Book - Chemical embryology 2-12 (1900)
Embryology - 25 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 12 The Metabolism of Lipoids, Sterols, Cycloses, Phosphorus and Sulphur
12-1. Phosphorus Metabolism of the Avian Egg
The study of the distribution of phosphorus in a Hving system is important for it exists in so many types of compound — Hpoids, phosphoproteins, nucleoproteins, hexosephosphates, etc. — that a remarkable survey of wide tracts of the mechanism of the egg can be obtained by simply observing where the phosphorus is. It is for this reason that the paper of Plimmer & Scott in 1909 may be called classical, for it threw more light on the complex transformations going on during the incubation of the hen's egg than any other single paper before or since. It is true that they were not the first to study the behaviour of the phosphorus fractions during development, for already in 1877 Hoppe-Seyler had estimated the phosphorus in the yolk, and had calculated thence the lecithin. He deserves much credit as being the first to approach these problems from the quantitative point of view, but his results are of little more than historical interest. Then in 1893 Maxwell went into the subject again. He seems to have been in the grip of a preconceived theory associated with work on plants, and he certainly had the misfortune to use untrustworthy methods, but he succeeded in showing that hpoid phosphorus preponderated at the beginning of development, and non-lipoid phosphorus at the end.
% of the total phosphorus
Ether-soluble Not ether-soluble
Beginning 58-5 41-5
End ... ... ' 27-0 73-0
The rest of Maxwell's figures compressed great variations into a minimum of data, and did not invite a beHef in their reliabiHty. Worse still, he counted the phosphorus in the vitelHn as "mineral" phosphorus. In 1908 a much better piece of work was done by Carpiaux, who observed an increase of inorganic phosphate during development at the expense of the ether-soluble phosphorus. He defined "inorganic" phosphorus as all the non-lipoid phosphorus, and the figures which he obtained were as follows :
Days o 6 7 Milligrams per egg non-lipoid phosphorus
75 128 127 137
Milligrams per egg lipoid phosphorus 150
146
135 108
About the same time Mesernitzki published in a short paper the results of some experiments in which he had followed the amount of ether-soluble phosphorus in the whole egg during incubation. His data, arranged graphically and expressed as lecithin in grams per 100 gm. dry weight gg of egg-contents, are to be found jj in Fig. 372. I 13
Before going on to discuss _£ ^^ Plimmer & Scott's data for the phosphorus distribution in the egg, we must touch on the question whether the contents of ^ the egg receive any accession of 6 phosphorus from the shell in ^ the case of birds, as has from time to time been supposed. In the analyses of Voit, Hermann, Forster, Feder & Stumpf, the yolk contained 203-86 mgm. of '^'
phosphorus at the beginning of development, while the white contained 7-04 mgm., a total of 210-9. At the end of development the egg-contents contained 237-5 i^g^i-j i-e. an increase of roughly 26 mgm., but Voit and his assistants did not mention this fact in their conclusions, and probably, if they noticed it, put it down to experimental error. In Pott & Preyer's experiments of 1882, the following figures were obtained :
In shell at beginning
In egg-contents at beginning
In shell at end
In egg-contents at end
PO,
mgm.
44 228
42 224
They concluded that the differences were within their experimental error, and that the shell did not provide any phosphorus for the egg-interior.
Carpiaux in 1908 made a certain number of shell-analyses with the following results :
Carpiavix Burke, Pinckney & Jones
% dry weight % dry weight
Unincubated Incubated Unincubated Incubated
Calcium oxide 54"30 54'86 52-69 52-77
Phosphorus pentoxide ... 0-31 0-32 0-371 0-403
The ratio calcium/phosphorus was therefore in the former case i75'i/i-o, and in the latter case i74-i/i-o. The amount of phosphorus taken from the shell must then be very minute, for according to other data of Carpiaux the shell loses 162-2 mgm. of calcium oxide to the egg-interior during the 21 days, and, as the calcium/phosphorus ratio in the shell is almost identical before and after, the phosphorus lost, if any, must bear the same ratio to the calcium lost, i.e. 175 to i, which would work out at not more than o-g mgm. of phosphorus pentoxide. Finally Harcourt & Fulmer in igoSjDelezenne & Fourneau in 1918, and Burke, Pinckney & Jones in 1925 showed absolutely no increase in total phosphorus of the egg-interior during the 2 1 days of development.
Plimmer & Scott made use of the following classification of phosphorus fractions :
(i) Inorganic phosphorus soluble in water and acids.
(2) Organic phosphorus compounds, soluble in water and acids, i.e. guanylic acid, inosinic acid, phosphocarnic acid, free glycerophosphoric acid, hexosephosphates and all similar bodies. This fraction would also include pyrophosphate.
(3) Compounds such as lecithin and kephalin soluble in ether.
(4) Nucleoproteins and similar bodies insoluble in water. (5! Phosphoproteins and similar bodies insoluble in water. Plimmer & Scott extracted the eggs and embryos with ether, alcohol,
I per cent, hydrochloric acid, distilled water, etc., and then estimated the inorganic, organic, and total phosphorus by the Plimmer-BayHss
1 It should be emphasised that organic phosphorus compounds of a labile character such as creatine-phosphate, which have been discovered since 1909, would appear in this fraction. An investigation of the appearance of these in the chick embryo is urgently needed.
modification of the traditional Neumann method on each fraction. The dry protein residue was split up into nucleoprotein and phosphoprotein by treatment with i per cent, sodium hydroxide at 37° for 24 hours, which converts the phosphoproteins, vitellin (and livetin?) into inorganic phosphorus, but does not hydrolyse the nucleoproteins. Then finally adding up the organic and inorganic phosphorus in the different fractions, Plimmer & Scott obtained their balance sheet of the phosphorus transformations. Thus they found 215-56 mgm. phosphorus pentoxide in an average egg as the total, and they recovered in the fractions 21 1-75 mgm. or 98-3 per cent., a result which gave them confidence in their technique. For the whole of development they found very notable changes, thus:
% of the total phosphorus
Inorganic P
Water-soluble organic P Ether-soluble organic P
Vitellin P
Nucleoprotein P
Beginning End
Trace 6o-o
6-2 8-6
64-8 19-3
27-1 o-o
1-9 12-0
The broad outline demonstrated, then, that the inorganic phosphorus had risen at the expense of the ether-soluble phosphorus, that the vitellin had also disappeared, and that the nucleoprotein had increased. The picture is better seen, however, on a graph constructed from Plimmer & Scott's data, in Fig. 373. It is composed of two principal change-overs, firstly, the "lecithin" or ether-soluble phosphorus, and the inorganic phosphorus, the former descending from 60 to 20 per cent., and the latter ascending from o to 60 per cent.; and secondly, the phosphoprotein and nucleoprotein phosphorus, the former descending from 30 per cent, to nothing and the latter rising from i to 1 5 per cent. During all this, the watersoluble organic phosphorus remains practically at a constant value, except for a dip on the 20th day, which probably has no significance. From the graph, then, it would seem as if the lecithin is transformed mainly into inorganic phosphorus, and the phosphoprotein into nucleoprotein, but we must not overlook the probability that a certain fraction of each precursor is devoted to each end product. Fourteen years after Phmmer & Scott's paper, Masai & Fukutomi undertook an exactly similar research. Their figures are assembled in Fig. 374, from which it can be seen that they confirmed Plimmer & Scott in every particular, except that they did not distinguish between phosphoprotein and nucleoprotein phosphorus. In both cases the facts agree excellently with the state of affairs long known to morphologists, namely, that the processes of ossification in the embryonic bones are proceeding during the last week of incubation. Obviously the lecithin phosphorus is transformed into phosphorus in the embryonic bones. As can be seen from Fig. 406, the curve for increase of calcium in the embryo runs almost exactly parallel to this curve for increase of inorganic phosphate. The increase in inorganic phosphate, however,
Fig- 374
is not confined to the embryo as Figs. 375 and 376 show, and this suggests that calcification of the bones is not the only destination for the inorganic phosphorus. The albumen becomes acid (see Fig. 211) towards the end of incubation, and, though from the work of Vladimirov it seems as if the increasing carbon dioxide elimination is not responsible for this, but rather some fixed acid produced by the embryo, yet the albumen never becomes more acid than pH 6- 1 , although its titratable acidity shows a rather greater proportionate increase. A buffer action is, in fact, indicated. If, then, the rise in inorganic phosphate is not confined to the embryo, it is not unreasonable to suppose that sodium or potassium phosphate may be the substances responsible for the buffer action.
Attention may now be directed to the protein phosphorus. As can be seen from both graphs (Plimmer & Scott; Masai & Fukutomi), the increase in nucleoprotein phosphorus does not account for more than a half or two-thirds of the vitelHn phosphorus disappearing. This is seen by the decrease which the total residual protein curve exhibits. Some of the vitellin phosphorus is therefore used to make something else besides nuclein phosphorus. It is very interesting to see that the nucleoprotein phosphorus increase is not only in the embryo, but also outside it, a fact due to the separation made by Plimmer & Scott being between the embryo and membranes, so that
Plimmer &i Scott P- distribution in embryo not including membranes
D Ether-soluble P
■ Inorganic P
i> Water-soluble organic P
® Nucieo-protein P
Plimmer &Scobb P-disbribufcion in remainder 70r- including membranes
□ Ether-soluble P ■ Inorganic P O Viteflin P ® Nucleo-probein P ® Vitellin and nucleo-protein P (not separated)
Days ■
Days-* 5
Fig. 375
Fig. 376.
the latter were included with the remainder of the egg. This is a demonstration that their content of nuclein is not insignificant. In the embryonic body the percentage of nuclein phosphorus seems to have a peak on the 17th day, but this is probably not real, as the curves are percentage curves and not curves of absolute magnitude. Doubtless the number of milligrams of nuclein phosphorus increase without a break in the embryo, but after the 17th day the enormous increase of inorganic phosphate in the chick's body causes a diminution in the percentage of nucleoprotein phosphorus. It has been suggested that the manufacture of nuclein proceeds externally to the embryo to some extent, and that the nuclein is absorbed afterwards, and, in view of Sendju's results on the purine content of the
I204 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
yolk, this is probably the case, so that the presence of the membranes in Plimmer & Scott's "remainders" will perhaps not wholly account for the rise in nucleoprotein phosphorus outside the embryo. The ether-soluble phosphorus outside the embryo behaves in a manner very similar to that seen on the curves for the whole egg, but inside the embryo it seems to show a fall towards the end of development. This may possibly be a chemical expression of cephalocaudal differential growth (see p. 583) ; in other words, the lecithin phosphorus in per cent, of the total phosphorus in the embryo may decHne owing to the fact that the brain and central nervous system of the embryo are declining in percentage of the total weight. Plimmer & Scott's figures for the phosphorus distribution in the embryo were not very numerous, and it would be exceedingly desirable to extend them in a backward direction, so that we might know the phosphorus distribution in an embryo 3 or 4 days old. It might be predicted that the ether-soluble phosphorus would be high and everything else low, except, perhaps, the water-soluble organic phosphorus. In this connection the results of Marza are of interest. Using the histochemical method of Romieu, which is said to indicate the presence of lecithin, he observed a distinct lag between the formation of the neural portions of the early chick embryo and the appearance of lecithin. A gradient was also perceptible, the amount of lecithin at a given time decreasing from cephalic to caudal end. This would suggest that the morphological pattern of the early neural elements is sketched out, as it were, in a protein-carbohydrate medium, while the lipoids, so essential a constituent of the fully developed central nervous system, are brought in later. All this fits in remarkably with what we know about the composition of white and yellow yolk (see p. 286). Another point of some interest arises out of Fig. 373, where the vitellin phosphorus of the whole egg seems almost as abundant on the 13th or 14th day of development as it was on the 3rd or 4th. This is another reflection of the fact already so often seen from different angles, namely, that the yolk is "a remoter and more deferred entertainment than the white".
