Book - Chemical embryology 2-11 (1900)
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Needham J. Chemical Embryology Vol. 2. (1900)
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Chemical Embryology - Volume Two
Section 11 Fat Metabolism
11-1. Fat Metabolism of Avian Eggs
One of the first definite pieces of information acquired about the chemical changes during incubation was that the "fats" diminished in quantity. As early as 1846 Prevost & Morin reported that the total ether extract diminished from 10-72 per cent, of the eggcontents at the beginning of development to 9-82 per cent, on the 7th day, 9-48 per cent, on the 14th day and 5-68 per cent, on the 2 1 St day. This was confirmed by Sacc in the following year, who made these interesting reflections: "Life is an ardent fire which needs nourishment incessantly; its activity is such that it will even devour its own hearth if it can find no other combustible. That is the reason why the same wisdom which we admire throughout nature has put at the disposal of the life in the egg this so abundant oil the destruction of which prevents that of the albumen. Without this oil of which the yolk is full (for it is in the yolk that the first traces of the embryo are formed) the albuminous matter would be burnt up by the oxygen of the air so that the development of the chicken could not go on",
Liebermann, in his important paper of 1 888, decided that the fatty acids of the egg-contents were distributed thus : 40 per cent, oleic, 38 per cent, palmitic and 15 per cent, stearic. During incubation in one experiment the amount of "ether-soluble fatty substances" diminished from 5-401 gm. to 2-729 gm., and his general conclusion was that 2-672 gm. of fat were lost by an egg during development. This confirmed the older observations of Parke in 1 866 and of Pott in 1879, but it is only since 1900 that data have been obtained of sufficient accuracy to allow of comparisons with those concerning protein and carbohydrate metabolism. Nevertheless Liebermann's figures were sufficiently definite to illuminate the subsequent respiratory researches of Bohr & Hasselbalch, and to lead to the conclusion that the expired carbon dioxide could be completely accounted for by the missing fat. It was here, of course, that the inaccuracy of the estimation methods for fat upset the calculations, and the quantitatively minor though very important participation of proteins and carbohydrates was overlooked.
Among the later workers, Tangl & von Mituch and Iljin simply estimated the amount of fatty acids in the whole egg before and after development.
Table 167.
Grams of fat per egg
A
Before
After
Loss
Tangl & von Mituch
■ Ul
5-00 6-33 6- 1 1 5-88
2-92 4-19
IS
I -80
VA
2-14
2-21 2-23
Average . . .
2- 1 I
Iljin
6-97 3-37
2-87 0-97
4:;o| Outside lin
Average...
3-25 -I-20 =2-05
Cahn
4-5
3-13 1-37 (average) (1-87 in yolk, 1-26 in embryo)
Iljin's figures are not comparable with those of Tangl and of Tangl & von Mituch, for he worked only on the yolk, and, though the exclusion of the egg-white does not matter, as there is practically no fat in it, yet the exclusion of the chick at the end of development would make a considerable difference. Judging from Murray's figures we might putitsfat content at i -2 gm., and this subtracted from Iljin's figure would make it agree with the rest. Tangl & von Mituch divided the fatty acids as follows: of the original 5-68 gm. in the average egg, 1-59 gm. went into the embryo, i.e. 28-0 per cent, of the original amount, 1-79 gm. remained in the yolk at the end of incubation, i.e. 31-5 per cent., leaving 2-11 gm. fat burnt, or 40-5 per cent. Sakuragi and Idzumi later studied the fat loss by the hen's egg in detail, both using a modified Kumagawa-Suto technique. Their results are plotted on Fig. 359, from which it can be seen that the utilisation of fatty acids follows a regular curve, descending more rapidly towards the end than towards the beginning of incubation. On the same graph are included the figures of Eaves ; Murray, and some already mentioned, and from the whole group a notable measure of agreement appears. It is difficult to draw exact conclusions from a curve which has been constructed from the data of so many different investigators, but it is certainly interesting to plot the milligrams of fatty acid disappearing each day from the egg, and this can easily be done by calculating the mean daily decrement. Such a curve is shown in Fig. 360, and on the same graph appears
7«0
O Parke (yolk only)
Q Drbge
'd Mendel 8c Leavenworth
© Pre'vosb ScMorln (1846)
O Eaves
® Idzumi
® Sakurag'i
e Murray (90°/ humidiby) ® Murray (65% » ) (3D TangI 8c v.Mibuch • ILjin
© Uebcrmann ® Cahn
•••A bheoreb'ical curve suggesbed by Murray
Fig- 359
a curve calculated by Murray from the carbon dioxide output, assuming that this was derived entirely from fat oxidation. There is evidently a discrepancy, for from
E p 400 -i
® Taken from chemical analyses O Calculated by H.A.Murray from
CO2 output assuming it all
came from fab
the 7th to the 14th day the fat lost, as determined by the averaged chemical analyses, is in excess of that lost as determined from the carbon dioxide output, even supposing that all the carbon dioxide was derived from fat, which is not true. The figures of Bohr & Hasselbalch for carbon dioxide output would give an even worse divergence, for during this particular period they were lower than those of Murray. The explanation for this missing fat must be that during that period it is used for other purposes than combustion. If the discrepancy is not simply an error due to imperfect technique, there is every probabihty that at this period the missing fat is transformed into carbohydrate, for a coincident increase in total carbohydrate occurs. This subject has been discussed in the Section on carbohydrate metabolism, to which reference may be made (pp. 1016-1018).
Table 168.
Day
Milligrams of
fat utilised per
Milligrams
of fat
day calculated
Grams of
utilised per day
by Murray from
fat in whole egg
Intervals of days
carbon dioxide output
Experimental
Smoothed
5-6
O-I
Q
I
5-6
1-2
2
5-6
2-3
3
5-6
3-4
4
5-6
4-5
3
i
5-6
5-6
6
5-6
6-7
II
7
5-6
7-8
20
20
20
8
5-58
8-9
§""
§°
33
9
5-53
9-10
80
80
45
10
5-45
lo-ir
100
94
60
II
5-35
11-12
100
105
80
12
5-25
12-13
120
116
105
13
5-13
13-14
120
127
132 lit
14 15
5-01 4-86
14-15 15-16
150 190
s
16
4-67
16-17
170
205
236
17
4-50
17-18
250
250
253
18
4-25
18-19
340
331
359
19
3-91
19-20
460
460
—
20
3-45
—
—
—
—
2150
We may now consider the growth of the fatty substances in the embryonic body. As Fig. 36 1 shows, there is a considerable divergence in the absolute value of the figures, but this is no doubt due to the various methods used, as will be shown below. One point of importance, however, emerges from this group of curves, namely, the inflection which they all possess about the 14th day. This corresponds with all that we know about the absorption intensity of fatty substances by the embryo (see Fig. 251), and supports the conclusion that there is a definite awakening of fat metabolism towards the end of development. Another closely related fact is the percentage constitution of the embryo, shown in Fig. 244, which indicates an important rise in the fat during the week before hatching — and it will be remembered that analyses of other embryos than the chick demonstrated fragments of the same set of curves. Facts of simple observation, too, confirm the enhanced activity of fat metabolism at the end of development. Metzner observed fat globules in the liver of the chick on the 1 2th day, not before, after which time they rapidly increased in number and in size, and this was but a confirmation of the earlier work of E. H. Weber. Nordmann, again found no fat in explanted liver cells from 8th or 9th day embryos but an abundance in those from the last week of incubation. He noted that fat added to the medium on which the former were growing would pass into the cells. Virchow, studying the yolk-sac of the chick histologically, observed fat drops after the loth day, but never before. On the quantitative side, Riddle's estimations of fat in the yolk towards the end of incubation, which will be mentioned again later, show a preferential utilisation of fatty acids by the chick embryo.
Fig. 361.
