THE LIPIDS OF THE PIG DURING EMBRYONIC DEVELOPMENT

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1 THE LIPIDS OF THE PIG DURING EMBRYONIC DEVELOPMENT BY WILLIS A. GORTNER* (From the Department of Biochemistry and Pharmacology, School of Medicine and Dentistry, The University of Rochester, Rochester, New York) (Received for publication, March 21, 1945) The bulk of the literature on embryochemical growth has been devoted to changes in the embryonic chick and fish. Unfortunately the unique position of the mammalian fetus, the only one not developing in a closed system, prevents any generalization of conclusions drawn from observations on the eggs of these lower forms of animals. Parallel growth changes are infrequent even between different mammalian species, since the placenta in one may offer a set of barriers to the passage of a particle which freely crosses the placental membranes in another species of mammal. The human placenta, for example, interposes three barriers between the fetal and maternal bloods, while in the pig no less than six membranes are interposed between the two circulatory systems. The only paper approaching completeness in dealing with the evolution of the various lipids in the mammalian fetus is that in which Boyd (5) studied the role of the placenta in the transfer of lipids to the rabbit fetus. For the purpose of the present study the pig fetus was selected as offering unique possibilities for an analysis of lipid development. Since the pig placenta offers a maximum number of barriers to the passage of particles from the maternal blood to the fetus, one might expect the fetal pig to exhibit a greater ability to synthesize its tissue components than is the case with other mammals, such as man and the rabbit. The complexity of the placenta in the pig, together with the pooling of fetuses from different litters for the various analyses, should reduce the effect of dietary variations in the mother. Methods of Analysis The pig has a gestation period of from 110 to 120 days, or approximately 4 months (17, 20). During the first 3 weeks, the fetuses are so small (15 mm. or less) that it has not been possible to obtain sufficient numbers for analysis. The period of most rapid growth rate, i.e. the 2nd and 3rd months, has been divided into approximately 5 day periods, the fetuses falling within a given age range being pooled until a sufficient number had been obtained for analysis; older fetuses were classed into groups with a 10 day age range. The samples were thus assigned to one of the following * Present address, School of Nutrition, Cornell University, Ithaca, New York. 135

2 136 FETAL LIPIDS groups (the figures representing the crown-rump length of the fetus in mm.) : 16 to 30, 31 to 50, 51 to 60, 61 to 80, 81 to 100, 101 to 110, 111 to 120, 121 to 140, 141 to 160, 161 to 175, 176 to 190, 191 to 200, 201 to 230, 231 to 260. Since the length of the pig fetus has been correlated with the intrauterine age by various workers (17, 20, 21, 23), it appears safe to conclude that fetuses of the same length are in the same phase of embryonic development. The specimens were collected within a half hour of the death of the mother, and usually showed life as exhibited by the fetal heart beat. The uterus was opened, the fetus dissected free from attaching membranes, dried with filter paper or gauze pads, and the crown-rump length recorded. After an immediate weighing, the abdominal and cranial cavities were opened to insure access of solvent to the tissues, and the specimens were placed in 95 per cent ethyl alcohol for dehydration and extraction. The collections were brought to the laboratory, ground to a fine pulp in a food chopper, care being taken to rinse all particles back into the flask with additional alcohol, and allowed to stand at room temperature for a minimum of 24 hours in an amount of alcohol at least equal to that of the volume of pulped tissue. The tissue was then exhaustively extracted with alcohol and ether and the combined extracts concentrated in vucuo at 40 to a small volume. This concentrate was in turn repeatedly extracted with redistilled petroleum ether. The clear petroleum ether solution, containing only the lipids, was made up to a suitable volume in a volumetric flask, from which aliquots were taken for the various analyses. The water-alcohol layer remaining after extraction with petroleum ether was evaporated to dryness and the caramel-like residue weighed. This tar, containing proteins, salts, carbohydrates, etc., soluble in the earlier, more watery extracts, was present in sufficient amounts to necessitate including it in calculating the dry weight of the fetuses. The extracted tissue was weighed after removal of the remaining solvent. This weight, plus the sum of the weights of the tar and the lipid, gave the dry weight of the sample from which the per cent of water in the fetuses was calculated. Most of the various lipid analyses were run in triplicate or quadruplicate (occasional determinations were run in duplicate only). Tot& lipid was determined gravimetrically on approximately 50 mg. samples. Free fatty acids were determined on these lipid samples by adding 10 ml. of hot, neutral 95 per cent alcohol and titrating with standard 0.05 N NaOH to an cr-naphtholphthalein end-point. The calculations for free fatty acids were made on the assumption (upheld by iodine number and molecular weight determinations) of a mean molecular weight equal to that of oleic acid (mol. wt ).