The water-soluble organic phosphorus is perhaps the most interesting of all the fractions. On both the graphs for the phosphorus distribution of the whole egg, it can be seen that this fraction, beginning at about 5 per cent., increases slowly to about 10 per cent, in the middle of development, and finally falls away again to its
SECT. 12] GYCLOSES, PHOSPHORUS, SULPHUR 1205
initial value, or rather below it. This must surely be due to the fact that, in the water-soluble phosphorus, we have the principal form in which phosphorus is transported from place to place. It would never be likely to make up a great part of the total phosphorus, for its concentration would always be kept low owing to its destruction as soon as it was formed, but yet a slight rise might be expected to hint at a more intense transport of phosphorus. The water-soluble organic phosphorus is perhaps especially associated with ossification, as an intermediate stage in the transformation of ether-soluble into inorganic phosphorus. In this fraction would be included hexosephosphates, glycerophosphates, inositolphosphates (phy tin-like bodies), etc. From the point of view of bone formation great interest attaches to these effects. Robison in 1923 discovered an enzyme in calcifying bone which had the power of breaking down hexosemonophosphoric acid to inorganic phosphate, and also attacked the glycerophosphoric esters, but no cyclose phosphorus compounds. It was not present in other tissues besides bone, except to a slight degree in the intestine and kidney (Robison & Soames; Robison & Kay; Robison & Goodwin), and Robison & Martland showed that there was concurrence between the onset of calcification and the appearance of the bone phosphatase. Subsequent work indicated that the bone phosphatase was exclusively concerned with calcification in growth, while the kidney phosphatase was concerned with normal functioning. Kay found that on the 12th day of development in the chick there was three times as much phosphatase in the leg bones as on the 21st day, and that on the 12th day the water-soluble organic substrate was present to a much greater extent than in the unincubated egg. Now in the curves for the embryo only (Fig. 375) it is noticeable that just during the period of most vigorous bone formation, i.e. from the 1 2th to the 21st day the water-soluble organic phosphorus is steadily decreasing. It is as if the water-soluble organic phosphorus was concentrated to a high level in the embryonic body during the first half of incubation, only to be transferred into the inorganic form by the activity of the bone phosphatase during the last half. Thus the lipoid phosphorus is transformed into a suitable substrate for the bone phosphatase, reinforcing the small amount of the substrate initially present, and then, as calcification proceeds, is deposited in inorganic form. Kay made some observations on rabbit embryos and young animals, from which the graph in Fig. 377 {a) has been con
i2o6 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
structed. The kidney phosphatase increases in activity before birth, and afterwards attains a constant level — the bone phosphatase decreases to a constant level.
Fig. 377 (b) shows the admirable experiments of Fell & Robison who studied the phosphatase in chick embryo bones, developing both in vivo and in vitro. It is evident that the activity of the enzyme (as measured by the phosphorus hydrolysed in 24 hours from sodium glycerophosphate) increases in the bones from chicks incubated normally. The same increase is seen in bone fragments differentiating
O mgms. P hydrolysed in 24 hrs.from sodium glycero-phosphate
i> » per mgm. dry weight of bone
'Femurs /'"^"^-ph^ (Fell S^ Robison)
» -> 5 10
E(>5r- Days of development
(a)
6 18 20 22 24 26
Fig- 377
'ij '\j I ^ zu 25 30
Days culture of 6 -day femurs in vitro
in vitro although it is slower. In both cases, a peaked curve is seen when the units of activity are referred to unit dry weight, no doubt because after a time the ash is laid down more rapidly than the phosphatase is formed. The almost perfect self-differentiation of the bones in these experiments was very striking; it was shown by 6th-day but not by 4th or 5th-day bones. In this connection the finding of phosphatase by Martland & Robison only in centres of active ossification in human foetal bones should be remembered.
It is worth remarking in connection with ossification that Hatchett in i8oo made the curious observation that "in the ova of those tribes of animals, the embryos of which have bones, there is a portion of oily matter, and in those ova whose embryos consist
SECT. 12] GYCLOSES, PHOSPHORUS, SULPHUR 1207
entirely of soft parts, there is none. Hence it is concluded that a certain portion of oil is necessary for the formation of bone". What Hatchett actually did we shall never know, for nothing but the bare statement given above is contained in the place referred to by Prout, and that not in a paper by Hatchett himself but in one by Sir Everard Home. But in spite of our ignorance of Hatchett's technique, one cannot help surmising that he had attained in some measure, however feeble, an estimate of the lipoidal constituents in eggs of different species, and that, in actual fact, more of these substances are present
in what Prout would call "the recent egg" where bones, with their calcium phosphate, have to be formed than where they have not. A study of Table 30 does not, unfortunately, lend weight to this view, but the analyses of eggs which included reliable estimations of lipoid phosphorus have been so few that no conclusion, either in favour of Hatchett or against him, can at present be drawn from them.
Other investigations besides those of Plimmer & Scott and Masai & Fukutomi have been made of the phosphorus in the
Lecithin, (Riddle)
• Yolk-sacs and contents O Liquid contents of yolk-sac ® Solid contents of yolk-sac O Intracellular yolk ^ Yolk-sac
5
Days-*
Fig. 378.
hen's egg. Thus Riddle estimated the ether-soluble phosphorus in the yolk and yolk-sac of the chick during the last half of incubation, and, expressing his results in terms of lecithin as per cent, of the dry weight, obtained figures from which Fig. 378 has been constructed. An extremely marked absorption of phosphatides from the yolk is indicated during the last week of incubation, for the percentage phosphatides to dry weight in the yolk falls with a rush, and this is just what one would expect from the evidence in PUmmer & Scott's experiments. The phosphatide-content of the yolk-sac seems to remain stationary, if anything can be deduced from two points only, and the composition of the more solid parts of the yolk would seem. N E II jy
i2o8 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
to be very variable. Riddle is certainly right in saying that after the 1 2th day the phosphatides are utiHsed more rapidly than the neutral fats, and the neutral fats more rapidly than the proteins. Riddle also found that yolk being absorbed from the foHicle which secreted it in the ovary shows a more rapid utilisation of the phosphatides than of the neutral fats, but this may hardly be more than a coincidence. Iljin in his turn estimated the lecithin and residual protein phosphorus in the yolk only, before and after development, obtaining the following figures :
Phosphorus in milUgrams per egg
Protein Lecithin phosphorus phosphorus
Yolk at beginning ... ... 150 65
" Spare yolk " at end 32 17
Absorbed by embryo... ... 118 38
Iljin's protein phosphorus figures do not correspond very well with those of Masai & Fukutomi, or of Plimmer & Scott, but his ethersoluble phosphorus ones do. Something must certainly have gone wrong with Iljin's vitellin analyses.
It was left for Cahn to make a proper series of lipoid phosphorus determinations on the embryo during its development. The results of his work were very interesting. Whether his absolute quantities were in good agreement with the figures of Plimmer & Scott cannot be stated, as the latter workers unfortunately omitted to give any absolute figures, confining their statements to percentages of the total phosphorus. Cahn found, as might be expected, that the total amount of lecithin phosphorus in the embryo rose with the growth of the body, but regularly, that is to say, not remaining very low till the 15th day and then suddenly rising rapidly, like the neutral fat. This difference in shape of the curves can be seen from Fig. 379, which is taken from his paper. When the lipoid phosphorus was related to wet and dry weight, however, more interesting curves appeared, as shown in Fig. 380. In per cent, of the moist tissue one finds a regular augmentation until the 15th day, after which a plateau supervenes up to hatching, though afterwards the curve again rises, and a chick 2 days after hatching contains as much as 660 mgm. per cent, wet weight. Then, as might be anticipated from a plateau on a wet weight curve, the dry weight curve rises to a peak, and thereafter falls away. This peak occurs, in the case of the
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR
1209
lipoid phosphorus, on the loth day of development, and it is probable that it simply represents the impingement of the law of cephalocaudal
Molecixles gra-ms.
40 00
3000
2000
1000
320
240
160
/•
I
• • LipoicL phosphorus / i ^ 0— ^Cholesterol / // X— — X Neutral fat / / /
/
i
)
/
/
k
l!
1
/
/
J
/i
/ .
//
/
/
/
/
/ / /
■^To---^
^,
/ ,y
OEmI^^i
14
16
18
20 21
Days
Fig. 379
differential growth on the partition of solid substances in the body. If the central nervous system continued to occupy all through development the important ponderal position which it occupies in the first
77-2
Lipoid Pin embryo, (Cahn) O
(nob including membranes) 2day old chick'^
I2I0 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
10 days, the lipoid phosphorus would continue to rise in per cent, of the dry weight, but this is not the case, and the loth day peak represents the point at which the cephalic end of the embryo ceases to dominate the chemical composition of the embryo. Cahn himself was more interested in the relation between water and lipoid phosphorus. In Fig. 38 1, the watercontent of the embryo per 100 gm. is placed on the same graph as the water-content
Days ^5
Fig. 380.
expressed in terms of grams per 100 mgm. of phosphatide phosphorus. It is evident that the two curves descend without evincing any close
? , ■ . I
Days->5
O Daily increment in mgms. lipoid Pin embryo (Cahn)
_^,5 O Daily increment in mgms/^ cholesterol in embryo(Cahn))
♦ Daily increment in
mgms. cholesterol in
em bryo (Roffo &^ Azaretti)
Fig. 382.
relationship, for the concavity of one lies towards the abscissa and that of the other towards the ordinate. Cahn next calculated the curve for daily increment of lipoid phosphorus, and found it to show (Fig. 382) an augmentation up to the 15th day, followed by a fall. This cor
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR 1211
responds in an interesting way with tlie data of Plimmer & Scott, for it looks as if the daily accretion of lipoid phosphorus in the embryo rises until a point at which great quantities of the lipoid of the yolk begin to be decomposed, and then falls off. Cahn thought that all the constituents of the embryo would show peaks on the daily increment curves, but, as may be seen from many of the graphs in this book, in actual fact this is not the case. He interpreted the peaks which do occur as being evidence of S-shaped curves of absolute magnitude. But, as we have seen, not all such curves are S-shaped in the time of the chick's development. Cahn finally calculated the percentage growth-rate of the lipoid phosphorus, and found that it followed the usual curves for percentage growth-rates of individual substances (see Fig. 364).
12-2. Tissue Phosphorus Coefficients
The conception of tissue coefficients, or ratio values constant for a given tissue for a given animal of a given age, first introduced by Mayer & Schaeffer, is interesting in this connection.
^^^ '^- Total fatty acids
Lipoid phosphorus'
can be regarded roughly as a measure of the relati\ e amounts of lipoid fatty acids and triglyceride fatty acids in the tissue. Mayer & Schaeffer obtained the following figures in the course of their experiments, all on the adult animal : , ^ . , . . ,
Total fatty acids/lipoid phosphorus
Man
36
25
Dog
21
22
Rabbit
20
22
Guinea-pig ...
16
25
Pigeon
33
27
Eel
126
34
Liver Kidney Lung Pancreas Muscle
— 55 213 22 27 69 25 — 19 19 — 38 16 — 53
— — 288
Thus only two tissues, the liver of the guinea-pig and the lung of the pigeon, had so little an amount of neutral fat that the ratio fell below 17, though in four or five cases it fell to or below 20. On the other hand, in certain cases the muscle showed an immense preponderance of neutral over lipoidal fatty acids. What happens in the chick embryo during its development? Cahn calculated this ratio from his data, with the result that it turned out to be well below the very lowest of the adult values. In other words, the fatty acids of the
I2I2 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
embryo, minute in amount as they are during the first lo days, must be almost wholly combined in the lipoid molecule, and only towards the end of development, when the fat content is beginning to rise sharply, does the coefficient rise too, showing an accumulation of neutral fat. Presumably by shortly after hatching the ratio would approach one of the lower levels seen in the table given above. Cahn's figures for the ratio are plotted in Fig. 383. JavilHer & Allaire later suggested the use of the following coefficients :
Purine phosphorus J Lipoid phosphorus
Total phosphorus — (lipoid phosphorus + purine phosphorus)
Total phosphorus which latter is equivalent to
Inorganic phosphorus + water-soluble organic phosphorus
Total phosphorus
X 100.
