Biochimica et Biophysica Acta. 1082 (1991) 57-62 iJ 1991 E'lsevier Science Publishers B.V. 0005-2760,/91/$03.50 ADONIS 000527609100103T
57
BBALIP 53588
Effect of dietary n - 3 and n - 6 fatty acids on fatty acid desaturation in rat liver E r l i n g N . C h r i s t i a n s e n , J o a n n a S. L u n d , T o r i l l R o r t v e i t a n d A r i i d C. R u s t a n lnstttute for Nutrition Research. Uml'erst(t" of Oxlo. Oslo (Nor~'ar) ( Received 29 June 1990)
Key words: Dietary fat: (n - 3) Fatty acid, ¢n - 6) Fatty acid: Fatty acid desaturation: Xticrosornc: (Rat liver)
To study the effect of high-fat diets with varying contents of n - 3 and n - 6 fatty, acids on the metabolism of essential fatty acids, the rat liver microsomal fatty acid desaturases were measured. The rats were fed for 3 weeks with diets high in linseed oil ( 1 8 : 3 ( n - 3)), sunflower seed oil ( 1 8 : 2 ( n - 6)) or fish oil ( 2 0 : 5 ( n - 3) and 2 2 : 6 ( n - 3)) (20%, w / w ) using pellet fed rats as a reference. The A6-desaturase using 18:2(n - 6) or 18:3(n - 3) as substrates was stimulated 1.5-2.5-fold by linseed or sunflower seed oil, compared to the pellet reference. The AS-desaturase was stimulated 3.5-fnld with linseed oil and 2.5-.fold with sunflower seed oil, while the Ag-desaturase was inhibited by all the high-fat diets. The z16-, s. and 9-desaturase activities were in all cases considerably reduced with fish oil as compared to linseed and sunflower seed oil diets. With pellet fed rats the rates were highest for zi~-desaturation and in decreasing order lower for AS-desaturation, A6-desaturation with 18:3 (n - 3) as substrate and finally A6-Desaturation with 18: 2(n - 6) as substrate. The content of 2 0 : 4 ( n - 6 ) in liver phospholipids increased with the diets rich in 1 8 : 2 ( n - 6 ) , and was reduced for the fish oil diet enriched in 20:5 and 22:6(n - 3) fatty, acids. The amount of 2 0 : 5 ( n - 3) in phosphnlipids was as high with linseed oil diet as with the fish oil diet, while the 22:6(n - 3) content was only increased with the fish oil diet.
Introduction It is of significant interest to know how the dietary content of polyunsaturated 'fatty acids, with different proportions of n - 3 and n - 6 fatty acids, affect the metabolism of essential fatty acids. Desaturation of polyunsaturated fatty acids is an important reaction for the synthesis of very-long-chain polyunsaturated fatty acids important for membrane functions and formation of eicosanoids [1]. A6-Desaturation is considered to be the rate limiting reaction for these sequences [2,3]. Fatty acid desaturation is regulated by a number of nutritional and hormonal factors [4] and the type and amount of dietary fat have been shown by Garg et al. [5-7] to be important factors. The fatty acids of the n - 3 and n - 6 families are considered to be metabolized by the same fatty acid desaturases, but the affinity
Abbreviations: LO, linseed oil diet; SO. sunflower seed oil diet: FO, fish oil diet; PEL, low-fat reference diet; GLC. gas-liquid chromatography; TLC. thin-layer chromatography. Correspondence: E.N. Christiansen, Institute for Nutrition Research, University of Oslo, P.O. Box 1046, N-0316 Oslo 3, Norway.
for the n - 3 family is greater than the affinity for the n - 6 family [8}. There is considerable evidence of the beneficial role of marine n - 3 fatty acids for reducing the incidence of cardiovascular and inflammatory diseases [9,10]. Related to these effects there are a number of important questions: for instance, what is the proper balance of the supplies of n - 3 and n - 6 fatty acids and to which extent is 18: 3 ( n - 3 ) providing sufficient amounts of long-chain n - 3 fatty acids? Therefore, it is important to know how diets with different {n - 3 ) / ( n - 6) fatty acid ratios and different n - 3 fatty acids affect fatty acid desaturation, and thereby the availability of longchain polyunsaturated fatty acids (e.g., arachidonic and eicosapentacnoic acid) for further metabolism. To obtain more information of the effect of n - 3 and n - 6 fatty acids on fatty acid desaturation in the liver, rats were fed high-fat diets with different amounts of n - 3 and n - 6 fatty acids. A linseed oil diet (LO) enriched with 1 8 : 3 ( n - 3 ) fatty acid, was compared with three other high-fat diets, one enriched with 18 : 2(n - 6) fatty acid (SO), another with equal amounts of n - 3 and n - 6 fatty acids ( L O / S O ) , and a third diet with fish oil (FO) containing high a m o u n t s of long-chain n - 3 fatty
58 acids (20 : 5 and 22 : 6). Animals fed on standard pellet (PEL) were used as a low fat reference. In the present study, we describe the effect of these diets on the in vitro activities of As-, A6- and Ag-desaturase and on the fatty acid composition of liver phospholipids. Materials and Methods
Materials The fish oil (Japanese) was a gift from D e N o F a and Lilleborg Fabriker A / S (Fredrikstad, Norway). Linseed oil and sunflower seed oil were gifts from Rett-trading (Oslo) (representing Henry Lamotte, Bremen, F.R.G.). The pelleted stock feed for rats was obtained from Ewos AB (S~Sdert~ilje, Sweden). Vitamin and salt mixtures of the semisynthetic diets were from ICN Nutritional Biochemicals (Cleveland, OH, U.S.A., Cat. No. 904610 and 904654). [1-14C]Stearic acid ( 1 8 : 0 ) (2.07 G B q / m m o l ) , [1-~4C]linoleic acid (18 : 2(n - 6)) (1.98 G B q / m m o l ) , [l~4C]linolenic acid (18 : 3(n - 3)) (1.99 G B q / m m o l ) and [l-~4C)dihomo-7-1inolenic acid ( 2 0 : 3 ( n - 6)) (1.77 GBq/mmol) were from A m e r s h a m I n t e r n a t i o n a l (Amersham, U.K.). Bovine serum albumin, essentially fatty acid free, and other fine chemicals were from Sigma Chemical (St. Louis, MO, U.S.A.). Animals and diets Male rats (approx. 160 g) of the Wistar strain were purchased from Mollegaard Breeding L a b o r a t o r y (Ejby, Denmark). The animals were fed a standard pellet diet for 5 - 9 days, then went on with standard pellet (PEL) or experimental semisynthetic diets for 3 weeks [11]. The composition of the semisynthetic diet was, in weight% of total: sucrose, 20; cornstarch, 33; casein (with 2% methionine) 20; cellulose, 1; vitamin mixture, 2; salt mixture, 4; and diet oil 20. The compositions of the diet oils were: LO, linseed oil (99%) and sunflower seed oil (1%); L O / S O , linseed oil (54%) and sunflower seed oil (46%); SO, linseed oil (21%) and sunflower seed oil (79%); FO, fish oil (92%) and sunflower seed oil (8%). The diet cAls were mixed to give a controlled scale of (n - 3 ) / ( n - 6) fatty acid ratios from 0.3 to 5.2. The L O / S O diet was mixed to give near equal a m o u n t s of n - 3 and n - 6 fatty acids. The fatty acid composition of the different semisynthetic diets are given in Table I. The pellet diet consisted of cereals, wheat germs, wheat middlings, fish protein concentrate, soya meal (toasted), fodder yeast, minerals, a n i m a l and vegetable fat, vitamin and trace element concentrates (protein, 22; carbohydrates, 52; fat 5 and others 21% of wet weight). The falty acid composition (%) of the fat according to the producer was: 1 6 : 0 , 21; 16:1, 2: 1 8 : 0 , 4; 1 8 : 1 , 22; 18:2(n-6), 42; 1 8 : 3 ( n - 3 ) , 7 and others, 2. All animals appeared healthy after the 3-week.~ experimental period. There were no significant differences in food consumption between the high-fat dietary groups, but
TABLE I Fatty acid composition of semts).nthetic diets (%) Fatty acid composition ','as determined by GLC as described in Materials and Methods. Antioxidants were 0.04% butylated hydroxytoluene (BHT) and 0.01% vitamin E. arty acids 14:0 16:0 16:1(n-7) 18:0 18:1(n-9) 18:1(n-7) 18:2(n -6) 18:3(n -3) 20:5(n -3) 22:l(n-ll) 22 : 5(n - 3) 22 : 6( n - 3) Others Total Saturated Monoenes Polyenes (n - 3)/(n -6)
LO 4.4 4.1 16.1 11.6 61.5 2.3
LO/SO
3.0 19.2 37.7 32.8 2.4
5.5 3.8 19.5 1.3 51.7 13.9 4.3
8.5 16.1 73.1 5.2
7.9 19.2 70.5 0.9
9.3 20.8 65.6 0.3
4.9
SO
FO 7.4 20.0 7.8 3.0 11.2 3.4 9.0 3.4 12.9 1.4 1.3 7.1 12.1 30.4 23.8 33.7 2.1
the pellet fed group had a slightly higher c o n s u m p t i o n ( P < 0,05). Weight gain, b o d y a n d liver weights for all dietary g r o u p s were similar (data not shown). The rats were housed in g r i d - b o t t o m e d cages, two in each cage, and they had free access to food a n d water. The r o o m - t e m p e r a t u r e was 2 4 ° C with 60% relative h u m i d i t y a n d a 12 h light period (07:00-19:00).
