Effects of dietary fats (fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver

E ec s o die a y a s ( ish,oli e andhigh-oleic-acid sun lowe oils) on lipid composi ion and an ioxidan enzymes in a li e Valen ina Ruiz-Gu ie ´ ez 1 , Alonso Pe ´ ez-Espinosa 1 , Ca men Ma ı ´aVa ´zquez 2 and Consuelo San a-Ma ı ´a 3 * 1 Ins i u o de la G asa y sus De i ados, CSIC, apdo 1078, Se illa, Spain 2 Depa amen o Fisiologı ´a y Biologı ´a Animal, Facul ad de Fa macia, c/ P o esso Ga cı ´a Gonza ´lez s/n, 41012, Se illa, Spain 3 Depa amen o de Bioquı ´mica, B oma ologı ´a y Toxicologı ´a, Facul ad de Fa macia, c/ P o esso Ga cı ´a Gonza ´lez s/n, 41012, Se illa, Spain (Recei ed 22 Sep embe 1998 – Re ised 12 Ap il 1999 – Accep ed 28 Ap il 1999) The e ec s o wo oleic-acid- ich die s (con aining oli e oil, OO, and high-oleic-acid sun lowe oil, HOSO) on plasma and li e lipid composi ion de oxi ica ion enzyme ac i i ies, we e compa ed wi h hose o a ish-oil (FO) die and a con ol die . Compa ed wi h he con ol die , plasma and hepa ic o al iacylglyce ol concen a ions we e inc eased in he animals ed on he HOSO and OO die s and dec eased in hose ed on he FO die . The animals ed on FO showed he highes le el o choles e ol in he li e and had lowe plasma choles e ol concen a ions when compa ed wi h hose ed on he wo oleic-acid- ich die s. In compa ison wi h he animals ed on he die s en iched in oleic acid, he FO g oup showed highe hepa ic le els o polyunsa u a ed a y acids o he n-3 se ies and lowe le els o a y acids o he n-6 se ies. Li e s o FO- ed a s, compa ed wi h hose o OO- and HOSO- ed a s showed: (1) signi ican ly highe ac i i ies o ca alase (EC 1.11.1.6) glu a hione pe oxidase (EC 1.11.1.9) and Cu/Zn supe oxide dismu ase (EC 1.15.1.1); (2) no di e ences in he NADPH-cy och ome c educ ase (EC 1.6.99.3) ac i i y. The HOSO die had a simila e ec on li e an ioxidan enzyme ac i i ies as he OO die . In conclusion, i appea s ha changes in he li e a y acid composi ion due mainly o n-3 lipids may enhance he e iciency o he an ioxidan de ence sys em. The wo monounsa u a ed a y acids oils s udied (OO and HOSO), wi h he same high con en o oleic acid bu di e en con en s o na u al an ioxidan s, had simila e ec s on he an ioxidan enzyme ac i i ies measu ed. Fa y acids: An ioxidan enzymes: Oleic acid Many clinical s udies ha e indica ed ha die s ich in ish a e associa ed wi h ca dio ascula heal h (K omhou e al. 1985; He old & Kinsella, 1986). The esponsible compo- nen o ish appea s o be he high con en o polyunsa u- a ed a y acids (PUFA) o he n-3 se ies (Bang e al. 1986). Howe e , he e is g owing conce n ha habi ual in ake o la ge quan i ies o PUFA may induce ca cinogenesis, p ob- ably because hey a e e y suscep ible o pe oxida ion and p oduc ion o ee adicals (Kok e al. 1994; Fang e al. 1996). The p oduc ion o ee adicals has been associa ed wi h ageing (Ha man, 1992); howe e , some s udies indi- ca e ha ish oil (FO) seems o ex end he li e span in animal models o au oimmune disease (Jeng & Fe nandes, 1989). The ole o he an ioxidan de ence sys em, which includes Cu/Zn supe oxide dismu ase (EC 1.15.1.1; Cu/ Zn SOD), ca alase (EC 1.11.1.6; CAT), and glu a hione pe oxidase (EC 1.11.1.9; GSH-Px), in p o ec ion agains oxida i e insul s is well cha ac e ized in he li e , and i has been sugges ed ha his an ioxidan de ence sys em may be in luenced by nu i ion (Huang e al. 1994). Besides FO, oli e oil (OO), an oil ich in monounsa u- a ed a y acids (MUFA), is also ela ed o ca dio ascula heal h. OO is bene icial in lowe ing LDL-choles e ol bu no HDL-choles e ol, and dec easing he suscep ibili y o he LDL o oxida ion, which in u n educes he a he ogenici y o he LDL and he de elopmen o CHD (Ma son & G undy, 1985). I has been epo ed ha oleic acid is no necessa ily he only componen esponsible o his e ec and ha o he an ioxidan compounds con ained in he non- glyce ide ac