Metabolism of Lipids in Rat Testes: Interconversions and Incorporation of Linoleic Acid Into Lipid Classes MASAMI NAKAMURA and O. S. PRIVETT, The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ABSTRACT

sideration of synthetic pathways defined through studies on the mode of their interconversions (3,7,11,14,29). Recently (13) we demonstrated that relatively large changes may occur in fatty acid composition and lipid classes more or less independently of each other in testicular lipids indicating that turnover of acyl chains and skeletal moieties of triglycerides and phospholipids occur at different rates. Studies on the metabolism of testicular lipids have been devoted mainly to the mode of interconversion of fatty acids (1,4,5,12,19) and the effect of nutritional (1,8,13) and hormonal deficiencies (9,10,20) on lipid class and fatty acid composition. The present study was undertaken to provide basic data for further studies along these lines as well as for general information on the mode of lipid synthesis in the testes.

Studies are reported on the mode of incorporation of linoleic acid into lipid classes of testicular lipids. 1-1'~C-linoleic acid was injected into the testes of adult rats of the Sprague-Dawley strain. Groups of animals were killed at 1, 3, 6, 12, 24 and 48 hr after injections of the radioactive linoleic acid. The testes of each animal and livers of some animals were excised. Fatty acid and lipid class composition of the extracted lipids of the testes of each animal were determined as well as the distribution of radioactvity in these compounds. Radioactive linoleic acid and fatty acids derived from it by interconversion and catabolism were incorporated into all the lipid classes. Incorporation of linoleic acid into the lipid classes was much faster than its interconversion or catabolism to other fatty acids. The importance of the fatty acid pool in the mode of incorporation of the fatty acids into the lipid classes is demonstrated.

EXPERIMENTAL PROCEDURES Materials and Methods

1-14C-Linoleic acid was obtained from Tracerlab Inc., Waltham, Mass., methylated with diazomethane (27) and purified by argentation TLC. Radio gas chromatography (6) showed that all the radioactivity was associated with methyl linoleate in the final preparation. The free acid obtained via saponification and acidification had an activity of 3.5 m C / r a M . F o r injections, the free acid was emulsified with a mixture of equal paris saline and rat serum; a 50 /d aliquot of this emulsion, the amount used for injections, had an activity of 1.4 • 105 c.p.m. Analysis of the radioactive linoleic acid in the emulsion showed that it remained unchanged and was stable at least for the period of the experiment. Radioactivity was measured by scintillation counting with a Packard Tri-Carb Model 3002 dual channel scintillation spectrometer with a scintillation solution consisting of PPOPOPOP in toluene or in dioxane-water solution described by Snyder (28). The latter solution was used for analysis of material recovered from chromatoplates in radio TLC. Counting efficiency for carbon-14 was 85% in the toluene solution and 71% in the dioxane-water solution. Values were not cor-

INTRODUCTION

I NTERRELATIONSIIIPS IN LIPID synthesis in mammalian tissue have received a great deal of attention and enzymatic steps in pathways for the net synthesis of common phosphatides and triglycerides have been fairly well defined (3,11,I4,29). Not so clear is the mode by which fatty acids are preferentially incorporated into these compounds. Diglycerides per se have been shown to be intermediates in the synthesis of triglycerides arid phosphatides (14); fatty acids apparently may be preferentially incorporated into phosphatides via acyl transferases (15,16,17, 18.24). Patton et al. (21, 22, 23) postulated that in lactating m a m m a r y tissue phosphatidylcholine serves as an intermediate in triglyceride synthesis in order to explain the preferential incorporation of short chain fatty acids into milk triglycerides. However, fatty acid composition and positional arrangement (structure) of individual lipids generally do not conform to patterns that are common to each other as might be expected from con41

