Fatty acid metabolism, pharmacological nutrients and hypertension

Biochimie (1l}97) 7~. 135-138

~_3S(~ci6t6 fi-angaise de biochhnie el biologic inoR6culah'e / Elsevier. Paris

Fatty acid nletabolism, pharmacological nutrients and hypertension M Narce, JM Freaoux, V Dardel, C Foucher, S Germain, MC Delachambre, JP Poisson Unit~; &" Nutritimt Celhdaire et Mdtaholique. EA DRED 186Z Facult6 des &'iem'es Miramte, Univetwiu~ de Bourgogne, BP 400, 21011 DOon cedex, Fram'e

(Received 7 October 1996; accepted IO January 1997) Summary ~ Tl~e purpose of the present study was to investigate tile effect of a concentrated preparation (EPA307 containing eicosapentaenoic acid (EPA. 20:5 la-3) and docosahexaenoic acid eDHA, 22:6 n-3) on the limiting desalu,'atio,i steps of the polyunsaturated fany acid biosynthesis in spontaneously Ilypertensivc rats ISHR). Adult SHR were divided into two groups: one group received a standard diet, and the experimental group the standard diet including 0.8% of EPA3() for 9 weeks. Blood pressure was measured at the end of the diets. The dcsaturase activities and fatty acid composition were determined iq isolated hepatocytes. The blood pressure did not decrease in the e×per)mental group. The desaturated products of the n-6 family (gamma-linolenic acid, 18:3 n-6 and arachidonic acid, 20:4 n-6~ were lowered m the EPA30 group, when their respective substrates ( 18:2 n-6 and 20:3 n-6) werc increased, EPA and DHA were higher in the experimental group. A6 n-3, A6 n-6 and A5 n-(~desaturase activities were depressed approxi,natcly 2l)'a m the EPA30 group. EPA30 being an active nutrient on the EFAs cascade, increasing the level of PG3 precursors and decreasing the level of PG2 precursors, favorable conditions have been established to reduce hypertension. The underlying mechanism ,'elated to the regulation of desaturase activities by these fatty nutrients remains to be elucidated.

hypertension / polyunsaturated fatty acids / eicosapentaenoic acid / docosahexaenoic acid / desaturation Introduction Essential fatty acids (EFAsT, litmlcic acid (I.A, 18:2 n-6) and ~-linolenic acid (ALA, 18:3 ~t-3) :trc converted withitt the manmlalian bodies into longer and more unsalttrated fatty acids, by an alternaled sequence of desalt, rations ;|lid elongations. This bmconvcrsion is dependent on numerous factors, which depress the ah'cady rate-limiting A6-tlcsaluration step I! 1. A(~ desaturasc is slowctl down during essential hypertensio,i 121, and consequently, the distribulion of polyunsaturated fatty acids (PUFAs} is altered in ntnncrous organs and tissues 13 I. As a result, deficits in EFA metabolites have been observed 14-61. The effects of diets rich in marine oils on hypertension have been particularly studied. Several animal and human studies have shown contradictory results concerning the effect o1' marine oils on hypertension [7-121. These contradictions may certainly be explained by design weaknesses, lbr example the doses of administered fats, the age of animals or the duration of the diets. The effects of these m a r n e oils may be due to the)," influence on 1he PUFA cascade, as PUFAs arc involved in important metabolic pathways, giving rise to vascular and renal vasoactive eicosa,mids and playing a role as structural constituents of celhtlar ntcmbrancs. Thcrelbrc, all these fatty nutrients have to be considered as pharmaceutical molecules and specific concentrations are probably required 1o allow optimum meiabolic cl'f~ci,,,. For such reasons, we chose to study the influence of an appropriate supple,nentation of the diet with a specific fatty

acid concentrate (EPA/DHA), extracted from fish oil, on spontaneously hypertensive rats (SIIR) blood pressure and on dcsaturase activities involved in the biogenesis of hepatocyte PUFAs.