ion o OO, such as s e ols and polyphenols, may con ibu e o hese bene icial esul s (Papadopoulos & Boskou, 1991). Al hough he adi ional sou ce o die a y MUFA is OO, o he sou ces a e now becoming a ailable such as he new high-oleic-acid a ie y o sun lowe oil B i ish Jou nal o Nu i ion (1999), 82, 233–241 233 Abb e ia ions: CAT, ca alase; FO, ish oil; GSH-Px,glu a hione pe oxidase; HOSO, high-oleic-acid sun lowe oil; MUFA,monounsa u a ed a y acids; OO, oli e oil; PUFA, polyunsa u a ed a y acids; SOD, supe oxide dismu ase. *Co esponding au ho : D Consuelo San a-Ma ı ´a, ax +34 95 423 3765, email [email p o ec ed] h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess (HOSO), which has a simila a y acid composi ion bu di e en an ioxidan con en (Pe ez-Jime ´nez e al. 1995). Compa ed wi h FO, he a e o pe oxida ion and p oduc ion o ee adicals in he MUFA oils is lowe . The aim o he p esen s udy was o compa e he e ec s o die a y FO, OO and he HOSO on plasma and hepa ic lipid composi ion and on some hepa ic an ioxidan enzyme ac i i ies. The esul s show ha he beha iou o CAT, Cu/Zn SOD and GSH-Px seems o be ela ed o he n-3 con en o lipid in he li e . The FO die p o ided he g ea es an ioxidan capabili y whils he OO and HOSO die s beha ed simila ly wi h ega d o he an ioxidan enzyme ac i i ies. Me hods Animals and die s Male Wis a a s (Le ica, Ba celona, Spain), weighing abou 80g a he beginning o he expe imen s, we e used. The animals we e housed in a well- en ila ed oom main ained a 22628on a 12h ligh –da k cycle. The a s we e andomly di ided in o ou g oups o en animals. Each g oup was ed on one o he ollowing die s o 12 weeks: a semipu i ied die (basal die ) con aining 20g unspeci ied lipid/kg, pu chased om Panlab SRL (Ba celona, Spain) (con ol g oup) o he basal die supplemen ed wi h 100g/kg OO (OO g oup) o HOSO (HOSO g oup) o FO (FO g oup). The composi ion o he expe imen al die s is shown in Table 1. To minimize oxida ion, all die s we e p epa ed once weekly and s o ed a 48unde an a mosphe e o N 2 un il needed. Changes in composi ion du ing s o age we e no de ec ed. The a y acid composi ions o he oils we e de e mined and a e shown in Table 2. The non- a y-acid componen s o he oils a e p esen ed in Table 3. Blood sampling and issue p epa a ions A comple ion o he s udy he animals we e killed by ce ical disloca ion. To minimize diu nal a ia ions he a s we e ou inely killed be ween 09.00 and 10.00 hou s. Blood samples we e emo ed om he hea and collec ed in o ubes con aining EDTA (1g/l). Plasma was sepa a ed by low-speed cen i uga ion a 1500ga 48 o 30min and was immedia ely analysed. Choles e ol, phospholipids and iacylglyce ols we e de e mined using an au oanalyse and con en ional enzymic me hods (Nelson, 1972; Bucolo & Da id, 1973; Allain e al. 1974). The li e s we e immedia ely insed in ice-cold 0⋅145M- NaCl, immed and quickly weighed. A 2g po ion o li e was used o lipid ex ac ion and he es was used o de e mine enzyme ac i i ies. All subsequen p ocessing p ocedu es we e ca ied ou a 0–48. Homogena es (100g/l) 234 V. Ruiz-Gu ie ´ ez e al. Table 1. Composi ion o expe imen al die s (g/kg) Ing edien s Con ol OO HOSO FO Casein 209 203 203 203 Suc ose 450 374 374 374 Maize s a ch 202 180 180 180 Lipids 20 20 20 20 OO – 100 – – HOSO – – 100 – FO – – – 100 Cellulose powde 52 56 56 56 Mine al mix* 57 57 57 57 Vi amin mix† 10 10 10 10 To al ene gy (MJ) 15⋅217⋅117⋅117⋅1 P o ein (% ene gy) 23⋅119⋅719⋅719⋅7 Lipid (% ene gy) 4⋅926⋅326⋅326⋅3 Ca bohyd a e (% ene gy) 71⋅953⋅953⋅953⋅9 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. *Mine al mix con ained (mg/g): NaCl 139⋅3, K 2 HPO 4 389⋅1, CaCO 3 381⋅4, MgSO 4 .7H 2 O57⋅3, FeSO 4 .7H 2 O27⋅0, MnSO 4 .H 2 O4⋅0, ZnSO 4 .7H 2 O1⋅25, KI 0⋅8, CuSO 4 .5H 2 O0⋅5, CoCl 2 .6H 2 O0⋅02. †Vi amin mix p o ided (/kg die ): e inol 5⋅9mg, calci e ols 0⋅15mg, hiamin 20mg, ibo la in 15mg, niacin 70mg, py