THE RELATION OF THE LIPIDS TO PHYSIOLOGICAL ACTIVITY. · THE RELATION OF THE LIPIDS TO PHYSIOLOGICAL ACTIVITY. I. THE CHANGES IN THE LIPID CONTENT OF THE CORPUS LUTEUM OF THE SOW.*

THE RELATION OF THE LIPIDS TO PHYSIOLOGICAL ACTIVITY.

I. THE CHANGES IN THE LIPID CONTENT OF THE CORPUS LUTEUM OF THE SOW.*

BY W. R. BLOOR, RUTH OKEY,? AND GEORGE W. CORNER.

(From the Departments of Biochemistry and Anatomy, The llniaersity of Rochester School of Medicine and Dentistry, Rochester, New York.)

(Received for publication, January 17, 1930.)

The earliest work having to do with the physiology of the fatty sub- stances other than fat was done on the brain by men who were interested in a possible relationship between chemical composition and mental ac- tivity (1). The complex nature of the lipid material of the brain prevented much progress in the physiology but stimulated interest in the chemistry of the brain lipids, and much time and effort for many years was devoted to the attempt to separate and identify the lipids of the brain and other tis- sues. Owing to the difficulty of separation of the lipids from each other and from other substances a large number of compounds were listed, most of which had no real existence, with the result that the chemistry of the lipids was made to appear discouragingly difficult. Recent workers, especially MacLean and Levene have however succeeded in eliminating many of these hypothetical substances and in reducing the chemistry of the group to a relatively simple basis so that there has been recently a renewed interest in their significance in the living organism.

Probably because of their ready oxidation, the possible role of the unsaturated phospholipids in cellular oxidations attracted attention first of Koch (a), then Friinkel (3), and more recently Thunberg (4), Warburg (5), Meyerhof (6), and Hopkins (7). The net results of the work of these investigators, while not definitely proving the participation of the lipids in oxidative processes in the living cell, showed that it was very probable.

* The studies reported in this and the following paper were undertaken in the hope that the data obtained might be of interest in connection with two independent lines of research already under way in the laboratories of the different members of the present group. Since the results obtained would appeal to two widely different groups of investigators it seemed desirable to present the results obtained in two papers and to discuss the findings separately in terms of the two problems.

t On leave from the University of California. 291

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292 Lipids of Corpus Luteum

The similarity in composition between the phospholipids and the fats led to the belief that t.he phospholipids were stages in the metabolism of fat. This conception was especially attractive because the miscibility of lecithin with water seemed to offer a means of transport of the insoluble fats. Loew who was the first to state this hypothesis, believed that the phospholipid was a machine for burning the fatty acids. Following him Leathes (8) and a series of later workers succeeded in connecting the phos- pholipids with some, at least, of the changes undergone by the fat molecule in metabolism. In addition to the work of Leathes and his collaborators may be mentioned the findings of Bang (9) and of Bloor (10) concerning the increases in blood phospholipid during fat absorption, the results of Meigs, Blatherwick, and Cary (11) which indicate that milk fat is formed from the phospholipid of the blood, and the recent work of Sinclair (12) which shows that the fatty acids of the phospholipids of the intestinal mucosa and the liver are promptly changed by the fatty acids of absorbed fat and therefore probably have an important part in fat absorption. The participation of cholesterol in fat absorption, as shown by the increased amount present in the blood during the absorptive period, has been demonstrated by a number of investigators (13).

The conception of the lipids as essential constituents of protoplasm which activated the earliest group of workers in this field has been revived by Mayer and his associates, especially Schaeffer and Terroine. The contributions of this group of workers may be summed up as follows: (a) Cholesterol and lipid phosphorus (by inference phospholipids) constitute together an integral and constant component (element constant) of tissues, the amount of which is not essentially altered by extreme changes in the nutritional status of the animal. These substances are therefore essential protoplasmic constituents-parts of the machine-and not merely fuel. The percentage amounts of these constituents-their “concentration’‘-is also considered to be characteristic of the tissue. Neutral fat on the other hand is the variable element, not an essential constituent of tissues, and dependent upon the nutritional status of the animal. (b) The ratio cho- lesterol-lipid phosphorus, and in most tissues (those which do not contain much fat), the ratio cholesterol-total fatty acids is constant for and char- acteristic of the tissue.

