appreciable amount of protein is used up during echinoderm early development. The only information we possess as to the corresponding end products is due to the work of Ashbel who estimated the ammonia produced in cultures of developing eggs, with the following results :
Cubic
Milligrams of
millimetres
ammonia pro
of oxygen
duced by i c.mm.
used by i c.mm.
of centrifuged
of centrifuged
Sphaerechinus
eggs per hour
eggs per hour
Unfertilised
0-289
263-53
Fertilised
More
More
Paracentrotus
Time after fertilisation 6 h. 20 min.
0-222
174-0
„ ,, 26 h. min.
0-287
525-0
,, ,, 50 h. 20 min.
0-377
400-0
The ammonia production could be inhibited by potassium cyanide. Ashbel apparently did not look for other end products.
9*13. Protein Utilisation in Mammalian Embryonic Life
The examination of the nitrogen excretion of the mammalian embryo was begun by Vauquelin & Buniva in 1800, who isolated a substance resembling uric acid from the amniotic liquid of a cow embryo ; to it they gave the name " amniotic acid ". In 1 82 1 Lassaigne isolated it also from the allantoic liquid of the cow, and called it "allantoic acid". Probably these early workers were dealing with impure allantoin containing traces of uric acid. Urea was not found till rather later, for Fromherz & Gugert were the first to report its presence in the amniotic liquid of man in 1827, but they were not sure that they had excluded the contamination of the maternal urine, so it was not until Rees in 1838 obtained pure amniotic liquid from a 7|-month foetus, and found urea in it, that the production of urea by the foetus was really demonstrated. Thenceforward a large number of reports appeared, e.g. those of Wohler; Regnauld; Picard; Majevski; Tschernov; Litzmann-Colberg; Beale; Siebert; Winckel; Grohe; Schlossberger; Mack; Vogt; Scherer, etc., in which the urea was for the most part estimated by the Liebig- Wohler method in the amniotic liquids of man, the cow, and other animals. The data of the majority of these observers are now of little interest, for they only gave their results in terms of urea per cent, of the amniotic or allantoic liquid, and omitted to mention either the weight of
SECT. 9] PROTEIN METABOLISM 1123
the embryo, the total volume of liquid, or some other detail essential for making any calculation or comparison. A tabular presentment of their figures is given by Prochovnik; the values vary from 27 to 420 mgm. per cent. Nevertheless, in certain instances, it is possible to calculate the amount of urea present per 100 gm. of formed embryo, thus:
Table 156.
Sheep
Cow
Cat
It is evident that in all cases the amount of urea produced by 100 gm. of foetus and excreted into the allantoic liquid declines as development proceeds.
It is interesting that Schondorff in 1899 concluded that the concentration of urea in the amniotic liquid in man was of much the same order as that in the blood and milk — another hint, perhaps, that such a diffusible molecule cannot be concentrated in a limited space, and so of interest in view of the discussion on p. 1 133. It is evident, of course, that the urea in the amniotic liquid might come from the maternal circulation, but, on the other hand, it is certain that the foetus contributes largely to it, for as early as 1843 Prout found urea and uric acid in the fluid from the ureters of a human 8-month embryo and later Virchow; Schwarz; Dohrn, and Panzer all found urea and uric acid in the embryonic bladder. Then Wohler found in 1846 a small uric acid calculus in a stillborn foetus, and uric acid infarcts in the kidneys of human embryos were reported by Hoogeweg; Virchow, and Salomonsen.
Gusserov in 1872 began a new line of investigation. His experiments proved that not only excretion of urine into the amniotic
Weeks
Weight of embryo (gm.)
Milligrams urea
per 100 gm.
embryo in
allantoLs
Investigator
6i- 9 10 -12A 12^18
1-62
10-25
52-10
207-00
17-90 4-71 2-54
2-72
Majevski
9 -12 12-21 21 -27
19-50 144-50 342-00
8-50
6-12
3-96
Majevski
I - 2
I-IO
f-p
Tschernov
V-t
6-8
12-34 40-15 98-29
0-48
0-34
II24 PROTEIN METABOLISM [pt. m
liquid occurred, but also genuine discontinuous micturition, at any rate during the late stages, for he often found (69 per cent.) the bladders of newborn human infants to be full. This had already been the view of Englisch; Dohrn; Betschler; and many other obstetricians. According to Keene & Hewer who experimented with vital dyes, and to Hewer who used differential staining methods, the excretory activity of the human kidney begins as early as the loth to the 1 2th week. Similarly Firket found that the cat's kidney can excrete sodium ferricyanide and ferric ammonium citrate before the glomeruli are fully differentiated. Mijsberg even has reasons for supposing that the human pronephros is functional (see also Fritschek) .
Gusserov reported the presence of allantoin in the urine of pregnant women, and therefore concluded that the foetus possessed an active uricase, but this idea was exploded by Wiechovski who found no traces of it, and finally Wells & Corper discredited Cannata's assertion of the presence of uricase in the human placenta. Gusserov's original observation must have been a mistake, but he has the merit of being among the first to investigate the subject. It is not necessary here to detail the work of those investigators who have published results for urea, uric acid content, etc., of the blood of newborn infants (e.g. Martin, Ruge & Biedermann; Dohrn; Hofmeier, etc.) for the processes of parturition introduce too many doubtful factors. An interesting piece of work was later done by Feis, who found that urea injected hypodermically is a strong poison for the embryo rabbit, which cannot withstand the same degree of uraemia as the mother. In 1902 Panzer, in examining an hydropic foetus, was able to collect embryonic urine unmixed with amniotic liquid. Of specific gravity i-oo8, it contained traces of protein, no glucose, keto-substances, or indican, and no creatinine. The nitrogen partition was peculiar, urea accounting for only 40 per cent, of the total nitrogen and uric acid for 15-5 per cent., the rest being in some unknown form.
Doderlein in 1890 made a detailed investigation of many aspects of the amniotic and allantoic liquids of the cow, and among his observations was a series on the non-protein nitrogen of both. Assuming that the preponderant part of this could be counted as waste nitrogen, it is easy to calculate it per 100 gm. wet weight of embryo, with the results which follow:
SECT. 9] PROTEIN METABOLISM 1125
Table 157.
MilHgrams of
non-protein nitrogen in amniotic and
Age of
embryo
Wet weight
allantoic liquids per
in months
of embryo
100 gm. embryo
3
276
138
4
Ifoo
95
5
67
6
7
5.123
Ts
8
6,690
47
6,700
48
8,300
52 66
11,300
9
14,900
96(?)