idoxine 10mg, inosi ol 150mg, cobalamin 50mg, ocophe ols 170mg, i amin K 40mg, choline 1⋅36g, p e oylmonoglu amic acid 5mg, p -aminobenzoic acid 50mg, bio in 0⋅3mg. Table 2. Fa y acid composi ion o die a y a s (g/100g o al a y acids) Fa y acids Con ol OO HOSO FO 14:0 – – – 3⋅8 16:0 13⋅211⋅84⋅313⋅9 16:1 n -7 1⋅20⋅90⋅115⋅1 17:0 – 0⋅40⋅1– 18:0 3⋅12⋅84⋅70⋅9 18:1 n -9 36⋅879⋅280⋅226⋅2 18:2 n -6 41⋅73⋅59⋅45⋅1 18:3 n -3 4⋅10⋅60⋅10⋅2 18:4 n -3 – – – 2⋅5 20:0 – 0⋅30⋅4– 20:1 n -9 – 0⋅20⋅22⋅4 20:2 n -6 – – – 1⋅4 20:3 n -6 – – – 0⋅4 20:4 n -6 – – – 0⋅6 20:5 n -3 – – – 13⋅8 22:4 n -6 – – – 0⋅3 22:6 n -3 – – – 12⋅7 24:0 – 0⋅40⋅4– OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. Table 3. Composi ion o he non- a y-acid componen s o he expe imen al oils OO HOSO FO To al unsaponi iable ac ion (g/kg) 15 10 11 To al s e ols (mg/kg) 1696 1754 1500 Choles e ol (%) 0⋅15 0⋅01 100 B asicas e ol (%) – 0⋅32 – Campes e ol (%) 3⋅80 10⋅25 – S igmas e ol (%) 0⋅81 11⋅59 – b-Si os e ol (%) 93⋅99 59⋅35 – D-5-A enas e ol (%) – 2⋅88 – D-7-S igmas e ol (%) 0⋅22 11⋅84 – D-7-A enas e ol (%) 0⋅15 3⋅27 – Squalene (mg/kg) 3000 90 7400 Tocophe ols (mg/kg) 47 10 300 a-Tocophe ols 34 10 300 g-Tocophe ols 13 – – F ac ion o polyphenols (mg/kg) 470 – – To al polyphenols (mg/kg) 430 – – O odiphenol (mg/kg) 40 – – OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess we e p epa ed in 0⋅25M-suc ose, 1mM-EDTA, 1mM-DL- di hio h ei ol and 15mM-T is HCl (pH 7⋅4), using an all- glass Po e El ehjem homogenize (Selec a, Ba celona, Spain). Each homogena e was cen i uged o 20min a 800g. The esul ing supe na an ac ion was used o de e mine enzyme ac i i ies. Ex ac ion and sepa a ion o lipids Quan i a i e ex ac ion o o al lipids om 2g li e was ca ied ou ollowing he me hod o Folch e al. (1957) in he p esence o bu yla ed hyd oxy oluene as an ioxidan . Tissue dissocia ion was achie ed by homogeniza ion in ice- cold chlo o o m–me hanol (2:1, / ) con aining 0⋅1g BHT/l using an Ul a-Tu ax model Type TP-18-1 (Ul a-Tu ax, Vineland, NJ, USA). The lipid ex ac was quan i ied g a ime ically and kep in s oppe ed ubes unde N 2 a −308un il he assays. Lipid composi ion was de e mined by means o he Ia oscan TLC echnique wi h lame ioniza ion de ec ion (De Sch ij e & Ve meulen, 1991). The Ia oscan MK-5 (Ia on Labo a- o ies Inc., Tokyo, Japan) was used in combina ion wi h Ch oma ods S, which ha e a p ecoa ed ac i e silica hin laye . Samples o o al lipids (3ml) we e spo ed on o each od, using a 10ml Hamil on sy inge. To sepa a e o al lipids, ods we e de eloped in hexane–die hyl e he – o mic acid (90:10:2, by ol.). Rods we e scanned unde he ollowing condi ions: H 2 low, 150ml/min; ai low, 1750ml/min; scanning speed, 47mm/s; cha speed, 42mm/min. An Ia oco de TC-11 in eg a o was used o eco ding and a ea in eg a ion. Fa y acid analysis Fa y acids (g/100g o al a y acids) we e de e mined by GC, as p e iously desc ibed (Molina e al. 1989). The samples we e saponi ied by hea ing o 25min wi h 5ml 0⋅2M-sodium me hyla e and hea ed again a 808 o 25min wi h H 2 SO 4 in anhyd ous me hanol (60g/l). The a y acid me hyl es e s hus o med we e elu ed wi h hexane and analysed in a Hewle Packa d 5890 se ies II GC equipped wi h lame ioniza ion de ec o and using an Omegawak 320 used silica capilla y column (30m× 0⋅32mm i.d., 0⋅25mm ilm). The ini ial column empe a u e was 2008, which was held o 10min, hen p og ammed om 200–2308a 28/min. Non- a y-acid componen s Fo he ex ac ion o he unsaponi iable ma e , 20g oil was saponi ied o 30min wi h 75ml o e hanolic KOH (100g/l). The solu ion was ans e ed o a 5000ml decan ing unnel, 100ml dis illed wa e was added, and he mix u e was ex ac ed wi h 100ml po ions o hexane. The hexane solu ion was e apo a ed o d yness in a o a y e apo a o a 308unde educed p essu e. The s e ol ac ion was analysed by capilla y GLC (Ga cia Reguei o e al. 1994). Tocophe ols we e analysed