Fatty Acid Composition of Embryonic Fat Organ Lipids

1851

CORN OIL AND EGG WEIGHT

lege of Agri., Storrs, Conn. Shutze, J. V., L. S. Jensen and J. McGinnis, 1958. Effect of different dietary lipids on egg size. Poultry Sci. 37: 1242. Shutze, J. V., L. S. Jensen and J. McGinnis, 1959. Further studies on unidentified nutritional factors affecting egg size. Poultry Sci. 38: 1247. Shutze, J. V., L. S. Jensen and J. McGinnis, 1962. Unpublished data, Washington State University. Snedecor, G. W., 1956. Statistical Methods, Iowa State College Press, Ames, Iowa. Treat, C. M., B. L. Reid, R. E. Davies and J. R. Couch, 1960. Effect of animal fat and mixtures of animal and vegetable fats containing varying amounts of free fatty acids on performance of cage layers. Poultry Sci. 3 9 : 1550-1555.

Fatty Acid Composition of Embryonic Fat Organ Lipids GERALD L. FELDMAN, HALDOR T. JONSSON, THOMAS W.

CULP

AND RAMONA H . GOWAN Biochemistry Section, Division of Ophthalmology, Baylor University College of Medicine, Houston, Texas and, Metabolic Endocrine Research Department, The Methodist Hospital, Houston, Texas (Received for publication April 9, 1962)

' n p H E PERIODICITY in the deposi•*- tion of lipid material in the fat organs of the embryonic chick has been previously reported in an earlier paper from the author's laboratory (Feldman et al., 1962a). The accumulation of lipid material in the fat organ was very rapid during the period of the thirteenth to the seventeenth day and was probably due to the transport of yolk lipids to the fat organs by means of serum lipoproteins. The metabolic change that occurs after this period includes the synthesis of triglyceride by the fat organ; and, in addition, the carbohydrate and amino acid metabolism is profoundly altered (Feldman et al., 1962b). It is not unreasonable to assume that if triglyceride synthesis did occur at approximately seventeen days of incubation, the

event would be marked by a change in the fatty acid content of the tissue. Furthermore, these changes in fatty acid content would be apparent in the phospholipids also undergoing periodic changes. To test the hypothesis, the fatty acids from the triglyceride and phospholipid fractions of embryonic fat organs were studied by gasliquid chromatography. The results of this study provide the basis for the present report. EXPERIMENTAL PROCEDURE Preparation of Fatty Acid Esters. The triglyceride and the four phospholipid fractions obtained in an earlier study were used as a source for the fatty acids. Samples of these fractions, providing a maximum of 20 mg. of lipid material, were placed in 13 X 100 mm. screw top tubes

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tion studies in the mature fowl. 2. Effect of soybean oil meal, organic and inorganic fractions of corn on production, fertility, and hatchability. Poultry Sci. 40: 1299-1305. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Harms, R. H., and P. W. Waldroup, 1961. The effect of composition of diet upon weight of eggs. Poultry Sci. 40: 564-567. Jensen, L. S., J. B. Allred, R. E. Fry and J. McGinnis, 1958. Evidence for an unidentified factor necessary for maximum egg weight in chickens. J. Nutrition, 65: 219-233. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal number of replications. Biometrics, 12: 307-310. Potter, L. M., and L. D. Matterson, 1960. The metabolizable energy of feed ingredients for chickens. Progress Report 39, Exper. Sta., Col-

1852

G. L. FELDMAN, H. T. JONSSON, T. W. CULP AND R. H. GOWAN

sample of the esters was injected into the machine with a Hamilton syringe in each case. RESULTS AND DISCUSSION

The data obtained in the present study may be considered with two thoughts in mind. The first consideration should be of the fatty acids which occur in each lipid fraction, as compared with the same lipid from other sources. The second point for consideration is the relationship of fatty acid composition to the metabolic changes associated with embryonic development. Triglycerides. Most of the earlier data of adipose tissue fatty acids are based on the total lipid extracted from the tissue. However, the fat extracted is largely triglyceride and hence most of the comparisons are germane to the topic of triglycerides. The fatty acids of embryonic fat organ triglycerides are distributed almost equally between saturated and unsaturated acids (Table 1). The C-16 and C-18 acids predominate in the former and the monounsaturated C-18 acid in the latter. The triglyceride fatty acids are relatively constant in composition throughout the development of the fat organ. Although day to day fluctuations are observed, they are too minute to be related to the very marked changes in lipid content occurring in development (Feldman et al., 1962a). Only a limited number of fatty acids have been reported to occur in the depot fats of animals (Deuel, 1951) and these are profoundly altered by diet. Generally, the C-16 and C-18 saturated acids and the C-18 unsaturated acids predominate. Feigenbaum and Fisher (1959) fed five different oils to hens and studied their effect on body fat fatty acids. Except in the case of coconut oil, the general effect was deposition of approximately twice as much unsaturated as saturated fatty acids. There is little similarity in the distribution