Mayer, Rathery, and Schaeffer (14) have shown that the granules and filaments in cytoplasm called mitochondria by the histologists, are lipid in nature, and contain especially unsaturated phospholipid. Thus the considerable information accumulated with regard to the mitochondria has been made applicable more specifically to the lipids. Mayer’s conception was that these cellular elements were concerned especially with oxidation. Terroine and Belin (15) considered that the nature of the phospholipid fatty acids was also constant and characteristic, a finding which however has recently been questioned by Sinclair (12).

The conception of a constant and characteristic content of lipids and especially of phospholipids for each tissue, is supported by the work of Bloor (16) who made analyses of various beef tissues of which enough

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material was used that the constituents were separated, weighed, and analyzed by standard gross methods, and so avoided the necessity of making assumptions regarding the nature of the compounds. In general it was found that the phospholipid content of a tissue was characteristic of the tissue but that a considerable allowance (30 per cent) must be made for individual variations.

A balanced relationship between cholesterol and phospholipid and less definitely between cholesterol and total fatty acids in blood has been noted by several workers (17-19) and implied by the work of Hueck and Wacker (20) who found that a change in one of the lipid constituents of blood (fat, cholesterol, lecithin) resulted in corresponding changes in the others. Further evidence of a probable balance between these two substances is the fact that cholesterol and lecithin (phospholipid) have been found to be antagonistic in some types of physiological reaction. Thus the hemolysis of red blood corpuscles by lysolecithin is prevented by cholesterol (21). Mayer and Schaeffer (22) found that the red corpuscles of various animals were the more easily hemolyzed as the “coefficient lipocytique,” cho- lesterol-lipid phosphorus, was greater, also that the imbibition of water by tissues varied with this ratio.

Thus not only the concentration of these substances but the balance between them appears to be of physiological significance. What the significance of the balance is can only be guessed, but the phospholipid seems to be the positive factor concerned with these activities and with the general activity of the cell, and cholesterol, therefore, may be considered to be the negative or inhibiting factor. The final degree of activity of a cell would be determined by (a) the phospholipid content, (b) the phospholipid- cholesterol ratio. The phospholipid content would be a measure of the potential activity; the ratio would indicate the actual or resultant activity.

If we grant that phospholipids and cholesterol are important and integral constituents of protoplasm and concerned with its activities, the next point to be considered is the nature of their relation. Some examples of specific functions of the phospho- lipids; oxidation, reduction, participation in changes in fats, etc., are mentioned above. Very little is known with certainty about the physiology of cholesterol in spite of the great amount of work which has been done on it. In normal tissues it is present mainly in the free form, while the cholesterol esters are found in notable amounts only in blood plasma and in degenerating tissues such as sclerotic arteries. As noted above, it appears to occur in

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294 Lipids of Corpus Luteum

definite relationship to phospholipids in tissues, to act antag- onistically to the phospholipids in some reactions and to be closely connected to them and to the fats in fat metabolism. Cholesterol prevents hemolysis of red blood corpuscles by free unsaturated fatty acids, possibly by forming combinations with them and it is interesting that the fatty acids in the cholesterol esters of plasma (beef) are highly unsaturated (23). The sug- gestion of Hanes (24) that cholesterol combines with the fatty acids set free by decomposing lecithin offers a possible explanation of the accumulation of cholesterol esters in degenerating tissues and in blood plasma.

Since we may concede the essentially lipid nature of mito- chondria the following conclusions of Cowdry (25) regarding mitochondria may be included here as evidence connecting the phospholipids with general physiological activity.

“In the first place the association of abundant mitochondria [is] with in- tense protoplasmic activity. In cytomorphosis for example, they are especially numerous in the active stages of the life of the cell and they diminish with senility in both plants and animals. There is a sharp in- crease in mitochondria with regenerative activity and in compensatory hypertrophy andi n many other conditions. There is a distinct recip- rocal relationship between the amount of mitochondria and the amount of fat. Where there are few mitochondria, there is much fat and vice versa. It seems probable that normal variations in the amount of mitochondria are in some way dependent on variations in the respiration of the cells containing them.”