M a l e r i a l s and methods SIIR adult rats (I 3owceks okl) purchased fror, l IFFA CREDO IDo° mat ne des Onci as, U Arbrcs le. France), were fed commercial stand° au'd pellets (Souriffarat, UAR, Villentoiss(m~suroOrge, France7 during I week for adaptation and then divided into two different groups. One group (standard7 received daily 25 t~ of u standard senti-synthetic diet (UAR. Villemoison-sur-Org~', France) contain° ing 5% of lipids (ISIO 4 Lesieur, France; a mixture o f sunflower oil, oleisol, soya and grape pips comaining in weight (%7: 16:O = 6.2; 18:(7 = 3.3; 18:1 n-9 = 4(); 18:2 n-6 = 45.0; 18:3 n6 = 1.3; 18:3 n-3 = 1,6; 20:0 = 0.3; 2(7:1 ;1-9 = 0.2:20:5 n-3 = O. 1; 22:(7 = O.8; 24:0 = 0,3), The other group (EPA3()) received daily 25 g of the standard diet containing 5% of lipids including 0.8% EPA 3(7 (Callanish Lid Breasclete, Isle x~I"Lewis. UK. containing in weight (%): 16:1 n-7 = 15. l; 18:1 n-9 -- 6.1; 18:2 n-6 = 3.7; 18:3 n-6 = 2.4; 18:4 n-3 = 7.4; 20:4 n-6 = 1.7; 20:5 n-3 = 37.7; 22:5 n-3 = 5.5:22:6 n-3 = 20.4, administered as ethyl-estersT. The duration of the diets was O weeks, and the animals received lap water ad lib)turn during the whole experiment. The systolic blood pressure of prewarmed and conscious rats was measured weekly in the morning, ttsing a tail pulse pickup device (M+M Electronic Inc. BaseI-Miincheenstein. Switzerland). Blood pressure wa:~ determined for each atfimal by means of five measurements.

~ v c n animals fnm~ each group were selected at the end of the diel, ar,d hel~,t~zytes were i~)lated from animals after anesthesia, as described Wevkmsly II 31. One millkm of living hepatocytes were incubated during i h with the appropriate labeled fatty acid in a total volume of 2 mL Krebs-Hen~leit solution (pH 7.4). with 3.5% fauy acid ~ a ~ m ~ i n , containing (~n~,d): L (+) lactate 20, D (+) glucose I0, NaHO.)~ %5 [ ! - ~ 1 LA (pn.~cursorof t ~ n-6 cascade), [ I- t'~CI ALAIpc~mso¢ of the n-3 ca~ade)and [ ! - ~ 1 DGLA L~cursor of at~aehidonic~id), ~ere diluted into albumin in presence of the corre~ d i n g u n l a b e ~ fatty acid, to obtain a specific activity of 5 ~ilmmcd, The e ~ n ~ t i c reaction was start~ by adding 200 nmol labeled precursor in the medium, The incubations were conducted i~ du~ic,:~, szoppcdby adding 15 mLof ehitwotbrm/methanol t vlv), arp~l~ frozen (-80"C) until analysis, After lipid extraction follow in.gthe l~ok,~hmeth~J 114]. the fatty acid methyl esters [ 14. 151 were ~ted by revere pl~a~ liquid chromatography according to the nl~|l~tKlof N:wce el a11161. The de~atura~e activities were then determi~d by the ratio of the radioactive products of desaturatioed~bs~rate, a ~ exlwessed as nmol of ~uhstrate converted/l(~' incubated living hepatocytes, The fatty acid composition of total lipids was determined by gasliquid chromatography from an aliquot of unincubated hepatocytes, ~nd exptes.~d in weight t~ ),

Results

Food i~;ta~' and body weight Food intake was siti~ilar in the two gtxmps and the average weight changed from 310 g to 350 g in the two groups during the trial, indicating that the diet had no major effects on the animal's growth.

Blood p~,ssure The blood pressure in the EPA30 group (202 ± 7 nltnHg) was not significantly different l~om lha! of the standard group ( 197 ± 6 mmHg).

Fatty ~wid composition o)' htTrmr'ytu~ The administration of IEPA30 was reflected in the decreased desaturation products of the , - 6 family lfig ! ), with correspoading increase of desaturation substrates. While G L A AA, 22:4 n-6 and 22:5 n 6 were decreased in the EPA30 group, LA and DGLA were increased, The administration of the EPA/DHA mixture was reflected in the increased EPA, dlx:osapentaenoic acid and DHA. Consequently, the ratio of the n-6 desaturated PUFAsh~-3 desaturated PUFAs was significantly decreased in the EPA30 group compared

St~aistieal m~alysis, Results are expressed as the arithmetical mean li~rcad~ group with their ~tar,dard error (mean ± SB). Data were analy~,edu~ing a twoway ANOVA tbllowed by Student's t,test.

30

*

**

I

10

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*

*

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~

C

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,

*

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~

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Fatty

C

~

C

C

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acids

I~tl~!, Fair) acid O,~U~|~sitilql(weight t~ }) uf hepatocyte t~)tal lipids, Standard: spontaneously hypertensive rats fed the standard dict. EPA30: sp~da~)usly hypertensive rats fed the experimental 12PA30diet, Gas chromatographic analysis was l~erfiwmcdon methyl esters as reported tn M(~teriuls amt meOwed,s, Results are sho~,,nas me;~nof 7 ± SEM, Significant differences from control rats: *P < 0.05; **P < t).01.