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with Teflon-lined caps. The samples were evaporated to dryness under a stream of nitrogen and then redissolved in a few drops of benzene. A methylating agent was freshly prepared by carefully adding 6 ml. of concentrated sulfuric acid to 94 ml. of absolute methanol. One ml. of this mixture was added to each lipid tube, the tubes were loosely capped and then warmed in a water bath at 65-70°C. for 10 minutes. This step is necessary to expand the air in the tube because the following step requires the tubes to be tightly capped and placed in an oven at 65-70°C. overnight. After the warming period, the caps were loosened and the reaction was stopped by adding 1 ml. of distilled water. The esters were then extracted with 1 ml. of hexane. Using a capillary pipette, the hexane extract was removed and transferred to a clean, dry test tube and dried over sodium sulfate for 30 minutes. The dried extract was then quantitatively transferred to a clean, dry, pre-weighed test tube, the hexane evaporated off under a stream of nitrogen, and the remaining esters weighed. Finally, hexane was added to make a 2% solution. Chromatography of the Esters. The fatty acid esters were separated with a BarberColman model 15 gas chromatograph with argon ionization detection. Chromosorb W (80-100 mesh) coated with 17% ethylene glycol succinate (Applied Science Laboratories, State College, Pa.) was used as the packing for an 8 foot glass column. The resolution of random samples was checked with a second column packed with the same support coated with 8% ethylene glycol adipate. The operational conditions for these columns were: Column temperature 182° to 195°C, flash heater temperature 270° to 280°C, detector temperature 245°C, argon pressure 25 to 30 pounds per square inch, and a flow rate of 40 to 70 ml. per minute. A 5 microliter

1853

EMBRYONIC FAT ORGAN LIPIDS

TABLE 1.—Fatty acid composition of the triglyceride fraction offal organ lipids from the developing chick embryo Days of incubation 14 Saturated Acids C-14 C-16 C-18 C-20 Total

Total Unidentified Components**

16

17

18

19-20

21

1.2% 35.2 12.3 0.5

1.1% 35.2 10.7 0.4

1.0% 37.7 11.6 0.7

0.9% 41.3 9.1 0.3

1.1% 35.9 9.9

—

—

—

49.2

47.4

51.0

51.6

46.9

54.3

50.3

2.3 34.9 3.2 0.9 4.7

2.6 36.4 4.5 0.3 3.0

2.9 37.3 2.4 0.7 1.7

2.8 37.3 4.0 0.6 1.3

3.4 39.8 5.7 0.6 1.5

1.0 35.8 2.8 0.2 1.5

3.4 37.1 3.1

46.0

46.8

45.0

46.0

51.0

41.3

45.4

4.8

5.8

4.0

3.3

2.1

4.4

4.3

1.1% 42.1 11.1

1.2% 39.2 9.9

—

1.8

* Number in parenthesis represents number of points of unsaturation, i.e. number of double bonds. ** Composite of all unidentified peaks in the recording of the chromatogram.

of the fatty acids of embryonic triglyceride as compared to the adult hen. This is true from a qualitative as well as quantitative standpoint. The Rutger's group (Feigenbaum and Fisher, 1959) reported five unsaturated acid fractions which they identified as oleic, linoleic, linolenic, arachidonic, and pentaenoic acids. Unfortunately, the gas chromatographic separation employed in the present study does not distinguish between the various cis-trans isomers and, hence, the acids are referred to by the number of carbon atoms and the number of centers of unsaturation. It is likely that the C-18 unsaturated fatty acids are mostly oleic, linoleic and linolenic respectively, but definite identification based on isomeric configuration must await more refined methods of separation. Fatty acids analogous to the "arachidonic" and "pentaenoic" acids reported by the Rutger's investigators did not occur in the fat organs at any time during their development. However, a C-20 acid with one double bond was found in embryonic triglyceride which did not occur in the hen fat.