42

MASAMI NAKAMURA AND O. S. PRIVETT

Fro. 1. TLC of rat testicular lipids. Plate 1 developed with petroleum ether-ethyl ether-acetic acid (80:20:1); plate 2 with petroleum ether-ethyl ether-methanol-acetic acid (90:20:5:2); plate 3 with chloroform-methanol-acetic acid (65:25:4:8); plate 4 with chloroform-water-acetic acid (25:15:2:4). CE = cholesteryl esters. GEDE = glyceryl ether diesters. TG = triglycerides, FA = fatty acids, DG = diglycerides. PL = polar lipids. NL = neutral lipids, PE = phosphatidyl-ethanolamine, PS =- phosphatidylserine, PI = phosphatidylinositol, PC = phosphatidylcholine, SPH = sphingomyelin, LPC = lysophosphatidylcholine, O = origin (unknown). rected to 100% efficiency because results obtained in the different solutions were not compared. Distribution of radioactivity among the lipid classes was determined by scintillation counting of bands of the components separated by TLC. The bands were scrapped directly from plates into vials of scintillation solution for counting. Fractionation of the lipids was 'carried out on four separate 5 X 20 cm chromatoplates containing a 0.25 cm layer of Silica Gel H (Brinkman Instruments Inc., Des Plaines, Illinois) as illustrated in Fig. 1. The positions of the bands were detected by exposing the plate to iodine vapors for just sufficient time to make them visible. The bands were marked and then the plates were placed in a chromatographic jar in a current of nitrogen for about 10 rain to evaporate LIPIDS, VOL. 4, No. i

most of the iodine. Four different solvent systems were employed in order to separate the major components completely from each other and provide duplicate checks on all components. Plate 1 in Fig. 1 was developed in petroleum ether-ethyl ether-acetic acid (80:20:1) for analysis of cholesteryl esters (CE), glyceryl ether diester ( G E D E ) , triglycerides ( T G ) , free fatty acid ( F F A ) , cholesterol (CH), diglyceride (DG) and polar lipids (PL) as a group. Plate 2 was developed with petroleum ether-ethyl ether-methanol-acetic acid ( 9 0 : 2 0 : 5 : 2 ) . It was used mainly for separation of the diglyceride fraction, but it also provided a check on the total of T G + CE + GEDE, FA and the PL fraction. Cholesterol separated with the diglyceride in this plate, but generally it had little activity, as measured on Plate 1, and could be disre-

INCORPORATION OF L1NOLEIC ACID INTO TESTICULAR LIPIDS

garded. Plate 3 was developed with chloroform-methanol-water-acetic acid (65: 25: 4: 8). This plate was used mainly for phosphatidylethalolamine (PE) which separates completely in this solvent system; it also served to check Plate 4. The system used in Plate 4 consisted of chloroform-methanol-water-acetic acid ( 2 5 : 1 5 : 2 : 4 ) . It has been described by Skipski et al. (25, 26) and its widely used for the separation of polar lipids. 1 Overall recoveries of radioactivity of approximately 96% were consistently obtained by the above technique. Specific activities were calculated from the amount of each lipid class determined on another aliquot of the sample by quantitative TLC by the charring-densitometry technique (2,13,20). Fatty acid composition was determined with an F & M Scientific Corp. Model 1650 flame ionization gas chromatograph on methyl esters of the total lipid and lipid classes prepared by interesterification with methanol-HCI. Nitrogen was used as the carrier gas, and separations were carried out on 6 ft. • ~/~ in. column packed with Gas Chrom P containing 16% EGSS-X (Applied Science Laboratory, State College, Pa.). Percent composition was determined on the basis of the direct proportionalities of the peak areas measured by triangulation. The average of triplicate analyses of standard mixtures of reference fatty acids of composition similar to those distributed by the N I H (purchased from the Lipids Preparation Laboratory of The Hormel Institute) agreed with the known composition within a maximum of _+ 2.6% and 4-5.4% relative error for the maior and minor components, respectively. Radio gas chromatography of methyl esters was carried out essentially by the collection technique described by Dutton (6) with an F & M Model gas chromatograph equipped with a thermal conductivity detector and a 6 ft. X 1A in. column packed with Gas Chrom P containing 16% EGSS-X (applied Science Lab, State College, Pa.). The collected samples were counted with a Packard Tricarb Model 3002 scintillation counter. Recoveries of radioactivity by this technique were approximately 80%. Specific activities were calculated from the amount of each component determined by GLC via pentadecanoic acid added to the esterification mixture as an internal standard. Application of this technique to a number of standard radioactive fatty acids gave values that agreed within +-1.5% between duplicate analyses.