137 with the standard group. Oleic acid (~8:1 ~-91, product of A9 dcsaturation, was also decreased in the EPA30 group when its corresponding substra|e, stearic acid, was increased. Desal;~rase activigh,,g

The msuhs pointed out tfig 2) that the A6 tt-3 desaturase activity was lower (-20%) in the EPA30 group as compared to the standard group. The n-6 desaturation steps were also decreased in the EPA30 group, the A6 n-6 desaturation by 22% and the A5 n-6 desaturation by 19%. Thus, both A6 desaturase activities were decreased in the same proportions under the influence of EPA30. The activity of A6 n-6 desaturation was lower tha,~ the activity of A6 ,-3 in both ,,rt ups, and the A5 n-6 desatnrase was the most active of these enzymes.

A6 n-3 desatura~ion r,

EPA30 [ dlg N E

20

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0

-

-

SHR

A6 n-6 desaturafion

]m stanC~rd [ ga,

Discussion

EPA30 did not decrease hypertension in the present experiment. It has been shown that the ratio n-61n-3 PUFAs in the diet may be related to the sensitivity of anintal.~ m st:'ess 117 I. In our experiment, this ratio was higher in the standard diet than in the EPA30 diet, with a consequent increase in their blood pressure. The present study demonstrates Ihc efficiency of dietary EPA + DHA on the hepatocyte bioconversion of EFAs in SHR and points out thai favorable conditions have been positioned for a decrease of blood pressure: LA increased in bepatocytes from the EPA30 group, while tire AAtLA ratio, as well as the content of AA. decreased significantly. These data suggest that EPA + DI t A inhibited the conversion of I.A to AA, as suggested in previous cxpe,'intents 1181. Therefore, it has been hypothesized thai I.A cotdd be increased aud AA decreased by :m inhibio !ion of A6 and A5 desa'urases 13 I. It has also been repo,'ted that marine oil lowers the activity of A5 and A6 desaturases in rodents 119,201. The desaturase activities evidenced that these changes in httty acid con!position are linked to the LA cascade. The observed desatttrase activities allow a belie," understanding of hepatocyte fatty acid compositions and arc coherent with the previous hypothesis. EPA30 induced a decreased A6 n-3 desaturase activity, certainly by at Icedback el't~ct of the desaturation products, and has a similar influence on the A6 n-6 and A5 n-6 desaturase activities. Multiple interactions among products of the AA cascade have been documented [211. Both EPA and DHA may exert their effects by competing with AA, thereby lowering the biosynthesis of AA metabolites. Moreover, the impaired release of DGLA in SHR 151 is helpful Ibr a better understanding of the reduced ability of SHR to produce eicosanoids from endogenous fatty acids 122, 231. Nevertheless, the exact mechanism underlying the effects of mari,le oils in hypertension has not yet been clarified. it seems that an increase of p,'ecursor of the series no

o

G I=

SHR

A5 n-6 desaturation --

80

m. .g:

60

=

40

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[ ~ sta.d~rd EPA~

20

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E

0 SHR

Fig 2, Desalurase activities. Standard: spontaneouslyhypeltcnsive l'als fed the standard diet. EPA30: spontaneouslyhypertensive rats fed the experiment'41 EPA30 diet. Desaturase activities were measured using I I-HC] sttbstrates of desaturation and an HPLC method as detailed in Materhds amt methods, alld expressed as nmol of substrate converted/10(~incubated living hepamcytes. Reo stilts are shown ;is mean of 7 ± SEM. Signific:mtdiflerences li'om control I'~.IIS::l,p < 0.(15; *:i: p < II.I) l.

3 proslaglandins is not sul'ficient to induce a decreased blood pressure. The availability of precursor of the series no I prostaglandins is still depressed by feeding rats with marine oils, and could explain the non-observed effect of marine oil on blood pressure. A study using GLA. precursor of DGLA. in combination with EPA may be c¢msidered, in

v~w to increase PGEI synthesis, known to provide a mechanism lot down+regulating vascular reactivity in normolens|re auimals+ a i ~ shown to be depressed in SHR. In conclusion, the present study evidenced that an EPA/DHA concentrate is a pharmacological active nutrient o n ~ F~A ca:"scade in SHR, and ~ the fatty acid composition of hepatocyt~, ir.creasi~ the level of ~ 3 precursors, decreasing the availability of subs|rote for TXA2 synthesis. Thus+ n-3 PUPAs may decrease blood pressure because they may interfere with eicosanoid production from n + PUPAs 1241. It appears that the~ changes are not sufficient to induce a decrease of hlood pressure. A combination of ~PA30 and GLA or DGLA, precursor of PGEi, known as down-regulator of vascular reactivity, has to be estimated+ Further studies are needed to elucidate the underlying mechanism related to the effects of EPA30 on desatura~ regulation.

Acknowledgments This work was supportedby a Grant from InternationalFoundation fo¢ the Prt~m(~ionof Nutrition Research and Nutrition Educatkm 0SFE) S~i~crland, and from the Ministry of ~uca;ion, France, The autho~ arc grateful In Doctor David F Ho~lbin from EFAMOt, Kentville, Canada, tot providing EPA30,

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