3 W. A. GORTNER 137 Unsaponijiable lipids were determined gravimetrically on 50 mg. samples according to the procedure recommended by Kelsey (16). The total and free cholesterol fractions were determined by the method of Kelsey (15). Phospholipids were determined by three methods, each focusing itself on either a different part of the phospholipid molecule, viz the phosphorus (18) or the fatty acids (2), or on the intact molecule (3). The comparative data for the three methods have been previously reported (9). The averages of the values obtained by the three methods have been used in the present paper. Iodine numbers were determined on the total and phospholipid fatty acids by the method of Yasuda (25). DISCUSSION The results of the analysis of twenty-six groups of pig fetuses, representing 438 fetuses from 66 litters, and ranging from 17 to 254 mm. in length, are presented in Tables I and II. Water-The water content of the fetuses at different developmental stages shows a decided downward trend throughout the gestation period, the most rapid changes occurring before the 50 mm. and after the 200 mm. stages. Wilkerson and Gortner (24) have correlated the changes in the water content of the pig fetus with anatomical differentiation in the fetal kidneys. These authors secured data indicating a rapid fall in water content at the 15 mm. stage, at which level it remained constant until the fetus reached a length of 160 mm. The values reported here show a more gradual initial fall, and give no indication of an equilibrium in water content between the 15 and 160 mm. stages; instead, they show a small but steady drop in the per cent of water during this period. Total Lipid-The lipid fraction remains constant during a considerable part of the gestation period. Calculated in terms of the entire fetal weight, the lipid content remains at slightly over 1 per cent until the last month of fetal life, at which time it rises slightly to a value of 1.4 per cent near term. This behavior of the lipid is in striking contrast to that observed in other mammals. The fetuses of rabbits (5, 8), dogs (19), and man (8, 14) exhibit a sharp rise of 300 to 800 per cent in the lipid content during the latter part of the gestation period. Of interest is the close parallelism of the nitrogen and lipid contents of the pig fetus. Wilkerson and Gortner (24) found the protein nitrogen to be constant after the 50 mm. stage; on a dry weight basis, total lipids remain at a constant level after the 60 mm. stage, indicative of a fairly rigid ratio of these substances in developing tissues. Free Fatty Acids-The data for free fatty acids in developing pig fetuses as presented in Table II indicate that unesterified fatty acids are present

4 138 FETAL LIPIDS in considerable amounts, especially in the earlier periods of prenatal life. The very definite trend in the free fatty acid content during embryonic development, together with the precautions followed to avoid autolysis, TABLE I Changes in Size and Water Content - of Pig - during Embryonic Development Estimated conception No. of fetuses No. of litters Water age of fetus - mm. duys km. per cent * * content * Samples from the same litter. minimize the possibility that these acids are artifacts arising from the decomposition of some other lipid. The presence of considerable amounts of fatty acids in unesterified form in the very small fetuses suggests the possibility that certain enzymes asso- 1 It is interesting to note the similarity in trends for free fatty acids and for basic nitrogen in terms of tissue solids. The basic nitrogen falls rapidly until the 40 to 50 mm. stage, after which it remains relatively constant (24). It is impossible to say whether the similar behavior of these two groups of compounds is other than coincidental. That the free fatty acids are in some way associated with the basic nitrogenous substances, however, seems probable. The existence of free fatty acids as

5 W. A. GORTNER 139 ciated with lipid metabolism are absent (or of very low potency) in the young fetuses, and are formed (or assume function) at about the 25 mm. stage. TABLE II Lipids of Pig at Various Stages of Embryonic Development The concentrations of the lipid fractions are expressed as per cent of the dry weight of the fetus. mm. Total lipid _- Free fatty acids.- P U nsaponi. fiable lipids s * Average of three methods of determination (cf. (9)). t By difference. $ Based on oxidation values for the fatty acid solutions. $ Samples from the same litter Cholesterol ;lyceiidest i- Total 2% Iodine No.$ hosphol.pld fatty acids Phospholipids-The average values for phospholipids, when calculated as per cent of the wet weight of the fetus, show little change at any time salt complexes with basic amino acids seems more logical than the presence of uncombined fatty acids as such in the tissues.