Important information on these questions has been gained by the use of the dye Sudan III, which attaches itself to fat molecules and indicates their presence. Sitovski was the first to make use of this substance, and by feeding it to moths was able to obtain their eggs deeply pigmented. Riddle then independently fed Sudan III to laying hens (3 to 25 mgm. of the dye per hen per day), and observed that the yolks of their eggs became red, or rather, that the yellow yolk became red, the rings of white yolk and the latebra remaining pale yellow, or becoming very pale pink, thus according with the chemical analyses (see p. 286). Riddle succeeded in pigmenting the eggs of turtles in the same way, and since that time his experiments on fowls have been confirmed by many investigators (e.g. Hainan) . Gage & Gage were the next to go into the matter, and, feeding hens on the dye, incubated the resulting eggs. "As the yolk softens during the process of incubation", they said, "the layered (pink and yellow) mass becomes homogeneous and of a uniform pink. This is marked from the 3rd day onwards. For the first 10 days the transparent embryo shows no sign of the colour, but as soon as the chick begins to deposit fat, at the 1 7 th day of incubation, a minute mass of fat lying in the loose connective tissue between the leg and the abdomen was found with the characteristic pink colour which deposited fat takes in adults fed with the stain. At this time the yolk-mass is of a nearly uniform dark red and almost enclosed within the body." These beautiful observations fit in exactly with the chemical evidence, and are in good agreement with the rest of our knowledge about fat metabolism. They were subsequently repeated and confirmed by Gage & Fish, but work is still required, for Hainan in some unpublished experiments has failed to obtain such clear-cut results as were reported by Gage & Gage. Cross and Rogers reported some curious facts relating to Sudan III. The usual banded appearance of the yolk disappeared by the 5th day of development, and shortly afterwards "the albumen near the embryo" took on a pink colour. On separating by coagulation in situ, Cross found the following distribution of fat :
Neutral fat % dry weight
Yolk 63-4
White albumen ... ... o-2
Pink albumen ... ... 55-0
For other work on Sudan III and the fat metabolism of the reproductive system see Riddle's paper of 1 910.
The technique used in the greater part of the researches hitherto referred to was crude, though perhaps uniform as far as it went. Thus Murray used the term "fat" to designate the extract obtained after washing the ground-up dried tissue with a mixture of equal parts of alcohol and ether followed by a 24-hour extraction with re-distilled anhydrous ether in a Soxhlet extractor, the material being re-ground with sand half-way through the process, and the extract being finally dried to constant weight. Such an extract would include a large number of substances besides pure triglycerides or neutral fats, and it was clearly necessary to make a more detailed investigation. This was done by Cahn, who obtained experimentally the amount of total fatty acids, and subtracted from this the amount associated with the Hpoid phosphorus on the one hand, and the amount present as cholesterol esters on the other hand. He differed completely from Murray in finding that, although the total fatty acids and the total alcohol-ether extract rose in grams per cent, wet weight, the latter remained quite constant in per cent, dry weight. On the other hand, the total fatty acids did show a rise, similar to Murray's. Fig. 362 shows this, but it only means that some differences between the techniques used by Murray and by Cahn, which, owing to the lack of detailed information in their papers, we cannot exactly define, caused discrepancies in the earlier stages. In any case, the
O Murray (bobal ether alcohol extract)
• Cahn f total ether alcohol extract)
♦ Cahn (total fatty acids) ® Cahn (triglyceride fatty acids)
Fig. 363
total alcohol-ether extract is not an entity of great interest. Cahn's figures for triglyceride fatty acids are shown in Fig. 362, expressed in per cent, of the dry weight of the embryo. Here the rise is undoubted, and it is significant that the usual 14th day inflection appears on the curve.
If the daily increment is plotted, an interesting bell-shaped curve appears. As Fig. 363 shows, 100 gm. add on to themselves more and more fatty acids (triglycerides) until the 19th day, after which there is a slackening off. This agrees well with the absorption intensity curve for fat as shown in Fig. 251. Cahn's interesting com parisons between the behaviour of the true fatty acids and the Hpoids, and his calculations of tissue constants will be discussed more easily in the Section on lipoids and sterols. He also calculated the percentage growth-rate of the fatty acids of the triglycerides, obtaining a curious result which he did not explain. The curves plotted in Fig. 364 are, firstly, the percentage growth-rate (Minot) of the wet weight of the embryo, secondly, a composite curve for the growth-rate of dry weight, calorific value, total carbohydrate, coagulable protein, and total ether-alcohol extract, all taken from the data of Murray and Needham. The dry weight growth-rate exhibits a plateau between the loth and 15th days because the dry weight is then most rapidly increasing, but Cahn's curve for growth-rate of triglyceride fatty acids has not a plateau, but a peak. On the other hand, his percentage growth-rate curves for lipoid phosphorus and for cholesterol follow approximately the usual course.
Fig. 364.
Something must next be said about the variations in the nature of the fatty acids in the egg at the different stages of development. Mottram showed in 191 3 that the mean iodine value of the fatty acids in the hen's egg was constant, and did not vary greatly. He studied the individual variations, and the effect of incubation on infertile eggs, and his results have already been discussed in Section i. The first week of incubation, he found, affected the iodine value but little in the fertile egg, but he sometimes got evidence of a slight rise, thus:
Day Iodine value
o 81
4 83
8 88
12 80
16 84
19 86
He was inclined to regard his results as demonstrating the possibiUty of desaturation outside the liver cells. More interesting were the experiments of Eaves, who estimated the iodine value of the fatty acids of the chick embryo and the rest of the egg throughout development. Her resuks are plotted in Fig. 365, from which it appears that there is little change during the first 10 days of development, but that after that time the iodine value of the fatty acids in the chick rises, while that of the fatty acids in the remainder of the egg falls. This decrease in the iodine value of the yolk-fat during development is highly suggestive of a primary absorption of the less saturated fats with a subsequent equal absorption of saturated and unsaturated fat. The increase in the iodine value of the embryonic fatty acids points to an absorption of the more desaturated fatty acids, and suggests that in the early stages the chick embryo does not possess the power of performing the desaturation. If the embryonic liver was incapable of desaturating fatty acids until, say,
(O Chick Eaves <• Remainder (3 Whole egg Days-*-5 To is 20"
the loth day of development, it P^ g
might be supposed that the unsaturated fatty acids would be preferentially absorbed, as the saturated ones would be of no use to the embryo. However, no a priori arguments are here of importance, and I accordingly arranged a series of experiments to test the hypothesis. An aqueous emulsion of embryonic tissues was mixed with the corresponding yolk and vigorously shaken, after which it was allowed to stand anaerobically under toluene for 5 days at 37°. The results were as follows (fatty acids being separated by the Mottram-Lemeland technique) :
Iodine value
6-day yolk alone
6-day embryo and 6-day yolk ... 2-day embryo and 12-day yolk ... 9-day embryo and 19-day yolk ...
Before After experiment experiment 76-0 66-8 69-8 66-4 72-8 qi-6 6i-2 8o-o
Thus where the 12th and 19th day embryonic cells were present there was a very marked desaturation occurring, but not in the case of the 6th day embryo, nor the 6th day yolk alone. If this set of experiments should be confirmed, it will show that in the earlier stages the embryo does not possess the power of desaturating the fatty acids it absorbs from the yolk. It is therefore forced to use the unsaturated ones, and possibly, as these are not present in great quantity, it is thus prevented from devoting much fat to combustion.