It is instructive, however, to compare the data of Javillier & Allaire, and of Javillier, Cremieu
Level of guinea-pig
& Hinglais, for various adult tissues, with the data at our disposal for the foetal tissues of the chick and the undeveloped egg, more especially as Javillier & Cremieu have investigated in this way the tissues of various invertebrates, and Javillier, Allaire & Rousseau the tissues of the whole white mouse from birth to 40 days of post-natal life. JavilHer & Cremieu, in comparing the invertebrate tissues with those of mammals, suggested using the total phosphorus minus the inorganic phosphorus as the denominator, instead of the true total phosphorus, a procedure which they hoped would make a better basis of comparison. This non-calcification phosphorus they
Days
Fig- 383
SECT. i2l GYGLOSES, PHOSPHORUS, SULPHUR 1213
•a
S
M
B
Pi
. .-«
'0 •
^
^
il "
'>
l3 ^ 3
^ O ►« E ^ A I
°o^x zziZ7Z^ iiim^Vp^^ 1 1 1 1 M 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 jo.«^.o 1 1 1 1 1 1
-J3JEM OTireSjo aiqEJS *
I
^^ sruoMdsoMd.Apov ^^^l^l^^^Jl | | | | M I I I I I I I I I I I I I M I I I I I -^^^^^1 Mil! snjondsond piodiq -^"^^mu^nn-tt r^-^-r-r^^ g
^n£oqdsoqdJAp^v.^^^^^^?J:r^l | [ | | | | M I I I I I I M I I I I I I I I I I ^^;?S^5jl Mill
stuoqdsoud uiapnjsj: m«mm«nm '^^ u
II I smoqdsoqdiB^ox ^rj V^^ 9 ^ V^ J^ r |; , | I I 1 1 I I 1 1 1 tf ^....0^^"^ ^
sruoqdsoqd aiqnjos
•Sa
-jajBM oiireSjo aiqeiS S
n « t-> o 00 ■*» •*'0 ^ 00 r^O lO i^ o o
a
sruoqdsoqd ibjox ^-^^"^^^^^^lllllllllll .ooo*o>m|||||'§
°°^^ smoqdsoqdo;uBg..oui 1^^^ ?8 8 '^S?: ' I I ' I I I I I I I ^.SR^S^' ' ' ' ' |
sruoqdsoqd Tbjo t oo t^o >n n t^oo n ^ P i i
OOI X ^ — ^~— ^ — 3=- S>nfO" f^oo o>-*o-oo r«o r^ ino>oovoo.OM>HN •+o oo^noji
sruoqdsoqd ptodiq nc^Sm5nn ' -<NT^' NNTt.inTj-M^t-)inin('5*"it-«o>voNi-i««MMMiHMM,(.Tr>oTrn g
sruoqdsoqd piodtq yiP'r^N^y'r"?*^, , , , , , , , , , ,
OOI X -^T -V-^-^ ^i- 00 OvQvO inN O I^t^
sruoqdsoqd ui3]3n>j nn (^ m» ^^t-^ni I ' 1 I I I I I I I
>c^^ 2
snjoqdsoqd JEJOX t^ r> o> tj- c> n m T^o ' sruoqdsoqd u;apn2«j 2 2"* S"^ 2 « "^ "
If il 1 =1
1 aoO t~ in OM3< N invO
^^""S^^^S^^^^^'^""
oi;ob £
l«t^OOO
.00 1
in c^ M N 11 t^oo N ■+ w r> t~ 11 o ooo
. . . -2
«
3^ «
•S-s-~,
lsislS§|pi.sl|"Jsl?||||||||lilra I 3
l-S-S-3
T3 iH
".Z
W.^'
■q
i
"•S
i".
§2
3 ^
O Plimmer S^ Scott (chick) ® Calculated from the nVjclein N data of LeBreton Sz Schaeff6r and Targonski, and lipoid P of Cahn (chick)
• Javillier, Allaire and Rousseau (mouse)
1214 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
called the "active phosphorus". As Table 175 shows, the mammals and invertebrates can hardly be compared except in this way, but when it is done it is found that the mammal comes inter- ^^^^r mediate between some of the invertebrates as regards its phosphorus distribution. It does not seem possible to draw any ontogenetic conclusions from this body of data, but it is as yet, of course, very restricted.
The undeveloped egg of the bird has, as would be expected, a phosphorus distribution very different from any tissue, and even the body of the 14-day chick is not very like any of the adult tissues of the bird (we only have figures q_
2
Days-5
Fig. 384.
for the pigeon). The nuclem phosphorus/lipoid phosphorus ratio is interesting. The values for the chick placed in Table 1 75 are taken from Plimmer & Scott's figures for the phosphorus distribution in the embryo, and these authors, as I have before remarked, did not give their figures in milligrams per embryo, or even per cent, of the weight of the material. Thinking therefore that a calculation based on percentages of the total phosphorus alone might be distorted, I calculated the nuclein phosphorus in the embryo (which has never been directly estimated) from the
Targonsk
Days
Fig- 385
data of LeBreton & Schaeffer and Targonski for the nuclein nitrogen in the embryo, and in this way obtained the nuclein phosphorus/lipoid phosphorus ratio for each day during the last fortnight of development. The result, given in Fig. 384, shows first a rise and then the beginning
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR 1215
of a fall. This fits in with the data for the mouse, as appears from the fact that in Javillier, Allaire & Rousseau's figures for the phosphorus distribution in white mice, the nuclein phosphorus/lipoid phosphorus ratio is above 100 at birth, and then falls. Apparently the nuclein phosphorus never greatly exceeds the lipoid phosphorus.
So far nothing has been said about the phosphorus in the allantoic liquid of the chick. It has indeed only been estimated by one worker, namely, Targonski. By measuring the total and inorganic phosphorus in the allantoic hquid during the last half of development, he was able to find the percentage of the total allantoic phosphorus existing there in inorganic form, and the ratio :
Total phosphorus Total nitrogen
The main results are plotted on Fig. 385. What happens to the inorganic phosphorus in per cent, of the total phosphorus is not very clear; it might be regarded as showing a distinct break at the i6th day, but more probably the relation remains constant. The phosphorus/nitrogen ratios, however, are very interesting, for both the total phosphorus/total nitrogen and the inorganic phosphorus/total nitrogen decline steadily, the former rather less rapidly than the latter. The phosphorus/nitrogen ratio never reaches that for vitellin, namely, 0-057. ^^ other words, there is always more phosphorus in the allantoic liquid, and sometimes much more, than would result from a simple removal of part of a vitellin molecule (if it was homogeneous) and excretion of the results of its combustion. These problems need much further investigation.
12-3. Choline in Avian Development
If now we return to the consideration of Plimmer & Scott's curves for phosphorus distribution throughout the egg, it will be remembered that the ether-soluble phosphorus diminishes greatly, i.e. that the lipoids of the yolk, lecithin and kephalin, are broken down, releasing inorganic phosphorus. The other substances released at the same time are worth consideration. From its formula, one would expect that a good deal of free choline would make its appearance during incubation. The amount of choline in the hen's egg has been studied by Sharpe and by Okada. The former investigator thought that the increase in total guanidine during incubation might be due to a decrease
I2i6 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
in total choline, and made a few estimations by means of his own method to see whether this was so. Choline, by reacting with urea, might be the precursor of guanidine, and the formulae of the three substances lent a certain colour to the suggestion :
CH,.CH.(OH)
I +
OH-N-(CH3)3
CO-NH,
i
CH^.CHaCOH)
N-CH3 I C=NH
I NH2
NH2
1 C=NH
NH2
Sharpe's results are seen in Fig. 386, from which it is clear that the total choline content of the hen's egg diminishes from 400 mgm. per cent, on the ist day of development to 220 mgm. per cent, at the 2 ist. This decrease, argued Sharpe, meant a loss of total choline of °° 130 mgm.; and Burns found a rise in the amount of total guanidine per egg of from 80 to 260 mgm., i.e. an increase of 180 mgm. Although, on the basis of the transformation pictured above, igm. of chohne should yield 2 gm. of guanidine, yet the correspondence was sufficient to justify Sharpe's remark that in all probability choline was here the precursor of guanidine.
The shape of Sharpe's descending total choline curve cannot be emphasised, as it is constructed from so few points,
Choline
• Total choline (Sharpe) O Free '> (Sharpe) O Free (OKada)
Days-*-5
Fig. 386.
but it may be remarked that it begins to fall much earlier than the lipoid phosphorus, either in the experiments of Plimmer & Scott or in those of Masai & Fukutomi. Is there here a possibility that the choline portion of the lecithin molecule may be detached before the phosphoric acid, so that the latter would remain ether-soluble?
The subject of choline in the egg was carried a stage further by Okada, who estimated the free choline only during development.
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR
1217
Nakamura • Total choline in whole egg
► » r> r> yolk
'ro " " » embryo
Sharpe had already published two estimations of this in his preliminary note, but Okada's curve amplified them, and demonstrated that, of the choline which is found in the G,gg at the end of incubation, more than two-thirds is uncombined in lecithin. The almost complete disappearance of the combined choline, seen in the work of Sharpe and of Okada, is a striking sidelight on the utilisation of the lipoids of the Ggg by the chick. It has since been confirmed by Fukutomi, but his figures are unfortunately not published. From the data of Sharpe and Okada, the combined choline can be calculated; this corresponds to 386 mgm. per cent, at o day, and 65 mgm. per cent, just before hatching. As there are about 187-8 mgm. per cent, of total phosphorus at the beginning and 62 per cent, of this is lecithin phosphorus (Plimmer & Scott), there must be 115-2 per cent, of lecithin phosphorus, i.e. 2880 mgm. of lecithin, and therefore 390 mgm. per cent, of choline — almost exactly what was obtained in Sharpe's direct estimation.
Unfortunately, the more recent investigations of Nakamura have not supported this apparently straightforward story, for, using a simple gravimetric method, in which
Days-* 5
Fig. 387.
the melting-point and other properties of the crystals were verifiable, he obtained results in some opposition to those already described. Fig. 387 shows them. The total choline in the whole Q.gg, according to him, rises by 10 or 15 per cent, from the initial value to a maximum of just over 90 mgm. per egg at the 9th day, after which it rapidly falls oflf, reaching what seems to be a steady level about the i8th day (of about 25 mgm. per c^gg). This contrasts with the Sharpe-Okada-Fukutomi results, according to which the whole egg contains some 200 mgm. of total choline before incubation, and some 1 10 mgm.. at the end of it. While in both cases there is a large fall according to Nakamura the
I2i8 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
free choline never reaches more than 3 mgm. in the whole egg by the end of development, whereas according to Okada it reaches at least 75 mgm., and according to Sharpe at least 15. It is difficult to see to what these large discrepancies can be due, but it is probable that the colorimetric method used by the other observers may be less satisfactory than the gravimetric one used by Nakamura. If, however, we are to accept Nakamura's figures as the best, we have to take seriously the initial rise which he found in the total choline, and neither he nor anyone else has been able to suggest anything to explain it. The egg-white and the amniotic and allantoic ^eoo liquids were worked up for .J , choline by Nakamura, but he -g never succeeded in finding 2 any there. Fig. 388, which ^ gives his results in per cent. ^' wet weight of the yolk and the f embryo, seems to show that, whereas the free choline of the yolk has large fluctuations, ^ that of the embryo remains at J a constant figure, and that the -g total choline declines in the S yolk and rises in the embryo. !!^ Nakamura made no specula- g tions as to the fate of the 65 f mgm. of combined choline lost from the egg during its development, except to suggest that it might provide the creatinine of the embryo. We have seen, however, in Section i o that the highest estimate of the creatine or total creatinine content of the embryo by the time of hatching is 25 mgm., so here also there is some discrepancy.
12-4. The Metabolism of Sterols during Avian Development We can now pass to the cholesterol metabolism of the egg. This substance was prepared from the yolk by Lecanu in 1829, and studied by Gobley in 1846 (see Plate XII). In 191 2 Hanes, while working with a chick embryo of 19 days' incubation, was attracted by the
-'^'^ Nakamura
-.0^^^* .
_/
-30 , ^~"^-^^^
^
-20 -"'0
-"r-*
-10 . , . . 1 . . , , 1 , , , . 1
, , . 1
Days -* 5 10 15
20
O Embryo • Yolk
/
/
/ o
\ /
/u
1 1 1 I'l 1 1 1 1 1 1 < 1 , 1
f 1 r 1 ,
Days -^ 5
Fig.
PLATE XII
YOLK OF HEN'S EGG AT THE SECOND DAY OF INCUBATION
Microphotographed by Dr V. Marza through crossed Nicol prisms (31°). The doubly-refracting esters of cholesterol are seen to be localised at the periphery of the vitelline globules. Magnification 8 x A.
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR
brilliant yellow colour of the liver, and, teasing some of it out, examined it between the crossed Nicol prisms of a polarising microscope. The result was a very large number of droplets showing double refractivity. On adding Sudan III, Hanes observed that the droplets stained a deep yellowish red, from which he concluded that the chick's liver contains a great deal of cholesterol esters towards the end of incubation. This led him to make a histochemical study of the liver throughout the incubation period. As the liver grows, he said, it assumes more and more the colour of the yolk. The liver cells of the 6th-day chick contain numerous small fatty globules, but under the polarising microscope these are isotropic. The globules gradually increase in size and number, and about the 14th day of incubation many of them begin to show double refraction. By the time of hatching the liver is very rich indeed in fat droplets, recalling Imrie & Graham's work on guinea-pig livers. Hanes then availed himself of Kawamura's critical study of the differential histochemical methods for identifying the various fatty substances, and applied them to the chick livers. His results were as follows :
Nicol prisms ...