6 140 FETAL LIPIDS during the gestation period. This is quite different from the trend in the fetal rabbit (5), in which the concentration of phospholipids increased throughout pregnancy, a 5-fold increase being effected during the period studied. Calculated on either a wet or dry weight basis, the phospholipids of the fetal pig are at a maximum during the early stages of intrauterine development. Unsaponijiable Lipids-The total unsaponifiable matter, as determined gravimetrically, was found to contain variable amounts of digitoninprecipitable sterols (55 to 76 per cent, calculated as cholesterol). In general the trend is toward a higher ratio of sterol to non-sterol unsaponifiable lipids as the fetus becomes more developed. Expressed as per cent of the dry weight of the fetus, the values for unsaponifiable lipid decrease during the developmental period to less than half the original value, falling on a smooth curve which shows the change to be more rapid during the early stages of embryonic growth. The cholesterol, as one of the constituents of the unsaponifiable material, exhibits a similar trend. The total and free cholesterol contents parallel, at lower levels, the changes in unsaponifiable matter. The bound cholesterol, while distributing itself over a fairly wide range of values, shows no change during fetal growth. While the latter is entirely in agreement with the findings of Boyd (5) on rabbit fetuses, the trend for free cholesterol is quite different. In the pig, little change is evident in the free cholesterol content of the moist fetal tissues during intrauterine development. In the rabbit fetus, however, the free cholesterol doubles in concentration during embryonic growth, increasing particularly in the early and in the very late stages. This again emphasizes the difficulty of generalizing in interpreting data on fetal composition without due regard to the type of placenta linking the fetus to the mother. At no time does there appear to be any appreciable demand or tendency for cholesterol to appear in ester form, an average of only 1 out of every 8 cholesterol molecules being in the bound fraction. In general, only small amounts of cholesterol esters are found in active tissues, the content varying inversely with the physiological activity of the organ (4, 12, 22). The low ester cholesterol values2 in the actively growing fetuses are in harmony with these findings. Neutral Fat- Glycerides were determined by difference, being that portion of the total lipid not accounted for as phospholipid, free fatty acid, unsaponifiable lipid substance, or cholesterol ester fatty acid.3 Since 2 Harms (10) notes that, while the liver of the fetal dog contains large amounts of anisotropic lipoid substance (cholesterol esters), he was unable to find such substances in the liver of the pig fetus in any of the stages of development. 3 Fatty acids combined as cholesterol esters were assumed to have a molecular weight of 283.

7 Vi. A. GORTNER 141 certain assumptions as well as analytical errors are necessarily made in calculating the amounts in each of these four fractions, the data for the glycerides cannot be considered other than as relative values. The scattering of the data is undoubtedly greater than the true fluctuations of the neutral fat; the trend, however, is readily apparent. The glycerides remain at a very low level until approximately the middle of the gestation period. After the 100 mm. stage is reached, the glycerides increase steadily. This increase in neutral fat during the last half of the gestation period is attained more gradually than the huge increase found in the fetal rabbit (5), and the absolute values are far lower in the pig, which shows less than half as much infiltrated fat as is found in the rabbit near term. In the rabbit, the curve for neutral fat has leveled off before parturition; in the pig, the peak has not been attained at term. This is confirmation of the observation of Engel and Bode (7) that pig fetuses have a minimum enrichment of fat stores before parturition, in contrast to the full depots of the rabbit (5) and man (8). Iodine Numbers of Fatty Acids-In Table II are listed the values for iodine numbers of the total and phospholipid fatty acids. The majority of the values for both fractions lie between an iodine number of 60 and 110, the average value for both the total and the phospholipid fatty acids being 82. In neither case is there any indication of a shift toward either a greater or lesser degree of unsaturation during progressive stages of fetal development, despite the fact that the total fatty acid fraction is made up of 70 per cent phospholipid and 25 per cent free fatty acids at one stage, and 45 per cent phospholipid and 45 per cent glyceride fatty acids at another stage of embryonic growth. The fatty acids have an average unsaturation equivalent to one double bond; the average analyses (iodine number 82 f 3.1, molecular weight 283 f 2.7*) compare closely with the values for oleic acid (90 and 282, respectively). The iodine numbers of the fatty acids in the fetus differ appreciably from those found in adult tissue. In general the fatty acids bound to phospholipids in adult tissue are considerably less saturated than those on the glyceride molecules; the phospholipid fatty acids of the fetal pig have an iodine number similar to the fatty acids on the other lipid components. In the present study, an average iodine number of over 80 for the glyceride fatty acids is indicated, a value well above the reported average of 63 for the back fat of the sow (1,13), which is representative of the entire body fat of the pig (6). This is in line with the observation of Hankins and Ellis (11) that the body fat of immature hogs is usually soft, becoming firmer as they approach maturity. 4 Mean (3~ standard error) of triplicate titrations of the fatty acids of ten lipid samples.