In a previous discussion of protein metaboHsm, the work of Riddle on the yolk-contents in the latter half of incubation was cited, and it is equally relevant here, for he determined the percentage (dry weight) of neutral fat in it from the 12th day onwards, using the method of Koch. His data, which are plotted in Fig. 366, demonstrate that the percentage of fat in the yolk-sac falls towards the end of development, just as the percentage of protein rises, indicating a preferential absorption of the former over the latter, and bearing out the absorption intensity curves shown in Fig. 251. The rest of the information is more difficult to understand, and concerns special fractions of yolk; thus the intracellular yolk (yolk-masses in the wall of the yolk-sac) seems to have a high fat concentration, and the solid masses in the body of the yolk itself a variable one. As for the yolk-sac, its fat-content rises greatly in the last few days of development. We shall see later that this preferential absorption of fatty acids is accompanied by a preferential absorption of phosphatides. This picture of the awakening of fat metabolism in the last days of incubation is to some extent mirrored in the results of Vladimirov & Schmidt on the blood fat of the embryo. As Fig. 367 shows, it rises to a peak on the 1 8th day of development, and then falls without a break at hatching. This peak corresponds well enough with the peak in the daily increment of fatty acids found by Cahn (Fig. 363), and occurs at the time when the absorption intensity of fatty acids (Fig. 251) is rising away from its 14th day trough, and taking the place of the protein absorption curve, which is descending. It must be admitted, however, that the individual variations in the curve of Vladimirov & Schmidt are considerable, and it would be worth while to repeat their measurements, using perhaps a better method (they employed that of Bang) .
Fig. 366.
Fig. 367.
11-2. Fat Metabolism of Reptilian Eggs
The fat metabolism of the reptile egg has only once been investigated — by Karashima in 1929, who worked with the turtle, Thalassochelys corticata. He found a diminution of some 30 per cent., rather low for a terrestrial egg, as the following table shows :
Days of
Grams fatty acid
development
per egg
15
\'A
30
1-36
45
Hatched
I-I2
Karashima also investigated the percentage of free fatty acids, watersoluble and water-insoluble volatile fatty acids, etc., in the egg-fat at the various stages, but these showed no definite changes : the firstnamed remaining at about 20 per cent, of the total fraction, the second at about 0-9 per cent, and the third at about 0-7 per cent. On the 15th day, however, there was a marked rise in the free fatty acids, no doubt to be interpreted as due to the action of a lipase, since the total fatty acids remained constant.
11-3. Fat Metabolism of Amphibian Eggs
The fat metabolism of the developing amphibian egg has been a subject of some difference of opinion between investigators. Parnas & Krasinska measured the total fatty acid content of frog embryos {Rana temporaria) using the Liebermann-Kumagawa-Suto method. As Table 169 shows, they found just as much at the time of hatching as at the beginning of development, and they therefore concluded that none was used as a source of energy. They did, however, find a decrease in lipoid phosphorus, as will be described in the following section, so that it seemed as if the amount of fatty acids in triglyceride form must even rise rather than fall. Parnas & Krasinska did not pursue their investigations into the free-swimming larval stage. The work of Faure-Fremiet & Dragoiu paralleled that of Parnas &
•004
-003
CO •D
J-' <D Q.
a> •OOl
E o
002
'015
• 010
Bialascewicz &^Mincovna Frog
— Probein ---Fab
End of developmenb
_o
^ 300
•S 200 £
^ 100
'005 - E^
i5r
10
r o ^*^
100 150 200 250
3C
It Fiske
8^Boyden,Needham, Murray
- S E
j\ and obhers , / ^ Chick / 7 \ !^ — Probein
Si o
- E-o
- o^
-•is
\--'
1 ,J'-'\ Hate
hing
- UXD J.
^ '
9.' '•
- r<^f
3^'
. . . 1 1 • 1 1 1 1 . 1 . 1
Days->5
10
15
20
Fig.
Krasinska, in that only the pre-natal period was considered, but it led to diametrically opposite results for they found a diminution in total fatty acids amounting to 0-113 mgm. per embryo. Two other researches, however, followed the development up to the end of the yolk-sac. Bialascewicz & Mincovna measured the fatty acids at the end of each period, and for the first of the two they confirmed Parnas & Krasinska, observing no change, or even an increase, in the fatty acid content up to hatching. Bialascewicz & Mincovna used two methods, that of Kumagawa-Suto and that of Bang, obtaining substantially the same results with each, while Faure-Fremiet & Dragoiu used that of Kumagawa-Suto alone. Still later Barthelemy & Bonnet fully confirmed the loss of fatty acids over the whole of development found by Bialascewicz & Mincovna, noting a loss of 0-102 mgm. per embryo, instead of o- 192. They did not, however, make any estimations on the just-hatched embryos, and so had nothing to say on the difference between Faure-Fremiet & Dragoiu and the other workers. Possibly the breed of frog used may account for this. In any case we shall assume here that practically no fat disappears from the amphibian egg before hatching, but that afterwards a considerable utilisation takes place. The rate at which the fatty acids disappear was sketched out by Bialascewicz & Mincovna in a few experiments, and is shown in Fig. 368, where in the upper graph the amount of fatty acids disappearing from one larva per day is plotted against the time. The curve rises more or less steadily. For the sake of comparison the curve of protein utilisation established by Bialascewicz & Mincovna is placed beside it, and we thus see how at a certain point the combustion of protein attains a maximum and afterwards falls away, giving place to the catabolism of fatty acids. Below is placed a composite graph showing the utilisation of protein and fat by the chick embryo (taken from Figs. 325 and 360). The likeness between the two pictures is quite striking. Not only do both embryos exhibit a peak of protein catabolism, but the peak comes at approximately the same epoch of development, and this although the frog derives 70 per cent, of its waste energy from protein and the chick only 5 per cent., and although the time of hatching in the two organisms is so completely different. For various reasons, which have already been stated, we cannot add curves for carbohydrate catabolism to the pictures, nor have any other embryos been investigated in sufficient detail to permit of parallel graphs being drawn for them. In the case of mammahan and selachian embryos, indeed, it is possible that we have already the descending limb of the peaked protein cataboHsm curve (see p. 1 1 17), and Hayes' account of the constitution of the embryo of the Atlantic salmon {Salmo salar) , which shows a rising fat-content up to the io6th day from fertilisation, followed by a fall, may indicate a late onset of fat catabolism in that case also. But to return to the fat metabolism of the frog embryo, the relations between fat and nitrogen depicted in Fig. 368 could be expressed in terms of a ratio. This Bialascewicz & Mincovna did, as follows :
Hours from Milligrams fatty acid in one larva/
fertilisation Milligrams nitrogen in one larva
o 3-61
72 3-96
96 4-26
140 4-13
143 4-07
164 3-49
239 2-86
284 2-50
The ratio tends to rise until hatching because the denominator is decreasing while the numerator is remaining constant, but in the later stages the numerator falls more rapidly than the denominator, so that the ratio falls.
Faure-Fremiet & Dragoiu studied the iodine value of the fatty acids of the frog's egg, as did Parnas & Krasinska. But again there was a certain contradiction, for whereas the Polish investigators only found a diminution of 2 units, the French ones found a diminution of 10 units. Faure-Fremiet & Dragoiu calculated that at the beginning of development 8o-8i per cent, of the total fatty acids were unsaturated (i double bond) and at the end (hatching) only 74-14 per cent. By multiplying the lipoid phosphorus by the coefficient 25-75, the percentages of phosphatide fatty acids were found, and this fell from 25-13 to 20-0 per cent. One embryo lost up to hatching, they found, 0-192 mgm. of unsaturated acids, and 0-06 mgm. of phosphatide fatty acid, but it must be remembered that none of the other workers found any loss of fat before hatching.
Barthelemy & Bonnet carried out their experiments at different temperatures, in order to see whether the rapidity of development would exercise any influence upon the amount of fatty acids lost throughout the whole period. Their results were as follows:
Fatty acids lost in %
Loss of fat/
Temperature (°) of initial fatty acids
Loss of nitrogen
8 48
0-75
10 32
0-73
14 41
0-75
21 38
0-75
showing that, however much the development may be speeded up, or damped down, the same amount of fatty acids have to be combusted. Moreover, the ratio of fat lost to nitrogen lost is identical, and compares in an interesting way with the ratio of Bialascewicz & Mincovna for the embryonic or larval body itself. This indicates that relatively much more protein is combusted than fat, as has been shown in Table 126.