43

Animals

Adult male rats of the Sprague-Dawley strain of 200-225 g were obtained from the Hormone Assay Laboratory, Chicago, IlL The animals were housed in individual metal cages and fed ad lib. a semi-synthetic diet consisting of 30% vitamin test casein, 50% sucrose, 4% cellulose2, 4% mineral mix 3. 2% vitamin mix 4 and 10% safflower seed oil. At the end of three weeks the animals were divided into six groups and one testicle of each animal in each ~zroup was injected with 50 M of the 1-1~C-linoleic acid emulsion (1.4 • 105 c.p.m.) in a sequential experiment. The animals were killed by exsanguination by withdrawal of the blood from the aorta, and the testes and livers were excised and frozen on dry ice. Groups were killed at 1. 3, 6, 12, 24 and 48 hr after injection of radioactivity. The testes of each animal in each group were decapsulated and weighed. The lipid was then extracted twice with chloroform-methanol ( 2 : I ) and once with a 1:2 ratio of these solvents, recovered in the usual manner as previously described (2,13,20). The distribution of the radioactivity and analyses of the lipid classes and fatty acids were determined as described above. RESULTS

The percent distribution of radioactivity among the lipid classes of the testes and general data on the different groups of animals are presented in Table I. These results show that 53.1% of the injected radioactivity were recovered in the testicular lioids of the animals in the first group and 30,2% were recovered from the testes of the last group of animals (48 hr after the injections of radioactive linoleic acid). In an accessory experiment, it was found that 80% of the injected radioactivity could be recovered in the testicular lipids of animals killed 15 min after injection of radioactive linoleic acid. No radioactivity was a:PC= phosphatidylcholine, PI = phosphatidylinositol, PS ~ phosphatidylserine. SPH = sphingomyelin, LPC ~lysophosphatidylcholine, DPG = diphosphatidylglycerol, NL = neutral lipid, PL = polar lipid. 2Non-nutritive cellulose, alphacel from Nutritional Binchemicals Corp., Cleveland, Ohio. aWesson modified, Osborne-Mendelsalt mix, from General Biochemicals,Chagrin Fails, Ohio. ~Vitamin mix, consisting of 0.25% vitamin A acetate crystals, 0.017% vitamin D: concentrate (400,000 U.S.P. #/g), 1.70% alpha-tocopherol. 0.06% i-inositol. 5.0% choline chloride 0.0085% menadione.0.0325% p-aminobenzoic acid, 0.43% niacin, 0.13% riboflavin, 0.035% pyridoxine HCI, 0.13% thiamine HCI, 0.43% calcium pantothenate, 0.001% biotin, 0.045% folic acid, 0.0002% vitamin B~., and 90.77% casein diluent. LIPIDS, VOL. 4, NO. 1

44

MASAMI NAKAMURA AND O. S. PRIVETT TABLE I Percent Distribution of Radioactivity Among the Testicular Lipids Of Animals Injected Intratesticularly With 1-~4C-Linoleic Acid

Time period (hr) No. of animals Testis wt. (g) % Lipid Recovery of radioactivity %