Preparation of liver microsomes The rats were sacrificed between 09:00 to 11:00 a.m. A 15% liver h o m o g e n a t e was prepared at 4 ° C in 250 m M sucrose c o n t a i n i n g 10 m M Hepes ( p H 7.4), using a Potter-Elvehjem homogenizer. The h o m o g e n a t e was centrifuged at 7 5 0 × g (2500 rpm) for 10 rain in a Sorvall R C 5 C highspeed centrifuge ( D u p o n t , Delaware), rotor SS34. The postnuclear fraction was centrifuged at 5100 × g (6500 rpm) for 10 min and 30900 × g (16000 rpm) for a further 10 min in a rotor as above. The microsomal pellet was o b t a i n e d at 63000 × g ( 2 9 0 0 0 rpm) for 60 min in a SorwaU O T D 55B ultracentrifuge, rotor T F T 70.38. Protein was d e t e r m i n e d according to Lowry et al. [12] using bovine serum a l b u m i n as standard. Analytical methods Microsomes from rats fed the different diets were assayed for activities of A5-, 6. and 9-desaturases m a i n l y as described by Mahfouz & H o l m a n and Svensson [13,14]. The o p t i m a l substrate (at s a t u r a t i o n level) a n d
59 microsomal protein concentrations (linear dependency) and incubation time (linear dependency) for the four desaturase assays were determined (data not shown). The incubation mix:ure consisted of 0.15 M KCI, 5 mM ATP, 5 mM MgCI 2, 0.25 mM CoA, 1.0 mM NADH, 1.5 mM glutathione, 45 mM NaF, 0.5 mM nicotinamide, I mg bovine serum albumin and 0.1 M potassium phosphate buffer (pH 7.4). In our experiments, we used 0.5-2.0 mg of microsomal protein and 75-150 nmol t4C-labelled fatty acid substrate (6-11 kBq) in 1 ml incubation volume (see legend to figures). The fatty acids were used as sodium salt/bovine serum albumin complexes. "[he incubation time used was 20 min at 37°C, and the reaction was terminated by addition of 1 ml of 2.7 M KOH in methanol. This mixture was heated at 65°C for 45 min ar.d acidified by 6 M HCI. Nonesterified fatty acids were extracted with hexane and methylated according to Hoshi et al. [15]. As- and 6-desaturation activities were determined using radioGLC (Varian 2100 equipped with a 10% SP-2340 on a Supelcoport 100/120 60 cm column (Supelco, BeIlefonte, PA, U.S.A.) with argon as carrier gas) connected to a nuclear radiodetector (ESI Nuclear, Surrey, U.K.) [16]. Ag-Desaturation activity was determined using silica gel TLC plates (Silica gel F 1500, Schleicher & Schuell, Dassel, F.R.G.) impregnated with 10% AgNO 3 ( w / v ) in acetonitrile. Carrier methyl esters (18 : 0 and 18 : l ) were applied and the TLC-plates were developed in h e x a n e / d i e t h y l ether (90:10, v / v ) for separation of saturated and monounsaturated fatty acids [7]. The spots were visualized by ultraviolet-light using 2,7-dichlorofluorescein (0.2%, w / v in ethanol) and counted in a Packard Tri-Carb 300 liquid scintillation spectrometer using 6 ml instagel I1 (Packard, Chicago, !L, U.S.A.). A control incubation was performed with microsomes in the absence of substrates and free fatty acids were measured. There was no change in their fatty acid composition before and after the incubation (unpublished data). This indicates that there is no contribution from endogeneous fatty acids affecting the results. The fatty acid composition of liver phospholipids was measured as methyl esters [171 with GLC (Carlo Erba Strumentazione, Fractovap Series 2150, Milan, Italy), equipped with a nonpolar capillary cohtmn (CP Sil 15CB, 50 m, diam. 0.2 ram) and using helium, 0.5 m l / m i n as a carrier gas [11]. The temperature was programmed to rise from 180 to 215°C at 0.5 C ° / m i n . Diheptadecanoylphosphatidylcholinewas used as an internal standard.
Statistical analysis Results are means + S.D. of six rats. Data were analyzed by the Mann-Whitney non-parametric test (two-tailed) (Minitab statistical vrogram, Minitab, State College, PA, U.S.A.L
Results
Effect of diets on desaturase actit,ities A~-desaturation. A6-desaturase using linoleic acid (n -6) as substrate was stimulated 2-2.5-times by the high fat L O / S O and SO diets as compared to the reference d:et (PEL) (Fig. 1). With the FO diet the activity was only 20% compared to the SO diet and 30% compared to the LO diet. There was a significant difference between the LO and SO diets, the LO being about 40% lower. With linolenic acid ( n - 3) as substrate, there was about 2.5-times stimulation with the LO, SO and L O / S O diets compared to the PEL diet (Fig. 2). With the FO diet the activity was 60% lower compared to the LO diet. The rate of /~6-desaturation with the PEL diet using linoleic acid ( n - 6) as substrate was about 50% lower than using linolenic acid (n - 3) as substrate (Fig. 1 and 2). ,~5-desaturation. ALdesaturase was measured using 20: 3 ( n - 6) as substrate (Fig. 3). The LO diet stimulated the activity by 4-times and the SO diet 3-times compared to the PEL diet. With the FO diet, the activity was 25% of the activity obtained with the LO diet. The rate of AS-desaturation with the PEL diet was nearly 3-times higher than for A6-desaturation with 18 : 2(n - 6) as substrate and about 7-times higher comparing the same desaturases with the LO fed rats (Figs. 1 and 3). A°-desaturation. A~-desaturase activity was decreased with all high-fat diets compared to the PEL diet; with the L O / S O and SO diets the activity was 40-50% lower and with the LO diet it was also a reduction, however
PEL
LO
LO/SO
SO
FO
diet
Fig. I. Effect of diets rich in n - 3 and n - 6 fatty acid~on rat liver microsomal A6-desaturationusing linoleic acid (l 8 : 2 ( n - 6)) as substrate. Activitiesare expressedas nmol 18:3(n -6) (7-1inolenicacid) synthesized per min per mg microsomal protein at 37°C. In this experiment 75 nmol [l-14C]linoleicacid (18:2(n-6)) was used as substrate and 2 mg microsomal protein. The substrate and product fatty acids were separated by radio-Gl C a~ described in Materials and Method~, Data represents means_+S.D. (n = 6). Bars without a common !crierare significantlydifferentat P < 0.05.
60 nmoles/mg x rain
Tb b
0.8
~ ,~
0.6
~~ ~a
0.4
O ~
PEL
~
LO
LO/SO SO FO diet Fig. 2. Effect of diets rich in n - 3 and n - 6 fatty acids on rat liver microsomal ~,6-desaturation using ]ino]enic acid (18:3(n -3)1 as substrate. Activities are expressed as nmol 1 8 : 4 ( n - 3) synthesized per min per mg microsomal protein at 37°C. In this experiment 100 nmol []-V~C]linolenic acid ( 1 8 : 3 ( n - 3 ) ) was used as substrate and 1.5 mg microsomal protein. The substrate and product fatty acids were separated by radio-GLC as described in Materials and Methods. Data represents means_+S.D. (n = 6). Bars without a common letter are significantly different at P < 0.01.