Evidently (setting apart the last value, which is doubtful) there is here to be seen the same fall in intensity of production of nitrogenous waste which appeared above from the data of Majevski and of Tschernov — the former set indeed, fit on, as it were, at the upper end of Doderlein's. But the mere determination of non-protein nitrogen in the liquids does not carry us very far, and we find a much more thorough attack on the nitrogen excretion of the mammalian embryo in the work of Lindsay. Table 158 summarises the more important points emerging from her data. They were confined to herbivorous animals, the sheep and the cow, and, as the figures show, they were quite concordant. In both cases there was no ammonia in the foetal urine, although it was present in that of the adult. The sheep's allantoic urine contained a good proportion of allantoin, but not the cow's, and a very notable thing was the high proportion of amino-acids other than hippuric acid. Creatine and creatinine were always present, and in much the same amount, but perhaps the most extraordinary finding was that the urea of the foetal urine was very low, its percentage value being about half that of the adult urine, a deficiency made up by a large amount of unidentified nitrogen. This unidentified nitrogen confirmed the earlier work of Panzer and of Paton, Watson & Kerr, and recalls the similar assertion of Targonski in the case of the chick. Lindsay made a determined attempt to discover its nature, but without much success, although she established the fact that it was all present in the phosphotungstic acid precipitate. It was therefore probably polypeptides or di-aminoacids, and it could not have been proteoses or peptones, for the
126
PROTEIN METABOLISM
[PT. m
trichloracetic acid filtrate was biuret-free. The dipeptide nitrogen, estimated by Henriques & Sorensen's method, did not account for more than a small proportion of it, nor by silver precipitation or other means was it possible to arrive at a clear idea of its nature.
Table 158. Lindsay s figures for percentage of the total nitrogen.
1
2
D
.2
<
1
X
u
5
Sheep. Adult
0-8
83-1
0-5
6-2
4-0
3-1
1-7
5-0
Foetal allantoic liquid, early
200-1000 gm
None
33-3
20-3
7-5
3-5
—
—
31-4
1000-1350 gm
None
15-2
12-4
IO-8
20-9
—
—
42-1
Full term liquid
None
21-6
12-3
14-7
12-0
—
—
40-2
Foetal anmiotic liquid
60-300 gm
None
64-8
7-0
5-5
I2-0
0-2
0-2
i8-9
21-6
300-1000 gm
None
63-4
IO-3 13-8
7-1
None
None
None
1000-1350 gm
None
6,-7
6-6
33
1-3
1-3
i8-3
Full termi
None
88-0
3-6
2-0
6-1
3-0
Cow. Adult
i-o
66-3
6-3
1-9
ii-i
5-0
3-2
8-8
Foetal allantoic liquid, early
1 00- 1 000 gm
None
38-4
5-2
25-3
1-6
4-6
2-6
24-6
1000-4000 gm
None
22-7 12-6
IO-2
25-3
1-6
—
40-0
Full term
None
180
27-1
—
6-6
6-0
42-2
Ox. Adult
1-3
8l-2
0-5
2-4
3-4
4-3
3-2
5-4
Foetal amniotic liquid
1 00- 1 000 gm
None
63-0
5-2
12-3
—
None
l-g
19-5
1000-4000 gm
4000-6000 gm
None
59-5
4-7
9-9
3-4
None
2-6
25-9
None
42-8
i6-3
25-4
—
—
15-3
One rather obvious consideration seems not to have been taken into account by Lindsay, namely, that products of the foetal protein metabolism are constantly passing through the placenta into the maternal circulation, even in the mammals with epitheliochorial placentas studied by her. There is therefore no guarantee that, when we determine the nitrogen partition in the early allantoic liquid, we are determining the nitrogen partition of the total nitrogen excretion of the embryo. Furthermore, it is obviously possible that if we knew exactly how much nitrogen the embryo was excreting and in what forms, through the placenta as well as through its own kidney, we should find such substances as amino-acids and Lindsay's unidentifiable fraction to be much less important quantitatively than would appear from her figures. And, on the other hand, there is the further possibiHty, though it may be remote, that the walls of the allantois
SECT. 9]
PROTEIN METABOLISM
1127
themselves contribute nitrogenous substances to the liquid within them. Lindsay's assumption, in fact, that the composition of the allantoic liquid in the early stages gives an undistorted picture of the foetal protein metaboHsm must be admitted only with reservations.
In this connection it is worth while anticipating the chapter on placental permeability to allude to the work which has been done on the concentration of nitrogenous end products in the maternal and foetal blood. By studying them, many investigators have hoped to discover the existence of a concentration gradient between the foetal and maternal organisms. The relevant figures are as follows (all on human blood) :
Table 159.
Urea nitrogen (mgm. %) Uric acid nitrogen (mgra. %)
aternal Foetal
Maternal
Foetal
Investigators
IO-5 IO-4
—
Slemons & Morriss
20-I 20-2
—
—
Morel & Mouriquaud
— —
3-5
3-5
Slemons & Bogert Kingsbury & Sedgewick
— —
3-1
31
Less 2 1 '5
Cavazzani & Levi
14-8 15-2
3-9
3-9
von Oettingen
120 I2-0
Howe & Givens
No difference
No difference
Caldwell & Lyle
No difference
Low
High
Plass & Mathew
Slemons & Morriss observed that rises and falls in the maternal blood urea were always accompanied by like rises and falls in the foetal blood urea, and they concluded that urea passed into the maternal circulation by diffusion. As the above table shows, the concentrations of nitrogenous end products in the maternal and foetal blood are almost identical, with a sHghtly higher level in some cases in the foetal blood. If a gradient exists, then, it is in the direction foetus ->mother. Kreidl & Mandl in 1904 summarised the reasons for believing that by far the greater part of the nitrogenous excretion of the foetus passes through the foetal kidneys. The interesting anatomical evidence for this, with its bearing on the function of the mesonephros, will be considered in the Section on placental permeability.
If, then, we cannot suppose that the nitrogenous end products which collect in the allantoic sac of the herbivorous mammal are the sum of all the nitrogenous end products of the embryo, there is not much use in relating them to its total weight, i.e. in attempting
N E II 72
I 128
PROTEIN METABOLISM
[PT. Ill
® Cow Ipa^on.WatsonScKerr o Sheep]
ffl Cow 1 |_j
a Sheep )
id say
to gain some notion of the relative intensity of its protein catabolism,
as was so straightforward in the
case of the chick. Nevertheless,
the enterprise is not without in- 2
terest, and Lindsay essayed it, :^
using her own figures in con- ^
junction with those of Paton, o
Watson & Kerr. The results are •D
plotted in Fig. 341, where an ^' unmistakably descending curve ^ is seen, becoming asymptotic to e the time axis. Exactly parallel f tothisarethedataofGriinbaum, s who worked with the cow, esti- §, mating the total nitrogen in the allantoic liquid at different stages of development. His figures, or rather the results
'^ Sheep 500
Cow 1000 2000 3000 4000 5000 6000
Weight of embryo in gms.