So far the eggs of the anuran branch of amphibia have alone been considered, but one chemical investigation exists which deals with those of a urodele, ihe giant salamander, Cryptobranchus allegheniensis.
This is that of Gortner, to which attention has already been given in Section 9-9, We have seen above that the general consensus of opinion leads to the view that no fatty acids are lost from the egg of Rana temporaria up to hatching. But, if Table 1 69 is carefully examined, it will be seen that Bialascewicz & Mincovna observed an actual increase in fatty acids during the pre-natal phase. The increase was not great, being about 11 per cent., but, if they were there dealing with a real phenomenon, it was interesting, for a synthesis of fatty acids was found by Gortner to be important in the salamander egg. Gortner's facts were as follows (he did not follow the development further than hatching):
Table 170.
Weight in
milligrams absolute
% dry weight
Dry weight
Total ether extract
Ether-insoluble but
alcohol -soluble ... Protein
I egg
... 58-25 ii-i8
6-63 40-26
I larva 57-28 12-75
6-18 38-28
Change
-0-97 + 1-57
-0-45 -1-98
Eggs
19-19
11-38 69-10
Larvae 22-25
10-78 66-82
Change -1-66 + 3-06
-o-6o -2-28
The loss of 1-66 per cent, of the dry weight was due to combustions to carbon dioxide and water. "Accompanying this loss in weight", said Gortner, "there is a very marked gain of fats equal to 3-06 per cent, of the egg weight and to an increase of 1 4 per cent, of the fat already present in the tgg. Gortner meant by "fat" the total ether extract dried to constant weight, in which a number of other substances would be included. It was not long before the fact of fat synthesis in this egg was confirmed, by the aid of an accurate method. McClendon in the following year applied the Kumagawa-Suto method to the same material. He observed a numerically identical loss of dry weight between fertilisation and hatching, and the figures for total extract compared as follows :
Total ether- and alcoholsoluble substances % of the dry weight Increase (% )
Eggs Larvae Dry weight Initial
Gortner 30-57 33'03 2-46 6-9
McClendon 35-0 37-8 2-8 8-0
The determination of the non-volatile fatty acids showed them to amount to precisely 50 per cent, of the total extract in each case, so that the increase in them corresponded with the increase in the total extracts. McClendon explained the slight lowness of Gortner's figures as compared with his own by referring to the extreme difficulty of removing by extraction the last traces of phosphatides from yolk, owing to their association with vitellin. McClendon found the MoHsch reaction negative on the whole egg, and could obser\'e no reduction of copper after total hydrolysis (experimental details not given), so he concluded that no carbohydrate groups were present, and that the increase in fatty acids must be due to the destruction of protein molecules. The evidence for this view was insufficient.
Some histochemical investigations have been made on the developing amphibian egg. The work of Konopacka and of Konopacki & Konopacka is especially detailed, and Hibbard has given an exhaustive study of the histochemistry of the development of the anuran, Discoglossus pictus. Unfortunately, as I have pointed out before, we have no guarantee that the substances which the histochemical worker studies are the same as those which we analyse and measure by purely chemical methods, though they may be, and usually are, called by the same names. For this reason it is very difficult to know what emphasis to lay on the findings of these workers. Thus Konopacki & Konopacka, who worked with the embryos of Rana temporaria, reported a disappearance of "fat" during the segmentation stages, and concluded that "the intensity of metabolism is parallel to the morphogenetic changes", though how this result could be arrived at from the study of stained sections is not clear. But Konopacki & Konopacka had less to say about neutral fats than about lipoids, as the Flemming-Ciaccio method is supposed only to reveal the presence of the latter. However, "in the early stages of development ", says Konopacka, " the fatty acids are utilised, for the number of droplets staining with osmic acid are more numerous and larger than those which stain with Sudan III after fixation according to Ciaccio's technique. In later (post-gastrula) stages, however, the two kinds of droplets are of the same size, and eventually the ' lipoid ' droplets disappear completely, once the structure of an organ is determined". Hibbard says that, during the early development of Discoglossus, there is little change in the distribution or appearance of the fat globules, but just before hatching they begin to increase in number and size, reaching a maximum at the time when the external gills are half covered over by the operculum, and afterwards falling away again. By the time the tadpole has reached a length of 17 mm. no fat droplets are to be found in the cells. She concluded that some of the fatty acids absorbed from the yolk were utilised in the cells of the tissues, but that most of them were eventually transferred to stock the newly formed liver cells.
Among the histochemical researches, one of the most interesting is that of Abe, who began by the not very hopeful statement that "the anuran yolk is a complicated lipoid-protein mixture containing phosphoprotein and iron", but went on to show that the utilisation of the substances in it could not be going on at a uniform rate, because in the early stages eosin stains the yolk light red or pink, later dark red, and finally deep violet-red. No direct physicochemical meaning can as yet be attached to this, but it gives a glimpse of the unequal utilisation of the yolk constituents which we have been discussing.
11-4. Fat Metabolism of Selachian Eggs
We can now consider the events which take place in the fish egg with respect to fatty acids during its development, and we shall begin with the ovoviviparous selachians. In these fishes pecuHar relations seem to exist between the liver of the "pregnant" fish and the embryos within their transparent envelopes in its uterus. Lo Bianco was the first to notice that whenever embryos were present the size of the liver was increased, and this observation was confirmed by Polimanti, who gave the following figures :
Table 171.
Liver weight in
Grams % dry
% weight of
weight fatty
animal
acids in liver
Pregnant females
Trygon violacea
12-42
92-95
Torpedo ocellata
5-29
83-43
Scyllium canicula
Trygon violacea
7-70 8-27 5-26
99-86
Males
85-23 76-49
Torpedo ocellata ...
Scyllium canicula ...
7-97
90-58
No explanation for this was suggested by Polimanti. A subsequent paper by Reach & Vidakovich went further into the matter, as far as the Torpedo was concerned, and Table 1 72 summarises the results they obtained. Their first set of data, taken from fishes caught in April, gives the figures for just after fertilisation, when the large yolked undeveloped eggs fill up the uterine cavity; their second set shows what has happened by the time the embryos have reached the length of about 4 cm., a dry weight of 4-45 per cent, and a nitrogen content of 1-95 per cent. Finally, their third set of data concerns the fish after the embryos have left the uterus. The first point of
Table 172. Reach & Vidakovich's figures (in grams) :
Liver Ovaries _
Description of animal
Weight in % of weight
animal
Fatty acids % of liverweight
Weight in % of weight
animal
Fatty acids % of ovaryweight
r
Weight fi^s^h
Weight each
Fatty acids
^fish^^^
Gm. %
foh
Fatty
acids
gm. per
egg
Gm.
% per egg
"Spring-torpedo"
" Summer-torpedo "Autumn-torpedo '
4-5
' 314 412
44-75 23-6
22-6
029
023 I-7S
1-59 013
52
632 7'02
711 6-95 Embryos born
562
or6-5(?)
