Influence of Diet on Conversion of 1 4C1-Linolenic Acid to Docosahexaenoic Acid in the Rat B.P. POOVAIAH, J. TINOCO, and R.L. LYMAN, Department of Nutritional Sciences, University of California, Berkeley, California 94720 ABSTRACT

fatty acids strongly suggests that these fatty acids do have specific functions in warmblooded animals. Members of the linolenate family are absorbed from the diet, incorporated into tissue lipids, elongated and desaturated efficiently, and, finally, are located specifically within certain lipid classes in particular organelles. For example, 22:6n-3 is the major unsaturated fatty acid in retinal rods of the dog, pig, sheep, bovine, rabbit, and human, and it is located especially in the phosphatidyl ethanolamine (PE) molecule (5). Large proportions of 22:6n-3 are also found in PE and phosphatidyl serines of many other tissues, such as rat liver, heart, rat brain synaptosomes, grey matter of the human brain, etc. This specific location of 22:6n-3 in particular phospholipids (PL) in specialized cells or organelles implies that 22:6n-3 should have some special function. P r e v i o u s l y , we attempted to produce symptoms of linolenate deficiency in rats by maintaining rats for three generations on a diet devoid of n-3 fatty acids. Although the levels of n-3 fatty acids in tissues became very low, small amounts of 22:6n-3 still remained, and the rats showed no abnormality in growth, reproduction, or appearance (6). This report describes the incorporation of radioactive linolenic acid into the rat, and the rates at which it is converted to longer chain homologs. In addition, we studied the effects of several dietary manipulations on these parameters.

] 4C1-Linolenic acid was incorporated into lipids of hearts, livers, and carcasses of male rats. We studied the influence of diet composition on extent and distribution of radioactivity. A CHOW diet, a purified, essential fatty acid (EFA)-deficient diet, a purified control diet, and EFA-deficient diets with four fatty acid supplements were used. Supplements of 18:2n-6, 20:4n-6, 18:3n-3, and 22:6n-3 were given as single doses. Radioactivities in liver phosphatidyl ethanolamines (PE), phosphatidyl cholines, and neutral lipids were measured. The distribution of radioactivity among the fatty acids in liver phospholipids was determined. Rats on CHOW diet incorporated far less radioactivity than any other group into lipids of hearts and livers. Most of the activity in livers was recovered as 20:5n-3 and 22:6n-3 in all rats. In EFA-deficient rats, the radioactivity in 22:6n-3 of liver PE was still increasing 36 hr after 14C 1linolenic acid had been administered. The n-6 supplements (18:2n-6 and 20:4n-6) seemed to reduce the conversion of 20:4n-3 to 20:5n-3 (desaturation), whereas the n-3 supplements (18:3n-3 and 2 2 : 6 n - 3 ) reduced the conversion of 20:5n-3 to 22:5n-3 (elongation). Formation of 22:6n-3 may be controlled by 2 2 : 6 n - 3 itself at the elongation of 20:5n-3 to 22:5n-3.

EXPERIMENTAL PROCEDURES INTRODUCTION

Experimental Design

No biological function has yet been assigned to docosahexaenoic acid (22:6n-3) or, in fact, to any member of the n-3 (a-linolenate) series of fatty acids, either in mammals or in birds. [In the abbreviations used, e.g., 22:6n-3, the first number is the chain length, second number is number of double bonds, and final number is position of first double bond counted from methyl end of chain.] In contrast, rainbow trout appear to require dietary linolenic acid or its homologs, whereas members of the linoleate family (n-6) appear to have no specific function (1-4). Nevertheless, the metabolic behavior of n-3

To determine the extent and distribution of labeling in lipids in normal rats, 14Clqinolenic acid was administered to rats which were sacrificed after 1, 3, 4, 6, 8, 12, 16, and 2 4 h r of incorporation. These rats had been maintained on Purina Laboratory Chow. Radioactivity in the total lipids of livers, hearts, and carcasses was measured. Liver lipids were fractionated, and the distribution of radioactivity among neutral lipids (NL), phosphatidylcholines (PC) and PE was determined. Fatty acids from liver PC and PE were converted to methyl esters, and the amounts and radioactivities of each of these were measured.