Fig. 341
calculated from them, are not readily incorporated in the graph of Fig. 341, but are given in Table i6o.
Table i6o.
Total nitrogen in the allantoic liquid in Weight of the cow milligrams per loo gm. foetus in grams of foetus weight
0-9
9300
2-7
2070
4
1880
22
690
285
245
870
177
1,150
175
1,200
120
2,050
128
5>50o
67
13,000
82
"The waste nitrogen", said Lindsay, "accumulates as the foetus grows, but per unit of weight it markedly decreases." This holds good for all the constituents of the urine, as found from the analysis of the allantoic and amniotic fluids, and a group of descending curves is seen in Fig. 342. Now, if the curve of Fig. 341 be compared with that given in Fig. 325 for the intensity of protein metabolism of the
SECT. 9]
PROTEIN METABOLISM
1 129
chick embryo during its development, a striking resemblance appears, if it is assumed that the curve for the sheep and cow is the descending limb of a peaked curve essentially similar to that occurring in the chick embryo. If this view were adopted, we should have to conclude that the protein utilisation peak was reached by the sheep embryo some time before it attained the weight of 8 gm. (i.e. about the 4th week out of 22), and by the cow embryo before it attained the weight of 400 gm. (i.e. about the 12th week out of 32). In this there is nothing improbable. On the other hand, as Lindsay herself suggested, there is nothing to show that placental excretion does not play a relatively larger part towards the end of development, so that the fall in intensity of protein combustion might be purely an artifact. If this were the case, a difference in urea and uric acid content of the foetal blood in early and late pregnancy would be worth looking for. But, whether this be so or not, it is at any rate suggestive that we have a curve for the intensity of protein combustion for the mammalian embryo which looks as if it might be in perfect correspond
ndsay)
Cow
1000 2000 3000 4000 5000 Weight of embryo ingms.
Fig. 342.
ence with that established for the oviparous avian embryo. It would be interesting to make calculations about the total quantity of waste nitrogen produced by the mammalian embryo during its intrauterine life, using the data for nitrogen content of amniotic and allantoic liquids, and for urea content of maternal and foetal blood, but such a calculation would be beset with so many difficulties, and would involve so many assumptions, that it is not worth beginning it in the present state of our knowledge. In view of the arguments to be brought forward at the end of this chapter, it might be predicted that the mammalian embryo would combust much more protein than that of the chick, but it would be difficult to know in what terms to express it, for the initial store could be regarded as almost unlimited, and the total material combusted would be extremely difficult to ascertain. Perhaps something could
II30 PROTEIN METABOLISM [pt. iii
be done on the basis of a calculation giving the maximum amount of protein which the maternal apparatus could supply to the embryo in the time.
The data of Table 158, mentioned above, are in some ways rather difficult to interpret. The urea concentration in the amniotic liquid of the cow and sheep does not change much during pregnancy, nor does its percentage in terms of total nitrogen, though here there is a slight decrease. In the allantoic liquid the absolute quantity of urea is very much greater. The decrease in the proportion of urea nitrogen is more marked in the allantoic liquid of the cow than of the sheep. The amount of allantoin present in both liquids is considerable, and increases with length of pregnancy in the cow, but not in the sheep. The amino-acids, including hippuric acid, were abundant, especially towards the middle of pregnancy, in the allantoic liquids of sheep and cow, and they increased more rapidly the younger the foetus. In the cow the amino-acids make up the same proportion of the total nitrogen in the allantoic fluid, but rise in the amniotic, while in the sheep exactly the reverse process takes place. In the allantoic fluid of the sheep there was a considerable increase of hippuric acid as pregnancy advanced, both absolute and proportionate, but in the amniotic fluid the tendency was the other way.
"The picture of foetal metabolism", said Lindsay, "thus shown by the chemical composition of the early allantoic liquid is one of low deaminising power as indicated by the low urea output, the absence of ammonia, and the high proportion of amino-acids." A final remark which might be made before leaving these questions is that the very high allantoin content of the early allantoic urine might be taken to indicate an intense nucleoprotein metabolism (for here only purines would be concerned, unlike the bird), in which case the findings of LeBreton & Schaeffer (p. 11 53) on the chemical nucleoplasmatic ratio would be seen in a new light.
Traces of proteins are found in the amniotic and allantoic liquids of mammals, but for this see Section 22.
9-14. Protein Utilisation of Explanted Embryonic Cells
The metabolism of mammalian embryonic cells in tissue culture has been studied by Holmes & Watchorn, in experiments which bear the same relation to the subject of this section as those of Cohn & Murray to the section on growth. They used kidney and
SECT. 9] PROTEIN METABOLISM 1131
brain tissue from embryo rats, maintained in vitro with precautions ensuring sterility, and on these they carried out ammonia and urea estimations, making special provision for a complicated series of controls necessitated by the autolysis of the medium when held at 37°. Comparing the results from "resting" tissue, i.e. tissue submerged and floating so that it cannot grow but can yet live, with those from tissue attached to cotton-wool fibres and vigorously growing, Holmes & Watchorn found much more urea and ammonia production from the latter than from the former. Unfortunately, it was not usually practicable to weigh the pieces of kidney selected for culture, so that we cannot tell what level of metabolic intensity this represented, but in one instance 33 mgm. wet weight of kidney tissue had produced after a time unstated 0-023 nigm. of urea and 0-039 mgm. of ammonia, or about 0-05 mgm. of total nitrogen, i.e. 0-15 mgm, per 100 mgm. wet weight of the original tissue. Holmes & Watchorn found no evidence for the activity of urease in their cultures. In brain cultures, on the other hand, there was a definite suggestion of the action of urease, and when good growth took place there was found a surprising result, namely, that there was a considerable fall in the ammonia and urea nitrogen. Possibly a synthesis of nitrogenous substances such as choline was occurring. In a later paper these investigators added glucose to the medium of culture, and found that it gave rise to a marked alteration in the metabolism of the tissue, bringing about a definite inhibition of ammonia and urea formation. This was the second in vitro demonstration of the protein-sparing action of the carbohydrates: for the first see Warburg, Posener & Negelein (p. 765). Still later, Holmes & Watchorn used their technique to study the relation of cyanate, hydantoinacetic acid, etc., to urea and ammonia production by embryonic kidney cells in vitro. As regards the nourishment of the mammalian egg-cell before its implantation into the uterine wall, Emrys-Roberts suggested that the secretion of the mammalian uterus was analogous to that of the avian oviduct, and supplied a kind of egg-white which the ovum could digest and dissolve. He made no experiments to prove the point, but he conceived that the gelatinous envelope which surrounds the embryo of the rabbit and the mole before implantation was produced by a fermentative and coagulating action on the part of the trophoblast. Sobotta and Gaffier have produced evidence in favour of such a mechanism.