by HPLC (K ame e al. 1997). Fo he assay o squalene, he hyd oca bon ac ion was sepa a ed om he oil by column ch oma og aphy on silica gel and analysed by capilla y GLC (Sulpice & Fe ezou, 1984). The composi ion o he polyphenols ac ion was de e mined by capilla y GLC (A ce e al. 1998). Enzyme ac i i ies CAT ac i i y was assayed acco ding o he me hod o Bee s & Size (1952). The inal concen a ions in he cu e es we e 0⋅5M-KH 2 PO 4 (pH 7), 100mm H 2 O 2 and 0⋅05–0⋅1mg issue sample. The dec ease in he abso bance a 240nm a e he addi ion o he subs a e was ollowed spec o- pho ome ically. GSH-Px ac i i y was assayed wi h a coupled enzyme sys em in which GSSG educ ion was coupled o NADPH oxida ion by glu a hione educ ase (EC 1.6.4.1; Law ence & Bu k, 1976). The assay mix u e con ained 0⋅1M-KH 2 PO 4 (pH 7⋅5), 5mM-EDTA, 2mM-NaN 3 ,1mM-GSH, 0⋅2mM- NADPH, 1U glu a hione educ ase and issue sample (0⋅05–0⋅2mg). A e 5min p e-incuba ion (20–258), he eac ion was ini ia ed by he addi ion o 0⋅05ml 5mM- H 2 O 2 ( inal olume 1⋅0ml). The dec ease in he abso bance a 365nm was ollowed spec opho ome ically. SOD ac i i y was measu ed using he xan hine oxidase (EC 1.2.3.2)–cy och ome cme hod as desc ibed by McCo d & F ido ich (1969). The inal concen a ions in he cu e es we e 50mM-KH 2 PO 4 (pH 7⋅8), 0⋅1mM-EDTA, 10mM- cy och ome c,50m M-xan hine, 50mMo 2mM-cyanide, 1U CAT and 0⋅05–1⋅0mg issue sample. The eac ion was ini ia ed by he addi ion o 1U xan hine oxidase. The inhibi ion o xan hine oxidase was ollowed spec opho o- me ically a 550nm. One uni o SOD ac i i y is de ined as he amoun o enzyme equi ed o inhibi he a e o cy och ome c educ ion by 50%. NADPH-cy och ome c educ ase (EC 1.6.99.3) ac i i y was measu ed as desc ibed by Ve milion & Coon (1978). The 1⋅0ml assay mix u e con ained he ollowing com- ponen s: 300mM-phospha e bu e (pH 7⋅7), 0⋅04mM- cy och ome c,0⋅1mM-EDTA, 0⋅2mM-NADPH and issue (0⋅05–0⋅2mg). The eac ion was ini ia ed by he addi ion o he NADPH, and he educ ion o cy och ome cwas ollowed spec opho ome ically a 550nm. All spec opho ome ic measu emen s we e ca ied ou in a Shimadzu 160 A ul a iole spec opho ome e (Shimadzu Co po a ion, Kyo o, Japan) wi h 1⋅0ml qua z cu e es wi h a ligh pa h o 1⋅0cm. All enzyme assays we e pe o med a 258. Speci ic ac i i ies we e exp essed as nmol/min pe mg p o ein. P o ein concen a ions we e de e mined by he me hod o Low y e al. (1951). S a is ical me hods All esul s we e subjec ed o one-way ANOVA, and ep e- sen means, wi h hei s anda d e o s, o en animals pe g oup. Di e ences in mean alues be ween g oups we e assessed by he wo- ailed S uden ’s es and we e con- side ed s a is ically di e en a P,0⋅05. Resul s In he p esen s udy, a s in he ou expe imen al g oups consumed simila amoun s o ood (Table 4). Animals ed on die s con aining OO o HOSO had simila body weigh s, 235E ec s o die a y a s on a li e h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess bu hese we e highe han hose o animals ed on he con ol o FO die s. Animals ed on he die con aining FO had signi ican ly lowe body and li e weigh s when com- pa ed wi h he o he g oups; howe e , he li e :body weigh alues we e simila in all he g oups. Li e lipid con en was highe in animals ed on he OO and HOSO die s han in hose ed on he con ol o FO die s. Table 5 shows he e ec o die a y a ea men on plasma lipid con en . When compa ed wi h he con ol die , he HOSO and OO die s led o signi ican inc eases and he FO die led o a signi ican dec ease in plasma iacylglyce ol le el. Consump ion o he FO die esul ed in a dec ease in he o al choles e ol and phospholipid con- cen a ions in plasma in compa ison wi h he high-oleic- acid die s. The HOSO and OO g oups did no di e in plasma lipid concen a ions, bu hese we e highe han hose in he con ol g oup. To al iacylglyce ol concen a ions in li e a e shown in Table 6. The OO- ed g oup showed he highes alues and he