PEL
LO
LO/SO
SO
FO
diet
Fig. 3. Effect of diets rich in n - 3 and n - 6 fatty acids on rat liver microsomal AS-desaturation osing dihomo-7-1inolenic acid (20: 3 ( n 6)) as substrate. Activities are expressed as nmol 2 0 : 4 ( n - 6 ) (arachidonic acid) synthesized per rain per mg microsomal protein. 150 nmol [l-t4Cl dihomo-y-linolenic acid { 2 0 : 3 ( n - 6)) was used as substrate and !.5 mg microsomal protein. The substrate and product fatty acids were separated by radio-GLC as described in Materials and Methods. Data represents means ± S.D. (n = 6). Bars without a common letter are significantly different at P < 0.01.
n o t s i g n i f i c a n t ( F i g . 4). W i t h t h e F O d i e t t h e Ag-de saturase activity was significantly lower than for the LO, L O / S O
a n d S O diets.
C o m p a r e d t o t h e p e l l e t fed rats, t h o s e fed L O a n d S O d i e t s h a d a l o w e r a m o u n t o f l b : 0 a n d a h i g h e r level
The monoene content (18:1) was significantly lower w i t h all t h e h i g h - f a t d i e t fed r a t s t h a n w i t h t h e p e l l e t fed. 1 8 : 2 ( n - 6 ) was considerably lower with the FO diet than the other diets. 1 8 : 3 ( n 3) w a s n o t d e t e c t a b l e i n h e p a t i c p h o s p h o l i p i d s i n t h e S O a n d F O fed a n i m a l s a n d even in the presence of large a m o u n t s of
o f 1 8 : 0 in l i v e r p h o s p h o l i p i d s ( T a b l e 11). F o r t h e F O
d i e t a r y 18 : 3 ( L O g r o u p ) , t h e c o n c e n t r a t i o n o f 18 : 3 i n
fed a n i m a l s t h e r e w a s a s l i g h t l y h i g h e r a m o u n t o f 16 • 0.
p h o s p h o l i p i d s is n o t m u c h h i g h e r t h a n i n p e l l e t f e d rats.
Effect of diets on .fatty acid composition of lioer phospho/ipids
TABLE I1
Faro' acid composition of liverphospholipids (mg/g liver) Fatty acid composition was determined by GLC as described in Materials and Methods. Data represent means +. S.D. {n = 6). Values without a common superscript are significantly different at P < 0.05. n.d., not detectable. PEL
LO
LO/SO
SO
FO
16 :0
4.99 + 0 . 1 9 ~
3.91 + 0 . 4 5 h
3.79+0.31 h
3.49+0.19 h
6.11:1:0.52 ~
18 : 0 18:1 (n - 9 ) 18:1 (n - 7 ) 1~ : 2 (n -- 6) 18:3 OI -- 3) 20:3 (n - 6 ) 20:4 (n - 6 ) 20 : 5 (n - 3) 22:5 (n - 3 ) 22:6 (n - 3 )
5.45 + 0.21 ~ 1.94+_0.38 ~ 0.89+0.09 ~ 3.45 5:0.21 a 0,4830,02 a 0.28+0.04 ~ 3.55±0.39 ~ 0,25 +:0.03 a 0.21 +0.04 a 0.96 + 0,16 a
7.44 + 0.51 h 1.1830.39 ~ 0.53+0.16 h 4.28 + 0.36 b 0.5330.13 a 0.32+0.05 a 3,49±0.95 ~ 1.72 +:0.26 b 0.53+0,10 ~ 0,97±0.30 a
7.95 + 0.63 b 0.79:t:0.15 h 0.47+0.11 b 4.63 + 0.59 b 0.36+0.03 h 0.2630.05 ~ 5.19+0.86 b 0.62 + 0,19 c 0.31 +0.05 a 0.86+:0.16 a
6.67 ± 0.37 ~ 0.63+0.11 n 0.46+0.05 b 3.35 ± 0.28 a n.d. 0,1630.08 a 5.40+:0.40 b 0.39 +:0.02 ~ 0.16+'0,02 a 0.82+:0.12 a
6.06 + 0.20 ~ 0.76+0.05 b 0.79+0,10 ~ 1.85 5:0.26 ~ n.d. 0.20+0.07 ~ 2,47 +0.27 ¢ 1 68 + 0,16 h 0.50+0.06 b 2.32+:0.26 b
Total Saturated Monoenes Polyenes (n - 3)/(n - 6 )
22.4 10.4 2.8 9.2 0.2
24.9 11.4 1.7 11.8 0.5
25.2 11.7 1.3 12.2 0.2
21.6 10.2 1.1 10.3 0.2
22.8 12.2 1.6 9.0 1.0
61 nmoles/mg x min
[
T~
0~
l ~ PEL
0s
~'~ LO
kO/$O
$O
FO
diet
Fig. 4. Effect of diets rich in n -3 and n -6 fatty acids on rat liver microsomal ,~9-desaturation using stearic acid 118:0) as substrate. Activities are expressed as nmol 18: l¢n-9) (oleic acid) synthesized per rain per nag microsomal protein. In this experiment 150 nmol [l-14Clstearic acid was used as substrate and 1 mg microsomal protein. The substrate and product fatty acids were separated by argentation TLC as described in Materials and Methods. Data represents means+S.D. (n = 6). Bars without a common letter are different at P < 0.05.
A large a m o u n t of 1 8 : 3 ( n - 3 ) in the LO group was transformed to 2 0 : 5 and 2 2 : 5 , the content of these fatty acids being as high as the F O diet, while the 22 : 6 level was 50% lower and similar to the pellet diet. The a m o u n t of 2 0 : 4 ( n - 6) in liver p h o s p h o l i p i d s increased with the L O / S O and SO diets, whereas it was reduced in the F O group as c o m p a r e d to the pel!et diet. The total a m o u n t of polyunsaturated fatty acids was similar for all dietary groups. The fatty acid composition of liver microsomal phospholipids was also measured, and revealed the same p a t t e r n as for total liver phospholipids (data not shown). Diseusslon
The A6- and AS-desaturation increased with the diets rich in linoleic and linolenic acids c o m p a r e d to the PEL reference diet. There appeared to be a difference in the response using linoleic (n - 6) or linolenic acid ( n - 3) as substrate for the ,~6-desaturase even if the enzyme is considered to be the same for both substrates [8]. ¢ / i t h linoleic acid as substrate there was a significantly higher desaturation rate with the SO diet than the LO diet (Fig. 1). With linolenic acid as substrate there was a small, but not statistically significant, increase in A6 desaturation with the L O diet than with the SO diet (Fig. 2). These differences could possibly be explained by substrate dilution caused by fatty acids from the diet. W e examined, however, free fatty acids in microsomes before and after incubating under the same conditions as for the desaturase measurements, and found no changes in the fatty acid pattern. Thus, the possibil-
ity that A6-desaturation of n -- 3 and n - 6 fatty acids are carried out by different enzymes should not be overlooked. In difference from our experiments, Choi et al. i19 I. showed that a6-desaturation using 18: 2 ( n - 6) as substrate promoted a higher activity with a linseed oil diet (rich in 18 : 3(n - 3)) than a n - 6 f~.zty acid rich diet as safflower oil. The feeding program was slightly different from ours using a 10% fat diet for 4 weeks, and the rats were sacrificed at 02:00 pro. This brings in the question of the circadian cycle for the A6-desaturase which may alter dietary responses [20]. Also for AS-desaturation there was a great stimulation with LO and SO, more with LO (4-fold) than with SO (3-fold) as c o m p a r e d to the PEL diet (Fig. 3). In conflict with these data, D a n g et al. [211 found that a n - 6 rich diet (20% safflower oil) gave a J%desaturation activity slightly lower than a low-fat chow diet, while in our experiments we got a 3-fold stimulation. It is, in this case, difficult to evaluate which dietary factors caused these differences, since we used a similar reference diet. However, the a m o u n t of arachidonic acid ( 2 0 : 4 ( n - 6 ) was high in liver phospholipids with the high-fat 18 : 2(n - 6) diets, both in this study (Table !1) and in the study of D a n g et al.