II32 PROTEIN METABOLISM [pt. m
9-15. Uricotelic Metabolism and the Evolution of the Terrestrial Egg
We have now examined all the existing knowledge about the protein metabolism in embryonic life, and it is time to turn to a few general considerations. One of the questions always asked by students in biochemistry is why some animals should excrete urea, some ammonia, some uric acid. As far as I know, they have not so far been accustomed to receive any reasonable reply, and the problem has been set down as one of those arbitrary dispositions of fate which make Elementary Classes despair of biochemistry, but I think an answer can be given.
Fiske & Boyden, in their memoir on the nitrogen metabolism of the hen's egg, raised an interesting point when they calculated that 1 5 per cent, of all the water in the egg at the beginning is needed to excrete the 5 mgm. odd of uric acid which are present in the allantoic liquid by the 1 1 th day of development. From that time onwards re-absorption of water vigorously proceeds, no doubt for the reason that, without it, all the water in the residues and in the body of the embryo would be required to get rid of the uric acid that is to be formed. It is as if the water acted as an endless belt conveyer, transferring uric acid from the cells of the embryo into the allantoic liquid, and then returning to transfer more. The fowl is always good at absorbing water from its excretions, for, as Wiener and Sharpe have shown, the glomerular urine in the adult is quite liquid, and the cloaca absorbs great quantities of water. All terrestrial animals do this to some extent, if the views of Cushny about the function of the mammalian kidney tubules are correct. But in the hen's egg it is obvious how closely the process as a whole is bound up with the properties of uric acid. "A substance as soluble and diffusible as urea ", say Fiske & Boyden, "could not possibly replace it as an end-product when the organism and its excretions are confined to a closed system, the walls of which are only permeable to matter in the gaseous state."
This is a very important consideration. There appear to be only three substances which are available in the animal kingdom for carrying away the nitrogenous waste resulting from protein breakdown — ammonia, urea and uric acid. The first two of these compounds are very soluble and diffusible; uric acid is not. Quantitative expression of this fact has been given by Chauffard, Brodin &
SECT. 9] PROTEIN METABOLISM 1133
Grigaut, who found a dialysis coefficient of 93 for urea, but only 74 for sodium urate. The haemato-encephalic barrier, according to them, allows urea to pass easily, but not uric acid. And the first substance retained in the mammalian circulation, if the kidneys are impaired, is uric acid. Shut up as it is in its closed box, the chick embryo would evidently find uric acid by far the most convenient excretory product, for the two former would tend to diffuse throughout the egg, and to establish themselves in equal concentration in all its constituent regions, instead of being packed into a small store. As it happens, the work of Kamei provides a striking verification of this viewpoint, for as we have seen, he showed that, in the amniotic liquid of the chick, although the uric acid concentration never rises above a certain very low level, the ammonia and the urea rise continuously throughout development. It is easy to guess, therefore, what would happen if all the nitrogen excreted by the embryo were in the form of urea. As an illustrative calculation we may take the uric acid present in the allantois at the end of incubation as 100 mgm. (data of Fiske & Boyden; Needham; Targonski and others) — i.e. about 33 mgm. of uric acid nitrogen or 66 mgm. of urea. This, distributed over an egg of contents approximately 40 gm., would be 165 mgm. per cent. The egg would be uraemic (in the strict, not the clinical, sense of the word). The normal figure for the ureacontent of human and bovine blood is about 25 mgm. per cent., and the highest figure on record obtained by ingesting solid urea is just under 100 mgm. per cent. In severe renal obstruction or nephritis, it rises above 100, and may reach 300 or 400, but 165 is undoubtedly of the pathological order of magnitude, and if the avian embryo had to suffer from a constant headache and other symptoms before hatching, natural selection would hardly have preserved it for our entertainment. These consequences could be avoided by the use of uric acid.
Such considerations lead to the suggestion that the form of excretion of nitrogen adopted by an animal depends principally on the conditions under which its embryo has to live. There is good evidence that the combustion of protein substances as a source of energy is much more marked in aquatic than in terrestrial embryos (Needham). Table 161, constructed from as much of the information as is trustworthy, shows the partition between the substances comprising the total material catabolised. Thus only 5 or 6 per cent, of the total
II34 PROTEIN METABOLISM [pt. in
matter combusted by the chick embryo is protein, but the frog embryo combusts as much as 71 per cent, during its embryonic Hfe. Everything points to very deep-seated differences between eggs which develop in the water and eggs which develop on land. Not only do aquatic embryos burn much more protein in per cent, of the total material burned, but also in per cent, of the initial store of protein.
Table 161. Material burned as source of energy in per cent, of the total material so burned.
Carbohydrate Protein Fat Investigators Terrestrial. Chick [Callus 3-02 5-57 91-4 Murray; Needham;
domesticus) Fiske & Boyden
Aquatic. Frog {Ram tern- 6-84 70-70 22-4 Barthelemy & Bonnet;
poraria) Faure-Fremiet &
Dragoiu; Bialascewicz & Mincovna; Needham
Aquatic. Trout {Savelinus — 63 37 Gortner
fontinalis) Aquatic. Plaice {Pleuronectes — 90 — Dakin & Dakin
platessa) Terrestrial. Silkworm {Bombyx — 10 64 Tichomirov; Farkas
mori) Aquatic(?) Turtle [Thalassochelys — 19 81 Tomita; Nakamura;
corticata) Karashima
The figures above the line are those most accurately known. Credit should be given to Halban as the first to suggest that there might be a fundamental difference between aquatic and terrestrial embryonic life.
Table 1 62 shows this very clearly. The embryos of the chick and the silkworm are the only terrestrial ones for which we have dependable figures, and they agree in burning about 4 per cent, of their initial store of protein. Among aquatic embryos, the frog, the trout and the plaice agree in burning about 25 per cent. In the case of embryos which hatch only half-way through their development, as most of the aquatic ones do, it is interesting to find that up to hatching their protein utilisation is not high, but that for the whole embryonic period it much exceeds that of terrestrial embryos. Thus there is reason for supposing that the terrestrial environment of the embryo has two effects on its protein metabolism ; firstly, to suppress the production of nitrogenous waste by removing the means of its easy disposal, and, secondly, to elevate uric acid to the place of importance as a means of excreting nitrogen. From this point of view, the invention of viviparity was a " back-to-the-sea " movement on
SECT. 9] PROTEIN METABOLISM 1135
the part of the embryo, for even if, as McCallum would have us believe, the maternal sea water is practically pre-Cambrian, it is at any rate as good as any other sea water for the disposal of nitrogenous waste products, and from the embryonic point of view, a boundless ocean. In other words, the continuous perfusion system of the vivipara provides an artificial sea, and avoids the necessity of a
Table 162. P;
rotein bi
irned in per cent, of the ;
initia
/ protein store.