Sudan III
Nile blue sulphate
Smith's stain ...
Ciaccio's stain
Fischler's stain
Neutral red ...
Salkovski's test for cholesterol
Liver from 6th to 14th day
Isotropic Yellowish red Faintly blue Dark bluish black Positive Negative Negative Faintly positive
Liver from 14th to 2 1st day
Anisotropic
Yellowish red
Uncoloured or faintly pink
Faintly bluish grey or uncoloured
Negative
Negative
Negative
Strongly positive
He concluded from these facts that the fat present in the chick liver up to the 14th day of incubation is not pure neutral fat, nor at that time are any soaps or free fatty acids present. This agrees exactly with Mayer & Schaeffer's ratio mentioned above (p. 12 12). Moreover, Smith's stain, which does not colour neutral fats or cholesterol esters, and does colour lecithin-like substances, is very positive in the early stages. The same remarks apply to Ciaccio's method. As incubation proceeds, the droplets in the liver gradually cease to react with Smith's stain, and by 4 or 5 days after hatching this method will not colour them at all. Ciaccio's stain is likewise negative during the last week of incubation, and after hatching. But it is precisely during this time that they give the reactions characteristic of cholesterol esters. Thus, if warmed to about 40°, they lose their
1220 METABOLISM OF LIPOIDS, STEROLS, [pt. m
doubly refracting property, and upon cooling they again become anisotropic. Cholesterol esters are remarkably resistant to autolytic change, and this was found by Hanes to be a property of the droplets in the liver also. They retained their anisotropism for 10 days when autolysed at 37°, and at last changed to long needlelike crystals, which showed the reversible anisotropism on heating. Hanes concluded that during development esters of cholesterol appeared in the chick's liver and the lipoids disappeared. He correlated these changes with ossification and the arrival of calcium for deposition in the bones as calcium phosphate, drawing attention to the fact that if, as Plimmer & Scott had demonstrated, the lecithin of the yolk was broken down to provide the inorganic phosphorus, something must happen to the fatty acids of the lipoid molecule. The lecithin, said Hanes, must be absorbed gradually by the vitelline vessels, and carried to the liver, together with neutral fat, cholesterol, vitelHn, etc. The lecithins then breaking down to yield glycerophosphoric acid, the latter or some closely related substrate for Robison's bone enzyme travels to the bones, and the phosphorus being liberated, is deposited as calcium phosphate. Meanwhile, the two fatty acid molecules left by the decomposition of the lecithin would esterify some or all of the cholesterol which had also been absorbed by the vitelline vessels. The only weak point about this scheme was that, if cholesterol was going to be esterified in the liver, why should it ever have been free, in view of the great amounts of fatty acids everywhere in the egg? However, no doubt Hanes had in mind some special activation of the cholesterol molecule which could take place only in the liver. He found that the cholesterol ester droplets disappeared from the liver after hatching, though a fortnight later they were still abundant, and they were not present in the liver of the adult hen.
Hanes also examined the fat droplets of the yolk-sac. Here he found a good many with the property of double refraction, but they had other characteristics which sharply distinguished them from the anisotropic droplets of the liver. Thus they did not lose the property when heated to 90°, they stained with neutral red, and exhibited myelin forms upon the addition of water. Nile blue sulphate stained them blue. They behaved in every way, in fact, like kephalin or sphingomyelin. However, at the time of hatching, the yolk-sac, now within the body of the chick, showed fatty droplets
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR 1221
which gave all the reactions of cholesterol esters. Hanes suggested that finally the yolk-sac, Hke the Hver, takes part in the decomposition of the lecithin molecule. Finally, he recalled that Windaus had reported the presence of a great deal of esterified cholesterol in areas of pathological calcification, such as atherosclerotic aortas. He mentioned that in examining the livers of foetal dogs and of new-born puppies he had found a large quantity of anisotropic droplets. On the other hand, he failed to find any in the liver of the foetal pig (stage of development not stated) .
Apparently Hanes was unaware of some work of Valentin published as early as 1871. Valentin investigated the doubly refracting droplets in chick embryos, and stated that none of the tissues of the 3rd, 4th, 5th or 6th day embryos showed them, but that on the 7th day a very few appeared. He obtained comparable results in a study of the embryonic liver of the frog. Chalatov also had reported anisotropic globules in liver cells of rabbits fed with egg-yolk for 5 months. After Hanes' report, work was continued on much the same lines by Yamaguchi, who examined the livers of the foetuses of man, the rabbit, dog, guinea-pig, bat, toad, snake and salmon. In the two latter cases no cholesterol esters ever appeared as in all the others. In the human foetal liver, the esters appeared about the end of the ist month, and increased in amount — as far as histochemical work could show — until the beginning of the 5th month, after which they disappeared.
Chemical estimations of free and combined cholesterol in the hen's egg during development have, generally speaking, confirmed the views of Hanes, and indeed widely extended them. Its absorption from the yolk was studied by Idzumi, who simply estimated the amount of unsaponifiable substance in the petrol-ether extract. It is impossible to decide what his data mean, for he obtained much more total unsaponifiable substance when he estimated the embryo and the remainder separately than when he estimated the whole egg. The work of Mueller was on a different level, but, before considering it, it will be convenient to discuss the question of whether cholesterol is synthesised at all by the developing chick embryo. To settle this it was only necessary to estimate the total cholesterol present at the beginning of development, and to compare it with the total cholesterol present at the end. The first workers to do this were Ellis & Gardner in 1908. They pounded up the eggs and embryos in mortars with plaster of paris, and allowed the mass to set, after which they powdered it, and
1222 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
extracted it for 12 days with ether in a Soxhlet. The cholesterol in the unsaponifiable fraction was estimated gravimetrically as cholesterol benzoate. They found that no increase took place during development, the cholesterol accounting for 382-7 mgm. per cent, of the wet weight of the egg, and for 369-3 mgm. per cent, of the wet weight of the hatched chicks, or 317-2 mgm. per cent, expressed in terms of the original weight of the eggs. At first sight it would seem as if there had occurred a loss of cholesterol from the egg, but Ellis & Gardner pointed out that the difference between the average percentage in eggs and in chicks, i.e. 66 mgm., was of much the same order of magnitude as the average deviation from the mean in the two cases, i.e. 57 mgm. for the eggs and 75 mgm. for the chicks. So, as they only analysed 8 eggs and 8 chicks, and as they were not very confident in their method, they preferred to conclude that in all probability neither loss nor gain took place during the incubation of the egg. In another experiment in which 6 eggs and 6 chicks were analysed together, 489 mgm. per cent, were obtained in the former case, and 467 mgm. per cent, in the latter.
This result substantiated the view held in all their investigations by Ellis & Gardner, namely, that cholesterol cannot be synthesised by the animal body. However, Channon and Dam demonstrated some years later that the reverse proposition is true for the chick after hatching, and it was not long before Thannhauser & Schaber, using Windaus' method, reported an increase of total cholesterol during the incubation period. Their data were as follows:
Days
Change
Thus the free cholesterol decreased by 26 per cent, of the initial value, the combined cholesterol increased by 128 per cent, of the initial value, and the total cholesterol, thus partially compensated, increased by 10-7 per cent, of its initial value. We see here clearly that production of cholesterol esters observed histochemically by Hanes. Roffo & Azaretti, the next workers on the sterols of the egg, found no marked change in the cholesterol content of the whole egg before and after incubation, there being 219 mgm. per egg at
Free cholesterol
Cholesterol esters
Total cholesterol
Milli- % dry
grams weight
173 1-316
128 1-065
-45 -0-251
Milli- % dry
grams weight
54-2 0-417
123-5 1-027 + 69-3 +0-61
Milligrams 227-2
251-5 + 24-3
% .dry weight 1-717 2-09 + 0-35
SECT. 12] GYGLOSES, PHOSPHORUS, SULPHUR 1223
the beginning of development (i-86 per cent, of the dry weight) and 282 mgm. per egg at 18 days. The admitted positive balance was probably not significant. The later work of Dam was designed to answer this question. In one of her experiments, the cholesterol per egg was 245-0 mgm., and that of the 2ist-day embryos 263-5 mgm.; in another series, that of the undeveloped eggs was 310-0 mgm. and of the hatching chick 343-0 mgm. In the former case the increase
■ Total cholesterol (Parke) ♦ " " (Mendel ^Leavenworth)
^ -W » " (Roffo fie Azaretfi)
A " " (Dam)
4- » » (Cahn)
200
▼ Total cholesterol |
vFree « / Kusui
^Cholesterol estersj
• Total cholesterol ^
O Free " yMueller
® Cholesterol esters]
on the initial value was 7-6 per cent, and in the latter case 10-6 per cent., and these amounts, though not considerable, were of the same order as those found by Thannhauser & Schaber, and were invariably found by Dam. In a subsequent paper, however, she gave further figures which showed no increase.
Analogous results to these were obtained by Mueller in 1915, who investigated the free and combined cholesterol content of the whole egg throughout incubation. From his figures, which are plotted in Fig. 389, it can be seen that the total cholesterol in the egg remains at a steady level throughout incubation. But the most striking thing about the graph is the way in which the free cholesterol diminishes up to hatching, and the cholesterol esters increase. Thus on the 3rd day of development, the free cholesterol makes up 89-9 per cent, of the total cholesterol, but on the 21st only 58-68 per cent. Nothing could fit in more strikingly with the results of Hanes. As for the question of a synthesis of cholesterol, Mueller's data do not show any such process to have taken place, but an increase of only 1 1 per cent, might, of course, be masked in any but the most careful researches specially designed to test the point. The significance of the fall in total cholesterol found by Mueller after hatching is not evident, but the fact that the cholesterol esters diminish then is in admirable agreement with Hanes' work. Mueller suggested that the bile acids might be formed from cholesterol, and this would explain Ellis & Gardner's decrease, but the process could hardly be operative on the basis of his own results and those of Thannhauser & Schaber. Mueller, in order to carry further the suggestions of Hanes, investigated some embryonic ^ livers separately, and reported '■ that by no means all the cholesterol esters were contained
(bWet weight (Cahn) Cholesberolj* Dry weight (Cahn) (♦Dry weight(Roffo&i Azaretti)
Fig- 390.
in the liver, nor was the cholesterol of the liver all in the combined form. Five 20th-day livers were analysed together, yielding 17-9 mgm. of free cholesterol and 51-6 mgm. of combined cholesterol, while in the combined bodies of the 5 embryos, plus the yolk-sac, still partially unabsorbed, there was about 300 mgm. of combined cholesterol. Mueller largely agreed with Hanes' theory, and went so far as to speak of a detoxication of the lipoid fatty acids by combination with the cholesterol (why should the released fat be toxic?), but he calculated that a gram of lecithin would produce much more free fatty acid than could be esterified by 100 mgm. of cholesterol. It is not likely that the explanation of the whole process lies in a "detoxication", although there is evidence, such as the
Kusui
oFree | whole
® Esters > Q^q
Total) ^^
experiments of Robertson, that a definite cholesterol-lecithin balance is necessary for the normal functioning of the systems in the living organism. This notion is akin to that contained in Mayer & Schaeffer's cholesterol/lipoid phosphorus ratio, which will presently be discussed. Mueller's findings were later fully confirmed by Dam who found 1 2 per cent, of the cholesterol to be esterified at the beginning of development, but 45 per cent, at the time of hatching, and by Kusui, who could observe no effect of injected adrenalin on the percentage of esters.
Estimations of the cholesterol in the whole egg were also made by Parke and by Mendel & Leavenworth — their results are included in Fig. 389. It was not until 1926 that estimations were made of the cholesterol in the embryonic body, namely by Roffo & Azaretti, using the method of Windaus. Two years later another set of data was obtained by Cahn (see Fig. 379) . The two investigations agree well enough, and the rise is continual, following the growth of the embryo. When related to wet weight, however, Cahn observed a rise, falling off in speed with age, and when related to dry weight, a rise to the mid-point of development, followed by a fall (Fig. 390). Roffo & Azaretti's data show this too. We thus find that 100 gm. of dry weight of embryo contain more cholesterol on the i ith day of development than at any other time. Cahn next calculated the daily increments, and found that they fell on a curve having a peak about the i8th day of development. This curve is plotted in Fig. 382 beside that for daily increments of lipoid phosphorus. Cahn's curve for percentage growth-rate of cholesterol is shown in Fig. 364.