I211. The A"- and /~S-desaturase activities were considerably lower with the F O fed rats than with the SO and LO diets. For the A6-desaturase, this is consistent with data from C h e i et al. [19] using 1 8 : 2 ( n - 6 ) as substrate. In addition, in our experiments we also observed a significantly lower activity with 18 : 3(n - 3) as substrate for the A6-desaturase (Fig. 2). G a r g et al. 15,61 also observed a lower A~ and 3 L d e s a t u r a s e activity with n - 6 fatty acids as substrates for a fish oil diet as compared to a diet enriched with 1 8 : 3 ( n - 3 ) . The reduced z~6-desaturase activity observed for the F O group c o m p a r e d to the LO a n d SO groups might be due to a feedback inhibition of the enzyme by the 20 : 5(n 3) and 22 : 6(n - 3) fatty acids in the F O diet a n d / o r by reduced enzyme synthesis [4]. in the case of AS-desaturation there is probably no need for increased activity in the FO group because sufficient a m o u n t s of long chain poly-unsaturateO n - 3 fatty acids already are available through the diet. Relatively high rates of the rate-limiting A6-desatura tion step with the L O and SO diets may explain an increased level of 20: 5 ( n - 3) in liver phospholipids with the LO diet and 2 0 : 4 ( n - 6) with the L O / S O and SO d:,ets even if these fatty acids were not introduced by the diet (Table I!). A t the same time there is a lower level of 2 0 : 4 ( n - 6 ) with the F O diet in accordance with the low As- and especially A6-desaturation activities observed with this diet. The high a m o u n t s of 20 : 5 and 22 : 6 probably derives from the dici. The At~-desaturase activity was significantly lower with the L O / S O , SO a n d F O diets as c o m p a r e d to the
62 low fat P E L diet, a n d the F O diet revealed the greatest reduction (Fig. 4). This finding is consistent with G a r g et al. [7] w h o also f o u n d a lower Ag-desaturase activity with fish oil t h a n with linseed oil, T h e F O oil diet increased the a m o u n t of 1 6 : 0 in liver p h o s p h o l i p i d s (Table 1I) in a c c o r d a n c e with G a r g et al. [71 a n d could be a t t r i b u t e d to the a d d i t i o n a l lowering of Ag-desaturase activity observed for this d i e t a r y g r o u p , but also to a higher de n o v o synthesis of 1 6 : 0 . T h e r e was a lower a m o u n t of 18: l ( n - 9) with the high fat diets t h a n with the low-fat reference (PEL), also reflecting a r e d u c e d ,~9-desaturation. 11 is interesting that the L O diet increased the a m o u n t of 2 0 : 5 a n d 2 2 : 5 ( n - 3) in p h o s p h o l i p i d s to the s a m e level as the F O diet, while the 2 2 : 6 ( n - 3 ) level w a s only 50% with the L O diet ( a n d similar to PEL) as c o m p a r e d to the F O diet (Table II). This indicates t h a t
the A4-desaturation activity in rat liver is low or insufficient to increase the tissue content of 22 : 6 even in the presence of sufficient amounts of the metabolic precursors in the L O fed animals. T h e most o b v i o u s o b s e r v a t i o n in this s t u d y is the general lowering effect of the fish oil diet o n d e s a t u r a tion activities, c o m p a r e d to diets with high a m o u n t s o f linolenic acid ( n - 3 ) a n d linoleic acid ( n - 6 ) . h is
difficult to evaluate if this effect is beneficial or not, but the important role of fish oil by reducing atherosclerotic risk factors [9], changing the immunological response of importance for inflammatory diseases [10] and reducing the c a r c i n o g e n i c r e s p o n s e [22] is m o r e a n d m o r e c o n vincing. A diet c o n t a i n i n g high a m o u n t s of 18 : 3(n - 3) fatty acids d o e s not seem to h a v e these beneficial p r o p erties [23]. T h e fatty acid p a t t e r n of v e r y - l o n g - c h a i n fatty acids in the tissues have to be b a l a n c e d between the n - 3 and n-6 fatty acids to secure a p r o p e r s u p p l y of e i c o s a n o i d s f r o m b o t h 2 0 : 4 ( n - 6 ) and 2 0 : 5 ( n - 31, a a d it also seems to be o f importance to h a v e a certain b a l a n c e b e t w - z n the n - 3 f a t t y acids 1 8 : 3 a n d 2 0 : 5 / 2 2 : 6 . T h e beneficial role o f fish oil m a y be c a u s e d by k e e p i n g a m o d e r a t e level of 2 0 : 4 ( n - 6) a n d i n c r e a s i n g the a m o u n t of 20 : 5(n - 3) in tissue lipids to ensure a p r o p e r b a l a n c e of the different eicosanoids. If a higher level o f 22 : 6 ( n - 3) is i m p o r t a n t o r not is presently not k n o w n , b u t it has been o b s e r v e d that h y d r o x y l a t e d p r o d u c t s f r o m this fatty acid are p o t e n t regulators of a r a c h i d o n i c acid m e t a b o l i s m [24]. T o f u r t h e r clarify the d i e t a r y role of l b : 3 ( n - 3 ) versus 2 0 : 5 / 2 2 : 6 ( n 3) fatty acids we are p r e s e n t l y feeding rats with p u r e c o n c e n t r a t e s of these fatty acids r a t h e r t h a n d i e t a r y oils to avoid side-effects o f o t h e r fatty acids in the n a t u r a l p r o d u c t s , e.g., the c o n t e n t o f
2 0 : 1 a n d 2 2 : 1 in fish oil k n o w n to affect fatty acid m e t a b o l i s m [25]. Acknowledgment T h i s w o r k was s u p p o r t e d b y the R e s e a r c h Society of the N o r w e g i a n Edible F a t P r o d u c e r s a n d the N o r d i c Insulin F o u n d a t i o n . W e t h a n k A a s e K o p s t a d for technical assistance. References 1 Budowski, P. (198g) World Rev. Nutr. Diet 57. 214-274. 2 Stoffel. w. (19611 Biochem. Biophys. Res. Commun. 6. 270-273. 3 Holloway, D.W., Peluffe, R. and Walkin. S.J. (1963) Biochem. Biophys. Res. Commun. 12, 300-304. 4 Brenner, R. R. (i989) in The Role of Fats in Human Nutrition (Vergroesen, A.J. and Crawford, M., eds.) 2nd edn., pp. 45-79. Academic Press. London. 5 Garg. M.L.. Sebokova. E., Thomson. A.B.R. and Clandinin, M.T. (1988) Biochem. J. 249. 351-356. 6 Garg, M.L, Thomson, A.B.R. and Clandinin, M.T. 0988) J. Nutr. 118, 661-668. 7 Gar[g, M.L., Wierzbicki. A.A., Thomson. A.B.R. and Clandinin. M.T. (19881 Biochim. Biophys. Acta 962, 330-336. 8 Brenner, R.R. and Pcluffo, R.O. (19661 J. Biol. Chem. 241, 52135219. 9 Leaf. A. and Weber, P.C. (19881 The New Engl. J. Med., 318, 549-557. 10 Kinsella, J.E.. Lokesh, n., Broughton, S. and Whelan, J. (19891 Nutr;tion 5, suppl. 24-44. I1 Thomassen, M.S.. Christiansen, E.N. and Nomm. K.R. (19821 Biochem. J. 206, 195-202. 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol Chem. 193, 265-275. 13 Mahfouz, M. and Holman. R.T. (1980) Lipids 15, 63-65. 14 Svensson, L. {19831 Lipids 18, 171-192. 15 Hoshi, M., Williams, M. and Kishimoto, Y. 0973) J. Lipid Res. 14, 599-601. 16 Christiansen. E.N., Thomassen. M.S., Christiansen, R.Z., Osmundsen. H. and Norum, K.R. 0979) Lipids 14, 829-835. 17 Mason, M.E. and Waller, G.E. (19641 Anal. Chem. 36, 583-586. 18 Reference deleted. 19 Choi, Y.-S., Golo, S., Ikeda. L and Sugano, M. 0989) Lipids 24, 45-50. 20 Gomez Dumm. I.N.T. de, Alaniz, M.J.T. de and Brenner, R.R. (19841 Lipids 19, 91-95. 21 Dang, A.Q.. Kemp, K.. Faas, F.H. and Carter, W.J. (19891 Lipids 24, 882-889. 22 Pariza, M.W. (1988) Annu. Rev. Nutr. 8, 167-183. 23 Harris, W.S. (19891 J. Lipid Res. 30, 785-807. 24 Salem, N.jr. (1989) in New provective roles for selective nutriticnts (Spiller, G.A. and Scala, J., eds.) Current Topics in Nutrition and Disease. Vol. 22, pp. 109-228, Alan R. Liss, New York. 25 Norum, K.R, Christiansen, E.N.. Christophersen, B.O. and Bremer, J. 0989) in The Role of Fats in Human Nutrition (Vergroesen, A.J. and Crawford, M., eds.), 2nd edn., pp. tl 1-149. Academic Press, London.