Protein nitrogen combusted
Aquatic or during development in % of
terrestrial the total protein nitrogen
Animal
embryo
present at the beginning
Investigator
Pisces. Brook-trout
A.
To hatching 3-4
Gortner
(Savelinus fontinalis)
To end of yolk-sac
period To end of yolk-sac
21*9
"
period
17-0
Pearse
Pisces. Plaice
A.
To end of develop
{Pleuronectes platessa)
ment
i8-3
Dakin & Dakin
Amphibia. Frog
A,
To disappearance of
{Rana temporaria)
external gills
25-7
Barthelemy & Bonnet
A.
To hatching g-i To end of yolk-sac period
40-0
Bialascewicz Mincovna
&
A.
To hatching 10.6
Faure-Fremiet &
To end of yolk-sac
period To hatching 9-2
Dragoiu
A.
23-1
Faure-Fremiet &
Amphibia. Salamander {Cryptobranchus allegheniensis)
Insecta. Silkworm
A. T.
To hatching 4-9 Whole development
3-9
du Streel Gortner
Russo
{Bombyx mori) Chelonia. Turtle
prob. A.
(By end products found
Tomita;
[lu! LIBRARY
( Thalassochelys corti
and nitrogen lost)
i6-5
Nakamura
cata) AvES. Chick
T.
(Indirect calculations)
5-8
Idzumi
(Callus domesticus)
T.
(By protein lost) (By end products
8-0
Sakuragi
(All figures are for the
T.
-■-^...^t ,.:-■'■
whole of develop
found)
i-i
Needham
ment)
T.
(By end products
found)
3-4
Fiske & Boyden
uricotelic metabolism. Is it surprising, in view of these facts, that fishes turn the ammonia from their protein breakdown into urea, birds and reptiles into uric acid, and mammals once more into urea ?
The thought may be stated in another way. Perhaps the sauropsida excrete their nitrogen mainly as uric acid because they had to learn how to do so in order to pack their embryos into solid- and
1136 PROTEIN METABOLISM [pt. m
liquid-tight boxes, and never afterwards forgot. Even the eggs of water birds, which might be supposed to have the opportunity of excreting substances into the water around them, have impenetrable fat-impregnated shells, as Loisel has shown. The highest avian groups, exemplifying as they do the most complicated form of nitrogen excretion, would thus represent the crowning achievements of the uricotelic line of evolution. And the fact that, between the 2nd and 5th days in the chick's development, it excretes ammonia and urea with no uric acid would thus be a recapitulation of its pre-terrestrial or aquatic ancestry, entirely analogous with its gill-clefts. Moreover, the coincidence is exact, for it is just between the 2nd and 5th days that the embryo manifests its morphologically piscine characteristics. Fig. 323 showed the milligrams of ammonia, urea and uric acid present in embryo, amniotic and allantoic liquid throughout incubation, expressed in terms of 100 gm. dry weight of embryo; in other words, it showed what 100 gm. dry weight of embryo has manufactured in the way of nitrogenous end products by any given time. Table 138 showed the relations between these substances in another way. Although ammonia, urea and uric acid are excreted by the chick embryo during its development, the two first-named molecules only account for an insignificant part of the total nitrogen excreted. There is a progression in ontogeny from the smallest to the largest molecule, and from the most to the least efficient excretory product. It could be argued, of course, that, if the chick can excrete urea early in its development, it ought to be able to return to this practice after hatching; but this would be to neglect one of the most characteristic features of embryonic life, namely, its continual tendency to lose pluripotence, and to move towards a stable, "crystalline", or set state. The chick embryo begins to excrete uric acid about the 6th day. Before that time, as we know from the work of Przylecki & Rogalski, it possesses uricase, but after that time it does not. Perhaps, then, when the reptiles came ashore they found it was as well to leave their uricase behind them. As for avian development, hatching is followed by a sudden burst of protein catabolism, as we have seen in Section 6- 11, and this is probably associated with the chick's enlarged opportunities for getting rid of nitrogenous waste.
There remains the consideration that no animal would excrete uric acid as its main nitrogenous end product unless it was driven to it,
SECT. 9] PROTEIN METABOLISM 1137
for of the three in actual use it is much the most wasteful. Ammonia is clearly the most efficient end product, for it involves no wastage of carbon, but of the other two the carbon/nitrogen ratio is i /2 in urea and i/o-g in uric acid. In other words, two atoms of nitrogen can be got rid of at the expense of only one carbon atom in urea, but only 0-9 in uric acid. These amounts may not be individually considerable, butcollectively they may make all the difference between an efficient and an inefficient species. Ackermann has compiled an interesting table showing the nitrogen-removing efficiencies of many urinary constituents, and has emphasised this point. Moreover, it is obvious that uric acid excretion involves the wastage of a great deal more chemical energy than urea, thus:
Heat of combustion
(cals. per gm. mol.) Ammonia 906
Urea 152-6
Uric acid 462-1
Uric acid, then, as the main end product of protein metabolism, may be said to be more ingenious than the other two, but less efficient. The proposition that the circumstances in which the embryonic life has to be passed ultimately govern the form in which the nitrogen is excreted is thus not so far-fetched as it sounds. During the last fifty years much attention has been paid to the comparative study of nitrogen excretion, but the methods of the older workers, such as Krukenberg and Griffiths, were so unreliable that the earlier literature may be neglected. More recently, the researches of Przylecki; Delaunay and others have begun the erection of a solid structure of knowledge about the forms in which nitrogen is excreted. We are thus acquiring, as it were, a wide series of phylogenetic base-lines on which ontogenetic phenomena can be superimposed. The general conclusions of these workers support the idea of an association between aquatic life and the excretion of ammonia and urea, on the one hand, and between terrestrial life and the excretion of uric acid, on the other hand. But the important point is that the life of the embryo is the key, not the life of the adult. An animal may live all its life in the sea, but if its eggs are laid and develop on land it may be predicted that its main nitrogenous end product will be uric acid. Mammals, from the chemico-embryological viewpoint, count as aquatic animals , since the excretion of nitrogenous waste products through the placenta is analogous to their excretion into water.
1138 PROTEIN METABOLISM [pt. iii
Another interesting fact which fits in closely with this point of view is that, according to the researches of Przylecki who investigated a large variety of organisms, no animal possesses both uricoligase and uricase. In other words, all those animals which possess the power of making uric acid from amino-acids cannot destroy it, and all those which can destroy it have no power of making it, other than from purines. This certainly looks as if the power of formation of uric acid jfrom amino-acids was an adaptation of evolutionary value, for if uricase and uricoligase were often present together, it would be difficult to suggest that any special advantage was to be gained in certain circumstances from the manufacture of uric acid.