194

METABOLISM OF 14C1-LINOLENIC ACID The e x t e n t and distribution of labeling was measured also in rats that had been fed a purified diet, deficient in essential f a t t y acids (EFA). Labeled linolenic acid was administered to rats that were sacrificed after 4, 6, 8, 12, 16, 24, and 36 hr of incorporation. A group of control rats was maintained on the E F A - d e f i c i e n t diet s u p p l e m e n t e d with linoleic acid. These were sacrificed 4 hr after administration of labeled linolenic acid. To determine the effect of a lipid load on the metabolic behavior of linolenic acid, groups of EFA-deficient rats were tube-fed a single dose (50 m g / 1 0 0 g b o d y vet) of linoleic, arachidonic, linolenic, or d o c o s a h e x a e n o i c acids. After 24 hr, labeled linolenic acid was administered, and the rats were sacrificed 4 hr later. AnimaLs and Diets

195 TABLE I

Composition of Essential Fatty Acid-Deficient Diet Major components

Parts by wt

Vitamin-free caseina 20.0 Powdered sucrose 66.5 Salt mix, b ICB-IRb 3.5 Choline chloride 0.28 Hydrogenated coconut oil c 10.0 Vitamins Amount per 100 g diet Vitamin A powder d 1690 units Vitamin D~ powder d 104 units Vitamin E u 7 units Thiamine hydrochloride d 0.50 mg Riboflavine 0.50 mg Folic acid a 0.020 mg Pyridoxine hydrochloride d 0.25 mg Calcium pantothenate e 2.0 mg Nicotinic acid d 2.5 mg Biotina 0.010 mg lnositol, meso d 10.0 mg Vitamin B12 a 0.0020 mg Vitamin K (menadione) d 0.050 mg

Male Sprague-Dawley rats (Horton Laboratories, Oakland, CA) were used throughout these studies. Three different diets were aGeneral Biochemicals, Chagrin Falls, OH. used: Purina Laboratory C h o w (CHOW; Purina bRef. 7. Co., Davenport, IA); an E F A - d e f i c i e n t diet Cplastin IC, Durkee Co., Berkeley, CA. ( E F A D ; Table I); and a control diet (CONdNutritional Biochemicals, Cleveland, OH. T R O L ) , which had the same c o m p o s i t i o n as the eAmerican Cyanamid Corp., Wayne, NJ. E F A D diet, e x c e p t that 1% of h y d r o g e n a t e d c o c o n u t oil was replaced by 1% of m e t h y l benzene solution was purchased f r o m Amerlinoleate. CHOW diet had 3.8% fatty acids (as sham/Searle C o r p o r a t i o n (Arlington Heights, measured by gas liquid c h r o m a t o g r a p h i c (GLC) IL). R a d i o p u r i t y was 97-99%, as s h o w n by gas analysis of a c h l o r o f o r m : m e t h a n o l , 2:1, exc h r o m a t o g r a p h i c (GC) analysis. The benzene tract) of which ca. 47% was linoleic acid was r e m o v e d under a jet of N2, and to the (18:2n-6), 5% a - l i n o l e n i c (18:3n-3), 2% residue was added 20% rat serum (rat fasted eicosapentaenoic (20:5n-3), and 2% was 22: 6n-3. overnight) in isotonic saline. The solution was To produce the EFA-deficiency, m o t h e r rats stirred 1 hr at 25 C. A rat was restrained in a were given the E F A D diet when litters were 1 cage, and the labeled solution (0.4 ml) was week old to reduce the supply of 18:2n-6 and injected into the tail vein over a period of 1 18:3n-3 in the m o t h e r s ' milk. This p r o c e d u r e m i n . T h e dose was 8.7 to 10.1 x 106 p r o d u c e d an E F A deficiency, as s h o w n by the d p m / 1 0 0 g rat. liver PE fatty acid c o m p o s i t i o n (Table II), in a b o u t 6 weeks. There was no mortality. Lipid Extraction and Analysis CHOW-fed rats were used when their wts Animals were sacrificed by decapitation and were ca. 200 g. E F A D rats were used w h e n t h e y weighed ca. 200 g, at which time t h e y were 7 their b l o o d was collected. Livers and hearts weeks old. C O N T R O L rats, also ca. 200 g, were were r e m o v e d , rinsed with distilled water and isotonic saline solution, b l o t t e d w i t h filter ca. 8 weeks old. S U P P L E M E N T E D rats (ca. 2 0 0 g ) were paper, weighed, and frozen on solid CO2. E F A D rats which had been tube-fed a single F r o z e n livers and hearts were lyophilized and dose (50 m g / 1 0 0 g b o d y wt) o f 18:2n-6, pulverized, and lipids were e x t r a c t e d as de18:3n-3, 20:4n-6 arachidonic acid, or 22:6n-3 scribed earlier (6). The rest of the animal, inf a t t y acids. A f t e r 24 hr, 14Cl_linolenic acid was cluding blood, was t e r m e d "carcass." Carcasses administered and allowed to i n c o r p o r a t e for 4 were digested in a saturated solution of K O H in 95% ethanol. Aliquots of the alkaline digest hr, after which the rats were sacrificed. F o o d and water were available to the rats at were acidified (H 2 SO4), and the liberated f a t t y all times. Livers averaged 4.5-5.8% o f b o d y wt, acids were e x t r a c t e d into p e t r o l e u m ether (br 30-55 C). All chemicals and solvents were reaand hearts were 0.30 to 0.38% of b o d y wt. gent grade. Administration of 14C1-kinolenic Acid Liver lipids were fractionated according to 1 4 C 1 - L i n o l e n i c acid (58 m C i / m m o l ) in the procedure of Skipski et al. (8). Bands were LIPIDS, VOL. 11, NO. 3