More recently Kusui has reinvestigated the cholesterol metabolism of the hen's egg, and has made a step forward by showing that except
Fig- 391
for stray traces, it is confined to the yolk and the embryo, i.e. none appears in the white, the amniotic, or the allantoic liquid. His figures for total cholesterol in the whole egg, graphically reproduced in Fig. 391, show a diminution followed by a rise, but the dimensions of the change are not sufficient to warrant a belief in a decomposition followed by a synthesis. They come within the zone of other workers' results, shown in Fig. 389. Apart from this, Kusui confirmed the earlier discovery of a decrease of free and an increase of combined cholesterol. It would seem that the increase of cholesterol esters is not confined to the embryonic body but also takes place in the yolk.
According to Dam about i-6 mgm. of cholesterol in each yolk is attached in some way to the proteins and cannot be removed by ether extraction unless they are first hydrolysed. No oxycholesterol exists in eggs or embryos.
12-5. The Relation between Lipoids and Sterols: the Lipocytic Coefficients
We are now in a position to consider the "lipocytic coefficients" of Mayer & Schaeffer. The first is:
Total cholesterol Total fatty acids
This was established by Mayer & Schaeffer for the following animals and tissues:
Electric
organ Lung Kidney Liver Muscle
Dog — 20 ID 7 —
Rabbit — 17 13 8 2
Guinea-pig ... ... — 15 8 6 7
Pigeon — 24 98 2
Eel — II 74 2
Torpedo ... ... 22 12 — I i
Thus as a general rule the lung has much more cholesterol in relation to its total fatty acids than the muscle. Mayer & Schaeffer then tabulated the water-content of a number of tissues of different animals, and made the interesting discovery that a parallelism existed between water-content and lipocytic coefficient. Wherever the latter was high, so was the former, and vice versa. This is illustrated by the following table:
gm. water associated with lOO gm. dry weight
1227
Electric
^'
organ
Lung
Kidney
Liver
Muscle
Brair
Dog
—
352
315
236
281
Rabbit
—
408
340
278
335
399
Guinea-pig ..
—
387
416
278
347
Rat
—
—
—
—
—
399
Pigeon
—
310
306
241
241
Torpedo
I112
—
70
445
—
The correspondences are by no means exact, and doubtless often obscured by the existence of other factors, but a general relationship seems clear. In a later paper, Mayer & Schaeffer studied the imbibition properties of tissues, and were finally able to reduce their data to the following expression:
Maximum water retained by i gm. dry weight of tissue
Total fatty acid
Total cholesterol
K.
The constant was always about 60, and Mayer & Schaeffer compared
this generalisation to Boyle's
Law, but for the details re- [ '"^.Tj^^'
' - on Sth.day
ference must be made to their original papers. It is now possible to calculate the lipocytic coefficient for the chick through its development, and it is shown in Fig. 392. The earlier values are far higher than anything found in the adult, but by the end of development adult values are reached, as is shown by the horizontal fines drawn to represent the lipocytic coefficient in the organs of the full-grown pigeon. Now according to Mayer & Schaeflfer's generalisation, the water-content of the embryo ought to be very high in the earlier stages, and should
Days
Fig. 392.
descend to nearly an adult level at hatching; and this, of course,
is exactly what so many investigators have found. It is instructive to compare this curve for the lipocytic quotient with the standard curve for water-content of the embryo shown in Fig. 220, for not only the general trend, but even the forms of the two curves are the same, both falling rapidly until the 1 7th day, and then coming to an almost steady state. The conclusion that the two processes are related can hardly be avoided, and it ^^^^^
must be admitted that, as .lo'r?'^ Cahn
far as the embryo of the chick is concerned, Mayer & Schaeffer's generalisation is strongly supported. If the subject were not experimentally rather difficult, it would be interesting to determine the maximum imbibition of water of which the embryonic body is capable each day, and to find out whether it would obey the equation of Mayer & Schaeffer. This has been done in the case of the lung of the foetal sheep by Faure-Fremiet & Dragoiu (see p. 1573) The other coefficient which they introduced was :
"F^^'^'^oo
Days -> 5
Fig- 393
Lipoid phosphorus Total cholesterol
X 100.
It ran as follows in adult tissues:
Whole Lung Kidney Liver Muscle body Investigator
Dog ... ... 4 2 I I - Mayer & Schaeffer
Rabbit ...4321- jj >?
Guinea-pig ... 3 2 i i - ,, ,,
Pigeon ... 4 2 2 I - >, ,,
Eel - 2 5 3 - „ „
Rat _ _ _ _ 2 Cahn
Cahn calculated this coefficient from his figures for cholesterol and lipoid phosphorus in the embryonic body, and found it to be very constant throughout development, tending perhaps to fall a little as development proceeded. This is shown in Fig. 393.
12-6. Cycloses and Alcohols in Avian Development
As regards ethyl alcohol, which has long been known to be a constant, if minute, component of animal tissues, only one investigation has been made, that of Aoki, who estimated it on the egg as a whole, using Yamakami's modification of Nicloux's method. His results, which are shown in Fig. 394, led to the conclusion that the alcohol-content of the egg rises steadily until the end of incubation, at which time it has reached an adult value, for the adult fowl has 0-0028 vol. per cent, in its blood and 0*0039 ^^lper cent, in its liver. Not the faintest indication is available as to the significance of these results, but Taylor proved in 1 9 1 3 that alcohol can be found in vertebrate tissues under the most aseptic conditions, and is probably due to the reduction of small quantities of acetaldehyde by the cells. It would be interesting to know more about the metabolism of these substances. Kobert reported in 1903 that crushed eggs of Testudo graeca, Sipunculus nudus, and Arbacia equituberculata would form alcohol from added glucose at 37°. The alcohol was identified qualitatively with the iodoform test, but the sterility of these experiments is doubtful.
The other alcohol with which we are concerned is a very diflferent one, namely, the polyhydric cyclose, inositol, or hexahydroxycyclohexane. In 1 908 Rosenberger reported that he had found traces of it in the fresh hen's tgg. It was known that on autolysis the inositol content of tissues rose, and the precursor was called inositogen — it is probably a phosphoric ester of inositol, such as the phytin of the plant — and Rosenberger reported that traces of inositogen also were to be found in the fresh egg. Later in the same year, he reversed his opinion on the former point. Then in 1909 Klein was unable to isolate any free inositol from fresh hen's eggs, but got plenty ("eine reichHche Menge") from the chicks at hatching. A couple of years later Starkenstein isolated 20 mgm. per cent, from the yolk of a fresh egg. The question remained in this state until 1924 when
Fig- 394
Needham applied a new and approximately quantitative estimation method to the egg during its development. The resulting figures (Black Leghorn eggs) are shown in Fig. 395. It will be seen that the total inositol in the egg rises from 7 mgm. per cent, at the beginning to over 60 at the end. The general shape of the curve is doubly peaked, the first occurring about the loth day and the second one being coincident with hatching. Between the two maxima there is a depression in which the total inositol descends to a level not greatly
Fig- 395
above that which it occupied at the beginning. If all the inositol present at the end of development had been in the form of phytin or some similar compound at the beginning, there would be some 62 mgm. per cent, of water-soluble organic phosphorus present at the beginning, while, in point of fact, Plimmer & Scott only found 27 mgm. per cent, of water-soluble organic phosphorus present in the fertilised but unincubated egg. So if phytin were the precursor of inositol in the egg, about three times as much water-soluble organic phosphorus as is actually there would be needed to account for it. Of course, the precursor might be a di- or a tri-phosphate of inositol, instead of a hexaphosphate, but at present there is no reason for supposing that such compounds exist in the body.
In order to get some insight into the precursor of inositol, Needham injected a series of unincubated eggs with various substances, phytic acid, hexamethylenetetramine (to test the hypothesis of Posternak who suggested that inositol might arise from the condensation of six molecules of formaldehyde) and glucose. Tests showed subsequently
Fig. 396.
the presence of formaldehyde in the egg-interior after injection of hexamethylenetetramine, but neither the eggs which had received phytic acid nor those which had received hexamethylenetetramine could be got to develop normally. Doubtless the highest concentration compatible with normal development could have been found by further searching, but, in view of the positive results obtained with glucose, this was not undertaken. For, as Fig. 396 shows, injection of glucose markedly increased the inositol content of the embryo, though not of the rest of the egg. It would therefore seem likely that the synthesis of inositol from glucose only takes place within the embryo. The existence of this synthesis in the developing embryo agreed with the conclusions of Greenwald & Weiss, who studied the composition of the urine in dogs after injection of inositol into the circulation, and with those of Needham, Smith & Winter, who studied the behaviour of the free inositol of the body in insulin convulsions. No information is as yet available concerning the way in which the inositol ring is formed from glucose, although, as is well known, rings can be formed by the body, e.g. kynurenic acid. The hen's egg would be a good material to choose in a study directed towards ascertaining the method of synthesis of the cyclose molecule.
12-7. Sulphur Metabolism of the Avian Egg
The presence of sulphur in the hen's egg is familiar on account of the deposit of ferrous sulphide at the boundary of yolk and white after long boiling (Tinkler & Soar).i The first work which might be included under this heading was that of Liebermann, who estimated the sulphur in the feathers during the last week of incubation, but his method was very questionable, and, as his figures show no definite gradation or constancy, little attention need be given to them. Hopkins' isolation of glutathione in 1921 made it essential to re-open the question of sulphur metabolism in the egg, and he himself observed that in the fresh egg the nitroprusside reaction was quite negative, although the blastoderm and germinal area of a 3 days' embryo gave a brilliantly positive result. This was confirmed by Tunnicliffe. Some years later Murray estimated the glutathione present in the chick embryo at diflferent stages, using Tunnicliffe's method. It was found to be nearly all in the reduced form, and the amounts obtained increased, as was to be expected, with the increase in weight of the whole embryo. When related to wet weight the glutathione rose to a peak on the 13th day of development, and when related to dry weight it fell all the time in a more or less S-shaped curve. This is shown in Fig. 397. Later on, Sagara injected glutaminic acid and taurine into hen's eggs at the beginning of development, and observed a rise in glutathione subsequently as they developed; but this was so slight that little confidence can be placed in his conclusions.
Fig- 397
- And the tarnishing of silver by egg-yolk was known to Pliny: luteo".
Cahn went further into the matter, estimating total, organic, and mineral sulphur in the embryo and the remainder of the egg. His values for glutathione in the embryo did not exactly confirm those of Murray, for the peak on his wet weight curve came a day or two later than the latter's, but nevertheless the two workers are in substantial agreement. A third investigator, Yaoi, has also found the 14th day peak in glutathione per cent, wet weight E ^o and was able to trace it in brain and muscle, as well as in the body as a whole.
Cahn's data for total, organic, and inorganic sulphur are shown in Fig. 398. Apparently the total sulphur in the egg-contents remains at a steady value during development, not receiving any accessions from the shell (though Cahn left this point open). The total sulphur in the embryo rises to about 60 mgm. at hatching, so there is a corresponding fall in the total sulphur of the yolk, the white and the membranes. As the curves for organic sulphur demonstrate, at least 90 per cent, of the sulphur in the embryo and the remainder is in organic form, chiefly, no doubt, combined with the proteins as cystine. In addition to all this, the organic sulphur of the whole egg shows a slow and gradual decline to the extent of some 10 mgm., and there is a corresponding rise in the inorganic sulphur, most of which seems to be in or associated with the embryonic body. Cahn calculated from his figures the following ratio:
which ran as follows :
Total
organic
sulphur
Total nitrogen
Day
Ratio
8
lO
13 15 17 21
8-5 8-8
Apparently therefore, the total organic sulphur in the embryo grows fairly closely parallel with the total nitrogen. This simple fact may involve so many contributory processes that a trite allocation of significance to it is hardly warranted.
Although strictly speaking out of place here, the work of Thompson & Voegtlin on the rat embryo may be mentioned. As it is the only work on the sulphur metabolism of the mammalian embryo, it can best be taken now. Thompson & Voegtlin estimated the amounts of glutathione by the Tunnicliffe method in rats of different ages, obtaining the following results :
Weights Milligrams glutathione
in grams per 100 gm. wet weight Embryos ... ... 0-07- o-8o 60
1-04- 1-97 58
2-32- 2-95 54
^^ -- 3-89- 4-67 44
Newly-born ... ... 4"65- 4-95 36
Nursing 23-00- 26-00 32
Weaned ... ... 30-00- 50-00 31
Adult 137-00-170-00 23
Thus it has been shown both for the chick and for the rat that the concentration of glutathione in the tissues declines with age both during the embryonic and post-embryonic periods.