In Table 163 are collected together a number of figures for nitrogen excretion in various animals. All the older work has been excluded, and, as far as possible, only quantitative investigations of the percentage distribution of the excretory nitrogen appear. As a general rule, the marine invertebrates excrete most of their nitrogen as ammonia — a simple and easy procedure, considering their environment. But with the increasing complexity of the body, ammonia excretion disappears, for it is incompatible with a kidney, even in a very undeveloped form. Excretory structures — structures which have to live, as it were, in an excretory atmosphere — cannot deal with highly alkaline liquids, and the great disadvantage about simple ammonia excretion is that a constant supply of acid is required to neutralise it. This acid is nothing but waste, and so among the marine invertebrates themselves we see urea superseding ammonia. Among the invertebrates the only ones at present known which have a high percentage of uric acid are the pulmonate gastropods, the snail and the slug, which live on land and have terrestrial embryos. In this connection the concretions of urates in certain snails, which, according to McKinnon, contain bacteria capable of breaking down uric acid, are of special interest. Delaunay himself pointed out that the invertebrates could be separated into an aquatic and a terrestrial group, the former excreting much ammonia and the latter Httle, and he also remarked on the association between uric acid and terrestrial life. But this might remain enigmatic if we did not consider the needs of the embryo; able, in the one case, to get rid of its nitrogenous waste easily into the surrounding water, and forced, in the other case, to keep it close at hand in very restricted quarters. Delaunay's generahsation alone would not explain the
SECT. 9]
PROTEIN METABOLISM
1 139
Table 163. Quantitative Data for Nitrogen Partition in Urine.
Protozoa
Paramoecium ... Didinium
Annelida
Sea-mouse {Aphrodite aculeata) Leech (Hirudo officinalis) Earthworm {Lumbricus agricola)
Gephyrea
Worm {Sipunculus nudus)
Arthropoda
Crustacea
Crab {Carcinus moenas) Spidercrab {Maia squinado)... Crayfish {Astacus fluviatilis) ...
Insecta
Silkworm {Bombyx mori) Clothes-moth {Tinea pellionella)
Clothes-moth ( Tiniella crinella)
Insects in general ...
MOLLUSCA
Gastropoda
Sea-hare {Aplysia limacina) ... Land snail {Helix pomatia) . . . Slug {Limax agrestis)
Lamellibranchiata
Clam {Mya arenaria)
Oyster {Gryphoea angulata) ...
Pond-mussel {Anodonta cygnaea) Cephalopoda
Octopus {Octopus vulgaris) ...
Octopus {Sepia officinalis)
ECHINODERMATA
Nitrogen Partition in " ,'^ of the total nitrogen excreted
gj
'
u^
0-3
s|
n
•c
I^S
2
a
^i
c
'y
iSl
1 =
.Sf
i
E
s
•c
ess
■|
>
<h
<
D
P
<:SS
fS-S
90-0
None
—
Weatherb
—
go-o
—
None
—
—
»
A.
8o-o
0-2
0-8
_
_
Delaunay
A.
76-4
5-4
None
3-2
3-6
,,
■?
20-4
38-1
Trace
15-8
9-3
„
50-0 9-7 None i6-6
A.
67-8
2-9 0-7
A. A.
42-9 59-6
5-2 2-7 11-2 0-8
T.
None 85-8
T.
IO-22
17-6 47-3
T.
20-7
1-8 77-5
T.
"Als characterisches
8-7 2-3
20-2 3-5
lo-i 37
0-51
produkt des Insektenorganismus ist die Harnsaure zu betrachten."
33-5 8-7 4-6 13-0
13-7 20-0 10-7 6-0 4-6 70-8 6-9
21-5 4-5 Trace i8-o
7-3 3-2 0-2 —
63-0 _ _ _
5-0
33-3 41-7 67-0
[5-0 1-7
1-4
12-5
20-7 7-8
Farkas Babcock &
McCollum Hollande &
Cordebard von Fiirth*
9-3 Delaunay 5-8 1-7 5-9
Przylecki
23-6 Delaunay — von Fiirth 1-9 Delaunay
Starfish {Asterias rubens) Sea-urchin {Paracentrotus
lividus) Sea-cucumber {Holothuria
tubulosa)
p. 295. See Schmieder.
A. 39-3 1 1-7 Trace 23-8 6-8 „
A. 28-1 7-5 i-o 28-0 lo-o „
A. 40-0 6-0 Trace — — ,,
Ackermann; Kawase & Suda; Kutscher & Ackermann; Roubaud; Poisson; and
1 140
PROTEIN METABOLISM
[PT. Ill
Table 163. Quantitative Data for Nitrogen Partition in Urine (cont.^
Nitrogen Partition in % of the total nitrogen excreted
M
1-.T3
11
H
•a •5
4 11h
1
"a
c c
^ 3
3 fc!
1
t
•n
III
i
1
<
<h
<
p
^
<S S
£•£
■S
ERTEBRATA
Pisces
Pipefish {Muraena helem) ...
A.
237
30-0
o-o
28-0 & 15-0
un
Edwards &
determined
Condorelli
Goosefish {Lophius piscatorius)
A.
36-8
l6-2
o-o
15-8 & 29-2
un
determined
jj jj
A.
0-5
0-7
0-4
46-3 & 52
I of
GroUmann
trimethylamine
oxide *
» »
A.
—
—
—
23-56 and a
great
Marshall &
deal of undeter
Grafflin
mined
JJ JJ
A.
13-0
62-0
o-i
_
—
Denis
Dogfish (Mustelus canis)
A.
7-3
8o-7
0-2
—
—
,,
Dogfish [Scyllium canicula) ...
A.
8o-o
O-O
—
—
Herter
Carp {Cyprinus carpio)1
A.
56-0
57
0-2
IO-6 & 28
2 un
Smith
determined
JJ JJ
A.
77-4
14-5
—
2-6
—
Delaunay
Sole {Solea vulgaris)
A.
53-0 631
166
—
— _
—
J,
Seahorse (Hippocampus)
A.
8-9
—
138
—
,,
Angler-fish
A.
2-0
280
5-0
Torpedo (Torpedo)
A.
3-8
9i'4
—
08
—
,,
Lung-fish (Protopterus aethio
picus)
A.
41-2
i8-5
0-8
7-0
Smith
Amphibia
Frog (Ram temporaria)
A.
15-0
82-0
Trace
—
—
Przylecki
JJ JJ
A.
5-1
87-5
0-2
7-25 undeter
Toda&
mined
Taguchi
Frog (Rana virescens)
A.
3-2
84-0
0-4
—
—
van der Heyde
Toad (Bufo vulgaris)
A.
84-5
Trace
—
—
Burian
ReptUia
Chelonia
T:\irt\e (Chrysemys pinta)
A.
153
39-0
188
1 1-5 & i8-6 undetermme
Wiley & Lewis
Alligator (Alligator mississipi
'
ensis)
A.