508
0-66 0-66
0-66I 0-499
9-41 7-i8
Embryos
Yolks
Description of animal
Weight fish
Weight each
Fatty acid
fish
Fatty
acid
per
embryo
% per embryo
Weight fish
Weight each
L^7
fi^s^h^
per yolk
per yolk
"Spring-torpedo" " Summer-torpedo
Not large • 9-2 095
enough to measure 0067 0-009 094
61-94
6-0
S02
049
8-2
Note. In the "Spring-torpedo" the eggs have just come into the uterus; the analyses were made in June, fertilisation probably having taken place in April. In the "Summer-torpedo" embryonic growth has proceeded considerably, the analyses being made in July. The "Autumn-torpedo" has just given birth to the 6-10 embryos and its ovary contains yolked eggs from 10-20 mm. diameter; the analyses were made in August.
interest to be drawn from the table is that the fatty acids decrease notably from 66 1 to 499 mgm. per egg — a loss of 24-5 per cent. There is no means of telling, of course, whether this loss is all due to combustion, but, even if it were, the amount must be much less than the corresponding loss in a terrestrial egg such as that of the chick, for the latter has 30 per cent, of its yolk as fat and the former only 9'4 per cent. It may therefore be supposed that the main source of energy in the selachian egg is protein. As the table shows, the percentage of fatty acids in the whole egg diminishes from 9-4 to 7-2. As regards the maternal liver and ovaries. Reach & Vidakovich did not confirm Polimanti and Lo Bianco, for the livers of their fishes hardly changed at all in per cent, of the total body-weight, and what change there was during "pregnancy" was in the reverse direction. Nor did the percentage of fatty acids in the liver show any of the changes suggested by Polimanti. Reach & Vidakovich's iodine number results were interesting.
Table 173.
Iodine no. Saponification no.
Embryo Em(without bryonic
Liver Ovary Muscle Yolk liver) liver Liver Ovary Yolk
"Spring-torpedo" i20 44 47 129 — — i95 — 186
"Summer- torpedo" 122 42 — 140 38 65 — — —
"Autumn-torpedo" loi 153 43 — — -~ '93 '53 —
From this it appears that the fat of the embryonic body has a low iodine value, about the same as that of the adult muscles, but that its Uver fat has one twice as high. The yolk fat is twice as unsaturated as that of the avian egg-yolk. Its presence in the ovaries of the "Autumn-torpedo" accounts for the high value found then.
Another selachian, Centrina vulpecula, was studied by Kollmann, van Gaver & Timon-David who obtained the following constants from a specimen which seemed to contain in its abdomen practically nothing but eggs and liver :
Liver oil
Egg oil
Density at 15°
Refractive index Iodine value Saponification value
0-9002 1-4689 73-4 132-3
0-9106 1-4744
"3-9
133-7
This agrees well enough with the results of Reach & Vidakovich in that the egg oil is particularly unsaturated, cf the state of affairs in the hen's egg, and the value of ready-made double bonds to the embryo. Apparently the fatty acids of the yolk in this fish have as long chains as those of the liver.
Similar work was done on Centrophorus granulosus by Andre & Canal, who obtained the following figures :
Composition of the crude oil %
Crude oil ,- '^ ^,
in % of Fatty acids wet (principally weight clupanodonic) SqUalene Cholesterol
Egg ■•• 29 45-5 55-1 4-7
Foetal liver 56 32-0 66-0 3-8
Liver of immature female 78 15-8 840 i-6
Liver of adult female ... 85 82 91-0 i-o
They naturally concluded that during ontogeny there was a passage from clupanodonic glycerides and cholesterol to squalene, a passage which was traversed in the opposite direction during the preparation of the eggs by the adult female. Squalene seems to play an important part in selachian metabolism (see p. 350).
11-5. Fat Metabolism of Teleostean Eggs
In the teleostean egg all is different. Tangl, in the course of his series of researches on the energy sources of eggs, had accustomed himself to regard fatty acids as the most usual material of this kind, and he received an unexpected surprise when, with Farkas, he investigated the egg of the trout. Estimating the fatty acid content before fertilisation and at hatching, they noted an unmistakable increase, as follows :
Amounts in milligrams per egg Change
Before
After
^
%of
develop
develop
Dry
initial
ment
ment
Absolute
weight
value
Wet weight
88-2
83-5
-4-7
—
—
Water
58-5
54-2
-4-3
—
—
Dry weight
29-9
29-1
-0-8
—
—
Fatty acids:
Liebermann's direct saponi
fication method ...
6-4
6-64
+0-24
+ 0-825
+ 3-75
Ether extraction (probably
unrehable)
282
3-55
+0-73
+ 2-51
+ 27-6
This was confirmed on the eggs of another trout, Savelinus fontinalis, by McGlendon in 19 15, who obtained very similar results, indicating a synthesis of fatty acids during development. These were his results:
Amounts in milligrams per egg
Before After
Change
develop- develop- Dry
ment ment Absolute weight Initial
Wet weight ... ... ... 62-0 — — — —
Dry weight ... ... ... 17-0 — — — —
Fatty acids (direct) ... ... — — — +0-99 +5'57
Ether extract — — — +1-3 +5*55
This curious process appeared again in the work of Dakin & Dakin on the egg of the plaice {Pleuronectes platessa) :
Values in milligrams absolute per 1 00 eggs
Wet weight ... Dry weight Fatty acids
before development 336-0
23-9 0-28
at hatching 331-9
20-8 1-02
From these figures it appeared that there was an increase of 0-74 mgm. per 100 eggs, or 264 per cent, of the initial store of fatty acids. Hayes, again, working on the eggs of the lumpsucker {Cyclopterus lumpus) found a rise in total fat from 475 to 5-25 per cent, of the wet weight during the 40 days before hatching. And whereas one egg of the Atlantic salmon {Salmo salar) contained 6 mgm. fat at the beginning of development it contained 7 mgm. at hatching (65 days).
1 1 -6. Fat Metabolism of Mollusc, Worm, and Echinoderm Eggs The synthesis of fatty acids occurs again in the metabolism of the snail egg [Limnaea stagnalis), and it was here indeed that it was first recognised. For in 1853 F. W. Burdach estimated the fat-content of the eggs of the snail from fertilisation to hatching, and the results were published in his thesis De Commutatione Substantiarum Proteinacearum in Adipem at Konigsberg in that year. His technique was, of course, that in general use at the time, and therefore probably very inaccurate, but the figures were as follows :
Before After
development development
Absolute weight in milligrams per egg Dry weight ... 335"5 2i8-o
Neutral fat ... 2-25 3-5
Fat % of dry weight o-66 i-86
On the other hand, Faure-Fremiet, in his comprehensive study of the Ascaris egg, observed a diminution of the fraction which he called "fat", but which was really a mixture of neutral fats and ascarylic acid. This fraction fell from 26 per cent, of the dry weight of the egg at the beginning of development to 22-5 per cent, at the end, i.e. a loss of 3*5 per cent, of the dry weight. Then the work of Ephrussi & Rapkine on the developing egg of the sea-urchin, Paracentrotus lividus, showed that there was a diminution in the total fatty acids, thus:
Hours from fertilisation
12 40
Total fatty acids o (gastrula) (pluteus)
% dry weight ... 2i-2 19-55 ^TA
% wet weight ... 4-81 4-43 3-69
And Payne has shown that fertilisation does not affect the iodine value of the fatty acids in the egg of^ Arbacia.
11-7. Fat Metabolism of Insect Eggs
The silkworm egg definitely shows a negative balance of total fatty acids. Tichomirov in 1885 obtained the following figures:
FAT METABOLISM
1 185
% wet
weight
% dry weight
Ch
ange %
Before
After
Before
After
Dry
develop- develop
develop
develop
weight
Initial
ment
ment
ment
ment
loo-o
88-84
—
—
—
ii-i6 (wet)
35-51
30-20
—
—
—
14-9 >.