Targonski, in his work on the allantoic liquid, measured the amount of total sulphur in it. Its amount and concentration, judging from his few analyses, do not seem to be changing in any very definite way, but he calculated the total sulphur/total nitrogen ratio, and found it to be slowly rising, i.e. 0-067 ^^ the 14th day, 0-095 on the 1 6th and 0-105 on the i8th. It was thus moving in the opposite direction from the total phosphorus/total nitrogen ratio. He could only conclude that towards the end of incubation the breakdown of nitrogenous sulphur-containing bodies was more intense than at the beginning. The sulphur/nitrogen ratio is shown on the same graph as the phosphorus /nitrogen ratio in Fig. 385.
Somewhat more extensive studies on the sulphur excretion of the chick embryo were made by Takahashi in 1928, who estimated the sulphates in the developing hen's egg, using Folin's method. In the yolk, white and amniotic liquid these were never found, but they were always present in the embryo itself and in the allantoic f ®T°'^' '"'.P^^^, ^, h U . u u •^ -g O Inorganic sulphate? Takahashi
liquid.
Fig. 399, which sum- S^°[r ©Ethereal sulphate j marises the results obtained by 5 Takahashi, is of much interest. Takahashi was mainly interested in the ethereal sulphates as an index of the capabilities of the embryo for detoxicating substances harmful to it. As Fig. 399 shows, the total sulphates in milligrams per cent, increase considerably in the allantoic liquid from the 9th day onwards, and this increase is shared equally by the inorganic and ethereal sulphates. It is puzzling that Takahashi
Fig- 399 found a good deal of total sulphate on the 9th day in the allantoic liquid, but almost no inorganic sulphate, so that, as the difference was taken to be the ethereal sulphate, there was much more of the latter on the 9th than on the 1 2th day. Takahashi himself seems to have thought that something was wrong with these figures. However, the main outlines of what is happening stand out clearly, for in the embryo itself the total sulphate remains constant, as do its two components, except for the preHminary irregularities which are associated with that just mentioned. Takahashi's conclusion was that ethereal sulphate as a mode of excretion is already in action by half-way through development. But where does the phenol so excreted come from? At that stage there are no bacteria in the gut of the embryo nor would there be much for them to act upon even if they were there.
The problem had already been raised in the case of man. Senator in 1880 had assured himself that indican and other ethereal sulphates were present in the amniotic liquid but not in the meconium. Then Shibayama ascertained in 1927 that foetal blood gives a more intense indican test than maternal blood ^, although bacteriological examination always shows the meconium to be sterile. B. coli does not appear in the intestinal tract until a day or two after birth. The indoxylsulphuric acid must therefore arise in the intermediary metabolism of the foetus, and that its main source always is intermediary metabolism is also the opinion of Kishi who has recently reviewed the question anew.
100
90
80
70
60
50
40
^30
1,0
S..0
<r>
E
E
20
Turtle, Thalassochelys corbicata Kusui •Total ^
OFree y Cholesterol
12-8. Phosphorus, Sulphur, Choline and Cholesterol in Reptile Eggs
Kusui, in his work on the cholesterol metabolism of the developing marine turtle, Thalassochelys corticata, found, as Fig. 400 shows, a diminution of the total cholesterol and a transfer of it from the free to the combined form. This is extremely interesting in view of what all workers have found to take place in the hen's egg (see Fig. 389) and it would seem as if the increase of esters of cholesterol, at the expense of the free substance, was a phenomenon common to many de\'eloping organisms.
Glutathione has also been estimated in these eggs : Tomita found 0-15 mgm. present on the 30th day of development and 0-8 1 mgm. on the 45th.
The phosphorus distribution is interesting, for just as PUmmer & Scott found a production of inorganic at the expense of organic phosphorus in the hen's egg, so Karashima found the same thing in that of the turtle :
^ This was confirmed and placed on a quantitative basis by Hensel.
Days 10 20 30
Takahashi •Total O Free
•
40 Choline
SECT. 12] GYCLOSES, PHOSPHORUS, SULPHUR 1237
Milligrams % P2O5 in egg-contents
Days , ^ ^
development Inorganic Organic Total
o 6 342 348
15 13 280 293
30 15 266 281
45 123 106 229
Hatched 365 52 417
The figures for total phosphorus are insufficiently regular to permit any conclusion as to whether any of it is deri\'ed from the shell. The inorganic phosphorus is practically confined to the body of the embryonic reptile, and the organic phosphorus to the yolk. Only minimal amounts of either are found in the egg-white or in the amniotic or allantoic liquids.
The choline has been estimated by Takahashi (see Fig. 400).
12-9. Lipoids and Sterols in Amphibian Eggs
If we first enquire as to what is known of the phosphorus distribution in amphibian eggs, we find that Plimmer himself made a step in this direction, collaborating with Kaya in 1909. He used the eggs of the frog [Rana temporaria) in two lots: [a) ovarian and {b) shortly after being laid. It is to be supposed that a certain amount of development had taken place. The figures were as follows :
% of the total phosphorus
Ether-soluble phosphorus ... Total water-soluble phosphorus Inorganic phosphorus Total protein phosphorus ... Phosphoprotein phosphorus Nucleoprotein phosphorus
Thus there was every evidence of much the same changes as we have seen take place in the egg of the chick. The ether-soluble phosphorus and the phosphoprotein phosphorus were declining, the nucleoprotein and the water-soluble organic phosphorus were rising. Only the inorganic phosphorus seemed to be maintaining a very low level. This general arrangement fits in with the few later analyses of Parnas & Krasinska, who estimated the lipoid phosphorus only, finding 0-00532 mgm. present in one ^gg and 0-0039 mgm. in one hatched larva : a loss of 26 per cent. This is not so great a fall as in the chick.
(«)
{b)
26-2
20-2
4-3
II-3
o-o
Trace
69-5
68-4
6i-9
41-0
7-6
27-4
but it must be remembered that hatching occurs very early in the frog. Faure-Fremiet & Dragoiu found that 25' 13 per cent, of the total fatty acids were in the form of phosphatides at the beginning and 20-o per cent, at hatching,
loss by % of
I egg (mgm.) initial value Unsaturated acids ... 0-0838 30-0
Myristic acid o-ooig 2-7
Phosphatides ... ... 0-0444 42'0
No determinations of lipoid phosphorus were made by Faure-Fremiet & Dragoiu at the end of the second period, i.e. at the time of disappearance of the yolk-sac. There is general agreement, therefore, that the lipoid phosphorus diminishes during the development of the amphibian egg, but little is known about the behaviour of the other phosphorus fractions.
The cholesterol of the frog's egg has been occasionally investigated. Faure-Fremiet & Dragoiu found the total unsaponifiable fraction of their ether-alcohol extract to amount to 3-13 per cent, of the total solid present, and this, after treatment with digitonin, separated into 1-43 per cent, of cholesterol and 1-7 per cent, of a body which FaureFremiet identified with the "unsaponifiable X" of Kumagawa, probably spinacene or squalene. Thus the dry weight composition of the ether-alcohol extract at zero hour of development was :
%dry weight egg Phosphatides ... ... ... ... 5-98
Neutral fatty acids ... ... ... 14-82
Cholesterol 1-43
" Unsaponifiable X " ... ... ... 1-70
23-93
Unfortunately Faure-Fremiet & Dragoiu seem to have forgotten to make any estimations of cholesterol and other unsaponifiable substances at later stages of development. Parnas & Krasinska made determinations of cholesterol only as follows :
Cholesterol
% of the total Milligram per
fatty acids individual
Egg 13-6 0-056
Hatched larva ... ... 167 0-056
Thus there was apparently no change in the total cholesterol, although in per cent, of the fatty acids it rose, owing to their utilisation. This again would agree with what goes on in the hen's egg. The only other researches which have been carried out on the lipoid and sterol metabolism of the amphibian egg are those which have involved histochemical methods alone (e.g. Yamaguchi, who found cholesterol esters in the liver of the toad at hatching) . Doubtless the most important of these are the memoirs of Konopacki & Konopacka, and of Hibbard, but it is difficult to tell whether what they call "hpoid" is the same thing as the material known to chemists. It is interesting that Rosenheim finds cholesterol prepared from frog's eggs to be richer in ergosterol than similar products from any other source. According to Kamiya, no glutathione is synthesised during the development of the frog's egg.
12-10. Lipoids, Sterols and Cycloses in Fish Eggs
As regards the fishes, we are almost completely ignorant of what happens to the phosphorus distribution during embryonic growth, but some unpublished work of Rosenheim, Girsavicius, Ashford & Stickland indicates that in the fish egg events occur very Hke those in that of the hen and the frog. In 1928 they obtained on the trout, Salmofario, the following figures:
% of the total phosphorus
Ether-soluble phosphorus ... Total water-soluble phosphorus ... Inorganic phosphorus Organic water-soluble phosphorus Nucleoprotein phosphorus...
If these are compared with those previously given for the frog by Plimmer & Kaya and for the hen by Plimmer & Scott and others, the correspondence will be seen to be considerable. It is evident, however, that in these experiments the ichthulin or phosphoprotein of the trout's egg came out into the watery extract. When this is taken into consideration, it will be seen that the fall in lipoid phosphorus, the rise in inorganic phosphorus, and the probable fall in phosphoprotein phosphorus, parallel events in other eggs.
For cholesterol there is a solitary observation of McClendon's that the unsaponifiable substance soluble in petrol ether formed i-2 per cent, of the dry weight of the eggs of the brook- trout, Savelinus fontinalis, and i-6 per cent, of the dry weight of its hatched larvae — an increase of 33 per cent. This fraction crystallised, he says, into a
N E 11 79
Unde
Fry almost at the end
veloped eggs
of the yolk-sac period
26-78
0-0
73-2
92-9
o-o
27-9
73-2
65-0
o-o
7-1
MG. SCYLLITOL OP. INOSITOL
YOLK (BRE^ Mor stated) STA^RKENSrElN [iQHl (£?fR£Cr BSr/MAT/CW)
WHITE
rOLK
WHITE
YOLK
(SREED NOr ST^TEt?) EASICOTT \\Q2^
(BLACK LEGHORN) NEEDHAN RQZ^I (£SriMA,TfON) *- -^ -■
-JiS:y EMBRYO oKa UNUSEP roLK
YOLK
CWHITE LEGHORN) ,^ r ^^1
(^5>r/Af/)770v; NEE-DMAM [192^]
E"MSRYO
YOLK
SCYLLIUM CAMCULA ACANTH I AS VUUaARlS
EMBF^YO
cma UNUSEP YOLK
Fig. 401.
SECT. 12] GYCLOSES, PHOSPHORUS, SULPHUR 1241
solid mass largely composed of cholesterol, but he did not quantitatively separate the cholesterol from the squalene or other unsaponifiable matter.
Nitrogenous bases associated with lipoids have been reported as existing in several kinds offish embryos. Thus Ackermann & Kutscher isolated in 1907 as much as 2-0 per cent, (dry weight) of betaine from embryos of Acanthias vulgaris, and o-oy per cent, from adult fishes. The formula of this substance shows it to be related to choline:
Choline (trimethyl /3-hydroxy Betaine (trimethylglycine) ethyl ammonium hydroxide)
CHa— COO GH^— CH2OH
N 1 N— OH
IgCHs
CH3 CH3 CH3 CH3 CH3 CH3
and it has been believed, though certainly wrongly, that betaine can replace choline in the lecithin molecule. Suwa in 1909 and Kutscher in 19 10 have also obtained betaine from embryonic fish muscle. Later Berlin & Kutscher isolated no less than 1 2 per cent, of betaine (dry weight) from the embryos of Acanthias vulgaris, and 0-5 per cent, of choline, as against 0-7 per cent, of choHne in the adult muscles and 0-3 per cent, in the adult liver. Nobody has any idea as to the significance of these findings.
Considerable interest attaches to the presence of scyllitol in elasmobranch fishes, for this stereoisomer of the /-inositol of animals might or might not be present in the undeveloped eggs. In 1929 Needham examined a number of eggs of Acanthias vulgaris and Scyllium canicula for scyllitol, using a method as quantitative as possible, and found that only traces were present before development, although at hatching a large amount was to be found. The dogfishes, then, have to synthesise their own particular form of cyclose, just as the birds have to synthesise theirs. Fig. 401 gives a survey of the relationships involved, and it will be seen from it that the cases are indeed quite parallel.
1 2-1 1. Phosphorus, Lipoids and Sterols in Arthropod Eggs
For insect development we know next to nothing. Tichomirov's balance-sheet of the silkworm &gg, made in 1882, old as it is, contains the only information available. He found that the eggs of Bombyx mori contained the following percentages of lipoid (estimation-method not given) and cholesterol:
After the diapause At hatching
Lecithin Cholesterol ...