Mayer and Schaeffer, while they emphasize the constancy in content and relationships of the lipids in tissues, give convincing experiments (26) to show that the “normal” values may be changed as the result of changes in environmental conditions, from which the inference may be drawn that the observed con- stancy is the result of relatively constant environmental con- ditions. Thus they found that the amounts of lipid phosphorus in the livers and lungs of rabbits were first reduced, then greatly increased as the result of changes in metabolism produced by chilling and overheating. In dogs similar changes were produced, but in the liver only. They quote Richet’s data which indicate a correlation between the lipid phosphorus of livers of animals and their size, hence their body surface and total metabolism. Ex- pressed in mg. of lipid phosphorus per kilo of animal Richet’s

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values are as follows: beef 0.24, man 0.34, dog 0.41, rabbit 0.61, guinea pig 0.62, mouse 0.69. The variations in phospholipid therefore run parallel with intensity of metabolism. Mayer and Schaeffer (22) and Weill (27) have also presented data to show that the lipid levei in parenchymatous tissues is much lower and more irregular in cold blooded animals than in warm blooded animals and lower in hibernants when dormant than when awake.

As the result of analyses of livers of animals under normal and various abnormal conditions Theis (28) found that the normal liver had a higher phospholipid and lower fat content than ab- normal liver.

Studies on the lipid content of the more important tissues of a single animal led Bloor to the conclusion that the phospholipid content of a tissue was an expression or a measure of the extent and variety of the physiological activities of that tissue. Thus the liver had a higher phospholipid percentage than the kidney because it had a greater variety of physiological functions; the heart muscle had a higher content of phospholipid than the muscle of the thigh because it was more continuously and strongly active.

The term “physiological activities” as used here is thus meant to include all the processes of the living cell. Of these, oxidation is the basic one, upon which the others depend to the extent at least of their energy requirement, and as noted above oxidation is the function most emphasized in discussions of the mitochondria and the phospholipids. But it should be borne in mind that it is only one form of activity and perhaps not the most important except in muscle where energy transformations are quantitatively the most significant. Yet the muscles (except the heart) have the lowest phospholipid and cholesterol content of all the tissues. In other tissues such as the brain, liver, and kidney having a much higher lipid content, energy transformations are relatively less important and other activities than oxidation must obviously bear greater signficance in the consideration of the total physi- ological activity of most cells.

In the desire to examine more closely the relation of activity to lipid content of tissues, a tissue was sought which passed through a well defined cycle of changes from inactivity through a period of activity to inactivity again. The corpus luteum of the SOW

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296 Lipids of Corpus Luteum

appeared to be a suitable tissue, and it had the advantage that a complete histological study of its cycle of changes has been made by one of us, both in the non-pregnant and in the pregnant animal (29) as a result of which it is possible to date specimens accu- rately, in terms of the cycle or of the developing fetus. Moreover, because of the fact that there are several corpora lutea in the same animal at a time, sufficient material for a complete microanalytic study of the lipids can ordinarily be obtained from a single animal.

Regarding the corpus luteum, Fenger (30) found that it con- tained a relatively high content of phospholipid-about 15 times as much as muscle and about the same as that of other endocrine organs except the thyroid. Chauffard, La Roche, and Grigaut (31) found that the cholesterol content increased continuously during the metabolic cycle of this organ and they suggested that it was one of the places where cholesterol was formed or at least stored. A study of the lipids of human corpus luteum was made by Hermstein (32) and although the amount of available material was small and the difficulty of accurate dating great, his results agreed with those reported below, in that the highest content of phospholipids was found at the time of full activity.

EXPERIMENTAL.

The uteri and ovaries from freshly slaughtered sows were selected at once after removal from the animals and taken to the laboratory where samples of corpus luteum and endometrium were taken (a) for histological dating (b) for analysis of lipid content and distribution. The details of the histological study and method of dating were reported in a previous paper by one of us (29). It may be stated that the limits of accuracy of the dating on a 21 day cycle is about 1 day except toward the end of the interestrual period, when it may be 2 days. For chemical exami- nation the contents of the corpora lutea, mainly from the same animal, but occasionally from more than one, were shelled out of the capsules, weighed to mg., transferred to a mortar containing about 3 times their volume of clean sand and the whole ground to a fine mud. The material was transferred quantitatively to 200 cc. flasks for extraction, the last traces of tissue and sand being removed from the mortar with the help of 1 or 2 cc. portions of eater and rubbing with the pestle. About 150 cc. of 95 per cent