57
BBALIP 53588
Effect of dietary n - 3 and n - 6 fatty acids on fatty acid desaturation in rat liver E r l i n g N . C h r i s t i a n s e n , J o a n n a S. L u n d , T o r i l l R o r t v e i t a n d A r i i d C. R u s t a n lnstttute for Nutrition Research. Uml'erst(t" of Oxlo. Oslo (Nor~'ar) ( Received 29 June 1990)
Key words: Dietary fat: (n - 3) Fatty acid, ¢n - 6) Fatty acid: Fatty acid desaturation: Xticrosornc: (Rat liver)
To study the effect of high-fat diets with varying contents of n - 3 and n - 6 fatty, acids on the metabolism of essential fatty acids, the rat liver microsomal fatty acid desaturases were measured. The rats were fed for 3 weeks with diets high in linseed oil ( 1 8 : 3 ( n - 3)), sunflower seed oil ( 1 8 : 2 ( n - 6)) or fish oil ( 2 0 : 5 ( n - 3) and 2 2 : 6 ( n - 3)) (20%, w / w ) using pellet fed rats as a reference. The A6-desaturase using 18:2(n - 6) or 18:3(n - 3) as substrates was stimulated 1.5-2.5-fold by linseed or sunflower seed oil, compared to the pellet reference. The AS-desaturase was stimulated 3.5-fnld with linseed oil and 2.5-.fold with sunflower seed oil, while the Ag-desaturase was inhibited by all the high-fat diets. The z16-, s. and 9-desaturase activities were in all cases considerably reduced with fish oil as compared to linseed and sunflower seed oil diets. With pellet fed rats the rates were highest for zi~-desaturation and in decreasing order lower for AS-desaturation, A6-desaturation with 18:3 (n - 3) as substrate and finally A6-Desaturation with 18: 2(n - 6) as substrate. The content of 2 0 : 4 ( n - 6 ) in liver phospholipids increased with the diets rich in 1 8 : 2 ( n - 6 ) , and was reduced for the fish oil diet enriched in 20:5 and 22:6(n - 3) fatty, acids. The amount of 2 0 : 5 ( n - 3) in phosphnlipids was as high with linseed oil diet as with the fish oil diet, while the 22:6(n - 3) content was only increased with the fish oil diet.
Introduction It is of significant interest to know how the dietary content of polyunsaturated 'fatty acids, with different proportions of n - 3 and n - 6 fatty acids, affect the metabolism of essential fatty acids. Desaturation of polyunsaturated fatty acids is an important reaction for the synthesis of very-long-chain polyunsaturated fatty acids important for membrane functions and formation of eicosanoids [1]. A6-Desaturation is considered to be the rate limiting reaction for these sequences [2,3]. Fatty acid desaturation is regulated by a number of nutritional and hormonal factors [4] and the type and amount of dietary fat have been shown by Garg et al. [5-7] to be important factors. The fatty acids of the n - 3 and n - 6 families are considered to be metabolized by the same fatty acid desaturases, but the affinity
Abbreviations: LO, linseed oil diet; SO. sunflower seed oil diet: FO, fish oil diet; PEL, low-fat reference diet; GLC. gas-liquid chromatography; TLC. thin-layer chromatography. Correspondence: E.N. Christiansen, Institute for Nutrition Research, University of Oslo, P.O. Box 1046, N-0316 Oslo 3, Norway.
for the n - 3 family is greater than the affinity for the n - 6 family [8}. There is considerable evidence of the beneficial role of marine n - 3 fatty acids for reducing the incidence of cardiovascular and inflammatory diseases [9,10]. Related to these effects there are a number of important questions: for instance, what is the proper balance of the supplies of n - 3 and n - 6 fatty acids and to which extent is 18: 3 ( n - 3 ) providing sufficient amounts of long-chain n - 3 fatty acids? Therefore, it is important to know how diets with different {n - 3 ) / ( n - 6) fatty acid ratios and different n - 3 fatty acids affect fatty acid desaturation, and thereby the availability of longchain polyunsaturated fatty acids (e.g., arachidonic and eicosapentacnoic acid) for further metabolism. To obtain more information of the effect of n - 3 and n - 6 fatty acids on fatty acid desaturation in the liver, rats were fed high-fat diets with different amounts of n - 3 and n - 6 fatty acids. A linseed oil diet (LO) enriched with 1 8 : 3 ( n - 3 ) fatty acid, was compared with three other high-fat diets, one enriched with 18 : 2(n - 6) fatty acid (SO), another with equal amounts of n - 3 and n - 6 fatty acids ( L O / S O ) , and a third diet with fish oil (FO) containing high a m o u n t s of long-chain n - 3 fatty
58 acids (20 : 5 and 22 : 6). Animals fed on standard pellet (PEL) were used as a low fat reference. In the present study, we describe the effect of these diets on the in vitro activities of As-, A6- and Ag-desaturase and on the fatty acid composition of liver phospholipids. Materials and Methods
Materials The fish oil (Japanese) was a gift from D e N o F a and Lilleborg Fabriker A / S (Fredrikstad, Norway). Linseed oil and sunflower seed oil were gifts from Rett-trading (Oslo) (representing Henry Lamotte, Bremen, F.R.G.). The pelleted stock feed for rats was obtained from Ewos AB (S~Sdert~ilje, Sweden). Vitamin and salt mixtures of the semisynthetic diets were from ICN Nutritional Biochemicals (Cleveland, OH, U.S.A., Cat. No. 904610 and 904654). [1-14C]Stearic acid ( 1 8 : 0 ) (2.07 G B q / m m o l ) , [1-~4C]linoleic acid (18 : 2(n - 6)) (1.98 G B q / m m o l ) , [l~4C]linolenic acid (18 : 3(n - 3)) (1.99 G B q / m m o l ) and [l-~4C)dihomo-7-1inolenic acid ( 2 0 : 3 ( n - 6)) (1.77 GBq/mmol) were from A m e r s h a m I n t e r n a t i o n a l (Amersham, U.K.). Bovine serum albumin, essentially fatty acid free, and other fine chemicals were from Sigma Chemical (St. Louis, MO, U.S.A.). Animals and diets Male rats (approx. 160 g) of the Wistar strain were purchased from Mollegaard Breeding L a b o r a t o r y (Ejby, Denmark). The animals were fed a standard pellet diet for 5 - 9 days, then went on with standard pellet (PEL) or experimental semisynthetic diets for 3 weeks [11]. The composition of the semisynthetic diet was, in weight% of total: sucrose, 20; cornstarch, 33; casein (with 2% methionine) 20; cellulose, 1; vitamin mixture, 2; salt mixture, 4; and diet oil 20. The compositions of the diet oils were: LO, linseed oil (99%) and sunflower seed oil (1%); L O / S O , linseed oil (54%) and sunflower seed oil (46%); SO, linseed oil (21%) and sunflower seed oil (79%); FO, fish oil (92%) and sunflower seed oil (8%). The diet cAls were mixed to give a controlled scale of (n - 3 ) / ( n - 6) fatty acid ratios from 0.3 to 5.2. The L O / S O diet was mixed to give near equal a m o u n t s of n - 3 and n - 6 fatty acids. The fatty acid composition of the different semisynthetic diets are given in Table I. The pellet diet consisted of cereals, wheat germs, wheat middlings, fish protein concentrate, soya meal (toasted), fodder yeast, minerals, a n i m a l and vegetable fat, vitamin and trace element concentrates (protein, 22; carbohydrates, 52; fat 5 and others 21% of wet weight). The falty acid composition (%) of the fat according to the producer was: 1 6 : 0 , 21; 16:1, 2: 1 8 : 0 , 4; 1 8 : 1 , 22; 18:2(n-6), 42; 1 8 : 3 ( n - 3 ) , 7 and others, 2. All animals appeared healthy after the 3-week.~ experimental period. There were no significant differences in food consumption between the high-fat dietary groups, but
TABLE I Fatty acid composition of semts).nthetic diets (%) Fatty acid composition ','as determined by GLC as described in Materials and Methods. Antioxidants were 0.04% butylated hydroxytoluene (BHT) and 0.01% vitamin E. arty acids 14:0 16:0 16:1(n-7) 18:0 18:1(n-9) 18:1(n-7) 18:2(n -6) 18:3(n -3) 20:5(n -3) 22:l(n-ll) 22 : 5(n - 3) 22 : 6( n - 3) Others Total Saturated Monoenes Polyenes (n - 3)/(n -6)
LO 4.4 4.1 16.1 11.6 61.5 2.3
LO/SO
3.0 19.2 37.7 32.8 2.4
5.5 3.8 19.5 1.3 51.7 13.9 4.3
8.5 16.1 73.1 5.2
7.9 19.2 70.5 0.9
9.3 20.8 65.6 0.3
4.9
SO
FO 7.4 20.0 7.8 3.0 11.2 3.4 9.0 3.4 12.9 1.4 1.3 7.1 12.1 30.4 23.8 33.7 2.1
the pellet fed group had a slightly higher c o n s u m p t i o n ( P < 0,05). Weight gain, b o d y a n d liver weights for all dietary g r o u p s were similar (data not shown). The rats were housed in g r i d - b o t t o m e d cages, two in each cage, and they had free access to food a n d water. The r o o m - t e m p e r a t u r e was 2 4 ° C with 60% relative h u m i d i t y a n d a 12 h light period (07:00-19:00).