196

B.P. POOVAIAH, J. TINOCO, AND R.L. LYMAN

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METABOLISM OF 14C1-LINOLENIC ACID

197

TABLE III Incorporation of Radioactivity from Linolenic Acid into Heart, Liver, and Carcass Lipids of Rats Fed Different Diets a Radioactivity

Diet CHOW

EFAD

CONTROL SUPPLEMENTED

Supplement b

Time after injection (hr)

none

none

none 18:2n-6 20:4n-6 18:3n-3 22:6n-3

Heart

Liver

in Carcass

(% of dose)

1 3 4 6 8

0.048 0.056 0.046 0,059 0.038

5.73 7.71 5.51 5.72 3.91

81.5 79.6 73.8 73.2 68.5

12

16 24 4 6 8 12

0.030

3.06

69.8

0.029 0.025 0.19 0.16 0.14 0.16

2.37 2.19 21.8

17.6 16.9 15.2

66.5 63.3 71.7 68.4 68.4 68.5

16

0.16

15.4

68.1

24 36 4 4 4

0.19 O.17 0.08 0.14 0.10 0.16 0.08

16.7 15.2 15.2 17.8 15.3 14.0 9.2

56.5 42.3 76.5 62.7 66.6 71.1 74.0

4

4

aCHOW and EFAD (essential fatty acid-deficient) diets, two rats/interval; SUPPLEMENTED and CONTROL diets, three rats/group. bSupplements (50 rag/100 g body wt) were tube-fed 24 hr before administration of radioactivity. scraped directly from thin-layer chromatography (TLC) plates into scintillation vials and counted with dioxane scintillation mixture. F o r analysis of radioactivity in individual fatty acids, PE and PC were eluted from the silica gel (9) and converted to methyl esters for analysis by GC (10). Gas Chromatography

GC analyses were performed on an F & M Chromatograph (Model 810) equipped with a Packard fraction collector (Model 830). The instrument was calibrated with a mixture of methyl esters of 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Other components (20: 3n-9, 20:4n-3, 22: 5n-6, and 22: 5n-3) were identified by comparison with published relative retention times and by log plots (11). Individual methyl esters were collected in glass tubes containing glass wool, which were placed in scintillation vials and counted with toluene scintillation mixture. An internal standard, 1 4C_heptadecanoi c acid of known specific activity (Nuclear Chicago Co., Des Plaines, IL), was used to determine both the total mass and the total radioactivity in each methyl ester. All fatty acids or methyl esters used in the diets and in the GC standard were obtained from The

Hormel Institute (Austin, MN) and were i> 99% pure by GC analysis. Radioactivity Measurements

Radioactivities were measured with a Beckm a n L S - 1 0 0 l i q u i d scintillation counter. Toluene mixture (0.5% 2,5-diphenyloxazole in toluene) gave 81% counting efficiency, and dioxane mixture (0.6% 2,5-diphenyloxazole plus 10% naphthalene in dioxane, to which is added 0.1 vol H 2 0 ) had 77% efficiency. Lipids in solution were evaporated to dryness and counted in toluene scintillation mixture. Precision of Radioactivity Data

Most of the values for percentage incorporation of label (Tables III and IV) are averages of data that agreed within 10% or less for replicate animals, but a few values represent a range of up to 25%. RESULTS