FO- ed g oup he lowes . To al hepa ic choles e ol was lowes in he con ol g oup and highes in he FO g oup. OO- and HOSO- ed g oups showed in e media e alues, and no di e ence was ound be ween hese wo high-oleic- acid die s. The hepa ic phospholipid con en was lowes in he animals ed on he con ol die . The animals ed on he oil-en iched die s showed simila hepa ic phospholipid con en s. Table 7 shows he p opo ions o a y acids (g/100g o al a y acids) in li e lipids in a s ed on he di e en die s. The highes p opo ions o he wo majo sa u a ed a y acids, palmi ic (16:0) and s ea ic (18:0) acids, we e ound in a s ed on he FO and con ol die s as compa ed wi h he high-oleic-acid oil g oups. Thus, he le els o o al sa u a ed a y acids in li e we e highe in he con ol and he FO- ed g oups in compa ison wi h he animals ed on he die s en iched in oleic acid. The p opo ions o 18:1n-9 we e simila in he OO- and HOSO- ed g oups and highe han in he con ol- o FO- ed animals. The MUFA:sa u a ed a y acids a io was consequen ly inc eased in animals ed on he wo oleic-acid- ich die s compa ed wi h he o he g oups. The FO g oup had a highe p opo ion o long-chain PUFA o he n-3 se ies (20:5, 22:5 and 22:6) han he con ol, OO, o HOSO g oups. The FO g oup also had lowe p opo ions o long-chain PUFA o he n-6 se ies (20:4, 22:4 and 22:5) wi h espec o he o he g oups. The e o e he n-6:n-3 a io and he 20:4/18:2 a io we e ma kedly lowe in he FO g oup. The animals ed on he FO die exp essed highe CAT ac i i y in he li e (5⋅06 (SE 0⋅27) U/mg p o ein) when compa ed wi h he o he g oups (Fig. 1(a)). The CAT ac i i y was simila in he OO and HOSO g oups (app oxi- ma ely 3⋅70U/mg p o ein). The GSH-Px ac i i y in animals ed on he con ol die was 0⋅63 (SE 0⋅01) U/mg p o ein. Feeding he FO die inc eased his ac i i y o 0⋅74 (SE 0⋅05) U/mg p o ein and eeding OO o HOSO dec eased he ac i i y o 0⋅39 (SE 0⋅02) U/mg p o ein and 0⋅47 (SE 0⋅08) U/mg p o ein espec i ely (Fig. 1(b)). The Cu/Zn SOD ac i i y was highes in he FO- ed g oup (3⋅51 (SE 1⋅12) U/mg p o ein) and was signi ican ly highe han in he o he g oups. The oleic-acid-en iched oils had no e ec on Cu/Zn SOD ac i i y compa ed wi h he con ol die (Fig. 2(a)). The ac i i ies o NADPH-cy och ome c 236 V. Ruiz-Gu ie ´ ez e al. Table 4. E ec o di e en die a y a s, ed o 12 weeks, on body weigh , ood in ake, li e weigh and li e lipid con en in a s* (Mean alues wi h hei s anda d e o s o en a s pe g oup) Die ... Con ol OO HOSO FO Mean SE Mean SE Mean SE Mean SE Food in ake (g/d) 20⋅81⋅121⋅00⋅922⋅81⋅919⋅11⋅2 Body w (g) a en y 85⋅12⋅377⋅63⋅278⋅33⋅186⋅82⋅4 a 12 weeks 302⋅4 a 7⋅8 325⋅9 b 6⋅1 331⋅1 b 13⋅2 260⋅4 c 6⋅6 Li e w (g) 9⋅3 a 0⋅810⋅4 a 0⋅89⋅4 a 1⋅27⋅5 b 0⋅6 Li e w (g/kg body weigh ) 31 2 30 3 31 2 29 4 Li e lipid con en (g/kg) 21 a 545 b 444 b 332 c 2 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. a,b,c Mean alues wi hin a ow no sha ing a common supe sc ip le e we e signi ican ly di e en : P ,0⋅05. *Fo de ails o die s and p ocedu es, see Tables 1–3 and pp. 234–235. Table 5. Plasma lipid concen a ions in a s ed o 12 weeks on die s con aining di e en a s* (Mean alues wi h hei s anda d e o s o en a s pe g oup) Die ... Con ol OO HOSO FO Plasma lipid Mean SE Mean SE Mean SE Mean SE T iacylglyce ols (mmol/l) 1⋅25 a 0⋅23 1⋅72 b 0⋅12 1⋅68 b 0⋅17 0⋅89 c 0⋅14 Choles e ol (mmol/l) 2⋅02 a 0⋅41 2⋅55 b 0⋅22 2⋅44 b 0⋅23 2⋅20 a 0⋅21 Phospholipids (mmol/l) 1⋅29 a 0⋅12 1⋅44 b 0⋅15 1⋅46 b 0⋅21 1⋅23 a 0⋅15 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. a,b,c Mean alues wi hin a ow no sha ing a common supe sc ip le e we e signi ican ly di e en : P ,0⋅05. *Fo de ails o die s and p ocedu es, see Tables 1–3 and pp. 234–235. h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess educ ase we e simila in li e s o a s ed on he oil- en iched die s, and we e highe han ha o he con ol animals (Fig. 2(b)). Discussion The p esen s udy was