Preparation of liver microsomes The rats were sacrificed between 09:00 to 11:00 a.m. A 15% liver h o m o g e n a t e was prepared at 4 ° C in 250 m M sucrose c o n t a i n i n g 10 m M Hepes ( p H 7.4), using a Potter-Elvehjem homogenizer. The h o m o g e n a t e was centrifuged at 7 5 0 × g (2500 rpm) for 10 rain in a Sorvall R C 5 C highspeed centrifuge ( D u p o n t , Delaware), rotor SS34. The postnuclear fraction was centrifuged at 5100 × g (6500 rpm) for 10 min and 30900 × g (16000 rpm) for a further 10 min in a rotor as above. The microsomal pellet was o b t a i n e d at 63000 × g ( 2 9 0 0 0 rpm) for 60 min in a SorwaU O T D 55B ultracentrifuge, rotor T F T 70.38. Protein was d e t e r m i n e d according to Lowry et al. [12] using bovine serum a l b u m i n as standard. Analytical methods Microsomes from rats fed the different diets were assayed for activities of A5-, 6. and 9-desaturases m a i n l y as described by Mahfouz & H o l m a n and Svensson [13,14]. The o p t i m a l substrate (at s a t u r a t i o n level) a n d
59 microsomal protein concentrations (linear dependency) and incubation time (linear dependency) for the four desaturase assays were determined (data not shown). The incubation mix:ure consisted of 0.15 M KCI, 5 mM ATP, 5 mM MgCI 2, 0.25 mM CoA, 1.0 mM NADH, 1.5 mM glutathione, 45 mM NaF, 0.5 mM nicotinamide, I mg bovine serum albumin and 0.1 M potassium phosphate buffer (pH 7.4). In our experiments, we used 0.5-2.0 mg of microsomal protein and 75-150 nmol t4C-labelled fatty acid substrate (6-11 kBq) in 1 ml incubation volume (see legend to figures). The fatty acids were used as sodium salt/bovine serum albumin complexes. "[he incubation time used was 20 min at 37°C, and the reaction was terminated by addition of 1 ml of 2.7 M KOH in methanol. This mixture was heated at 65°C for 45 min ar.d acidified by 6 M HCI. Nonesterified fatty acids were extracted with hexane and methylated according to Hoshi et al. [15]. As- and 6-desaturation activities were determined using radioGLC (Varian 2100 equipped with a 10% SP-2340 on a Supelcoport 100/120 60 cm column (Supelco, BeIlefonte, PA, U.S.A.) with argon as carrier gas) connected to a nuclear radiodetector (ESI Nuclear, Surrey, U.K.) [16]. Ag-Desaturation activity was determined using silica gel TLC plates (Silica gel F 1500, Schleicher & Schuell, Dassel, F.R.G.) impregnated with 10% AgNO 3 ( w / v ) in acetonitrile. Carrier methyl esters (18 : 0 and 18 : l ) were applied and the TLC-plates were developed in h e x a n e / d i e t h y l ether (90:10, v / v ) for separation of saturated and monounsaturated fatty acids [7]. The spots were visualized by ultraviolet-light using 2,7-dichlorofluorescein (0.2%, w / v in ethanol) and counted in a Packard Tri-Carb 300 liquid scintillation spectrometer using 6 ml instagel I1 (Packard, Chicago, !L, U.S.A.). A control incubation was performed with microsomes in the absence of substrates and free fatty acids were measured. There was no change in their fatty acid composition before and after the incubation (unpublished data). This indicates that there is no contribution from endogeneous fatty acids affecting the results. The fatty acid composition of liver phospholipids was measured as methyl esters [171 with GLC (Carlo Erba Strumentazione, Fractovap Series 2150, Milan, Italy), equipped with a nonpolar capillary cohtmn (CP Sil 15CB, 50 m, diam. 0.2 ram) and using helium, 0.5 m l / m i n as a carrier gas [11]. The temperature was programmed to rise from 180 to 215°C at 0.5 C ° / m i n . Diheptadecanoylphosphatidylcholinewas used as an internal standard.
Statistical analysis Results are means + S.D. of six rats. Data were analyzed by the Mann-Whitney non-parametric test (two-tailed) (Minitab statistical vrogram, Minitab, State College, PA, U.S.A.L
Results
Effect of diets on desaturase actit,ities A~-desaturation. A6-desaturase using linoleic acid (n -6) as substrate was stimulated 2-2.5-times by the high fat L O / S O and SO diets as compared to the reference d:et (PEL) (Fig. 1). With the FO diet the activity was only 20% compared to the SO diet and 30% compared to the LO diet. There was a significant difference between the LO and SO diets, the LO being about 40% lower. With linolenic acid ( n - 3) as substrate, there was about 2.5-times stimulation with the LO, SO and L O / S O diets compared to the PEL diet (Fig. 2). With the FO diet the activity was 60% lower compared to the LO diet. The rate of /~6-desaturation with the PEL diet using linoleic acid ( n - 6) as substrate was about 50% lower than using linolenic acid (n - 3) as substrate (Fig. 1 and 2). ,~5-desaturation. ALdesaturase was measured using 20: 3 ( n - 6) as substrate (Fig. 3). The LO diet stimulated the activity by 4-times and the SO diet 3-times compared to the PEL diet. With the FO diet, the activity was 25% of the activity obtained with the LO diet. The rate of AS-desaturation with the PEL diet was nearly 3-times higher than for A6-desaturation with 18 : 2(n - 6) as substrate and about 7-times higher comparing the same desaturases with the LO fed rats (Figs. 1 and 3). A°-desaturation. A~-desaturase activity was decreased with all high-fat diets compared to the PEL diet; with the L O / S O and SO diets the activity was 40-50% lower and with the LO diet it was also a reduction, however
PEL
LO
LO/SO
SO
FO
diet
Fig. I. Effect of diets rich in n - 3 and n - 6 fatty acid~on rat liver microsomal A6-desaturationusing linoleic acid (l 8 : 2 ( n - 6)) as substrate. Activitiesare expressedas nmol 18:3(n -6) (7-1inolenicacid) synthesized per min per mg microsomal protein at 37°C. In this experiment 75 nmol [l-14C]linoleicacid (18:2(n-6)) was used as substrate and 2 mg microsomal protein. The substrate and product fatty acids were separated by radio-Gl C a~ described in Materials and Method~, Data represents means_+S.D. (n = 6). Bars without a common !crierare significantlydifferentat P < 0.05.
60 nmoles/mg x rain
Tb b
0.8
~ ,~
0.6
~~ ~a
0.4
O ~
PEL
~
LO
LO/SO SO FO diet Fig. 2. Effect of diets rich in n - 3 and n - 6 fatty acids on rat liver microsomal ~,6-desaturation using ]ino]enic acid (18:3(n -3)1 as substrate. Activities are expressed as nmol 1 8 : 4 ( n - 3) synthesized per min per mg microsomal protein at 37°C. In this experiment 100 nmol []-V~C]linolenic acid ( 1 8 : 3 ( n - 3 ) ) was used as substrate and 1.5 mg microsomal protein. The substrate and product fatty acids were separated by radio-GLC as described in Materials and Methods. Data represents means_+S.D. (n = 6). Bars without a common letter are significantly different at P < 0.01.