1 5

3 5

1.49• a 2.7 4-0.1

1.57• 3.0 4-0 1

53.1 4-1.0

48.5 4-1.4

Neutral lipids (% distribution of radioactivity) Composition b (wt. % ) CE .... tr e tr GEDE tr tr TG 915 5.7 4-0.2 5.0 4-0.04 FA 3.0 20.2 4-1.1 20.6 4-0.9 DG 1.8 8.0 4-0.4 6.9 4-0.3 Chol . . . . . . . tr tr Total Neutral lipids (NL) 35.9 4-1.2 34.4 4-1.7 Polar lipids (% distribution of radioactivity) DPG 1.4 4-0,2 PE 2714 11.7 +0.3 PI+PS 4.3 3.2 __+0.2 PC 27.4 44.3 4-1.0 Sph .... 0.6 4-0.02 Lyso 1.8 +_0.2 Total polar lipids (PL) 64.0 4-l.2

2.4 14.2 4.2 42.1 0.8 0.6

4-0.2 4-0.3 4-0.1 __+1.6 4-0.02 4-0.1

65.5 4-1.7

6 5 1.51_.+_.0.03 3.1 -4-0.1

12 6

24 6

48 6

1.64• 3.2 -4-0.1

1.59__0.03 3.2 -+-0.04

1.534--0.07 3.3 4-0.1

46.7 4-0.5

47.5 4-3.2

43.8 4-2.4

30.2 4-1.4

0.5 4-0.04 tr 7.9 4-0.8 19.0 4-0.9 6.4 4-0.2 tr

0.6 4-0.04 tr 6.1 4-0.6 20.0 4-1.0 7.1 4-0.3 tr

0.9 0.7 12.2 19.5 6.3

1.0 0.9 11.0 16.0 6.0

33.5 4-1.5

35.2 4-1.5

40.4 4-0.7

36.2 4-0.6

1.5 16.3 5.2 40.7 0.5 0.5

1.5 19.1 7.6 33.2 0.6 0.5

1.4 17.6 8.0 30.3 1.1 0.3

1.6 18.6 9.3 31.4 1.4 0.6

4-0,3 4-0.5 4-0.6 +__0.3 4-0.1 -4-0.1

66.5 4-1.5

4-0.2 4-0.6 4-0.4 4-0.5 4-0.1 4-0.1

62.8 -+-1.5

+0.1 4-0.1 4-0.5 • +0.4 tr

+0.3 4-0.5 4-0.2 4-0.6 +0.1 4-0.3

59.3 4-0.7

4-0.2 +0.1 4-1.5 +0.8 +0.4 tr

___0.1 +0.4 4-0.3 +__0.6 4-0.1 4-0.1

63.8 4-0.6

aM+SE. bMajor radioactive components. eTrace = under 0.4%.

found in the lipids of the livers or testicle not injected with 1-14C-linoleic acid during the course of the experiment. Thus, no data are reported on these organs and it may be concluded that the radioactive linoleic acid was metabolized in the testicle into which it was injected. Further evidence to this effect was the relative constancy of the percentage distribution of the radioactivity among the lipid classes in view of the fact that approximately 70% of it was dissipated over the course of the experiment. This observation also indicates that linoleic acid undergoes transformations among the lipid classes in a very orderly pattern, because the percentage composition (by weight) of the components does not change during the period o f the experiment. The changes that did occur in the percentage distribution of the radioactivity among the lipid classes were probably due primarily to conversion of linoleic acid to other fatty acids that were transformed among the lipid classes and catabolized at different rates. More information of the mode of transformations of the fatty acids among the lipid classes was obtained from a consideration of the data on the specific activities of the lipid classes presented in LIPIDS,