designed o de e mine whe he eeding a PUFA oil (FO) o wo MUFA oils, p epa ed om wo di e en sou ces (OO and HOSO), a ec s he li e lipid composi ion and he an ioxidan de ence sys em; he plasma lipid composi ion was also s udied. Li e lipid iacylglyce ol le els we e lowes in FO- ed a s (Table 6). This may ha e been due o inhibi ion o hepa ic iacylglyce ol syn hesis (Wong e al. 1984; F oy- land e al. 1997), and s imula ion o hepa ic pe oxisomal b-oxida ion (Yamazaki e al. 1987) p oduced by he FO die . Dec eases in lipop o ein lipase (EC 3.1.1.34) and iacylglyce ol lipase (EC 3.1.1.3) ac i i ies in he li e a e FO consump ion ha e also been desc ibed (Hu e al. 1993), and his may be an adap i e esponse o he low concen a ions o subs a es ( iacylglyce ols) o hese enzymes. The low le els o iacylglyce ols in li e a e FO consump ion may also be ela ed o he educed le els o plasma iacylglyce ols ound in ou s udy (Table 5) and also desc ibed by o he s in man (Phillipson e al. 1985) and a s (Yamazaki e al. 1987). The OO die esul ed in a highe li e iacylglyce ol con en han he HOSO die . This ac has been ecen ly co obo a ed by o he s udies om ou g oup (Pe ona & Ruiz-Gu ie ez, 1998), in which i has been epo ed ha he 237E ec s o die a y a s on a li e Table 6. Li e lipid concen a ions in a s ed o 12 weeks on die s con aining di e en a s* (Mean alues wi h hei s anda d e o s o en a s pe g oup) Die ... Con ol OO HOSO FO Li e lipid Mean SE Mean SE Mean SE Mean SE T iacylglyce ols (mg/g) 5⋅5 a 0⋅812⋅3 b 1⋅78⋅3 c 0⋅91⋅9 d 0⋅6 Choles e ol (mg/g) 3⋅1 a 0⋅34⋅4 b 0⋅64⋅7 b 0⋅56⋅7 c 0⋅6 Phospholipids (mg/g) 13⋅4 a 1⋅329⋅6 b 3⋅732⋅3 b 3⋅826⋅5 b 3⋅2 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; FO, ish oil. a,b,c,d Mean alues wi hin a ow no sha ing a common supe sc ip le e we e signi ican ly di e en : P ,0⋅05. *Fo de ails o die s and p ocedu es, see Tables 1–3 and pp. 234–235. Table 7. Fa y acid composi ion (g/100g o al a y acids) o he li e lipids o a s ed o 12 weeks on die s con aining di e en a s* (Mean alues wi h hei s anda d e o s o en a s pe g oup) Die ... Con ol OO HOSO FO Fa y acid Mean SE Mean SE Mean SE Mean SE 14:0 0⋅40⋅10⋅30⋅10⋅40⋅10⋅40⋅1 16:0 21⋅1 a 0⋅415⋅4 b 2⋅017⋅3 b 0⋅722⋅3 a 1⋅0 16:1 n -7 1⋅80⋅31⋅10⋅11⋅50⋅11⋅10⋅1 18:0 18⋅5 a 3⋅313⋅9 b 0⋅713⋅3 b 2⋅119⋅7 a 1⋅8 18:1 n -9 13⋅1 a 1⋅327⋅7 b 0⋅524⋅6 b 2⋅78⋅4 c 0⋅2 18:1 n -7 1⋅40⋅11⋅50⋅41⋅70⋅11⋅00⋅1 18:2 n -6 13⋅11⋅711⋅90⋅513⋅31⋅113⋅91⋅6 18:3 n -3 0⋅3 a 0⋅00⋅2 b 0⋅00⋅2 b 0⋅00⋅3 a 0⋅0 20:0 0⋅5 a 0⋅10⋅2 b 0⋅10⋅3 b 0⋅00⋅3 b 0⋅0 20:1 n -9 0⋅1 a 0⋅00⋅4 b 0⋅10⋅3 b 0⋅00⋅1 a 0⋅0 20:4 n -6 23⋅2 a 1⋅621⋅9 a 1⋅521⋅6 a 1⋅615⋅6 b 0⋅7 20:5 n -3 0⋅2 a 0⋅00⋅1 a 0⋅00⋅1 a 0⋅03⋅0 b 0⋅9 22:0 0⋅6 a 0⋅10⋅5 a 0⋅10⋅7 a 0⋅10⋅9 b 0⋅0 22:4 n -6 0⋅5 a 0⋅00⋅4 a 0⋅10⋅3 a 0⋅10⋅6 b 0⋅0 22:5 n -6 0⋅6 a 0⋅20⋅4 a 0⋅10⋅4 a 0⋅10⋅2 b 0⋅0 22:5 n -3 0⋅7 a 0⋅10⋅3 b 0⋅10⋅4 b 0⋅11⋅7 c 0⋅4 22:6 n -3 3⋅8 a 0⋅63⋅6 a 0⋅83⋅3 a 0⋅411⋅1 c 1⋅8 SFA 41⋅1 a 3⋅430⋅3 b 2⋅831⋅9 b 2⋅943⋅5 a 2⋅8 MUFA 16⋅5 a 1⋅730⋅8 b 1⋅128⋅1 b 2⋅410⋅6 c 0⋅5 PUFA 42⋅4 a 3⋅439⋅0 b 2⋅339⋅7 b 1⋅345⋅9 a 1⋅7 To al n -6 37⋅4 a 3⋅934⋅7 a 2⋅235⋅7 a 3⋅729⋅9 b 2⋅2 To al n -3 5⋅0 a 0⋅44⋅3 a 0⋅44⋅0 a 0⋅416⋅1 b 2⋅9 n -6: n -3 7⋅5 a 1⋅98⋅1 a 1⋅78⋅9 a 2⋅31⋅9 b 0⋅6 MUFA:SFA 0⋅4 a 0⋅31⋅0 b 0⋅30⋅9 b 0⋅20⋅2 a 0⋅1 20:4/18:2 1⋅7 a 0⋅41⋅8 a 0⋅51⋅6 a 0⋅31⋅1 b 0⋅2 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil;FO, ish oil; SFA, sa u a ed a y acids; MUFA, monounsa u a ed a y acids; PUFA, polyunsa u a ed a y acids. a,b,c Mean alues wi hin a ow no sha ing a common supe sc ip le e we e signi ican ly di e en : P ,0⋅05. *Fo de ails o die s and p ocedu es, see Tables 1–3 and pp. 234–235. h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess consump ion o OO esul s in a g ea e amoun o li e iacylglyce ol when compa ed wi h consump ion o HOSO. These esul s sugges ha ac o s o he han he oleic acid con en o he oils may be esponsible o enhancing iacylglyce ol syn hesis in he li e o