Table III shows the distribution of radioactivity in lipids of hearts, livers, and carcasses. Hearts incorporated very little activity under any conditions and were not analyzed further. Most of the activity was recovered in carcasses, LIPIDS, VOL. 11, NO. 3

B.P. POOVAIAH, J. TINOCO, AND R.L. LYMAN

198

TABLE IV Incorporation of Radioactivity from Linolenic Acid into Liver Lipids from Rats Fed Different Diets a

Diet

Supplement b

CHOW

none

EFAD

none

CONTROL SUPPLEMENTED

none 18:2n-6 20:4n-6 18:3n-3 22:6n-3

Time after injection (hr)

PC

Radioactivity in c PE (% of dose)

NL

1 3 4 6 8 12 16 24 4 6 8 12 16 24 36 4 4 4 4 4

1.1 2.1 1.3 1.2 1.2 1.0 0.64 0.80 8.3 6.9 5.4 5.5 6.0 4.8 4.2 4.6 7.5 7.7 7.5 4.8

0.95 1.3 0.96 1.0 0.80 0.56 0.47 0.53 5.5 6.8 5.7 4.2 6.1 9.4 8.8 6.2 7.8 5.2 3.4 1.6

3.4 2.5 2.8 2.6 1.5 1.1 1.0 0.63 2.6 2.1 1.5 1.7 1.3 1.7 0.96 2.1 1.4 1.7 1.9 2.2

aCHOW and EFAD (essential fatty acid-deficient) diets, two ratsfinterval; SUPPLEMENTED and CONTROL diets, three rats/group. bSupplements (S0 mg/100 g body wt) were tube-fed 24 hr before administration of ra d i o a c t ivit y .

cpc = phosphatidyl cholines; PE = phosphatidyl ethanolamines; NL = neutral lipids. n e a r l y all as u n c h a n g e d l i n o l e n i c acid ( d a t a n o t p r e s e n t e d ) . M a x i m u m i n c o r p o r a t i o n i n t o liver lipids was o b s e r v e d at 3 h r i n CHOW rats, a n d b y 4 h r in E F A D rats. I n c o r p o r a t i o n s i n t o h e a r t a n d fiver lipids were m u c h h i g h e r (2- t o 5-fold) in t h e rats fed p u r i f i e d diets t h a n in t h o s e fed CHOW ( T a b l e III). Carcass i n c o r p o r a t i o n s were ca. e q u a l in CHOW a n d E F A D rats, so t h a t m o r e a c t i v i t y was r e c o v e r e d f r o m E F A D rats t h a n f r o m t h e CHOW-Fed. Rats o n C O N T R O L or S U P P L E M E N T E D diets i n c o r p o r a t e d slightly less a c t i v i t y i n t o h e a r t s a n d livers t h a n was f o u n d in E F A D rats. In particular, t h e C O N T R O L diet a n d t h e 22:6n-3 SUPPLEMENT reduced incorporation i n t o h e a r t s a n d livers in c o m p a r i s o n w i t h E F A D rats (4 h r ) a n d d i v e r t e d t h e label i n t o t h e carcass. D i s t r i b u t i o n o f r a d i o a c t i v i t y a m o n g liver lipid classes is s h o w n in T a b l e IV. In CHOW rats, r a d i o a c t i v i t y was i n c o r p o r a t e d fastest a n d t o t h e g r e a t e s t e x t e n t i n t o NL). PC possessed t h e n e x t h i g h e s t p r o p o r t i o n a n d PE c o n t a i n e d a l m o s t as m u c h . M u c h smaller a m o u n t s were r e c o v e r e d in o t h e r lipids ( d a t a n o t s h o w n ) . In sharp c o n t r a s t , E F A D rats i n c o r p o r a t e d m o s t o f LIPIDS, VOL. 11, NO. 3