VOL. 4, NO. 1

Figures 2 and 3. The fatty acid fraction had the highest specific activity, as expected. The specific activity of this fraction gradually decreased as the fatty acids were catabolized and new fatty acids entered the pool to dilute the radioactivity. The concentration of the radioactivity (specific activity) in PC and D G followed the same pattern as the fatty acid fraction but since these compounds contained no radioactivity originally, the specific activity apparently reached a maximum at some time prior to 1 hr, the time at which the first group of animals was killed. The concentration of radioactivity in P E appeared to reach a maximum next among the lipid classes at between 6 and 12 hr. The peaks in the concentrations of the radioactivity of the other lipid classes came at approximately 24 hr. Analyses were not made at short enough time intervals to indicate the maxima of the specific activities with great precision. Nevertheless, it was apparent the specific activities reached maximal values first in D G and PC, then in P E and later in the other lipid classes. The percentage distribution of the radioactivity among the fatty acids in the different

INCORPORATION OF LINOLEIC ACID INTO TESTICULAR LIPIDS

.•

~

45

TOTAL LIPID 5

--o-CE --x-GEDE --A-TG ~- (scale)

2

-~ -~-

FFA DG

20

(scale)-~

Q

'~--

0 1 3 Hrs

6

"~

12

24

48

FIG. 2. Specific activities of neutral lipids of the testicular lipids of animals injected intratesticularly with 1-1~C-linoleic acid. C E = cholesteryl esters, G E D E = glyceryl ether diester, T G = triglycerides, F F A = free fatty acids, D G = diglycerides.

TOTAL ~~ E .> E

LIPID

; PC -o-PE - x - p I + PS

~ 4 X

-

r

.~_ 2

~/ O "--------

0 1 3 HPS

6

12

24

48

FIG. 3. Specific activities of polar lipids of the testicular lipids of animals injected intratesticularly with 1-"C-linoleic acid. PC ~ phosphatidylcholine, P E ---- phosphatidylethanolamine, PI -- phosphatidylinositol, PS ---- phosphatidylserine. LIPIDS, VOL. 4, No. 1

MASAMI NAKAMURA A N D (.). S. PRIVETT

46

"(scale)

TOTAL LIPID

16:0 20:4 22:4

5

18:2 20:3 (scale) -~

22:5

xlO 5

~,3

2

='1

18:2 -020:3 ~16:0 22:5 48

/~j / 7"

.~ 0 1 HPs

~-

6

5

x, 24

,. 12

FIG. 4. Specific activities of fatty acids in total lipid. Shorthand designation for fatty acids; number before colon ---- number of carbon atoms in chain; number after colon - number of double bonds. groups is summarized in Table II. As the percentage distribution of radioactivity of linoleic acid decreased that in the other fatty acids increased showing that linoleic acid underwent interconversion to other members of this family of acids. However, linoleic acid still contained the highest percentage of radioactivity even in the last group of animals in which all but approximately 30% of the radioactivity had been dissipated. The radioactivity

in patmitic acid may be presumed tO arise by de novo synthesis from radioactive acetate produced in the catabolism of radioactive acids. More information on the interconversion and catabolism of the fatty acids was indicated from the plot of their specific activities in the total l i p i d (Fig. 4). Figure 4 shows the specific activities of the major fatty acids determined in the total lipid. N o results were

TABLE II Per Cent Distribution of Radioactivity Among the Fatty Acid of the Testicular Lipids of Animals Injected Intratesticularly With 1-14C-Linoleic Acid % Radioactivity l i m e period (hr) Composition (wt %) 16:0 18:2 20:3 20:4 22:4 22:5

29,1-:-2.6 6.1 ~0.4 1,54-0.1 15.34-I.6 2.7-4-0.02 20.8___1.8

a Pooled samples. bM -~ SE.