75-3
7-2
131
—
—
Hopping
Turtle (Che lone my das)
A.
i6-i
45' I
19-1
—
—
Lewis
Tortoise ( Testudo graeca)
A.
—
90-0
Trace
—
—
Clementi
Sauria
Snake (Boa constrictor)
T.
—
—
8o-o
—
—
Boussingault
Snake (Python)
T.
—
—
8o-o
—
—
J,
Snake (Python)
T.
8-7
—
89-0 :t
2-3
—
Bacon
Grass-snake ( Tropidonotus
natrix)
T.
Trace
Trace
8o-o
—
—
Girod
It has been suggested that this extraordinary nitrogen partition may be an adaptation; trimethylamine as an odorous substance being used to attract the prey. Grollmann's figures apply only to the product of the kidneys and do not include that of the gills.
t Fishes were found by H. W. Smith to excrete 6-10 times as much nitrogen through the gills as through the kidneys : the former dealt with the readily diffusible end products and the latter with the rest. According to Delaunay, Selachians excrete mostly urea with little ammonia and teleosteans mostly ammonia with little urea.
X In these cases the ammonia present was calculated to be just sufficient to combine with the uric acid as ammonium urate.
SECT. 9] PROTEIN METABOLISM 1141
Table 163, Qiiantitative Data for Nitrogen Partition in Urine (cont.).
Nitrogen Partition ir
% of the t
otal
nitrogen excreted
(S
•0
4
11
2 Si
■3
•- on
1
«
iS-S
01 3
•s
e
'c
E
.g
•J
1
<
<h
<
D
D
<S3S
£-5
,5
ERTEBRATA {cont.)
Reptilia {cont.)
Sauria {cont.)
Lizard {Lacerta viridis)
T.
—
—
91-0
—
—
von Schreiber
Horned lizard {Phrynosoma
cornutum)
T.
20-0
None
980*
—
—
Weese
Aves
Hen {Gallus domesticus)
T.
1-5
0-9
70-0
—
—
Salaskin & Kovalevski
)5 J)
_
i-i
62-9
—
—
Minkovski
,, ,,
—
17-3
10-4
62-9
9-4
—
Davis
5> ,,
—
—
65-7
6-0
—
Mayrs
Swan {Cygnus)
T.
i5~8
2-6
68-7
—
—
Salaskin & Kovalevski
Duck {Anas)
T.
3-2
4-2
71-9
—
—
Szalagyi & Kriwuscha
Goose {Anser)
T.
13-5
—
8o-o
—
—
Paton
Mammalia
Man {Homo sapiens)
A.
4'3
87-5
0-8
—
Folin
Dog {Canis vulgaris)
A.
3-0
89-0
i-ot
—
—
Osterberg &
Wolf Hammett
Gat {Felis vulgaris) ...
A.
4"9
68-1
o-i
Badger {Toxidia texus)
A.
—
8-0
—
—
Himter, Givens & Guion
Raccoon {Procjon lotor)
A.
—
—
3-0
—
—
)) >>
Guinea-pig {Cavia)
A.
—
—
3-5
—
—
}) >>
Rat {Mus rattus)
A.
—
—
5-0
—
—
Opossum {Didelphys vir
giniana)
A.
—
—
1-5
—
—
>j j>
Horse {Equus caballus)
A.
—
—
3-2
—
—
M J>
Sheep {Ovis vulgaris)
A.
—
—
3-3
—
—
,, ,,
Whale {Balaena mysticetus) ...
A.
1-5
90-0
3-0
—
—
Schmidt-Nielsen & Holmsen
,, ,,
A.
3'5
91-0
0-3
i"5
—
Ichimi, etc.
Egyptian bat {Xantharpyia
collaris)
A.
0-6
77-8
1-2
—
Popp
Dromedary {Camelus drome
darius) ...
A.
12-3
55-5
0-3
i9"3
—
Smith & Silvette
Camel {Camelus bactrianus) ...
A.
0-5
97-1
2-4
—
Petri
A.
4-1
62-7
i-o
i8-5
—
Smith & Silvette
Weasel {Putorius vulgaris) ...
A.
0-9
85-2
0-2
2-1
06
Fuse
Tiger {Felis tigris)
A.
3-7
891
0-04
1-9
0-2
,,
Hyaena {Hyaena crocuta)
A.
3-8
89-3
O-I
1-5
0-2
,,
Llama {Auchenia huanacos) ...
A.
2-2
67-7
0-8
lo-o
—
Smith & Silvette
Alpaca {Auchenia vicunna) ...
A.
4-5
59-6
0-3
8-0
—
,, „
Monotreme
Spiny anteater {Echidna
aculeata)
p
6-9
8l-2
None
Neumeister
M >;
?
88-0
Some
—
—
Robertson
In these cases the ammonia present was calculated to be just sufficient to combine with the uric acid as ammonium urate.
t In mammals other than the primates the purine ring is excreted in the form of allantoin but this is not shown in the Table.
I
II42 PROTEIN METABOLISM [pt. iii
mammals. It is the conditions under which the embryo has to live that govern what form of nitrogen shall be excreted throughout the life-span. As Table 163 shows, the fishes and mammals (even the whale) with their urea and the birds with their uric acid fit in with the theory here propounded. The insects also are in perfect accord, for, although we have no quantitative data concerning the nitrogen partition of their urine, yet it has been generally recognised for many years that uric acid is the most prominent constituent of their excreta (see von Fiirth) , and there is doubt if urea has ever even been shown to be present. A quite parallel case is that of the hymenoptera and diptera, which excrete uric acid during metamorphosis into their fat bodies, according to Fabre; Schmieder and other entomologists. Thus the insects, coming to live on land earher than the reptiles, had the same chemico-embryological problem to face, and solved it in the same way. This is a good biochemical instance of the phenomenon known to zoologists as "convergence". It is interesting that ovoviviparity occurs among the insects, but never true viviparity (Holmgren; KeiUn), and one would like to know why they never invented the placenta, and dropped their uricotelic qualities, for they did invent the "private pond", or amnion. The reptiles themselves form an interesting group in Table 163, for the chelonia, standing as they do phylogenetically near the amphibia, show a high ammonia and urea excretion, while the sauria with their terrestrial embryos show a high uric acid excretion. It is, of course, among the reptilian group that such a diflference would be likely to show itself, as they were the first vertebrates to conquer the land. In this connection it may be recalled that Tomita found urea in the imperfectly cleidoic egg of a marine turtle. Clementi, again, has found arginase in chelonian livers, an enzyme which does not occur in the livers of uricotelic animals.