PEL
LO
LO/SO
SO
FO
diet
Fig. 3. Effect of diets rich in n - 3 and n - 6 fatty acids on rat liver microsomal AS-desaturation osing dihomo-7-1inolenic acid (20: 3 ( n 6)) as substrate. Activities are expressed as nmol 2 0 : 4 ( n - 6 ) (arachidonic acid) synthesized per rain per mg microsomal protein. 150 nmol [l-t4Cl dihomo-y-linolenic acid { 2 0 : 3 ( n - 6)) was used as substrate and !.5 mg microsomal protein. The substrate and product fatty acids were separated by radio-GLC as described in Materials and Methods. Data represents means ± S.D. (n = 6). Bars without a common letter are significantly different at P < 0.01.
n o t s i g n i f i c a n t ( F i g . 4). W i t h t h e F O d i e t t h e Ag-de saturase activity was significantly lower than for the LO, L O / S O
a n d S O diets.
C o m p a r e d t o t h e p e l l e t fed rats, t h o s e fed L O a n d S O d i e t s h a d a l o w e r a m o u n t o f l b : 0 a n d a h i g h e r level
The monoene content (18:1) was significantly lower w i t h all t h e h i g h - f a t d i e t fed r a t s t h a n w i t h t h e p e l l e t fed. 1 8 : 2 ( n - 6 ) was considerably lower with the FO diet than the other diets. 1 8 : 3 ( n 3) w a s n o t d e t e c t a b l e i n h e p a t i c p h o s p h o l i p i d s i n t h e S O a n d F O fed a n i m a l s a n d even in the presence of large a m o u n t s of
o f 1 8 : 0 in l i v e r p h o s p h o l i p i d s ( T a b l e 11). F o r t h e F O
d i e t a r y 18 : 3 ( L O g r o u p ) , t h e c o n c e n t r a t i o n o f 18 : 3 i n
fed a n i m a l s t h e r e w a s a s l i g h t l y h i g h e r a m o u n t o f 16 • 0.
p h o s p h o l i p i d s is n o t m u c h h i g h e r t h a n i n p e l l e t f e d rats.
Effect of diets on .fatty acid composition of lioer phospho/ipids
TABLE I1
Faro' acid composition of liverphospholipids (mg/g liver) Fatty acid composition was determined by GLC as described in Materials and Methods. Data represent means +. S.D. {n = 6). Values without a common superscript are significantly different at P < 0.05. n.d., not detectable. PEL
LO
LO/SO
SO
FO
16 :0
4.99 + 0 . 1 9 ~
3.91 + 0 . 4 5 h
3.79+0.31 h
3.49+0.19 h
6.11:1:0.52 ~
18 : 0 18:1 (n - 9 ) 18:1 (n - 7 ) 1~ : 2 (n -- 6) 18:3 OI -- 3) 20:3 (n - 6 ) 20:4 (n - 6 ) 20 : 5 (n - 3) 22:5 (n - 3 ) 22:6 (n - 3 )
5.45 + 0.21 ~ 1.94+_0.38 ~ 0.89+0.09 ~ 3.45 5:0.21 a 0,4830,02 a 0.28+0.04 ~ 3.55±0.39 ~ 0,25 +:0.03 a 0.21 +0.04 a 0.96 + 0,16 a
7.44 + 0.51 h 1.1830.39 ~ 0.53+0.16 h 4.28 + 0.36 b 0.5330.13 a 0.32+0.05 a 3,49±0.95 ~ 1.72 +:0.26 b 0.53+0,10 ~ 0,97±0.30 a
7.95 + 0.63 b 0.79:t:0.15 h 0.47+0.11 b 4.63 + 0.59 b 0.36+0.03 h 0.2630.05 ~ 5.19+0.86 b 0.62 + 0,19 c 0.31 +0.05 a 0.86+:0.16 a
6.67 ± 0.37 ~ 0.63+0.11 n 0.46+0.05 b 3.35 ± 0.28 a n.d. 0,1630.08 a 5.40+:0.40 b 0.39 +:0.02 ~ 0.16+'0,02 a 0.82+:0.12 a
6.06 + 0.20 ~ 0.76+0.05 b 0.79+0,10 ~ 1.85 5:0.26 ~ n.d. 0.20+0.07 ~ 2,47 +0.27 ¢ 1 68 + 0,16 h 0.50+0.06 b 2.32+:0.26 b
Total Saturated Monoenes Polyenes (n - 3)/(n - 6 )
22.4 10.4 2.8 9.2 0.2
24.9 11.4 1.7 11.8 0.5
25.2 11.7 1.3 12.2 0.2
21.6 10.2 1.1 10.3 0.2
22.8 12.2 1.6 9.0 1.0
61 nmoles/mg x min
[
T~
0~
l ~ PEL
0s
~'~ LO
kO/$O
$O
FO
diet
Fig. 4. Effect of diets rich in n -3 and n -6 fatty acids on rat liver microsomal ,~9-desaturation using stearic acid 118:0) as substrate. Activities are expressed as nmol 18: l¢n-9) (oleic acid) synthesized per rain per nag microsomal protein. In this experiment 150 nmol [l-14Clstearic acid was used as substrate and 1 mg microsomal protein. The substrate and product fatty acids were separated by argentation TLC as described in Materials and Methods. Data represents means+S.D. (n = 6). Bars without a common letter are different at P < 0.05.
A large a m o u n t of 1 8 : 3 ( n - 3 ) in the LO group was transformed to 2 0 : 5 and 2 2 : 5 , the content of these fatty acids being as high as the F O diet, while the 22 : 6 level was 50% lower and similar to the pellet diet. The a m o u n t of 2 0 : 4 ( n - 6) in liver p h o s p h o l i p i d s increased with the L O / S O and SO diets, whereas it was reduced in the F O group as c o m p a r e d to the pel!et diet. The total a m o u n t of polyunsaturated fatty acids was similar for all dietary groups. The fatty acid composition of liver microsomal phospholipids was also measured, and revealed the same p a t t e r n as for total liver phospholipids (data not shown). Diseusslon
The A6- and AS-desaturation increased with the diets rich in linoleic and linolenic acids c o m p a r e d to the PEL reference diet. There appeared to be a difference in the response using linoleic (n - 6) or linolenic acid ( n - 3) as substrate for the ,~6-desaturase even if the enzyme is considered to be the same for both substrates [8]. ¢ / i t h linoleic acid as substrate there was a significantly higher desaturation rate with the SO diet than the LO diet (Fig. 1). With linolenic acid as substrate there was a small, but not statistically significant, increase in A6 desaturation with the L O diet than with the SO diet (Fig. 2). These differences could possibly be explained by substrate dilution caused by fatty acids from the diet. W e examined, however, free fatty acids in microsomes before and after incubating under the same conditions as for the desaturase measurements, and found no changes in the fatty acid pattern. Thus, the possibil-
ity that A6-desaturation of n -- 3 and n - 6 fatty acids are carried out by different enzymes should not be overlooked. In difference from our experiments, Choi et al. i19 I. showed that a6-desaturation using 18: 2 ( n - 6) as substrate promoted a higher activity with a linseed oil diet (rich in 18 : 3(n - 3)) than a n - 6 f~.zty acid rich diet as safflower oil. The feeding program was slightly different from ours using a 10% fat diet for 4 weeks, and the rats were sacrificed at 02:00 pro. This brings in the question of the circadian cycle for the A6-desaturase which may alter dietary responses [20]. Also for AS-desaturation there was a great stimulation with LO and SO, more with LO (4-fold) than with SO (3-fold) as c o m p a r e d to the PEL diet (Fig. 3). In conflict with these data, D a n g et al. [211 found that a n - 6 rich diet (20% safflower oil) gave a J%desaturation activity slightly lower than a low-fat chow diet, while in our experiments we got a 3-fold stimulation. It is, in this case, difficult to evaluate which dietary factors caused these differences, since we used a similar reference diet. However, the a m o u n t of arachidonic acid ( 2 0 : 4 ( n - 6 ) was high in liver phospholipids with the high-fat 18 : 2(n - 6) diets, both in this study (Table !1) and in the study of D a n g et al.