he animals ed on he OO die . Fu he mo e, he same au ho s in he p esen s udy obse ed di e en dis ibu ions o iacylglyce ol molecula species in a li e ; OO led o a highe con en o dioleoacylglyce ol species while he HOSO die led o an inc ease in sa u a ed iacylglyce ol species, sugges ing ha hese e ec s may be due o an enhancemen in he syn hesis o such molecula species. In plasma we did no obse e di e ences in iacylglyce ol con en o he wo high-oleic-acid die s s udied. Choles e ol le els in li e we e inc eased in animals ed on he lipid-supplemen ed die s compa ed wi h he con ol g oup (Table 6). The FO die led o highe alues han he HOSO and OOdie s. The inc eased li e choles e ol con en in he FO- ed a s migh ha e been due o he inc eased squalene con en in his die compa ed wi h he o he s (Table 3). The die a y e ec o his p ecu so o choles e ol biosyn hesis is no clea , bu i has been shown o inc ease bilia y choles e ol sec e ion (Ulloa & Ne i, 1985). In plasma we ound a lowe choles e ol le el in he FO g oup han in he OO and HOSO g oups. I has also been desc ibed ha FO consump ion educes choles e ol le els in blood, and ha i is mo e hypocholes e olaemic han OO (Masi e al. 1986). Ga g e al. (1988) epo ed ha PUFA lowe choles e ol le els in man and animals due o a edis ibu ion o choles e ol om blood o issues. We ound high choles e ol le els in bo h li e and plasma ollowing oleic acid eeding compa ed wi h he con ol die . This hype choles e olaemic e ec o a die high in OO in se um and li e o a s has been epo ed p e iously by Yaqoob e al. (1995). Je e y e al. (1996) con i med he 238 V. Ruiz-Gu ie ´ ez e al. Fig. 1. Speci ic ac i i ies o (a) ca alase and (b) glu a hione pe oxi- dase in he li e s o a s ed o 12 weeks on a con ol die o die s con aining 100g/kg o oli e oil (OO), high-oleic-acid sun lowe oil (HOSO) o ish oil (FO). Values a e means o en a s pe g oup, wi h s anda d de ia ions ep esen ed by e ical ba s. a,b Mean alues no sha ing a common le e we e signi ican ly di e en : P ,0⋅05. Fig. 2. Speci ic ac i i ies o (a) Cu/Zn supe oxide dismu ase and (b) NADPH-cy och ome c educ ase in he li e s o a s ed o 12 weeks on a con ol die o die s con aining 100g/kg o oli e oil (OO), high- oleic-acidsun lowe oil (HOSO) o ish oil (FO). Values a e means o en a s pe g oup, wi h s anda d de ia ions ep esen ed by e ical ba s. a,b Mean alues no sha ing a common le e we e signi ican ly di e en : P ,0⋅05. h ps://doi.o g/10.1017/S0007114599001415 Published online by Camb idge Uni e si y P ess e ec on se um o al choles e ol concen a ions in a s ed on die s con aining OO o HOSO. In man, howe e , hypocholes e olaemic e ec o MUFA is well es ablished (Ma son & G undy, 1985) al hough he mechanism by which i is b ough abou emains unclea . Analysis o he a y acid composi ion in a li e homo- gena es showed a signi ican inc ease o 18:1 in li e lipids ob ained om OO- and HOSO- ed a s. We also ound lowe sa u a ed a y acid le els in bo h g oups. This inc ease in oleic acid con en was p obably ela ed o he highe con en o oleic acid in he die o hese animals. I has been epo ed ha when endo helial cell cul u es a e di ec ly supplemen ed wi h oleic acid, an inc ease in i s con en is ound, accompanied by a dec ease in he sa u a ed acid con en (Spec o & Yo ek, 1985). Wi h espec o he FO die , we ound a ma ked inc ease in he p opo ion o o al n-3 a y acids (mainly 20:5 and 22:6), and a concomi an dec ease in n-6 (20:4, 22:4 and 22:5) a y acids in he li e . As a consequence, a signi ican educ ion in he a io n-6:n-3 was ound in hese animals. O he au ho s ha e epo ed a dec ease in a achidonic acid in li e (Venka aman e al. 1994) and lung (A che e al. 1987) in animals ed on FO. Animals ed on he FO die had highe ac i i ies o GSH- Px, CAT and Cu/Zn SOD compa ed wi h hose ed on he con ol die and he oleic-acid-en iched die s. Because he lipid composi ion o he li e o a s e lec