Although the embryonic fat was not quantitatively similar to adult fat in fatty acid composition, this was not true when compared with egg fat. Feigenbaum and Fisher (1959) found that the influence of diet did not have as marked an effect on the egg fat as it did on body fat. The six dietary treatments resulted in from 35.9 to 54.0% saturated fatty acids and from 46.0 to 64.1% unsaturated fatty acids in the egg fat. It is interesting to note that all of the values obtained for the embryonic saturates are included in the range of the egg fat saturates. The embryonic unsaturated acids do not compare as favorably, because of the arithmetical influence of the unidentified components on the relative percentage composition of the other components. The "arachidonic" and "pentaenoic" acids of the hen fat were also seen in the egg fat along with the other fatty acids. The qualitative differences between hen fat and embryonic triglycerides are also evident between the embryonic and egg lipids. Cephalitis. Stearic, oleic, linoleic and

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Unsaturated Acids* C-16 (1) C-18 (1) C-18 (2) C-18 (3) C-20 (1)

15

1854

G. L. FELDMAN, H. T. JONSSON, T. W. CULP AND R. H. GOWAN

TABLE 2.—Fatty acid composition of the cephalin fraction of fat organ lipids from the developing chick embryo Days of incubation 14 Saturated Acids C-14 C-16 C-18

15

16

17

18

19-20

21

1-6% 44.9 18.1

1.2% 40.9 16.6

1.3% 37.9 16.0

1.3% 31.6 18.8

1.4% 26.7 13.3

1.5% 32.0 12.8

56.8

64.6

58.7

55.2

51.7

41.4

46.3

2.3 29.4 2.8 1.8

1.9 21.7 1.3 1.3

1.9 26.3 2.8 1.6

2.1 26.2 3.2 2.6

2.2 23.7 2.1 3.3

3.4 36.1 8.3 2.0

3.5 34.5 7.7 1.7

36.3

26.2

32.6

34.1

31.3

49.8

47.4

All others

4.0 2.9

4.9 4.3

6.5 2.2

8.2 2.5

6.3 10.7

2.3 6.5

2.4 3.9

Total

6.9

9.2

8.7

10.7

17.0

8.8

6.3

Total Unsaturated Acids* C-16 (1) C-18 (1) C-18 (2) C-18 (3) Total Unidentified Components

* Number in parenthesis refers to number of points of unsaturation. ** A single major component aside from the minor composite of all unidentified peaks.

arachidonic acids have all been reported to occur in the cephalin molecule (Deuel, 1959). Many of the earlier workers contended that stearic was the sole saturated acid of cephalin and that oleic was the predominant unsaturate. The fatty acid composition of the embryonic fat organ cephalin partially supports this contention (Table 2). The monounsaturated C-18 acid predominates among the unsaturates and this acid is probably oleic. However, the saturates do not conform to the earlier contention. In fact, the predominant saturated acid was palmitic, the C-16 acid, with only half as much stearic as palmitic. Recent studies of rat liver cephalin (Hanahan, 1960) indicated that stearic and palmitic acids were present in equal quantities and, when combined, accounted for 45.5% of the total fatty acids. A large, single, unidentified fatty acid was observed in the embryonic cephalin. This component had a retention time which suggested that it was a C-20 acid. The failure to identify the component may have

been due to an atypical retention time as the result of chain branching or unusual unsaturation. This component occurred in all of the phospholipids. Studies to determine its identity are in progress. From a quantitative standpoint the fat organ cephalin fatty acids decrease in saturation after the fifteenth day of incubation with a corresponding increase in the unsaturates. It is interesting to note the sharp drop in saturated fatty acids after the eighteenth day. This shift is accompanied by an almost equal rise in unsaturates and occurs simultaneously with a marked cephalin depletion in the fat organ (Feldman et al., 1962a). It should also be noted that the shift is preceded by a marked elevation of the unidentified components and followed by a sharp drop. The significance of this rearrangement is unknown, but may be a reflection of profound metabolic changes brought about as a result of pipping the shell. Lecithin. Earlier investigators have reported the occurrence of palmitic, stearic,

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1.5% 33.9 21.4

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EMBRYONIC FAT ORGAN LIPIDS

become most marked on the seventeenth day and again after nineteen days. These two periods correspond to the apparent onset of triglyceride synthesis (Feldman et al., 1962a) and pipping of the shell respectively. The nature of the relationship to these two events is not known. It is interesting to note that these two periods of marked fluctuation also occur when the amount of lecithin per milligram of tissue is at its lowest (Feldman et al., 1962a). Sphingomyelins. These compounds differ from the other phospholipids in that the molecule contains no glycerol and but a single fatty acid. This fatty acid is linked through an amino group of sphingosine rather than the usual linkage of the glycerol esters. Most of the available data on the sphingomyelins are based on brain and spleen. The fatty acids that have been reported to occur are palmitic, stearic, lignoceric, and nervonic acids (Deuel, 1951; Hanahan, 1960). The latter two acids are not found in the sphingomyelins of the embryonic fat organ (Table 4).