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Bloor, Okey, and Corner 297

alcohol were added to the flasks and the mixture boiled for 5 to 10 minutes after which the hot liquid was poured through a filter into 200 cc. graduated flasks. The extracted material, after the alcohol had been drained off, was reextracted several times by boiling out with small amounts of ether, enough ether being used to bring the volume of liquid in the volumetric flasks up to 200 cc. when cooled. From this mixed extract aliquot samples were taken for the determination of phospholipid, total fatty acids, and free and ester cholesterol. Methods for phospholipid (33)

FIG. 1. Lipids of corpus luteum.

and total fatty acids (34) have already been described in this Journal. The method for free and bound cholesterol used is new and depends on the oxidimetric measurement of the cholesterol- digitonin precipitate. A preliminary report of the method has already appeared (35) and a full description will be published soon. Comparative determinations by calorimetry were attempted but given up because of the presence of some substance giving an atypical color.

From the values so obtained the ratios phospholipid-free

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298 Lipids of Corpus Luteum

cholesterol, phospholipid-ester cholesterol, and phospholipid- total cholesterol were calculated. The value for neutral fat was obtained by subtracting from the value for total fatty acids, the

TABLE I.

Corpora Lutea. Non-Pregnant Animals. Results are expressed in per cent.

30-33 (corn- 0.2270.2400.4671.4

I I I I

3 1 6.11 5.811.161 Normal. 2 posite).

5 27-A 0.1940.1320.3261.4: 4.3 7.3 10.82.06 Hemor- rhagic.

6 35 0.2480.2420.4902.3( 4.4 8.3 9.52.23 Cystic. 7 X37 0.2640.2000.4642.3( 4.9 9.011.5 1.76 7 3 0.1730.2620.4351.4: 3.26 8.2 6.0 9 5 4.6 10.8 7.6 Partv,lost in

extrac- tion.

91 15 0.2580.2000.4582.3 5.8 9.011.50.48 11 26 0.2420.1170.3594.0 11.5 16.534 1.55 13 4 0.3110.2850.5964.4! 7.5 14.3 16.02.3 14 X36 0.3140.2380.5524.2 7.6 13.317.62.24 14 25 0.2490.2090.4584.0( 8.3 18.018.62.18 15 16 0.2230.1050.3283.11 9.7 14.230.2 1.8 161 22 0.4370.4720.9092.4! 0.6 5.6 5.12.19 16-1 23 0.3890.5450.9342.3 2.4 5.9 4.23.27 19 24 0.52 0.64 1.16 1.6f 1.4 3.2 2.67.6 192 x29 0.4720.5781.04 1.7! 1.7 3.5 3.03.73 19-2 28 0.3890.3240.7132.4 3.4 6.2 8.63.52 21 32-33 0.45 0.77 1.22 1.8 1.4 4.0 2.23.86 Corpus al-

bicans. 21-2 17 0.41 0.91 1.34 1.11 0.88 2.6 1.21.7 “ “ 24-2 27B 0.43 0.59 1.02 0.7. 0.69 1.6 1.20.1 “ “ 25-3 7 0.4381.75 2.19 1.0 0.45 2.3 0.6 “ “

Notes.

-- --

amount of fatty acid in combination in the phospholipid (assumed to be two-thirds of the weight), plus the amount of fatty acid in

combination with cholesterol as cholesterol esters 282 386 Or ap-

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Bloor, Okey, and Corner 299

proximately three-fourths of the weight of the ester cholesterol). This value of residual fatty acids may be multiplied by 1.05 to convert it to glyceride but probably nothing is to be gained by the conversion since the errors of all the determinations are carried by the residual fatty acid. The results are given in Tables I and II and shown graphically in Fig. 1.

TABLE II.

Corpora Lutea during Pregnancy.

Results are expressed in per cent.

;j

2 2 ; 0 d

a* 2 : .$ % 7 2 .t: 3 4 d z 8 2 5

.-

hl mm.