LIPIDS, VOL. 4, No. I

1

3

6

12

24

48

2.8 a 81.8 3.9 3.5 0.6 0.2

3.4• b 75.2--'0.5 4.1• 8.5_+0.3 0.9• 0.3-+-0.1

4.5 a 71.5 4.9 8.7 0.9 0.4

6.0-+0.2 60.1 ~0.9 5.8___+0.2 12.84-0.5 1.6-+0.1 0.9-+__0.1

11.2-+1.1 4 2 . 7 ~ 1.8 5.9• 17.04-1.1 3.2-+0.3 2.3_+0.3

13.0+0.8 35.3~3.5 6.0-+-0.5 20.I• 4.3-+0.3 4.1_+0.3

INCORPORATION OF LINOLEIC ACID INTO TESTICULAR LiPIDS

t0* I *,,, I

18:2

I

47

--'-Tot", -o-DG

o

1 3 Hrs

6

12

24

48

FIG. 5. Specific activities of linoleic acid (18:2) in the lipid classes. DG ---- diglycerides, F F A = free fatty acids, TG = triglyceride, PC = phosphatidylcholine, PE = phosphatidylethanolamine. obtained on 18:3 (gamma linolenic) or 20:2 ( l l , 1 4 - e i c o s a d i e n o i c acid) members of the linoleic acid family, because in addition to being in small concentrations they could not be separated quantitatively for counting. Specific activities of the major fatty acids in PC, PE, T G and F F A fractions were also determined. These showed the same general pattern as for the total lipid except that there were some differences in the order of magnitude of the values for the different fatty acids in each lipid class. In general, the specific activities of the 22:4, 20:3 and 20:4 were highest in the polar lipids, reached maximal concentrations and were decreasing by the end of the experiment. The 16:0 and 22:5 were the lowest and, except for the 16:0 in TG, did not exhibit a peak in concentration of radioactivity. Conversions to 22:5 were relatively slow. Linoleic acid exhibited a decay curve in all of the lipid classes except PE where it appeared to peak at about 12 hr. (Fig. 5.) Since there was no radioactive linoleic acid in the lipid classes originally (except in the F A fraction) its specific activity

reached a maximum in these compounds before the first group of animals was killed. Thus, the incorporation of linoleic acid into the lipid classes is much faster than its conversion to other fatty acids. The fatty acids derived from ]inoleic acid probably also were incorporated into the lipid classes very quickly but the rates of incorporation could not be determined because the measurement of their specific activities depended on their conversion from radioactive ]inoleic acid. However, the specific activities of all fatty acids containing radioactivity in the various lipid classes were determined. Typical of these data are those shown for arachidonic acid in Fig. 6. The specific activities of all of the fatty acids were highest in the free fatty acid fraction and next highest in D G (Fig. 6). There were some differences in the order of magnitude of the specific activities of the individual fatty acids from one lipid class to another but they were probably related to the rate of their interconversion; that of arachidonic acid was highest in PC of the polar lipids (Fig. 6). LIPIDS, VOL. 4, NO. 1

48

MASAMI NAKAMURA AND O. S. PR1VETT

x+o+

20:4

- o - F FA -o-DG

2

o

4

3 1

0 1 Hrs

,3

6

12

24

48

FIG. 6. Specific activities of arachidonic acid (20:4) in the lipid classes. F A A = free fatty acids, DG =- diglycerides, TG = triglycerides, PC = phosphatidylcholine and PE = phosphatidylethanolamine. DISCUSSION

The distribution of radioactivity among the entire spectrum of fatty acids shows that linoleic acid undergoes interconversion and is also catabolized. Evidence for catabolism is the presence of radioactive palmitic acid. The radioactivity in this fatty acid must arise by de novo synthesis from radioactive acetate derived from injected linoleic acid. These processes (catabolism and interconversion) go on more slowly than the incorporation of linoleic acid into the lipid classes. Evidence to this effect is that, in addition to its rapid incorporation into the lipid classes, linoleic acid contained most of the radioactivity ( 3 5 % ) , remaining 48 hr after injection of the animals. The relatively slow interconversion of linoleic acid is not because this process is performed only in the liver. Several reports (4,12,19) demonstrate the interconversion of fatty acids by testicular tissue. I n the present study the amount of radioactivity in the lipid of livers and the testicle not injected with radioactive 1-t4C-linoleic acid was insignificant, even after 48 hr. Thus, it may be concluded that inter-