9-52
6-46
26-8
21-4
5-4
20-1 (dry)
8-o8
4-37
22-7
12-2
IO-5
46-1 „
1-04
1-74
—
—
—
—
0-40
0-35
—
—
—
—
Wet weight
Dry weight
Total ether extract
Neutral fat fatty acids..,
Phosphatide fatty acids
Cholesterol
Thus 20 per cent, of the initial amount of ether extract in the silkworm egg disappears between the end of the diapause and the time of hatching, and when the neutral fat fatty acids are alone considered the fall reaches 46 per cent, of the initial value. This was later confirmed by Farkas who found a fall of 49 per cent. The only other data which we possess for the silkworm are those of Vaney & Conte, but it is likely that their methods were inadequate and they isolated less than half the amount of total fatty acid found by Tichomirov. Their figures were as follows : 17 • j • 0/
° Fatty acids m %
dry weight of egg 4 days after laying ... ... ... ... 7-21
6 days after laying ... ... ... ... 6-68
End of the diapause (g months after laying) ... 5-35
Hatching (lo months) ... ... ... ... 4-88
and suggested a loss of 2-33 per cent, of the dry weight, or 32-3 per cent, of the initial weight. Kaneko has made a histochemical study of the fat in the silkworm egg, especially concerning himself with its localisation. Another piece of information concerning fat metabolism in insect eggs is due to Weinland, who made a ^'Brei" of the eggs of the blowfly, Calliphora vomitoria, and adding Witte's peptone, found after some days of anaerobic action a decrease of about 30 per cent, in total fatty acids. This stood in the sharpest contrast to the behaviour of "Breis"" of larvae and of pupae, which have a remarkable power of converting peptone and even protein into fatty acids. It would thus appear that Calliphora vomitoria embryos consume a large amount of fat during their development, just as do those of Bombyx mori. Then for the grasshopper, Melanoplus differ entialis, we have the careful work of Slifer, who found 0-352 mgm. total fatty acid at the beginning of development and 0-14 mgm. at the end; each egg, therefore, lost 54-3 per cent, of its initial store. This fits in well with the respiratory quotients of 0-7 to o-8 which, according to Bodine, are always found for this material.
Moth,(Rudolf5)
UNE JULY AUG. SEPT. OCT. NOV. DEC. JAN. FEB.
1186 FAT METABOLISM [pt. iii
An interesting study of the fat metabolism of the eggs of the lackey moth or tent caterpillar, Malacosoma americana, was made by Rudolfs. Fig. 369, constructed from his data, shows the progressive decUne in total fatty acids during the development of this insect. Evidently a very large proportion is used up, probably by combustion, in agreement with the findings on ^ the silkworm and the grasshop- : per. As the chorionic coverings constitute 52 per cent, of the whole egg-mass, the fat-content of the G.gg itself on laying would be about 9-0 per cent, dry weight. As the water-content Y\^. 369.
decreased only slightly during
development (see Fig. 369), the fat/water ratio decreased a good deal; thus at laying there would be 9 mgm. of fatty acids per 100 mgm. of water, but at hatching only about 2 mgm.
11-8. Combustion and Synthesis of Fatty Acids in Relation to Metabolic Water
It is exceedingly interesting to note the similarity between the insect c^g and the egg of the chick. All these terrestrial eggs burn relatively large amounts of fat, and, though this is perhaps associated with the ease with which fatty acids can be stored in a small space, yet it may also be related to the fact that fat, when it burns, leaves behind all its original weight in the form of water. We have already seen that one of the most difficult problems confronting the earliest terrestrial animals must have been the proper supply of water for their embryos, and it is therefore likely that the use of fat as the principal source of energy is associated with the importance of the water balance. Aquatic embryos which have no trouble in this direction do not, as far as we know, burn large quantities of fat. Babcock and others have calculated that 100 gm. of fat on combustion yield 107-1 gm. of water, 100 gm. of carbohydrate, 55*5 gm. of water, and 100 gm. of protein only 41-3 gm. of water. From Table 126 of Section 7 it is clear that the chick burns much more fat than the frog (approximately 80 per cent, in the former case and 20 per cent, in the latter case, of the total material combusted), and this may be regarded as a consequence of their special needs. Energy is no doubt the same from whatever source it comes, but one source may be more convenient than another. In the hen's egg, according to Murray's data, 6-32 gm. of water are lost from the system during its development, and 2-01 gm. of solid. As 91 per cent, of the solid lost is fat, about 2-1 gm. of "metabolic water" are added to the egg, so that 8-4 gm. of water would have been lost if fat had not been burnt, or 40 per cent, more than what actually is lost. The chick in its closed terrestrial box cannot afford to despise this extra two grams of metabolic water.
Table 174.
Fatty acids combusted during
development in % of the total Aquatic or fatty acids present in the egg Terrestrial at the beginning. (All figures
Animal
embryo ft
)r the whole of development) Investigator
Chick
T.
60
Murray; Idzumi; Saku ragi, etc., etc.
Silkworm ...
T.
48
Tichomirov; Vaney & Conte; Farkas
Lackey moth
T.
85
Rudolfs
Grasshopper
T.
54
Slifer
Marine turtle
... A.(?)
34
Karashima
Frog
A.
28
Parnas & Krasinska; Barthelemy & Bonnet, etc
Torpedo
A.
24-5
Reach & Vidakovich
Plaice
A.
<io
Dakin & Dakin
Sea-urchin ...
A.
20
Ephrussi & Rapkine
It is difficult to ascertain exactly how much fat is combusted by different animals; for as it burns away, leaving no measurable incombustible residues, its combustion-rate cannot be calculated from them, and no one can say a priori what proportion of the carbon dioxide eliminated comes from fatty acids. Then in some animals, as we have seen, there is a veritable synthesis of fatty acids during embryonic development, obscuring any combustion of them. One could therefore predict that the difference between terrestrial and aquatic embryos would not be so clear-cut in the case of fat combustion as it is in the case of protein combustion. However, Table 1 74, constructed in the same way as Table 162 in Section 9-15, shows that embryos do divide into the two classes according to their environment in this case also. Terrestrial eggs burn between 60 and 70 per cent, of their initial fat stores, and aquatic ones only about 20 per cent. The difference is really even more striking than it seems from the table, for terrestrial eggs store the greatest amount of fat, so that 20 per cent, of the fat in the hen's egg is a good deal more proportionately to the embryonic weight than 20 per cent, of the fat in the sea-urchin's egg. But any direct comparison between the terrestrial or aquatic environment and the amount of fat stored in the egg is not possible, for, as Table 31 (in which the fat/protein ratios of many eggs are given) shows, there is no strict correlation. The terrestrial sauropsid egg contains much more fat than any of the others, relatively to the protein, but the insect egg, which is also terrestrial, contains very httle (e.g. silkworm and lackey moth). In other words, the correlation between environment and material used as energy source does not appear until one knows the exact amount of material combusted during development, and expresses that in terms either of the amount of it originally present, or of the total material combusted.
Now we have seen that, in six separate instances, an absolute increase in fatty acids has been observed during development, one of the animals in question being a urodele amphibian, two being teleostean fishes, and one a pulmonate gastropod (all aquatic embryos). It is likely, in view of the discussion on p. 350 about the oil drops in fish eggs, etc., that this list would be much prolonged if more investigations were made of their chemical embryology.