7-8 0 11 0.2770.1710.448 2.54 20 3 38 0.2130.2200.433 4.6 17-18 10 0.2890.0560.345 4.8:

32 9 0.1890.0940.283 3.1( 60 18 0.2360.1030.339 4.2 85 19 0.2220.1040.326 3.5: 90 1 0.3010.0700.371 3.7:

190 2 0.2400.1320.372 3.3 230 8 0.2460.1020.348 3.32

-- ---

150 (dead fetus). 12 0.2580.0140.272 3.51 160 “ “ 20 0.2980.00 IO.298 3.62

DISCUSSION.

5.6 9.C 10.6 14.3 13.6 16.i 10.4 15.5 12.7 17.: 10.8 16.C 10.6 13.1 8.9 13.8 9.5 13.E

--

12.9 13.6 12.1 12.1

15.0 3.4 20.9 1.25 86 0.00 32 1.38 40 2.33 34 2.13 56 0.93 25.4 1.43 33.0 2.0 ~-

106 1.77 03 2.48

The changes in lipid content of corpus luteum in this animal may best be followed if they are correlated with the changes in histological structure described by Corner (29). These are briefly as follows: After rupture of the graafian follicle and escape of the egg, the walls of the follicle collapse usually around a central blood clot. The layer of cells surrounding this central cavity then begins to enlarge, apparently by the taking up of material which contains considerable lipid. Within a week the corpora lutea have increased from a diameter of about 4 to 6 mm. to a diameter of 8 to 9 mm. If pregnancy ensues there is a further growth to

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300 Lipids of Corpus Luteum

10 to 11 mm. By the 7th day the corpora lutea are usually solid and their cells have become fully differentiated. On cutting the capsule the contents bulge out, are of a yellowish pink color, and in consistency are not unlike pig liver. It is a fact of the greatest importance that the changes which lead up to the formation of the corpus luteum take place with equal completeness whether impregnation has taken place or not. It is not possible to dis- tinguish the corpus luteum of pregnancy from the corpus luteum of unfertilized ovulation during the first 2 weeks after the dis- charge of the ova. Beginning on the 14th to 15th day a sudden change takes place in the corpus luteum of the unfertilized ani- mals. Within 2 or 3 days the diameter has decreased from 9 mm. to 6 mm.; its color has changed from the pink of active capillary circulation to the whitish tone of scar-tissue and its texture has become much tougher and firmer. Microscopic examination shows that a very rapid degeneration has taken place with complete breakdown of the cells (granulosa) which make up the bulk of the organ and collapse of the capillaries. By the time of the new ovulation (about 21 days) the corpora lutea have diminished in diameter to 6 mm., by the mid-estrual period (30 days) to 4 mm., and by the next ovulation to 2 mm., after which they disappear slowly. The changes may be summed up as follows: 2nd to 8th days rapid growth to full size with beginning function, 8th to 14th days slow or no growth with full functioning; then in the non-pregnant animal, 14th to 21st days retrogression and reabsorption, at first very rapid. In the pregnant animal the corpus luteum remains large and continues its function for a considerable period, perhaps throughout the whole period of gestation, although the diminution in the lipid phosphorus content as observed by Corner (36) may mean diminu- tion of function.

Changes in the Lipid Content.

Phospholipid (Lecithin and Cephalin) .-The changes in phos- pholipid content follow quite closely the gross and histological changes and the functional changes noted above. In the early days the percentage increase is small, then from the 5th to the 10th day the increase is great, while from the 10th to the 14th day, during the period of active function of the gland, there is very little

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percentage change. From the 14th to the 17th day in the non- pregnant animal the decrease in phospholipid percentage is very rapid, running parallel to the retrogression as observed histologi- cally. After that the decrease is slower. If an animal becomes pregnant the phospholipid content of the corpus luteum does not fall on the 14th day. There is apparently an increase up to the time of implantation (17th to 18th day) after which time the phospholipid percentage falls somewhat and then continues at a level slightly below the maximum during as much of the period of pregnancy as our specimens cover. The functional activity and phospholipid content of the gland are both greatest from the 8th to the 14th day in the non-pregnant animal and from the 8th day throughout the gestation in the pregnant animal. The correlation between functional activity and phospholipid percentage is thus seen to be close. The statement of Corner (29) “that by the 7th day the corpus luteum is solid and the cells fully differentiated” apparently does not quite fit in with the facts found about phos- pholipid, which does not reach its highest values until on or after the 8th day. The discrepancy may however be explained as due to the fact that the cells, although of full size, do not at once reach their maximum content of phospholipid. The percentage of phospholipid in the active gland is about the same as in the liver, perhaps a little higher, which indicates that the corpus luteum may be one of the potentially most active glands of the organism. This high content of phospholipid has been found by Fenger (30) to be characteristic of the endocrine glands with the exception of the thyroid.