LIPIDS, VOL. 4, NO. 1

conversions and catabolism as well as incorporation of radioactive linoleic acid into the lipid classes occurs entirely in the testes in the present study. It is well established that lipid in living tissue is in a dynamic state; turnover measured by changes in composition m a y be a fairly slow process varying from several months in some tissues to several days or weeks in more active metabolizing tissues. In the present work no measurable changes in composition of the lipid classes would be expected in the 48 hr period of the experiment. However, the individual reactions involved in the overall process of lipid metabolism appear to be rapid, judging by the fact that approximately 70% of the radioactivity was dissipated in a period of 48 hr, and the radioactive linoleic acid was incorporated into all the lipid classes in less than 1 hr. The mode of incorporation of fatty acids into the lipid classes is important in determining composition and positional arrangement of the fatty acids. The present study indicates the importance of the fatty acid pool in these

INCORPORATION OF LINOLEIC ACID INTO TESTICULAR LIPIDS processes. It m a y be calculated that 1 h r after injection o f the radioactive linoleic acid, 6.1 /~/~ curies o f this acid is p r e s e n t as free fatty acid fin the f a t t y acid p o o l ) . H o w e v e r , after 48 h r 13.3 /~/~ c o f fatty acid derived f r o m linoleic acid is p r e s e n t in the lipid classes. If, as generally a s s u m e d , i n t e r c o n v e r s i o n s and catabolism involve f a t t y a c i d - C o A derivatives as obligatory i n t e r m e d i a t e s in these processes, t h e n linoleic acid m u s t have b e e n released f r o m the lipid classes to a c c o u n t for the synthesis o f these acids. This, and the general p a t t e r n of distribution o f radioactivity in T a b l e I, indicate that the acyl chains o f the lipid classes are in d y n a m i c equilibrium w i t h the fatty acid pool. A c c o r d i n g l y , fatty acids m a y be t a k e n f r o m the pool and d i r e c t e d p r e f e r e n t i a l l y into n o t only the lipid classes b u t also into specific positions in the molecules of these c o m p o u n d s . M o l e c u l a r species as well as fatty acid c o m position o f the lipid classes m a y be regulated via the action o f specific e n z y m e s in these reactions. C h a n g e s in the p e r c e n t a g e o f distribution of the radioactivity a m o n g the lipid classes were r e l a t i v e l y small c o m p a r e d to the large c h a n g e in total a m o u n t o f radioactivity d u r i n g the course o f the e x p e r i m e n t . T h e s e c h a n g e s are believed to o c c u r m a i n l y as a result o f conversions o f linoleic acid to o t h e r fatty acids that are distributed in a different m a n n e r and rate f r o m that of linoleic acid. T h e precise p a t h w a y s w h e r e b y fatty acids are i n c o r p o r a t e d into the lipid classes h a v e n o t b e e n c o m p l e t e l y elucidated. F a t t y acids m a y u n d e r g o t r a n s f o r m a t i o n s a m o n g the lipid classes in a c c o r d a n c e with the K e n n e d y p a t h w a y ( 1 4 ) , b u t this process appears to apply m o r e to t h e m o d e o f net synthesis o f lipid t h a n to a d y n a m i c state of equilibrium. W h e t h e r the greater i n c o r p o r a t i o n o f radioactive linoleic acid into p h o s p h a t i d y l c h o l i n e r a t h e r t h a n triglycerides r e p r e s e n t s a deviation f r o m the K e n n e d y p a t h w a y or a ramification o f it is n o t known. It a p p e a r e d that diglycerides w e r e int e r m e d i a t e s in these reactions because o f their high specific activity. H o w e v e r , the radioactivity in the d i p h o s p h a t i d y l g l y c e r o l fraction w h i c h s h o u l d also c o n t a i n p h o s p h a t i d i c acid a p p e a r e d to be too low for p h o s p h a t i d i c acid to be an i n t e r m e d i a t e in these r e a c t i o n s in a c c o r d ance with the K e n n e d y p a t h w a y . T h e a m o u n t and c o n c e n t r a t i o n of radioactivity in the lysop h o s p h a t i d y l c h o l i n e f r a c t i o n also a p p e a r e d to p r e c l u d e it as an i n t e r m e d i a t e f o r existence o f an i m p o r t a n t acyl t r a n s f e r a s e system (7,1 5,1 7,18) or as a p r o d u c t o f the release o f fatty acids into the fatty acid pool. H o w e v e r , if the