I211. The A"- and /~S-desaturase activities were considerably lower with the F O fed rats than with the SO and LO diets. For the A6-desaturase, this is consistent with data from C h e i et al. [19] using 1 8 : 2 ( n - 6 ) as substrate. In addition, in our experiments we also observed a significantly lower activity with 18 : 3(n - 3) as substrate for the A6-desaturase (Fig. 2). G a r g et al. 15,61 also observed a lower A~ and 3 L d e s a t u r a s e activity with n - 6 fatty acids as substrates for a fish oil diet as compared to a diet enriched with 1 8 : 3 ( n - 3 ) . The reduced z~6-desaturase activity observed for the F O group c o m p a r e d to the LO a n d SO groups might be due to a feedback inhibition of the enzyme by the 20 : 5(n 3) and 22 : 6(n - 3) fatty acids in the F O diet a n d / o r by reduced enzyme synthesis [4]. in the case of AS-desaturation there is probably no need for increased activity in the FO group because sufficient a m o u n t s of long chain poly-unsaturateO n - 3 fatty acids already are available through the diet. Relatively high rates of the rate-limiting A6-desatura tion step with the L O and SO diets may explain an increased level of 20: 5 ( n - 3) in liver phospholipids with the LO diet and 2 0 : 4 ( n - 6) with the L O / S O and SO d:,ets even if these fatty acids were not introduced by the diet (Table I!). A t the same time there is a lower level of 2 0 : 4 ( n - 6 ) with the F O diet in accordance with the low As- and especially A6-desaturation activities observed with this diet. The high a m o u n t s of 20 : 5 and 22 : 6 probably derives from the dici. The At~-desaturase activity was significantly lower with the L O / S O , SO a n d F O diets as c o m p a r e d to the
62 low fat P E L diet, a n d the F O diet revealed the greatest reduction (Fig. 4). This finding is consistent with G a r g et al. [7] w h o also f o u n d a lower Ag-desaturase activity with fish oil t h a n with linseed oil, T h e F O oil diet increased the a m o u n t of 1 6 : 0 in liver p h o s p h o l i p i d s (Table 1I) in a c c o r d a n c e with G a r g et al. [71 a n d could be a t t r i b u t e d to the a d d i t i o n a l lowering of Ag-desaturase activity observed for this d i e t a r y g r o u p , but also to a higher de n o v o synthesis of 1 6 : 0 . T h e r e was a lower a m o u n t of 18: l ( n - 9) with the high fat diets t h a n with the low-fat reference (PEL), also reflecting a r e d u c e d ,~9-desaturation. 11 is interesting that the L O diet increased the a m o u n t of 2 0 : 5 a n d 2 2 : 5 ( n - 3) in p h o s p h o l i p i d s to the s a m e level as the F O diet, while the 2 2 : 6 ( n - 3 ) level w a s only 50% with the L O diet ( a n d similar to PEL) as c o m p a r e d to the F O diet (Table II). This indicates t h a t
the A4-desaturation activity in rat liver is low or insufficient to increase the tissue content of 22 : 6 even in the presence of sufficient amounts of the metabolic precursors in the L O fed animals. T h e most o b v i o u s o b s e r v a t i o n in this s t u d y is the general lowering effect of the fish oil diet o n d e s a t u r a tion activities, c o m p a r e d to diets with high a m o u n t s o f linolenic acid ( n - 3 ) a n d linoleic acid ( n - 6 ) . h is
difficult to evaluate if this effect is beneficial or not, but the important role of fish oil by reducing atherosclerotic risk factors [9], changing the immunological response of importance for inflammatory diseases [10] and reducing the c a r c i n o g e n i c r e s p o n s e [22] is m o r e a n d m o r e c o n vincing. A diet c o n t a i n i n g high a m o u n t s of 18 : 3(n - 3) fatty acids d o e s not seem to h a v e these beneficial p r o p erties [23]. T h e fatty acid p a t t e r n of v e r y - l o n g - c h a i n fatty acids in the tissues have to be b a l a n c e d between the n - 3 and n-6 fatty acids to secure a p r o p e r s u p p l y of e i c o s a n o i d s f r o m b o t h 2 0 : 4 ( n - 6 ) and 2 0 : 5 ( n - 31, a a d it also seems to be o f importance to h a v e a certain b a l a n c e b e t w - z n the n - 3 f a t t y acids 1 8 : 3 a n d 2 0 : 5 / 2 2 : 6 . T h e beneficial role o f fish oil m a y be c a u s e d by k e e p i n g a m o d e r a t e level of 2 0 : 4 ( n - 6) a n d i n c r e a s i n g the a m o u n t of 20 : 5(n - 3) in tissue lipids to ensure a p r o p e r b a l a n c e of the different eicosanoids. If a higher level o f 22 : 6 ( n - 3) is i m p o r t a n t o r not is presently not k n o w n , b u t it has been o b s e r v e d that h y d r o x y l a t e d p r o d u c t s f r o m this fatty acid are p o t e n t regulators of a r a c h i d o n i c acid m e t a b o l i s m [24]. T o f u r t h e r clarify the d i e t a r y role of l b : 3 ( n - 3 ) versus 2 0 : 5 / 2 2 : 6 ( n 3) fatty acids we are p r e s e n t l y feeding rats with p u r e c o n c e n t r a t e s of these fatty acids r a t h e r t h a n d i e t a r y oils to avoid side-effects o f o t h e r fatty acids in the n a t u r a l p r o d u c t s , e.g., the c o n t e n t o f
2 0 : 1 a n d 2 2 : 1 in fish oil k n o w n to affect fatty acid m e t a b o l i s m [25]. Acknowledgment T h i s w o r k was s u p p o r t e d b y the R e s e a r c h Society of the N o r w e g i a n Edible F a t P r o d u c e r s a n d the N o r d i c Insulin F o u n d a t i o n . W e t h a n k A a s e K o p s t a d for technical assistance. References 1 Budowski, P. (198g) World Rev. Nutr. Diet 57. 214-274. 2 Stoffel. w. (19611 Biochem. Biophys. Res. Commun. 6. 270-273. 3 Holloway, D.W., Peluffe, R. and Walkin. S.J. (1963) Biochem. Biophys. Res. Commun. 12, 300-304. 4 Brenner, R. R. (i989) in The Role of Fats in Human Nutrition (Vergroesen, A.J. and Crawford, M., eds.) 2nd edn., pp. 45-79. Academic Press. London. 5 Garg. M.L.. Sebokova. E., Thomson. A.B.R. and Clandinin, M.T. (1988) Biochem. J. 249. 351-356. 6 Garg, M.L, Thomson, A.B.R. and Clandinin, M.T. 0988) J. Nutr. 118, 661-668. 7 Gar[g, M.L., Wierzbicki. A.A., Thomson. A.B.R. and Clandinin. M.T. (19881 Biochim. Biophys. Acta 962, 330-336. 8 Brenner, R.R. and Pcluffo, R.O. (19661 J. Biol. Chem. 241, 52135219. 9 Leaf. A. and Weber, P.C. (19881 The New Engl. J. Med., 318, 549-557. 10 Kinsella, J.E.. Lokesh, n., Broughton, S. and Whelan, J. (19891 Nutr;tion 5, suppl. 24-44. I1 Thomassen, M.S.. Christiansen, E.N. and Nomm. K.R. (19821 Biochem. J. 206, 195-202. 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol Chem. 193, 265-275. 13 Mahfouz, M. and Holman. R.T. (1980) Lipids 15, 63-65. 14 Svensson, L. {19831 Lipids 18, 171-192. 15 Hoshi, M., Williams, M. and Kishimoto, Y. 0973) J. Lipid Res. 14, 599-601. 16 Christiansen. E.N., Thomassen. M.S., Christiansen, R.Z., Osmundsen. H. and Norum, K.R. 0979) Lipids 14, 829-835. 17 Mason, M.E. and Waller, G.E. (19641 Anal. Chem. 36, 583-586. 18 Reference deleted. 19 Choi, Y.-S., Golo, S., Ikeda. L and Sugano, M. 0989) Lipids 24, 45-50. 20 Gomez Dumm. I.N.T. de, Alaniz, M.J.T. de and Brenner, R.R. (19841 Lipids 19, 91-95. 21 Dang, A.Q.. Kemp, K.. Faas, F.H. and Carter, W.J. (19891 Lipids 24, 882-889. 22 Pariza, M.W. (1988) Annu. Rev. Nutr. 8, 167-183. 23 Harris, W.S. (19891 J. Lipid Res. 30, 785-807. 24 Salem, N.jr. (1989) in New provective roles for selective nutriticnts (Spiller, G.A. and Scala, J., eds.) Current Topics in Nutrition and Disease. Vol. 22, pp. 109-228, Alan R. Liss, New York. 25 Norum, K.R, Christiansen, E.N.. Christophersen, B.O. and Bremer, J. 0989) in The Role of Fats in Human Nutrition (Vergroesen, A.J. and Crawford, M., eds.), 2nd edn., pp. tl 1-149. Academic Press, London.