TABLE 3.—Fatty acid composition of the lecithin fraction of fat organ lipids from the developing chick embryo Days of incubation 14 Saturated Acids C-14 C-16 C-18 C-20

15

16

17

18

19-20

21

1.5% 40.7 28.4

1.8% 33.8 27.5

1.7% 35.1 32.7 1.8

2.3% 21.6 23.2

2.0% 27.9 29.2

1.5% 22.7 25.1

1.7% 28.1 20.6 1.8

70.6

63.1

71.3

47.1

59.1

49.3

52.2

1.5 15.2 2.4 1.9

1.9 14.3 4.9 1.9

1.7 8.9 1.4 2.8

3.9 17.5 8.8 5.0

2.3 10.2 2.4 4.7

2.4 19.5 8.8 3.1

2.7 21.3 13.0 2.5

21.0

23.0

14.8

35.2

19.6

33.8

39.5

All other

4.7 3.7

6.0 7.9

9.1 4.8

5.3 12.4

3.1 18.2

3.5 13.4

2.3 6.0

Total

8.4

13.9

13.9

17.7

21.3

16.9

8.3

Total Unsaturated Acids* C-16 (1) C-18 (1) C-18 (2) C-18 (3) Total Unidentified Components V**

* Number in parenthesis refers to number of points of unsaturation. ** A single major component aside from the minor composite of all unidentified peaks.

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oleic, linoleic, linolenic and arachidonic acids in the lecithins from various natural sources (Deuel, 1951; Hanahan, 1960). All of these acids except arachidonic are found in embryonic fat organ lecithin (Table 3). A saturated C-20 acid occurs in the fat organ lecithin, but its presence can be demonstrated only on the sixteenth day and at hatching. The unidentified component "X" also appears in fat organ lecithin along with large amounts of other unknown materials. Several reports of the occurrence of uncommon fatty acids in lecithins are cited in Deuel's (1951) treatise so that this observation is not without precedent. It is a commonly held opinion that the saturated and unsaturated fatty acids of lecithin occur in equal quantity (Deuel, 1951). This concept has been challenged in recent years and the lecithins from embryonic fat organs represent an additional challenge. Although the fatty acids fluctuate widely from day to day they do not approach a 1:1 ratio. The fluctuations

1856

G. L. FELDMAN, H. T. JONSSON, T. W. CULP AND R. H. GOWAN TABLE 4.—Fatty acid composition of the sphingomyelin fraction of fat organ lipids from the developing chick embryo Days of incubation

INU. v^ctrDon i\r.oms Saturated Acids C-14 C-16 C-18 C-20 C-21 C-22

Unsaturated Acids* C-16 (1) C-18 (1) C-18 (2) C-18 (3) Total Unidentified Components X** All others Total

1.3% 46.1 26.8 1.9 4.2

15 2.2% 46.9 19.9 1.8 3.2

16 3.1% 35.7 21.4 3.7 6.8

17 3.8% 29.0 16.4 2.5 5.0

18 2.0% 32.4 25.5 2.3 3.9

19-20 2.1% 32.9 24.6 2.4 6.5

21 2.1% 38.6 17.7 3.7 7.0 4.3

—

—

—

—

—

—

80.3

74.0

70.7

56.7

66.1

68.5

73.4

1.3 3.6 1.3 1.9

1.4 3.9 1.7 2.7

4.0 2.9

—

—

2.6 4.5 3.9 6.2

2.1 6.6 2.4 4.2

2.4 8.9 5.2 1.7

2.7 4.7 2.1 3.3

8.1

9.7

6.9

17.2

15.3

18.2

12.8

5.3 6.3

8.9 7.4

8.2 14.2

8.0 18.1

3.4 15.2

1.9 11.4

2.7 11.1

11.6

16.3

22.4

26.1

18.6

13.3

13.8

* Number in parenthesis refers to number of points of unsaturation. ** A single major component aside from the minor composite of all unidentified peaks.