t h e a c t i v i t y i n t o PC a n d PE, at levels 5-fold or m o r e h i g h e r t h a n in t h e CHOW rats. N e u t r a l lipids, h o w e v e r , h a d a m o u n t s o f r a d i o a c t i v i t y m u c h like t h o s e f o u n d in CHOW rats, The changes i n a c t i v i t y w i t h t i m e i n PE a n d PC o f E F A D rats also c o n t r a s t e d w i t h t h o s e in CHOW rats. In CHOW rats, all lipids gradually lost activity a f t e r 4 hr, b u t in E F A D rats t h e PE b e g a n t o regain a c t i v i t y a t 16-24 hr. The C O N T R O L diet r e d u c e d i n c o r p o r a t i o n i n t o PC b u t n o t i n t o PE, in c o m p a r i s o n w i t h E F A D rats. However, a single large dose o f 1 8 : 2 n - 6 , 2 0 : 4 n - 6 , or 1 8 : 3 n - 3 h a d little effect o n i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o PE or PC. I n c o n t r a s t , 2 2 : 6 n - 3 greatly r e d u c e d i n c o r p o r a t i o n i n t o b o t h PE a n d PC. F a t t y acid p a t t e r n s in liver PE are s h o w n in Table II. PE f r o m r a t s given CHOW diet reflect e d t h e dietary f a t t y acids in t h a t t h e r e were considerable proportions of 20:5n-3, 22:5n-3, a n d 2 2 : 6 n - 3 . PE f r o m C O N T R O L rats, w h o s e diet c o n t a i n e d n o n-3 f a t t y acids, h a d levels o f n-3 f a t t y acids even l o w e r t h a n PE f r o m E F A D rats. PE f r o m C O N T R O L rats also c o n t a i n e d a n unusually high proportion of 22:5n-6, a p h e n o m e n o n we have seen b e f o r e (6). E F A D diet produced the well-known increase in

METABOLISM OF 14CI.LINOLENIC ACID 2 0 : 3 n - 9 and, despite t h e deficiency, tile PE still retained considerable 22:6n-3. S u p p l e m e n t a t i o n w i t h a single dose o f f a t t y acid p r o d u c e d various degrees o f r e s p o n s e in f a t t y acid p a t t e r n s of liver PE. S u p p l e m e n t a t i o n w i t h 1 8 : 2 n - 6 h a d little e f f e c t ; o n l y 2 0 : 4 n - 6 was increased a few p e r c e n t a b o v e t h e p r o p o r t i o n in u n s u p p l e m e n t e d E F A D rats, w i t h very m i n o r changes in o t h e r c o m p o n e n t s . S u p p l e m e n t a r y 2 0 : 4 n - 6 p r o d u c e d greater changes, causing a r e d u c t i o n in 2 0 : 3 n - 9 a n d i n c r e a s i n g 2 0 : 4 n - 6 t o m o r e t h a n t w i c e t h e E F A D value: 2 2 : 5 n - 6 was also increased. S u p p l e m e n t a t i o n w i t h 1 8 : 3 n - 3 reduced the 20:3n-9 and increased the 20:5n-3 a n d 2 2 : 6 n - 3 greatly. F e e d i n g 2 2 : 6 n - 3 p r o d u c e d t h e m o s t d r a m a t i c effects b y r e d u c i n g 1 8 : l n - 9 and 20:3n-9 and by increasing 22:6n-3 almost 5-fold. T h e r e was a small increase in 2 0 : 5 n - 3 , w h i c h suggests r e t r o c o n v e r s i o n o f 2 2 : 6 n - 3 (12). Ca. 25% o f t h e 2 2 : 6 n - 3 s u p p l e m e n t was recovered in liver PE a n d PC as 2 2 : 6 n - 3 . F a t t y acid p a t t e r n s in liver PC ( n o t s h o w n ) were similar t o t h o s e in PE, a n d r e s p o n s e s t o t h e s u p p i e m e n t s paralleled t h o s e in PE. Figure 1 shows t h e d i s t r i b u t i o n o f radioactivity a m o n g t h e i m p o r t a n t n-3 f a t t y acids in liver PC of E F A D rats as t h e c o n v e r s i o n o f 1-14C-linolenic acid progressed w i t h time. Very little activity was r e c o v e r e d as 1 8 : 3 n - 3 itself. T h e 2 0 : 5 n - 3 c o n t a i n e d t h e highest p e r c e n t a g e o f t h e dose at 4 hr, a n d this a c t i v i t y d e c l i n e d w i t h time. Its p r e s u m e d p r e c u r s o r , 2 0 : 4 n - 3 , h a d m u c h less activity a n d m a i n t a i n e d a s t e a d y level

i[

I~

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o 9 A 9

;

199

20:4 n3 20:5 n3 22:5 n3 226 n3

,~

,~

2'4

3'0

3'~

TiME (HR) FIG. 1. Radioactivity in n-3 fatty acids of liver phosphatidyl cholines from essential fatty acid-deficient rats. Averages of data from two rats per time period.