8 142 FETAL LIPIDS SUMMARY The lipid fractions of 438 fetuses, representing 66 litters and covering 70 per cent of the gestation period of the pig, have been investigated. The water content of the pig fetus exhibits two rapid falls during growth, a phenomenon previously correlated with changes in the fetal kidneys. The total lipid and the lipid to protein ratio remain constant for a large part of the embryonic growth period. Evidence is presented that a considerable portion of the non-phospholipid fatty acids, often considered neutral fat, is actually present in unesterified (free) form. On a dry weight basis, the phospholipid content is at a maximum in the very young fetus, which has twice as much of this lipid as does the fetus at term. The phospholipid fatty acids, in common with the other acid fractions, have an average iodine number of 82. The unsaponifiable lipids in the dry solids progressively decrease in their percentage content during embryonic growth, the total and free cholesterol fractions roughly paralleling this fall. At no time is there any notable tendency for cholesterol to appear in ester form. The fetal glycerides gradually increase beginning about the middle of the gestation period, but even at term they account for only a minor part of the total lipid substance. Considerable differences exist in the development of the lipids in the fetal pig in comparison with the fetal rabbit. BIBLIOGRAPHY 1. Banks, A., and Hilditch, T., Biochem. J., 26, 298 (1932). 2. Bloor, W. R., J. Biol. Chem., 80, 443 (1928). 3. Bloor, W. R., J. Biol. Chem., 82, 273 (1929). 4. Bloor, W. R., Okey, R., and Corner, G. W., J. Biol. Chem., 66, 291 (1930). 5. Boyd, E. M., Biochem. J., 29, 985 (1935). 6. Ellis, N. R., and Isbell, H. S., J. Biol. Chem., 69, 219 (1928). 7. Engel and Bode, Monatschr. Kinderh., 8, 618 (1909). 8. Fehling, H., Arch. Gyniik., 11, 523 (1877). 9. Gortner, W. A., J. Biol. Chem., 169, 97 (1945). 10. Hanes, F. M., J. Exp. Med., 16, 512 (1912). II. Hankins, 0. G., and Ellis, N. R., U. S. Dept. Agr., Bull (1926). 12. Haven, F. L., Am. J. Cancer, 29, 57 (1937). 13. Hilditch, T., and Stainsby, W., Biochem. J., 29, 90 (1935). 14. Iob, V., and Swanson, W., Am. J. Dis. Child., 47, 302 (1934). 15. Kelsey, F. E., J. BioE. Chem., 12 7, 15 (1939). 16. Kelsey, F. E., J. Biol. Chem., 130, 187 (1939). 17. Koch, M. L., J. Biol. Chem., 14, 267 (1913). 18. Kuttner, T., and Lichtenstein, L., J. BioZ. Chem., 86, 671 (1930). 19. Liesenfeld, F., Dahmen, H., and Junkersdorf, P., Arch. ges. Physiol., 216, 712 (1927).

9 W. A. GORTNER Mendel, L. B., and Mitchell, P. H., Am. J. Physiol., 20, 81 (1907). 21. Mendel, L. B., and Leavenworth, C., Am. J. Physiol., 20, 117 (1907). 22. Okey, R., Bloor, W. R., and Corner, G. W., J. Biol. Chem., 86, 307 (1930) 23. Warwick, B. L., J. Morphol. and Physiol., 46, 59 (1928). 24. Wilkerson, V., and Gortner, R. A., Am. J. Physiol., 102, 153 (1932). 25. Yasuda, M., J. Biol. Chem., 94, 401 ( ).

10 THE LIPIDS OF THE PIG DURING EMBRYONIC DEVELOPMENT Willis A. Gortner J. Biol. Chem. 1945, 159: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1