49

i n c o r p o r a t i o n a n d e q u i l i b r i u m processes involved only the 2 position o f l y s o p h o s p h a t i d y l choline, n o large a m o u n t o f radioactivity w o u l d a c c u m u l a t e in this molecule. Thus, a l t h o u g h the p r e s e n t study p r o v i d e s e v i d e n c e f o r the imp o r t a n c e o f the fatty acid p o o l in d y n a m i c equilibrium w i t h the lipid classes, m u c h f u r t h e r e x p e r i m e n t a t i o n is r e q u i r e d to delineate the reactions i n v o l v e d in the general process of lipid synthesis in the testes. ACKNOWLEDGMENT This investigation was supported in part by PHS research grants AM 04942 and HE 08214 from the National Institutes of Health, Public Health Service. B. D. Westra gave technical assistance. REFERENCES 1. Ahluwalia, B., G. Pincus and R. T. Holman, J. Nutrition 92, 205 (1967). 2. Blank, M. L., J. A. Schmit and O. S. Privett, JAOCS 41, 371 (1964). 3. Bremer, J., P. H. Figard and D. M. Greenberg, Biochim. Biophys. Acta 43, 477 (1960). 4. Davis, J. T., R. B. Bridges and J. G. Coniglio, Biochem. J. 98, 342 (1966). 5. Davis, J. T., and J. G. Coniglio, J. Biol. Chem. 241, 610 (1966). 6. Dntton, J. R., JAOCS 38, 631 (1961). 7. 1~lofson, R., Biochim. Biophys. Acta, in press. S. Gambal, D., J. Nutrition 89, 203 (1966). 9. Gambal, D., and R. J. Ackerman, Endocrinology 80, 231 (1967). 10. Goswami, A., and W. L. Williams, Biochem. J, 105, 537 (1967). 11. Gibson, K. D., J. D. Wilson and S. Undenfriend, J. Biol. Chem. 236, 673 (1961). 12. Hail, P. F,, E. E. Nishizawa and K. B. Eik-Nes, Can. J. Biochem. Phys. 41, 1267 (1963). 13. Jensen, B., M. Nakamura and O. S. Privett, J. Nutrition 95, 406 (1968). 14. Kennedy, E. P., Federation Proc. 20, 934 (1961). 15. Lands, W. E. M., and P. Hart, J. Biol. Chem. 240, 1905 (1965). 1,5. Lands, W. E. M., M. L. Blank, L J. Nutter and O. S. Privett, Lipids 1, 224 (1966). 17. Lands, W. E. M,, J. Biol. Chem. 231, 881 (1958). 18. Lands, W. E. M,, JAOCS 42, 465 (1965). /9. Morin, R. J., Proc. Soc. Exptl. Biol. Med. 126, 229 (1967). 20. Nakamura, M., B. Jensen and O. S. Privett, Endocrinology 82, 137 (1968). 2l. Patton, S., R. D. McCarthy and P. S. Dimick, J. Dairy Sci. 48, 1389 (1965). 22. Patton, S., R. O. Mumma and R, D. McCarthy, "An Active Role of Lecithin in the Synthesis of Milk Fat," presented at the AOCS Meeting, Philadelphia, October, 1966.

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