The eggs of aquatic animals seem to divide into two classes. The frog and the plaice develop within membranes which readily allow the nitrogenous end products to escape; the trout and Ascaris (Kozmina) do not. But hatching always occurs long before the end of development in these aquatic forms, so that the permeability of the egg-membranes is unimportant from the point of view of nitrogen excretion. The trout at hatching gets rid of what has accumulated, and for the rest of its embryonic life can excrete directly into the water. The elasmobranchs, which excrete their urea into their yolk
SECT. 9] PROTEIN METABOLISM 1143
(Needham & Needham), would form a third class, but they are notable exceptions in many ways, and to describe their behaviour in terms of the language here used would be to say that they use their own blood and yolk as the sea, and pile up the end products of their protein metabolism within themselves.
The amphibia never broke loose from the fishes; they always retained a piscine larval stage and laid their eggs in water. But when the first reptiles^ left the sea, they were faced with one or two very difficult embryological problems. To begin with they had to find out how to abandon metamorphosis, and to discover a way of arranging a water supply for their embryos. As Gray has shown, aquatic embryos depend upon their environment for a supply of water; in other words, the fertilised egg contains enough solid, but not enough water, to make the finished larva. The first terrestrial eggs, therefore, had to contain enough water as well as enough solid, and, as arrangements to prevent undue evaporation were essential, the closed-box system inevitably developed. The mechanism by which a constant pressure-head of water was provided in the terrestrial egg, namely, the egg-white, can be seen functioning at the present time in the yet unidentified acid, which, introduced by the embryo's metabolism into the egg-white, as Vladimirov has shown, gradually brings the latter to its isoelectric point, and liberates water by degrees from the colloidal albumen. All the economy of the successful terrestrial egg had to be directed towards conserving the water, and while a great bath would have been required to keep the urea concentration down within bearable limits, if all the nitrogen was excreted in that form, only 20 per cent, of the water in the egg need be set aside for handling uric acid. Another way out of the diflSculty would have been to burn no protein at all, and therefore to avoid all incombustible residues, and it is possible that some of the extinct saurians explored the possibilities in this direction, but I suspect that some factor which as yet we cannot quite define dictates from within the cells that life without protein combustion is not possible. It is therefore likely that such reptilian experiments did not proceed very far. In this way the closed-box system with its partial suppression of protein metabolism and its uricotelic qualities came into being
No doubt the first terrestrial vertebrates laid their eggs in water, and probably, like the trout, they hatched early. But although this
1 Or rather, their stegocephalic predecessors.
N E II 73
II44 PROTEIN METABOLISM [pt. iir
system may suffice in the sea, an embryonic reptile with a bag of yolk almost as big as itself would have, owing to obvious difficulties of locomotion, very little chance of survival on land. Such imperfectly mobile eggs would have been too tempting for adults of other species. The terrestrial egg had therefore to be constructed in such a way that the young organism could stay inside a long time and hatch out substantially mature. It had no chance, therefore, either to get rid of nitrogenous excreta at an early stage by hatching, or to excrete them through a semipermeable membrane. The only solution of the problem was uric acid. When, at a still later date, the prototheria and metatheria branched off from the reptiles in the mammalian direction, acquiring at last true viviparity, the need for a uricotelic metabolism ceased. For it is to be noted that there is no reason why an adult land animal should not excrete urea, if it drinks sufficient water, or even ammonia, if it has enough acid to spare. Thus Ambard found that a cat or dog on a meat diet, if left to itself, drinks exactly enough water to excrete urea at its maximum normal concentration, i.e. just to avoid the slightest uraemia. This is Ambard's "volume obligatoire". But what the adult does will depend on what it had to do as an embryo ; in other words, on what its facilities then were for absorbing water and for getting rid of waste nitrogen. The existence of an albuminous solution round the yolk of the terrestrial egg is, as Gray says, an admirably adapted mechanism for providing the growing embryo with water. The use of uric acid — insoluble, non-diffiisible — instead of urea or ammonia, is an equally well adapted mechanism for dealing with incombustible waste, and the re-absorption of water through the allantoic wall is the mechanism which unites the two. There is no need, however, to postulate any reversion from uricotelic to ureotelic metabolism in the case of mammals, for the palaeontological evidence admits of the possibility that they arose from some early reptile ^ which laid non-cleidoic eggs. True viviparity may thus have been a genuine alternative to uric acid production.
A final reference may be made to certain special cases, e.g. the Prototheria and the Elasmobranchs. The Elasmobranchs are at one and the same time the only marine animals which have evolved the closed-box system or somiething approaching it and the only ones which have found out a way to withstand high concentrations of urea. There are, perhaps, two possible explanations of their behaviour.
^ Probably one or more lines of cynodont (Therapsid) reptiles.
SECT. 9] PROTEIN METABOLISM 1145
They may first have discovered how to become permanently uraemic without suffering from it, and then have utiUsed the associated advantage of protecting their embryos until a late stage of development. Or they may have adopted a protective closed box, and then become adapted in some way to withstand the consequent uraemia. In any case, they offer an interesting comment on the terrestrial egg, for they seem to have found out a way of avoiding the uricotelic qualities of the closed-box system— a way, however, which appears to have been only suitable for a very restricted class of animals.^
As for the Prototheria, their excretory nitrogen as Table 163 shows, works out at a typically mammalian partition. As tho. Echidna is, strictly speaking, oviparous, this would seem at first sight to be much in opposition to the general views here suggested, but closer examination shows that this is not so. The Echidna lays eggs, it is true, but, according to Caldwell, who gives all the literature in his well-known paper, it picks them up and immediately places them in its pouch. Once there, they emerge at a very early stage from the thin shells, and suck up the milk which exudes from scattered pores inside the pouch. There seems no reason why the shells themselves should not be permeable to excretory products (for they are not hard), and after hatching there would not even be this difficulty — in both cases the epithelium of the pouch would be able to absorb the foetal excreta. The pouch, in fact, may be regarded as a uterus located in an unusual position, and, from the standpoint of this discussion. Echidna would be viviparous, and therefore "aquatic". It would be very interesting to investigate the nitrogen partition in the urine of Ornithorhyncus which allows its eggs to develop outside the body (Wood-Jones).
A generalisation might then be provisionally enunciated as follows : The main nitrogenous excretory product of an animal depends on the conditions under which its embryos live, ammonia and urea being associated with aquatic pre-natal life, and uric acid being associated with terrestrial pre-natal life. Up to the present time there has been no trace of order or system among the facts obtained by comparative investigations on nitrogen excretion, and no answer to the question of why a uricotelic metabolism should exist at all. The answer here suggested is that terrestrial oviparous animals would have been impossible without it.
1 In this class the Dipnoi may perhaps be included. Smith has shown that the lungfish Protopteriis, aestivating in its burrow, accumulates relatively enormous amounts of urea in its tissues.
Cite this page: Hill, M.A. (2024, April 19) Embryology Book - Chemical embryology 2-9 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-9_(1900)
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