Cholesterol (Free Cholesterol).-The percentage of free cho- lesterol in corpus luteum rises slowly up to the 18th day, then in the non-pregnant animals remains at a fairly constant level, while in the pregnant animals it falls somewhat, and continues through- out pregnancy at about the level found at the 10th to the 14th day. The rise during the development and active life of the gland is much less marked than that of the phospholipid, the maximum difference between high (0.45 per cent) and low (0.27 per cent) values being about 66 per cent of the low value while for phospho- lipid the difference is about 300 per cent.

Bound or Ester Cholesterol.--In the non-pregnant animals the percentage of ester cholesterol in the corpus luteum remains

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Lipids of Corpus Luteum

practically the same up to the 14th day, then rises rapidly with the retrogression of the gland. In the pregnant animals the per- centage of bound cholesterol is at first the same as in the non- pregnant corpus luteurn, then at the end of the 3rd week it falls to a low level and remains so low throughout pregnancy that in some cases it is a question whether any at all is present. In the two samples where there were dead fetuses which were being absorbed (Samples 12 and 20) the ester cholesterol was practically absent.

Total Cholesterol.-As was found by Chauffard, La Roche, and Grigaut (31), total cholesterol increases in percentage through the non-pregnant cycle of the corpus luteum. In the period up to and including the time of maximum size and activity of the corpus luteum the increase is slow and due to free cholesterol. During the degeneration the percentage increase is rapid, probably because the cholesterol ester is less readily absorbed and is left behind while the remaining material of the gland is being absorbed (see below).

The notable facts in connection with the choIestero1 in this gland are (a) the constancy of the free cholesterol percentage while the gland remains active, (b) the abrupt rise in ester-bound cholesterol during retrogression. These findings are taken to mean that the free cholesterol percentage is relatively constant in the growing and active gland, and that free cholesterol is supplied by the blood as needed for growth. During retrogression the free cholesterol is either removed or combined with fatty acids which may originate in the lecithin. The failure of the free cholesterol percentage to keep pace with the rapidly rising cholesterol-ester percentage during the period of degeneration may indicate either of these possibilities. The possibility of a deposition of esters from the blood such as Schijnheimer (37) finds in arteriosclerosis seems un- likely from a consideration of the change in size of the corpus luteum during about the week’s time after retrogression begins. According to Corner’s figures the change in diameter is from 9 mm. down to 6 mm. corresponding to a change in volume of from 382 c.mm. to 113 c.mm., or to about 1:3.37. The increase in ester cholesterol percentage is from about 0.2 to 0.7 or about 3.5 times. This would indicate that no deposition from the blood need be assumed, and that the cholesterol and cholesterol esters

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found there were residual. The ester cholesterol is thus seen to be low in the corpus luteum during growth, pregestational activity, and in pregnancy (and in the endometrium during its whole life). Its percentage is high in the degenerating corpus luteum ap- parently because it is left behind during the process of reabsorp- tion. These findings are in agreement with the statements in the literature to the effect that cholesterol esters are present in normal tissue only in small proportions except in the adrenals and the blood plasma. Their presence in considerable amounts in tissues appears to be associated with retrogression in which case they may be either residual or the result of deposition from the blood. Low cholesterol-ester percentage appears to be a sign of activity, high cholesterol-ester a sign of inactivity or retrogression.