What is the nature of this fat synthesis? Tangl & Farkas, who were the first in recent times to establish it experimentally, thought that the trout egg contained "glycoproteins" which were broken down to carbon dioxide and water, but which also went to form glycogen and fat. They believed, in fact, in a predominance of protein metabolism and in a formation of fat from protein, the waste nitrogen being retained in the egg in the form of urea, and liberated at hatching to the exterior. Tangl & Farkas, however, made no attempt to demonstrate the presence of urea. Then Gortner, in his studies on the protein metabolism of the trout and salamander egg, concluded that this could not be the case, as no urea or uric acid was to be found in the eggs at the time of hatching. Unfortunately, he neither identified the urea and uric acid specifically nor brought forward evidence in disproof of their presence by the use of appropriate tests. In view of the importance of the subject, it would be highly desirable for a new investigation to be made of the protein metabolism of the trout egg. The fact of the matter is that neither Tangl & Farkas' hypothesis nor Gortner's experiments throw any Hght on the origin of the synthesised fat in these eggs. Apparently the only reason why Tangl & Farkas suggested a glucoprotein as the energy source was because they could not imagine fat being synthesised from anything else. Each egg, they found (by analysis), expended 6-68 cal. during development, and they reasoned that to produce this energy from 518 eggs (their experimental number) 1-67 gm. of glucoprotein (9-7 Cal.) must be broken down to 0-38 gm. of fat (3-5 Gal.) and 0-30 gm. of glycogen (1-3 Gal.), and all of the nitrogen retained in the form of urea (0-57 gm., i.e. 1-40 Gal.), the difference between these heat values being carbon dioxide and water with a heat value of 3-5 Gal. As they found experimentally a heat loss of 3-46 Gal., they considered that their theory covered the facts sufficiently well, but it would be easy to think of several others equally convincing. They found, moreover, that the loss of carbon, 46-3 per cent., did not agree with the expected loss, 68 per cent., and they suggested that perhaps it was not all eliminated as carbon dioxide, but retained in some other form.
The problem is undoubtedly a difficult one and at present there is no solution for it, but there are two points which seem to have been overlooked by all those who have so far considered it. In the first place, even when the increase in "fat" has been demonstrated to be an increase of fatty acids, and not of total ether extract, it has always been assumed that no breakdown of these can have been occurring. Yet it is possible that a catabolism of fatty acids might exist masked by a reverse process, so that, as the fats were destroyed, more were formed from some other source so as to over-compensate for the fat combustion. The second suggestion is that substances such as spinacene (see p. 350), which have recently been found in fish eggs, may play a very important part as energy sources. So far there is no experimental foundation for this idea, but it is quite conceivable that spinacene or squalene might be oxidised directly for energy, or that such substances might be the origins of the synthesised fat in certain aquatic eggs. It is indeed difficult to see any biological reason why this fat synthesis should go on, and it is noticeable that it never reaches great dimensions — 8 per cent, in the salamander, 55 per cent, in the snail (if we may trust Burdach's figures), and 5 per cent, in the trout. It is true that the increase in the plaice is 264 per cent., but the total quantities in question there are exceedingly small relative to the total amount of substance in the eggs. It will be admitted that the whole subject of fat synthesis in these aquatic eggs urgently needs re-examination.
11-9. Fat Metabolism of Mammalian Embryos
It has often been asked how fatty acids arise in the mammalian embryo, since there is no yolk from which they can be transferred. The obvious answer is that the foetal fat is suppHed through the placenta, but this has to meet the objection that there is a good deal of evidence against the existence of this transportation. It would perhaps be more suitable to delay the consideration of this question till the Section on the placental barrier, but it is too intimately associated with fat metabolism. That the fat-content of the foetal blood is independent of the fat-content of the maternal blood was suggested by the experiments of Oshima, who in 1907 studied the ultra-microscopic particles of the foetal blood. Neumann had shown some years before that the concentration of these in blood ran closely parallel to the fatcontent as determined chemically, and they are probably identical with the "chylomicrons" of Gage & Fish. Oshima found that in the guinea-pig the maternal blood has always a " massiger, " " zahlreich ", or "massenhaft" number of fat-particles, but the early foetal blood "sparlich". As the embryo developed, however, the number of fatparticles in its blood rose greatly, and at birth nearly, if not quite, equalled that in the maternal blood. It was significant that the condition of the foetal blood seemed to be independent of the mother, for fasting, Oshima found, would easily reduce the fat-particles in the mother's blood to few or none, without having any effect on the foetus, which was always as rich in fat-particles as it was scheduled to be at the time of development in question. On the other hand, if fat was fed to the mother in large amounts there^ was no effect on the embryonic blood, although a large one on the maternal blood. Oshima naturally concluded that fat was not transported from the maternal to the foetal circulation.
This received later a quantitative backing from various investigators. Ahlfeld very early had reported large and inconstant differences between maternal and foetal blood-fat in the dog, and had shown that bacon feeding affected the maternal circulation only. Kreidl & Donath in 1910 estimated the fat-content of guinea-pig foetal blood in normal conditions (development time not stated) at 746 mgm. per cent, as against 314 mgm. per cent, in maternal blood, in both cases nearly all in the plasma. They thought it likely that a synthesis of fat by the placenta must occur, but they could not demonstrate the presence of any lipolytic enzyme in it. The other attempts which have been made to gain knowledge about the lipolytic enzymes of the placenta will be discussed in the section on that organ. More recently Slemons & Stander obtained the following figures for foetal and maternal blood at term in man :
Average
milligrams %
Whole blood Maternal ... 908
Foetal ... 707
Plasma Maternal ... 942
Foetal ... 737
and concluded that the fatty acids of the embryo were not directly derived from the mother. MurHn & Bailey found the same relation in man at term, though their figures were lower than those of Slemons & Stander. .
Average milligrams % Whole blood Maternal ... 550
Foetal ... 266
Murlin & Bailey believed that an association existed between fatcontent of foetal blood and severity of labour.
Another line of evidence against the passage of fatty acids from maternal organism to embryo arises from the fact that, when fats are earmarked in one way or another, they do not subsequently appear in the fat depots of the foetal tissues. Two principal methods have been used for this purpose, firstly, the marking of fatty acids by stains and dyes, and, secondly, the use of special or highly unsaturated fats which can easily be recognised at the other end. Hofbauer was perhaps the first to utilise these methods, for, in his many-sided study of the metabolism of the placenta in 1905, he fed fats stained in various ways to pregnant animals, and stated that he observed traces of the colour afterwards in the foetal fat. The colour can only have been in traces, for in the hands of subsequent experimentalists the results of this type of experiment have been uniformly negative. Thus Gage & Gage in 1909 fed fat stained with Sudan III to various kinds of pregnant animals, and never observed the slightest indication of a passage through the placenta. The maternal fat depots would be brilliantly pink or red, while those of the embryo would be perfectly colourless. Similar experiments were carried out by Mendel & Daniels in 191 2, using Sudan III and Biebrich scarlet, and working on pregnant rats and cats, but always the embryos remained absolutely colourless, although the maternal fat was brightly coloured. Baumann & Holly later still obtained the same quite negative results. There is some likelihood that the placenta could detach the dye from the fatty acids.
Then Hofbauer in 1905 fed cocoa butter to dogs, and claimed to have identified lauric acid in the foetal fat after some days, but its presence was not really estabhshed, and the experiments might well be repeated. Thiemich's work was better, although it antedated that of Hofbauer by some years. Thiemich fed certain dogs on cocoa butter and others on linseed oil cake, with the following results:
Iodine value Iodine
of fat value of
in food foetal fat
Cocoa butter 8 7i"3-73'i
Linseed oil 120 ^dS-Jo-^
from which he naturally concluded that the foetal fat maintained its accustomed iodine number quite unchanged, no matter what was the degree of unsaturation of the fatty acids entering the maternal body. Thiemich did not estimate the iodine value of the maternal fat, but assumed that it would vary closely with the food fed. In a later paper, however, he modified his conclusions somewhat, because he estimated simultaneously the iodine value of the maternal fat, and found that it did not change as much as he had expected, e.g. only from 30 to 50. Becker afterwards discussed the question further without adding anything to it. Wesson in 1926 using the method of bromine addition compounds, fed cod-liver oil to one set of pregnant rats, and ordinary butter to another set, and found, although the bromine number of the fed fats was extremely different, hardly any difference was to be seen in the foetal fat.
Cod-liver oil (bromine number 0-210) Butter (bromine number 0-0031)
This is probably the most reliable, as it is the most recent, work on this subject. Wesson suggested a choice between three possible explanations: (i) that the placenta has a selective action, rejecting all
Bromine number
of body fat
Foetal
0-021
Maternal .