% wet
weight
I-04
0-40
% dry
weight
2-93
I'I2
% wet
weight
1-74
0-35
% dry weight
III
What process explains the rise in lecithin we may well enquire, though at present there is no hope of an answer till more work is done. The cholesterol remains constant, much as in other eggs. Kaneko could not find any cholesterol esters in the silkworm egg by examination in the polarising microscope, but Kitamura and Kamiya affirm that the developing larva, as is the case with the chick, synthesises glutathione :
% wet wt. Freshly laid eggs 0-062
Hatching larvae o-o88
The same remarks apply to the phyllopod crustacean Artemia salina, for Hopkins obtained negative nitroprusside tests on the egg-contents and positive ones on the hatched nauplii.
Needham & Needham investigated the phosphorus-metabolism of this brine-shrimp in 1929, obtaining the following figures:
Ethersoluble P
Watersoluble inorganic
Pyrophosphate P
Stable watersoluble organic P
Phospho
protein
P
- Nucleo protein
P
Total P
0-302 0-564
0-937 0-738
1-64 1-24
0-33 1-23
None None
1-95 1-41
5-i6 5-16
5-9 10-9
18-1 14-3
31-9 24-1
6-4 23-9
o-o 0-0
37-9 27-4
1 00-0
lOO-O
Mgm. per gm. dry weight:
Undeveloped eggs
Hatched nauplii* % of the total phosphorus
Undeveloped eggs
Hatched nauplii*
The egg-shells left behind weigh a considerable amount and were included in the weighings. They contain practically no phosphorus.
It is clear from these figures that no absorption of phosphorus takes place from outside the tgg, and that there is, as noted in Section 10, a sufficiency of nuclein in the beginning for the needs of the embryo. In view of Tichomirov's results on another arthropod egg, it is of much interest to find the lipoid phosphorus rising. These same
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR
1243
authors also worked with another crustacean, the sand-crab, Emerita analoga, obtaining the following results :
Ethersoluble P
Watersoluble inorganic
Pyrophosphate P
Stable watersoluble organic
Phospho protein
P
■ Nucleo protein
P
Total P
VIgm. per gm. dry weight: Undeveloped eggs 3-27 Eye-spots visible 2-94 Embryo larger than 2-14
yolk About to hatch i -83
/o of the total phosphorus : Undeveloped eggs 28-1 Zoea larvae i6-o
2-24 1-59 2-33
1-55 2-70 1-26
3-36 316 3-88
Trace None None
1-26 1-25 I 50
11-62 11-65
II-IO
2-6o 19-3
22-8
2-31
13-3 20-3
3-06
28-9 26-8
None
1-59
10-82 1395
11-40
1 00-0 1000
Here again, then, we have an egg which is independent of its environment as regards phosphorus and one in which only a small synthesis of nucleins takes place. On the other hand, its lipoid phosphorus diminishes considerably.
12-12. Phosphorus, Lipoids and Sterols in Worm and Echinoderm Eggs
Faure-Fremiet's analyses of the undeveloped eggs of the polychaete Sabellaria alveolata showed i-8i per cent, dry weight of cholesterol and 2- 1 6 per cent, dry weight of " unsaponifiable X", but he made no determinations on hatched worms. On the other hand, in his study of the development of the nematode Ascaris megalocephala, he did investigate the distribution of phosphorus before and after the development of the eggs. The phosphorus is here expressed as phosphorus pentoxide.
% weight % of the
(wet
Before 0-90 o-i8 0-23 0-49
or
dry?)
After o-go o-i8 0-28 0-44
total phosphorus
Total phosphorus ... Inorganic phosphorus
Ether-soluble phosphorus
All other kinds of phosphorus * ...
Before After 1 00-0 lOO-O 20-0 20-0
25-6 31-1 54-4 49-0
Called improperly by Faure-Fremiet "nuclein phosphorus". The inorganic phosphorus remains constant, then, the Hpoid phosphorus gains and the rest of the phosphorus loses by some 5 per cent. This state of affairs so contrary to what we find to take place in the hen's egg recalls the similar finding in the case of the silkworm and
1244 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
the brine-shrimp. But it is difficult to picture what may be going on in the egg of the worm to produce these effects. It is, of course, to be remembered that Ascaris is an organism living in very peculiar circumstances^.
Gephyrean eggs were investigated by Needham & Needham, on the Californian echiuroid worm Urechis caupo.
Stable
Water
Pyro
water
Ether
soluble
phos
soluble
Phospho
Nucleo
soluble
inorganic
phate
organic
protein
protein
Total
P
P
P
P
P
P
P
Mgm. per gm. dry weight:
Undeveloped eggs 2-41
1-43
1-71
1-86
Trace
1-39
8-8
Trochospheres 0-32
2-40
0-27
0-I3
Trace
3-68
6-8
% of the total phosphorus :
Undeveloped eggs 27-4
i6-3
19-4
21-2
—
15-8
1000
Trochospheres 4-7
35-3
39
2-0
—
54-1
1000
Here the lipoid phosphorus declines markedly, the total phosphorus receives no accession from outside, and the nuclein phosphorus increases considerably.
When we come to the phosphorus metabolism of the echinoderm embryo, we find ourselves in a region where a good deal of work has been done, yielding at first unsatisfactory and controversial results. Interest in the phosphorus distribution of the echinoderm egg arose in the first instance out of the great increase in nuclear substance during the early stages of echinoderm development, noted by Boveri, Loeb and Godlevski. Thus the ratio of the total volume of the cell to the volume of the nuclear material in the unfertilised egg is 550/1 but in the blastula stage 6/1. There must then, said Loeb, be an increase in absolute and relative amount of nucleoprotein, i.e. in purine bases and nuclein phosphorus, and if the nuclein phosphorus increases, something else must decrease, probably either phosphoprotein or lecithin. Loeb chose the latter substance as being the most likely precursor, and was supported by various investigators, notably Robertson, who wanted the choline from the disintegrating lecithin for his choline surface-tension theory of cell-division. However, the first investigations which were made did not lead to favourable results, for Masing, taking the eggs of Arbacia pustulosa, applied the methods of Plimmer & Scott to unfertilised eggs and morulae. Of the unfertilised eggs
1 The eggs of another parasitic worm (the trematode, Distomum cygnoides) are said by Schmidt to contain considerable amounts of esterified cholesterol.
SECT. 12] CYCLOSES, PHOSPHORUS, SULPHUR 1245
I gm. of dry substance gave 3-53 mgm. of nucleoprotein phosphorus and no phosphoprotein phosphorus. Fertilised but undivided eggs yielded 3-95 mgm. per gm., and embryos at themorula stage 3-80 mgm. per gm., again with no phosphoprotein phosphorus. 100 mgm. of total nitrogen were associated with 3-6 mgm. of nucleoprotein phosphorus in the unfertilised egg, 4-1 mgm. in the fertiUsed but undivided egg, and 4-05 mgm. in the morula. From these data, which agreed well among themselves, Masing concluded that there could be no synthesis of nuclein during the period covered by his experiments. Moreover, as has already been mentioned (p. 1157), he isolated the purine bases from his material, and could not find any increase or decrease.
Shackell's work, which was published not long after Masing's, confirmed it. He estimated the ether-soluble and water-soluble organic phosphorus under much the same conditions as Masing, and observed no change in either. He was also unable to find any increase in the nucleoprotein phosphorus, as determined by the protein residue left after treatment in a standard peptic digestion.
Both these papers were severely criticised by Robertson & Wasteneys in 1 9 1 3, who considered that Masing's material must have been greatly contaminated by the spermatozoa. If this had been the case, his values for the just-fertilised eggs would have been too high, and would perhaps have obscured a real rise in nuclein phosphorus. They also affirmed that the conditions under which his eggs had developed had been inadequate, and that his extraction methods were faulty. Nor did they fail to bring much the same criticisms against Shackell. Later writers, such as LeBreton & Schaeffer, have followed Robertson & Wasteneys in their opinion of the work of Masing and of Shackell, but it must be remembered that Masing published a defence of his methods against the American workers, in which he pointed out that contamination with excess spermatozoa could not explain his results, some of which were derived from unfertilised eggs. Robertson & Wasteneys' own data were very erratic and difficult to interpret as they only estimated alcohol-soluble, water-soluble, and insoluble phosphorus, i.e. mixtures of compounds. They concluded that "the proportion of phosphorus which is present in the form of lecithin, etc., in Strongylocentrotus eggs diminishes progressively as development proceeds". Nothing could be said on the basis of their experiments about the nucleoprotein phosphorus.
1246 METABOLISM OF LIPOIDS, STEROLS, [pt. iii
The subject was evidently ripe for further work. Needham & Needham in 1929, working on the Cahfornian sand-dollar, Dendraster excentricus, and on a starfish, Patiria miniata, estimated the phosphorus micro-colorimetrically in each of Plimmer & Scott's fractions, either after incorporation of the material with anhydrous calcium sulphate, or after extraction with trichloracetic acid. Table 176 summarises their results :
Table 176.
Plaster-of-paris method
WaterEther- soluble Pyrosoluble inorganic phosphate P P P
Dendraster excentricus (mgm. per gm. dry weight) :
Unfertilised eggs 0-422 2-04
Gastrulae 0-77 323
Plutei 0-75 319
% of the total phosphorus:
Unfertilised eggs 6-05
Gastrulae 8-75
Plutei 7-90
Patiria miniata (mgm. per gm. dry weight) : Unfertilised eggs 2-14 1-32
Bipennaria larvae 1-09 i-ii
% of the total phosphorus :
Unfertilised eggs 32-20 19-80 Bipennaria larvae I7*75 18-05
29-4 36-8 33-6
0-950
o-8o
1-53
13-65 9-1 16-1
085 1-07
12-8
17-4
Stable watersoluble organic P
0-27 0-29 1-29
39 3-3 13-6
1-28 1-30
19-2 21-1
Total
water- Phospho- Nucleo soluble protein protein Total P P P P
3-26 4-32 6-01
46-95 49-19 63-30
3-45 3-48
51-8 56-5
0-84 037
0-20
4-2
Trace
None
Trace
None
2-22 3-36 2-54
32-0 38-2
26-7
1-04 1-56
1565 25-40
6-94 8-80 9-50
lOO-O
1000 loo-o
6-64 6-15
lOO-O lOO-O
Trichloracetic acid method
Watersoluble inorganic P
Unstable watersoluble organic p*
Dendraster excentricus (mgm. per gm. dry weight) : Unfertilised eggs 1-645 0-036
Gastrulae 1-274 0-067
Plutei 0-382 0053
Stable watersoluble organic P
0-941 0-665 1-360
Total watersoluble P
2-62 2-02 1-80
Possibly creatine phosphate.
Attention may be first directed to the rise in total phosphorus which the sand-dollar egg exhibits during its development. The figures given in Table 176, which are the sum of the various fractions, were supplemented by direct estimations of total phosphorus, which demonstrated the same phenomenon, for from an average of 760 mgm. per cent, dry weight before fertilisation, it rose to 990 mgm. per cent.
at the gastrula and 1230 at the pluteus stage. This might be regarded as being due to a removal of other dry substances from the egg by combustion, but such an explanation cannot be correct, for as Ephrussi & Rapkine showed, the total dry soHd in echinoderm eggs tends to rise slightly during development owing to the intake of ash from the sea- water. And even if there was a decrease of dry weight it could not, judging from the respiration data given in Section 4-2, amount to more than 5 per cent, of the initial dry weight. Yet the phosphorus increases by 62 per cent, of its initial value.
There is, indeed, nothing against the view that phosphorus is absorbed from the sea-water by certain marine invertebrate eggs, for we know what remarkable accumulations of other elements, such as strontium, can occur in foraminifera. As will be shown, moreover, in Section 13-4, inorganic constituents are absorbed from the seawater by many marine eggs. If now we ask how the absorption of phosphorus takes place we come upon several points which merit consideration. It is usually surmised that echinoderm eggs hatch so early and the larvae swim about so vigorously owing to the need for wide dispersal of the species in the plankton, but there may also be a chemical reason for this, the egg not being supplied with all that it needs for its proper development and being therefore under the necessity of going to fetch it. An analogous instance among aquatic vertebrates might be found in those teleostean eggs which begin to rotate within their envelopes at a very early stage, setting up intraovular currents and more readily absorbing the water which they require. In order to gain some idea of the speed of movement of echinoderm embryos Needham & Needham measured the time taken for them to traverse a course on a micrometer scale, with the following results :
Metres per hour
Dendraster
Blastulae i -39
jj
Plutei 1-71
Arbacia
Gastrulae i • 1 7
Asterias
Gastrulae i -83
Assessing thus the average speed ^ at 1-5 metres per hour we may consider an echinoderm gastrula passing for one hour through a tube of sea-water of its own diameter. As this is 0-196 mm. such a tube would contain 4-515 c.c. of sea-water, and in this there would be 1-56 X io~*mgm. of phosphorus, if we take the phosphorus-content
^ See also Runnstrom.
of sea-water to be, for purposes of calculation, 0-034 mgm, per litre. Now during development we find in round numbers an increase of from 7-5 to 12-5, i.e. 5-0 mgm. per gm. dry weight of eggs, and this, reckoning about 4,000,000 eggs to the gram, would be 0-012 x io~* gm. Comparing this estimate with that for the amount of phosphorus in the sea-water, it will be evident that the requirements of the embryo will be amply met by the water with which it comes in contact. In addition to this, turbulence effects must be remembered, and it is probable that under favourable conditions the embryo could dispense with a great deal of its activity as far as the phosphorusintake is concerned. But if it floated motionless in a perfectly still medium containing phosphorus in as high concentration as sea-water, it is likely that the supply would be deficient.