Neutral Fat.-Neutral fat is a derived value obtained by sub- tracting the fatty acids combined in phospholipids and cholesterol esters from the total fatty acids. The value so obtained although it includes small amounts of other fatty acid compounds, is mainly fat. In the non-pregnant corpora lutea as shown by Tables I and II, the percentage of neutral fat is variable but low up till the time of retrogression. Then it increases markedly up to about the 21st day, after which it falls off sharply. The increase prob- ably means that during early degeneration the reabsorption of fat is less rapid than that of some of the other constituents of the degenerating tissue so that the percentage increases. Later, absorption takes place, as shown by the drop in values of fat after the 21st day, while the percentage of cholesterol esters, probably because of less ready absorption, continues to increase. During pregnancy the fat content of the corpus luteum is variable but relatively low.

The corpus luteum has not proved altogether ideal for the study of possible relations between function and lipid content, for the reason that growth and assumption of function and still more the cessation of function and retrogression take place so rapidly. ,Also the retrogression is really a degeneration followed by ultimate disappearance. The rapidity of the changes has undoubtedly resulted in overlapping of stages in the number of animals which it was necessary to use, and this would explain the divergence of values for given days in the cycle. Nevertheless the results obtained support to a satisfactory degree the hypothesis that the

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304 Lipids of Corpus Luteum

phospholipid content of a tissue is a function of its activity. As the corpus luteum grows and becomes active, the percentage content of phospholipid increases until at the time of functioning the percentage is at its highest. This high content persists if pregnancy ensues with consequent continued functioning. If pregnancy does not ensue and functioning ceases, the fall in phospholipid is rapid, until in the gland remnant the content is less than one-fourth of what it was at full activity.

The only other lipid that compared with phospholipid in rapidity and extent of change was the cholesterol ester. But the increase in percentage of this substance occurred only during the degenera- tion and probably largely because it was left behind in the absorp- tion of the gland material, although possibly also because there was new formation from the free cholesterol and the fatty acids from the disappearing phospholipid. During pregnancy the cholesterol ester content of the corpus luteum was much below what it was in the non-pregnant condition while the free cholesterol was higher. The relation of cholesterol ester to functional activity in this organ was thus negative; its percentage decreased with the activity of the gland and increased with its retrogression, With the de- crease of cholesterol esters during pregnancy the free cholesterol in- creased, indicating that the active gland may keep the cholesterol stripped of fatty acids. The same increase in phospholipid and decrease in cholesterol ester content with activity was noted but to a lesser degree in the endometrium (see following paper). In this organ cholesterol esters were almost lacking during the whole cycle probably because the proportion of degenerating cells in the endometrium of the pig is never great.

The free cholesterol percentage in corpus luteum was about 50 per cent higher during the period of growth, assumption of func- tion, and throughout pregnancy than it was at t,he beginning, so that its changes do not compare in extent with those of the phospholipid, and its relation to function is therefore not so definitely shown. It may be noted, however, that the percentage content is relatively high compared with that of the liver and kidney.

Before the simple relation of activity to phospholipid and possibly cholesterol content can be accepted certain other factors must be taken into account. These are the relation of the lipids

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to growth of the organ and to the hormone formation. Regarding the first of these, the period of growth, according to histological evidence, is usually complete by the 7th day while the increase in percentage of phospholipid continued until about the 13th day, being then nearly double what it was at the 7th day. If pregnancy ensued the high values persisted. The relation of the phospho- lipid to the functioning of the gland rather than to growth thus seems definite.

With regard to the relation of the phospholipid to the hormone nothing can be said since the nature of the corpus luteum hormone is not known.

SUMMARY.

The percentage content of phospholipid of the corpus luteum of the sow has been found to vary markedly with the activity of the gland, being 2 to 3 times as high during the period of activity (previous to estrus and during pregnancy) as at the time of formation or after retrogression. Free cholesterol increases also up to and during the period of active functioning but to a much smaller extent.

Cholesterol esters were found to vary inversely with the activity of the gland, a high content being characteristic of the degenerated organ.

These results support the hypothesis that the phospholipid content of this tissue is a function of its physiological activity. The relation of free cholesterol to activity is less clear, but appears to be similar to phospholipid. ‘Cholesterol esters on the contrary seem to be related to inactivity or retrogression.

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CornerW. R. Bloor, Ruth Okey and George W.

THE CORPUS LUTEUM OF THE SOWCHANGES IN THE LIPID CONTENT OF

PHYSIOLOGICAL ACTIVITY: I. THE THE RELATION OF THE LIPIDS TO

1930, 86:291-306.J. Biol. Chem. 

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