61 f 5

0 20:4 n3 9 22:5 20:5 n3 n3 9 22:6 n3

u_ o

O!

•

12

24

}8

30

36

TiME (HR) FIG. 2. Radioactivity in n-3 fatty acids of liver phosphatidyl ethanolamines from essential fatty aciddeficient rats. Averages of data from two rats per time period.

TABLE V Distribution of Radioactivity Among n-3 Fatty Acids in Liver Phospholipids from Rats a Fed Different Diets: Percentage of Activity Recovered in n-3 Fatty Acids 4 hr after Administration of 14c1.Linolenic Acid Diets Fatty acid

CHOW

EFAD

CONTROL

SUPPLEMENTED with b 18:3n-3

18:2n-6

20:4n-6

22:6n-3

P h o s p h a t i d y l cholines 18:3n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3

17 6 67 5 5

3 25 51 5 16

3 13 24 20 40

3 40 33 8 16

2 35 37 8 18

4 18 63 4 10

5 15 64 5 12

3 l 1 37 11 39

2 5 11 25 57

2 33 30 13 23

2 14 34 18 34

3 6 52 14 24

3 6 51 8 32

Phosphatidyl ethanolamines 18:3n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3

24 3 41 t8 13

aThere were three rats in the SUPPLEMENTED and CONTROL groups, and two rats each in CHOW and EFAD (essential fatty acid-deficient) groups. bSupplements were fed 24 hr before administration of radioactivity. LIPIDS, VOL. 11, NO. 3

200

B.P. POOVAIAH, J. TINOCO, AND R.L. LYMAN

of activity after 12 hr. The amount of activity 18:3n-3 reduce these activities to normal levels in 22:5n-3 remained low throughout the experi- after 2-7 days (13,14). Probably our SUPPLEment. By 4 hr, 22:6n-3 had already incor- MENTED rats had insufficient time to reduce porated ca. 1% of the dose, and this fatty acid their enzyme activities to normal levels. Howgained activity gradually until 24 hr when ac- ever, CONTROL rats also incorporated more tivity leveled off. In CHOW rats (data not radioactivity than CHOW rats did. This suggests presented), similar behavior was seen, although either that n-3 fatty acids (in the CHOW diet) can suppress enzyme activity more effectively activities in each fatty acid were nmch lower. Figure 2 shows the incorporation of activity than 18:2n-6 can, or that some other factor is from linolenic acid into fatty acids of liver PE responsible. The dietary carbohydrate can also from EFAD rats. This lipid class, although only influence fatty acid incorporation, inasmuch as half as large by wt as PC, incorpoated much Macdonald found higher levels of 18:2n-6 in more of the administered radioactivity. As in liver Iipids of rabbits fed sucrose in comparison PC, 20:4n-3 and 22:5n-3 maintained a low with those given starch at the same level in the steady level of activity throughout the experi- diet (15). Our purified diets contain sucrose, ment. The activity in 20:5n-3 fluctuated with whereas CHOW diet contains polysaccharide. apparent maxima at 6 and 24 hr. 22:6n-3 Thus, the sucrose may have increased the incorgained activity with t i m e and appeared to be poration of linolenic acid and its homologs into the livers of all rats fed purified diets. The prostill increasing its activity even at 36 hr. The effects of dietary differences on distri- found differences between CHOW-fed rats and bution of radioactivity among n-3 fatty acids of those given purified diets illustrate dramatically liver PL are shown in Table V. CHOW rats the fallacy of comparing metabolic changes in incorporated very little activity into PC and PE, CHOW-fed animals with those in animals fed and the largest proportions were recovered in well-defined diets (16). The major path of chain elongation and 20:5n-3 and 18:3n-3. CONTROL rats, which might be expected to resemble the CHOW rats, desaturation of linolenic acid is as follows: instead incorporated the greatest proporations of activity into 20:5n-3 and 22:6n-3 of PC and 18:3n-3 --+ 18:4n-3 --+20:4n-3 -~ 20:5n-3 --+ into 22:5n-3 and 22:6n-3 in PE. Rats fed single 22:5n-3 --~22:6n-3 -+ ? supplements of 18:2n-6 and 20:4n-6 produced We found very little radioactivity in 18:3n-3 very similar distribution patterns of activity in PC; most activity was in 20:4n-3 plus 20:5n-3. of liver PL even at the shortest periods of incorA similar but less pronounced effect was pro- poration, although there already was activity in duced in PE. Animals fed supplements of 22:6n-3 (Table V). This indicates that 18:3n-3 18:3n-3 and 22:6n-3 incorporated the greatest is not well incorporated into liver PL, although proportion of activity into 20:5n-3 in both PE its metabolic products are. This observation is consistent with data obtained from feeding and PC. experiments. When rats were fed diets containing flaxseed oil (52% 18:3n-3), the amounts of DISCUSSION 18:3n-3 in liver PE and PC were negligible, The phenomena visible in the liver lipids are whereas the proportion of 20:5n-3 rose 10-fold the result of many concurrent processes in the (17). rat. These include uptake of the label from the Similarly, very little activity corresponding circulation by tissues, incorporation of the to either 18:4n-3 or 20:3n-3 was found. Thus, linolenic acid molecule into lipids, acyl CoA these intermediates, if they are formed, must be derivatives or other products, and, of course, converted very rapidly to 20:4n-3. elongation and desaturation of the fatty acyl There was little accumulation of radioacchains. Our data show the combined effects of tivity in 20:4n-3 of EFAD rats (Figs. 1 and 2). all these processes. Its formation and removal nmst occur at equal Table III shows that EFAD rats incorporated rates because activity did not remain high in into heart and liver !ipids far more radioactivity 20:4n-3, nor does its mass accumulate to than CHOW rats did. This effect can also be measurable levels even when excess linolenic seen in CONTROL rats and rats supplemented acid is fed in the diet (17). with a single dose of fatty acid, which also In contrast, 20:5n-3 always contained a large incorporated much more activity than CHOW proportion of the radioactivity (Figs. 1 and 2, rats did. These differences can be attributed Table V). Its rate of removal must have been p a r t l y t o elevated activities of lipogenic lower than its rate of formation and incorporaenzymes, which occur in rats given fat-free or tion. When flaxseed oil is fed, the amount of EFAD diets. Dietary 18:2n-6, 20:4n-6, or 20:5n-3 in rat liver PL rises ca. 10-fold (17). A LIPIDS, VOL. 11, NO. 3