Numerous saturated fatty acids are observed including a C-21 acid which is not observed in any of the previously discussed lipids. A C-22 acid is also noted, which appears just before hatching time. The saturated acids predominate in the fat body sphingomyelins, a fact in excellent agreement with existing literature. There is a marked drop in saturated acids on the seventeenth day, but they return to the higher values after twenty-four hours. The unidentified component "X" decreases in content very sharply during the same period, but does not return to its original high value. It is of interest that the saturated fatty acids are in greatest abundance when the sphingomyelin content is low and less abundant when the sphingomyelins increase. The reason for this response must await further study. Lysolecithins. The authors were unable to find data based on the fatty acid com-

position of the lysolecithins with which to compare the findings from the embryonic fat organs. The fatty acid composition is qualitatively similar to lecithin, differing only in the presence of a C-21 acid (Table 5). . The saturated fatty acids predominate, but fluctuate from day to day. The lowest levels are seen on the seventeenth day and at hatching time. The amount of unidentified components is particularly striking, being greater than in the other lipids studied. It should be pointed out that the lysolecithin of the sixteenth day is undetectable after fractionation (Feldman et al., 1962a). However, sufficient material was present for esterification and could be detected because of the extreme sensitivity of the gas chromatograph. SUMMARY AND CONCLUSIONS (1) The fatty acid composition of embryonic adipose tissue bears a striking re-

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Total

14

1857

EMBRYONIC FAT ORGAN LIPIDS

TABLE 5.—Fatty acid composition of the lysolecithin fraction offat organ lipids from the developing chick embryo Days of incubation 14 Saturated Acids C-14 C-16 C-18 C-20 C-21 Total

Total

3.5% 32.9 15.0 6.4

16

17

18

19-20

21

3.8% 32.6 14.9 1.8 1.7

4.2% 25.1 12.6 2.1 4.1

5.2% 28.6 19.9 2.3

3.2% 29.1 16.0 4.1 5.7

3.7% 26.9 12.8 2.0

68.2

57.8

54.8

48.1

56.0

58.1

45.4

3.0 5.2 4.8 4.8

2.4 4.1 5.2 7.5

3.7 7.2 2.3 3.3

2.8 2.7 5.1 10.0

5.3 6.1 4.1 7.0

4.2 9.7 3.3

5.7 10.8 7.2 3.0

17.8

19.2

16.5

20.6

22.5

17.2

26.7

7.8 6.2

11.8 11.2

5.0 23.7 -

8.0 23.3

4.6 16.9

4.0 20.7

27.9

14.0

23.0

28.7

31.3

21.5

24.7

27.9

Unidentified Components All others Total

* Number in parenthesis refers to number of points of unsaturation. ** A single major component aside from the minor composite of all unidentified peaks.

semblance to that of egg fat and not to the fat of the adult chicken. (2) The triglyceride fatty acids are relatively constant and do not reflect the changes associated with embryonic development. However, the phospholipid fatty acids fluctuate widely and appear to be related to the onset of triglyceride synthesis and pipping of the shell. (3) An unidentified fatty acid occurs in all of the phospholipids, in which it comprises a major component. ACKNOWLED GMENT Support for this work was provided in part by the following: The Fondren Foundation, The Eyes of Texas Sight Foundation, Public Health Service Grants B-2297, B-3172 from the National Institute of Neurological Diseases and Blindness,

H-S230 from the National Heart Institute, and National Science Foundation Grant No. G-1084S. REFERENCES Deuel, H. J., 1951. The Lipids, Vol. 1, Chaps. I l l and V. Interscience Publishers, New York. Feigenbaum, A. S., and H. Fisher, 1959. The influence of dietary fat on the incorporation of fatty acids into body and egg fat of the hen. Arch. Biochem. Biophys. 79: 302-306. Feldman, G. L., L. M. Churchwell, T. W. Culp, F. A Doyle and H. T. Jonsson, 1962a. The lipid content of the subcutaneous fat organs of the chick embryo. Poultry Sci. 4 1 : 1232-1240. Feldman, G. L., F. A. Doyle, M. R. Lawler, Jr., R. S. Rodgers and L. M. Churchwell, 1962b. Enzymatic activity of the developing subcutaneous fat organs of the chick. Poultry Sci. 4 1 : 1423-1428. Hanahan, D. J., 1960. Lipide Chemistry. Chaps. 3, 5, 6, and 7. John Wiley and Sons, New York.

AUGUST 9-15. SIXTH INTERNATIONAL CONGRESS ON NUTRITION, EDINBURGH, SCOTLAND

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Unsaturated Acids* C-16 (1) C-18 (1) C-18 (2) C-18 (3)

3.6% 36.0 24.0 4.6

15