0-042
Foetal
0-025
Maternal .
o-oii
fatty acids except those of a certain degree of saturation, (2) that the embryo can alter fats arriving at it by hydrogenation or dehydrogenation, or (3) that fatty acids do not pass the placenta at all, and that the embryo forms its suppHes from carbohydrate, or possibly from protein. In spite of the difficulties inherent in the third possibility, it was the one which Wesson himself considered most likely. For the further discussion of this problem, see the Section on placental permeability.
As regards the fat in the embryonic body of the mammal, Derman has stated, as a result of the employment of histochemical methods, more or less specific, that, up to the first month, the fat depots of the human embryo contain mostly neutral fat and cholesterol esters, less phosphatides and almost no free fatty acids, but, in the later stages, neutral fat and cholesterol esters only. For other histochemical work see Froboese and Berberich & Bar. Raudnitz found that the melting-point of human fat was higher the younger the body, thus:
8-5 month foetus
47-2
2 days after birth
43-8
I year
30-2
26 years ...
27-0
but no explanation is available for this phenomenon. Another curious observation, due to Dobatovkin, is that as growth proceeds the percentage of fatty acids liquid at room temperature increases. Thus, of human fat from the shoulder region :
Liquid
Solid
/o
%
At birth
52-7
39-6
6 years old ...
82-2
IO-7
The same fact, expressed more scientifically, is to be found in the data of Egg and of Jaeckele & Knopfelmacher, the latter of whom estimated the percentages of the three most important fatty acids as follows :
At birth
Adult
%
%
Oleic
65-04
86-21
Palmitic
27-81
7-83
Stearic
3-15
1-93
It is striking, from a comparative point of view, that the fat of the egg-yolk of the chick should contain only 5 or 6 per cent, of oleic acid. The data of Jaeckele & Knopfelmacher do not extend back
76-2
^94
into pre-natal life, but it may be presumed that the process is continuous.
An interesting investigation which involved several aspects of embryonic fat metaboHsm was made by Imrie & Graham, who estimated the amount of fat and its iodine value in the embryonic and maternal Hvers of guinea-pigs during gestation, using Leathes' method. Fatty infiltration of the liver in the non-pregnant animal is known to be due to the entry of tissue fat into the liver, for the iodine value characterises the fat normally present in the liver and in the other tissues, being high in the former and low in the latter. The question which Imrie & Graham set out to solve was ^ whether the embryonic liver | behaved in the same way, or ^ whether it evinced any special ^ physiological characteristics in its reactions to disturbances of fat metaboHsm. "It seemed reasonable to suppose", they said, ' ' that the embryonic tissue | might possess an avidity for 1 food material such as fat and ^ that this might evidence itself i» by a greater accumulation of fat in the embryonic than in the maternal liver, if the fat were mobilised experimentally." Obviously it was first necessary to estabhsh normal curves for the embryonic liver fat, and this in itself brought some interesting results, as may be seen from the curves plotted in Fig. 370. It was found that during development the guineapig embryo accumulates considerable stores of fatty acids in its liver. At first (up to body-weight 30 gm.) the percentage of fatty acids in the embryonic liver does not differ much from that in the maternal liver, but after that point the percentage in the former rises sharply to reach a maximum at about 80 gm. body-weight (roughly 65 days conception age), after which there is no further change until birth. Immediately upon birth, however, the fatty acid percentage drops
Fig. 370.
rapidly, and after 80 hours of post-natal life has reached a level very like that of the maternal liver. As the figure shows, the fatty acid concentration in the maternal liver is unaffected by gestation. "The livers obtained from young embryos", said Imrie & Graham, "resembled histologically and chemically adult hepatic tissue, whereas in embryos fully developed the liver was yellow in colour and showed chemically a large accumulation of fat. ... A fatty infiltration of a physiological kind occurs in the livers of embryonic guinea-pigs." The synchronous behaviour of the iodine value was very interesting, for, as Fig. 370 shows, it remained throughout the process in the neighbourhood of no, i.e. much above the iodine value of the embryonic connective tissue fat, which had an iodine value of about 85. The rises and falls on the curve are probably not significant, but the fat of the embryonic liver seems always to be a little less unsaturated than that of the maternal liver. The fact, however, that its iodine value was no precluded all possibility of the infiltration being one of tissue fat, as in the adult. It seems likely also that there is a slight decline in the iodine value of the embryonic liver fat; for embryos under 40 gm. it could be said to be generally iii, and above 40 gm. nearer 103 or 104. The difference between the average maximum and minimum fat values of the embryonic liver is 9-94 per cent. If we assume that this was fat coming from the connective tissue and having an iodine value of 85, and added to the 3-16 per cent, having an iodine value of 109, the resulting iodine value should be 91, but instead it is at its lowest 103. It would be very interesting to have a parallel set of data for the liver of the embryonic chick, for, as we have already seen, there are ocular evidences for a fatty infiltration there also.
It is generally admitted that the liver desaturates fatty acids brought to it, and that when it is infiltrated with connective tissue fat it falls behind in this work, with the result that the low iodine value is found. One must therefore conclude either that the liver of the embryonic guinea-pig can desaturate relatively much larger amounts of imported fat than is the case in extra-uterine life, or that the fat taken up by the embryonic liver is fat which has already been desaturated by the maternal liver or by some other tissue. As desaturation is the preliminary to combustion, Imrie & Graham's results raise the suspicion that fatty acids may play a more important part in the provision of energy for the mammalian embryo than has been supposed in the past. Imrie & Graham, however, in view of the sudden and rapid decrease in the Hver fat after birth, thought it likely that it was utilised for some special purpose then, which may well be the
• Embryonic o Maternal
They next proceeded to mobilise the fatty acids of the body by giving phloridzin and by subjecting the animals to a fast. The results were as shown in Fig. 371. Evidently phloridzin and fasting will produce a fatty infiltration of the embryonic liver. The graph also shows that treatment which will not very greatly affect the maternal liver will have a considerable effect on that of the embryo. From the data on the iodine value it can be seen that, simultaneously with the entry of fresh fat into the embryonic liver, the iodine value of the liver fat falls, just as would be expected if such fat was coming in from the other tissues. Here again the maternal liver is hardly influenced. Experiments similar to the foregoing were carried out on pregnant guinea-pigs with embryos over 40 gm. in weight, and from these it appeared that the physiological infiltration of about 15 gm.
Fig. 371. The normal data are here represented by continuous lines; the phloridzin data by points not joined together.
per cent, is by no means the maximum capacity of the embryonic liver, for by means of phloridzin the amount was raised to as much as 30-49 per cent, (wet weight). These experiments showed that phloridzin must pass the placenta, and make it highly probable that the physiological fatty infiltration is not derived from the tissue fat but some other source. Perhaps this other source is that postulated by Wesson in his explanation of the increasing fat-content of the embryonic body and the impermeability of the placenta to fatty acids.
One of the statements which has entered the literature without much justification is that embryonic cells (and carcinoma cells too) will not show fatty infiltration when treated with phosphorus. This was said to be the case by Hess & Saxl who made in vitro experiments on guinea-pig and rabbit tissues (liver and kidney) . Some years later doubt was thrown on this by Schwalbe & Miicke who could not confirm it, using very similar technique, and finally Rosenfeld, who worked with whole animals, found that foetal cells showed just as much fatty infiltration as adult ones.
% fatty acids % dry weight maternal liver foetal liver
Dog Normal 28-5 2-91
Phosphorus poisoning ... 40'0 io-o6
Whether carcinoma cells would also show fatty infiltration he did not try, but as he once obtained 2-3 per cent, of fat from a fiver metastasis in man, he was of the opinion that they would.
Cite this page: Hill, M.A. (2024, April 27) Embryology Book - Chemical embryology 2-11 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-11_(1900)
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