METABOLISM OF 14CI-LINOLENIC ACID small quantity of this acid may also be formed by r e t r o c o n v e r s i o n of 22:6n-3 (Table II, 22:6n-3 SUPPLEMENTED) (12). Figures 1 and 2 show that the radioactivity in 22:5n-3 remained low and constant, and its mass was low under various dietary conditions (Table II). Even in flaxseed oil-fed rats, 22:5n-3 does not rise above control levels in liver PE and PC (17). Its rates of formation and incorporation must be practically equal to its rate of removal from PL. Radioactivity was present in 22:6n-3 at 4 hr and continued to increase up to 24 hr in PC, and was still rising in PE at 36 hr (Figs. 1 and 2). The fact that 22:6n-3 is usually the major n-3 fatty acid in PL suggests that its rate of removal must be slow. Experiments with single-dose supplements were designed to show how dietary fatty acids would influence accumulation of intermediates in the elongation-desaturation sequence. Influences of the supplements can be separated into two effects, an effect on total incorporation of radioactivity into PL and another effect on distribution of radioactivity among the n-3 fatty acids. Table IV shows the effects of different diets on total incorporation into PL. The n-6 fatty acid supplements had little effect on early incorporation into PC and PE in comparison with E F A D rats, but 18:3n-3 reduced incorporation into PE and 22:6n-3 reduced incorporation into both PE and PC. The distribution of radioactivity among the n-3 fatty acids probably reflects the effects of diet composition on chain elongation and desaturation (Table V). The many competitions b e t w e e n various fatty acid structures for desaturation, elongation, and incorporation, mostly observed in vitro, have been reviewed by Brenner (18). Usually, a fatty acid with more double bonds and greater chain length will be desaturated faster than a more saturated, shorter chain under equivalent conditions in vitro. In the living animal, concentrations of the reacting species are unknown, as are the contributions from side reactions. Table V shows that radioactivity from linolenic acid was often concentrated in 20:5n-3 but rarely in 22:5n-3. This suggests that conversion of 20:5n-3 to 22:5n-3 is the rate4imiting step in the formation of 22:6n-3, in vivo. This idea is supported by evidence from feeding experiments in which 20:5n-3, but not 22:5n-3, accumulated when high levels of 18:3n-3 were fed (17). Furthermore, the conversion of 20:5n-3 to 22:5n-3 appeared to be controlled by the amount of 22:6n-3 in liver PL (Table II). This is shown by the high proportions of radioactivity in 20:5n-3 found when the liver PL contain high levels of