These facts have a bearing on the problem of the origin of freshwater fauna. The classical theory of Sollas attributes the paucity of fresh-water species mainly to the custom, usual among marine invertebrates, of hatching early in a larval form and dispersing in the plankton. These minute ciliated larvae, he pointed out, cannot swim against currents of any magnitude and are ill-adapted for river life, so that even if a marine animal solved the osmotic difficulties associated with existence in fresh-water it would see its eggs and larvae swept out to sea again as fast as it produced them. The well-known phenomena of poecilogony (see p. 316) favour this view. But such a theory has no explanation to give for the cases (e.g. the cephalopods) where the embryo spends a long time in its egg before hatching out substantially mature, and yet the group has never penetrated into fresh- water (see p. 317). Now if eggs such as these depend on the salts of the sea-water as a kind of additional yolk, the inorganic environment may become an important limiting factor, and in the case of phosphorus in particular, the margin between supply and demand in normal sea-water may be much reduced. Thus the plankton may use up nearly all the available phosphorus, for as Atkins has shown, there is a seasonal variation in the English Channel, surface water containing 0-0162 mgm. P per litre as a winter maximum and only 0-0032 as the summer minimum, the difference being associated with the growth of the phytoplankton. Corresponding figures for the Clyde area, given by Marshall & Orr, are 0-032 and 0-0022 mgm. P per litre respectively. In the Pacific Ocean the same cycle goes on, according to Stanford and Moberg. Finally, the analyses of Juday,
Birge, Kemmerer & Robinson show that for Wisconsin lake waters, while the dissolved phosphorus may occasionally rise to a typical marine level such as 0-015 mgm. P per litre, it may often be completely absent, and in most cases amounts to about 0-003 mgm. P per litre. Miiller again finds hardly detectable traces in the water of Lake Balaton in Hungary. Thus the phosphorus requirements of developing marine eggs may be an important factor prohibiting their colonisation of lake and river waters.
Phosphorus entering an echinoderm egg from sea-water must do so in the form of orthophosphate. Bertolo, using Pollacci's histochemical reaction (which is said to give a blue coloration with all phosphorus compounds, but which in fact only does so with inorganic phosphate) , found with Strongplocentrotus and Sphaerechinus eggs that a blue zone was produced round the periphery. If this observation has any physiological meaning, it must be that the concentration of inorganic phosphate is greatest at the periphery, as would be expected if it were being absorbed through the surface and then turned into something else. Herbst, in his work on the development of echinoderms in artificial saline environments, at first stated (see p. 1273) that phosphorus in the form of calcium phosphate was necessary for normal differentiation and assumed that it was absorbed by the eggs. In a later paper, however, he reported that perfectly normal development would go on in the complete absence of phosphorus and attributed his previous results to the precipitation, and hence the detoxication, of traces of copper in his distilled water. Loeb about the same time also reported that the eggs required no phosphorus from sea-water. Both these workers, however, relied on tests of inferior delicacy, and it is probable that under the conditions used by them, ample phosphorus was available for the developing eggs.
Returning now to Table 1 76, it can be seen that two methods were used, the first of which (plaster of paris) included the phosphorus of the spicules in the plutei, while the second (trichloracetic acid) did not. Accordingly they give different results from which it can be seen that the phosphorus of the spicules is quite appreciable in amount, being in fact about equal to the total phosphorus taken in from the environment, and almost all in inorganic form. Prenant regards echinoderm pluteus spicules as composed of calcite, and this is especially relevant since calcite originates fi-om an amorphous form of calcium carbonate which tends to be stabilised in the presence of phosphate when the P2O5/CO2 ratio is o-i. Biitschh, moreover, reports that adult echinoderm spicules contain phosphate.
The distribution of phosphorus in the Dendraster egg is, it may be noted, widely different from that in the hen's egg. Instead of the great stores of lipoid and phosphoprotein phosphorus, there is only a small proportion of these bodies, while a prominent place is taken by the various forms of water-soluble phosphorus and nuclein phosphorus. The phospho-protein phosphorus falls during development, perhaps indicating that it is playing a similar part in both cases. The figures for the starfish egg in Table 1 76 show no absorption of phosphorus from the sea-water, so that the practice cannot be universal among echinoderms, but both organisms provide almost enough nuclein phosphorus in their eggs for the requirements of the embryo. This fact has already been considered in Section 10. The constancy in lipoid phosphorus in Dendraster and the fall in Patiria may perhaps be related to the fact that the initial amount is small in the former case and large in the latter. Many instances of this have been given in this section, and we mav summarise them as follows :
Lipoid phosphorus
Loss of
lipoid
Mgm. per
%of
phosphorus
gm. dry
the total
in % of the
weight
phosphorus
initial value
Dendraster (Needham & Needham)
0-42
609
00
Patiria ,, „
2-14
32-2
m
Urechis ,, ,,
2-41
28- 1
Emerita ,, ,,
3.27
44-0
Artemia ,, ,,
030
59
00
Salmo (Rosenheim, Girsavicius, etc.)
27-0
goo
Gallus (Plimmer & Scott)
576
648
70-4
This probably indicates two main appropriations of lipoid [a] a fundamental quota which no egg can do without, and which is built up into the cell-membranes and other structures of the finished embryo without change, and {b) a further store, which in terrestrial eggs may be very great, which is broken down during development yielding phosphorus in a form available for calcification or other uses.
It is interesting to calculate the contribution of phosphorus made to one echinoderm e.gg by one spermatozoon. In the case of Dendraster we found i gm. dry weight of egg to contain 8 mgm. of total and 2 mgm. of nucleoprotein phosphorus, and this value may be accepted as being of the same order in various echinoderms. According to Page one million Arbacia eggs weigh when dried 0-124 gm., so that I gm. dry weight would contain 8,100,000 of these eggs, or 4,000,000 of those of the sand-dollar. Egg-size is variable :
but may be averaged at 75/z, and spermatozoon size is fairly constant, say 2/x, leaving the tail out of account. The cubic contents would therefore be, of the egg 1 13,000 cubic /x and of the sperm 4-18 cubic fx. One egg contains, therefore, 0-99 x lO"^ mgm. of total and 0-25 X io~® of nucleoprotein phosphorus. Calculation from the relati\ e volumes of egg and spermatozoon, assuming the same watercontent, gives 218,000,000,000 sperms contained in i gm. dry weight of sperm. The best values for phosphorus-content of echinoderm spermatozoa are still those of Matthews, who obtained 28-6 mgm. phosphorus per gm. dry weight in the case oi Strongylocentrotus. The total phosphorus in one spermatozoon would thus be 0-00013 x io~^ and the nucleoprotein phosphorus could hardly be more than 85 per cent, of this. So the total phosphorus brought in by the spermatozoon which fertilises the egg is about one ten-thousandth part of the total phosphorus which is there already, a computation which agrees roughly with the fact that one spermatozoon is about one thirtythousandth part of an egg in size.
Little is known from a dynamic point of view about the sterols of the echinoderm egg, although, as has been stated in Section i , there is a certain amount of information about the sterols as components of the unfertilised ovum. But Ephrussi & Rapkine in 1928 obtained the following figures for the total unsaponifiable fraction of developing Strongplocentrotus eggs :
Hours from % dry % of the total
fertilisation weight ether extract
o 3'3 15-7
12 (blastulae) 3-0 i6-i
40 (plutei) 2-7 15-6
From this it would seem that the total unsaponifiable fraction always bears a constant relation to the total fatty acids, and as they decline per lOO gm. dry weight, so does the former. It was made up as follows :
" Unsaponifiable X and Y" Cholesterol
Hours from f ■ — ■ — ^ , ^ ^
fertilisation % dry weight % ether extract % dry weight % ether extract o 1-5 7-0 1-8 8-7
12-13. Lipoids and Sterols in Mammalian Development
A certain number of quantitative determinations of lipoid phosphorus in individual tissues have been made, but these will be reserved for consideration in Section 23. Baumann & Holly found 560 mgm. per cent, phosphatides in a 20th-day rabbit embryo and 650 in a 29th-day one, also corresponding values of 240 and 230 mgm. per cent, for cholesterol. Vignes found 205 mgm. per cent, in whole rats (maternal) and 654 mgm. per cent, in their embryos. If we pass to nucleoprotein phosphorus we meet immediately with Masing's work. In 191 1 this investigator estimated the nucleoprotein phosphorus in rabbit embryos at different stages of development, but as he used no accurate time scale it is impossible to plot his data. All that can be done is to reproduce the table in which he summarised his results :
Table 177.
Embryos \X.o\\ cm. long, from the ist half of gestation
Embryos 21*5 gm. weight, about 4th week
Embryos a little older than the last lot
Embryos i or 2 days before birth
Fully developed embryos
Newly born rabbits
Rabbits 1 1 days after birth
Liver from beginning of 4th week
Liver from a little later stage ...
Livers from embryos i to 2 days before birth
Livers of new-born rabbits
Livers of rabbits 1 1 days old ...
Livers of rabbits 22 days old ...
Adult livers
Nucleoprotein phosphorus
associated with 350 mgm.
of total nitrogen
20-3
17-4
14-7
13-0
I2*0
II-7 1 1-9
2i2-8 20'4
180
I7-0 i6-o
I2-0
IO-7
The nucleoprotein phosphorus thus decreases relatively to the total nitrogen (i.e. approximately to the dry weight), and correspondingly the nucleoplasmatic raUo decreases. This is in agreement with what has already been said in the Section on purine metaboHsm.
This is all that we know about the phosphorus metabohsm of the mammalian embryo, if we except, firstly, the researches which have been done on the distribution of phosphorus compounds in the maternal and foetal blood, and which will more conveniently be reviewed in the Section on placental permeabihty; and, secondly, the suggestion made by Parat that the meconium is not a waste product, but a "veritable embryo trophe". Basing his conclusions entirely on histological evidence, Parat decided that the methods ordinarily used in histology "surprennent la cellule intestinale du foetus humain de 3 a 8 mois en plein travail d'absorption". In other words, the meconium was to be regarded as one of the means of foetal nutrition, though Parat did not explain why the foetus should secrete food for itself. Parat & Delaville subsequently made a few fragmentary analyses of the organic and inorganic phosphorus in the meconium at different stages of development, but the figures were erratic. The subject merits fuller examination than it received from these writers, and might well be joined to a chemical examination of the "uterine milk" (seep. 1467) about which at present we know nothing.
The cholesterol metabolism of the mammalian embryo is again quite uninvestigated, apart from the histochemical observations of Yamaguchi. It is not possible to follow him in his far-reaching conclusions about the functions of cholesterol esters. A certain amount of work has been done on the sterol content of the foetal and maternal bloods, but this will be discussed under the heading of Placental Permeability. The cholesterol content of the foetal suprarenals in man has also been estimated:
Man
% wet weight
% dry
weight
As esters
Free
Total
Investigators
6th month
1-28
Chauffard, Laroche & Grigaut
gth month
—
—
1-34
5> ))
6th month
0-053
0-227
Beumer
gth month 6th month
0-043
0-247
—
,,
i"335
Alamanni
7th month
—
—
2-172
J,
8th month
—
—
2-20
jj
gth month
—
—
4-20
.-•
MiHigrams % dry
weight
Suprarenals
Kidney
Liver
Investigators
3rd month
260
214
_
Chauffard, Laroche & Grigaut
4th month
329
222
—
)> J.
-th month
816
_
250
„ »
gth month
1488
255
251
y, „
Cite this page: Hill, M.A. (2024, April 25) Embryology Book - Chemical embryology 2-12 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-12_(1900)
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