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22:6n-3, that is, in rats supplemented with a single dose of either 18:3n-3 or 22:6n-3 and in those on the CHOW diet (Table II). Likewise, when the liver PL contained extremely low amounts of 22:6n-3 (CONTROL diet, Table II), there was minimum accumulation of activity in 20:5n-3 (Table V). PL that contain 22:6n-3, therefore, seem capable of suppressing their own formation at the elongation of 20:5n-3 to 22:5n-3. These PL may inhibit this elongation by releasing 22:6n-3 (for example, through the action of phospholipase A 2), which is known to inhibit elongation of 18:3n-3 in vitro (I 9). Desaturation of 20:4n-3 to 20:5n-3 appeared to be impaired in animals given supplements of 18:2n-6 or 20:4n-6 (Table V). This must have been a temporary effect caused by the sudden large dose of fatty acid because ~he CONTROL animals, fed small but adequate amounts of 18:2n-6 throughout the experiment, showed no suppression of this step. Evidently, the rat has great ability to form 22:6n-3 from precursors and to incorporate large amounts of this acid into PL, especially PE. In addition~ the amount of 22:6n-3 in liver PL of the rat appears to be controlled by 22:6n-3 itself for reasons as yet unknown. These elaborate systems for biosynthesis and i n c o r p o r a t i o n of 22:6n-3 imply that this molecule should have some important function in the cell, but at present we have no specific suggestions. ACKNOWLEDGMENTS This work was supported by USPHS Grant AM 10166. P.G. Miljanich developed the internal standard technique for measuring the specific activity of individual methyl esters, and Ruth Babcock prepared the illustrations. REFERENCES 1. Castell, J.D., R.O. Sinnhuber, J.H. Wales, and D.J. Lee, J. Nutr. 102:77 (1972). 2. Castell, J.D., R.O. Sinnhuber, D.J. Lee, and J.H. Wales, Ibid. 102:87 (1972). 3. Castell, J.D., D.J. Lee, and R.O. Sinnhuber, Ibid. 102:93 (1972). 4. Yu, T.C., and R.O. Sinnhuber, Lipids 7:450 (1972). 5. Anderson, R.E., Exp. Eye Res. 10:339 (1970). 6. Tinoco, J., M.A. Williams, I. Hincenbergs, and R.L. Lyman, J. Nutr. 101:937 (1971). 7. Williams, M.A., L.-C. Chu, D.J. Mclntosh, and I. Hincenbergs, Ibid. 94: 37q (1968). 8. Skipski, V.P., R.F. Peterson, and M. Barclay, Biochem. J. 90:374 (1964). 9. Arvidson, G.A.E., Eur. J. Biochem. 4:478 (1968). 10. Tinoco, J., S.M. Hopkins, D.J. Mclntosh, G. Sheehan, and R.L Lyman, Lipids 2:479 (1967). 11. Ackman, R.G., and R.D. Burgher, JAOCS 42:38 (1965). 12. Schlenk, H., D.M. Sand, and J.L. Gellerman, Biochin~ Biophys. Acta 187:201 (1969). LIPIDS, VOL. 11, NO. 3

13.P. POOVAIAH, J. TINOCO, AND R.L. LYMAN

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13. Chu, L.-C., D.J. McIntosh, I. Hincenbergs, and M.A. Williams, Ibid. 187:573 (1969). 14. Musch, K., M.A. Oiakian, and M.A. Williams, Ibid. 337:343 (1974). 15. Macdonald, I., J. Physiol. 162:334 (1962). 16. Greenfield, H., and G.M. Briggs, Ann. Rev. Biochem. 40:549 (1971). 17. Lyman, R.L., G. Sheehan. and J. Tinoco, Can. J.

Biochem. 49:71 (1971). 18. Brenner, R.R., Mol. Cell. Biochem. 3:41 (1974). 19. Christiansen, K., Y. Marcel, M.V. Gan, H. Mohrhauer, and R.T. Holman, J. Biol. Chem. 243:2969 (1968). [ R e c e i v e d S e p t e m b e r 23, t 97 5 ]

A Guide for Authors is Located in Lipids 11(January):85(1976) LIPIDS, VOL, 11, NO. 3