ed he lipid composi ion o he die , he li e lipids o he a s ed on he FO die con ained highe le els o PUFA. This ac migh ende he li e s o a s ed on he FO die mo e suscep ible o lipid pe oxida ion and he ac i i y o an ioxidan enzymes migh be induced. A po en ial mechanism o he induc ion o hepa ic an ioxidan s ollowing FO eeding migh be an inc ease o he exp ession o hei genes; induc ion o he exp ession o an ioxidan enzymes has been epo ed in ci cums ances whe e an inc ease in ee adicals is p o- duced, such as ageing (De Haan e al. 1992) o se e al pa hologies (Ce iello e al. 1996; La ea e al. 1998). I is also well known ha ee adicals may egula e he an- sc ip ion o many o he genes (C ame e al. 1995; Roche & Rome o-Al i a, 1995). This g ea e ac i i y o an ioxidan enzymes may con- ibu e o he hypo hesis ha consump ion o FO ex ends li espan. This ac has been desc ibed in animal models o au oimmune disease (Jeng & Fe nandes, 1989). An ioxi- dan s ha e been closely linked wi h he p ese a ion o heal h and longe i y in bo h mice and a s (Semsei e al. 1989; Rao e al. 1990). Yamazaki e al. (1987) ound ha eeding FO o animals inc eased he ac i i ies o some li e enzymes ( a y acyl- CoA syn he ase (EC 2.3.1.85), CAT and GSH-Px) when compa ed wi h sa lowe -oil- ed animals. Venka aman e al. (1994) desc ibed an inc ease in he ac i i ies and mRNA exp ession o CAT, GSH-Px and SOD in mice ed on a die ich in FO compa ed wi h o he die s ich in n-6 lipids. Recen ly, he same g oup has epo ed inc eases in CAT and GSH-Px ac i i ies in seden a y and exe cised Fische -344 a s espec i ely, a e consump ion o FO, and non-signi i- can changes in memb ane lipid pe oxida ion (Venka aman e al. 1998). In con as , L’Abbe e al. (1991) epo ed ha die s high in n-3 a y acids ele a e he lipid pe oxida ion in hea and li e , due in pa o dec eased SOD and GSH-Px ac i i ies. Nalbone e al. (1980) epo ed ha he GSH-Px ac i i y o a s ed on FO die s emained unchanged in li e . Be ge & Thomassen (1985) showed ha CAT ac i i y was a ec ed only sligh ly by a FO die , and Van Noo den (1995) desc ibed a mode a e inc ease in CAT ac i i y. Less a en ion has been paid o he e ec o oleic-acid- en iched die s on an ioxidan enzymes. In ou s udies we ha e no ound di e ences in he ac i i ies o he an iox- idan enzymes be ween he wo high-oleic-acid die s es ed. The non-glyce ide ac ions o hese oils a e di e en (Table 3); polyphenols, which ha e ee- adical-sca enging p op- e ies, a e only p esen in OO, and ocophe ols, which also ha e an an ioxidan e ec , a e highe in OO. These di e - ences in he na u al an ioxidan con en in he wo oils seems no o a ec he ac i i y o he hepa ic an ioxidan enzymes s udied. NADPH-cy och ome c educ ase pa icipa es in he de oxi ica ion o d ugs and xenobio ics. Simila ac i i ies o NADPH-cy och ome c educ ase we e obse ed in a s ed on he OO, HOSO and FO die s in he p esen s udy, and each oil die suppo ed highe ac i i y han he con ol die . Hepa ic NADPH-cy och ome c educ ase ac i i y was no signi ican ly a ec ed by he FO die in compa ison wi h a low- a die o die s en iched in n-6 PUFA (Van Noo den, 1995). The concen a ion o cy och ome P450 and a es o oxida i e d ug me abolism we e g ea e when polyunsa u- a ed a s we e inco po a ed in o he die han when a - ee die s o sa u a ed a s we e gi en (Smi h & Willis, 1981). In summa y, i appea s ha changes in he li e a y acid composi ion, due mainly o n-3 lipids, may inc ease he ac i i y o some an ioxidan enzymes, compensa ing he isk o ca cinogenesis due o he pe oxida ion o PUFA and ee- adical p oduc ion. On he o he hand, he wo MUFA oils s udied (OO and HOSO), in spi e o hei di e en con en s o na u al an ioxidan s, ha e simila e ec s on he an ioxidan enzyme ac i i ies s udied. Acknowledgemen s This s udy was suppo ed by G an ALI96-0456 om he Comision In e minis e ial de Ciencia y Tecnologı ´a, Spain. We wish o hank Koipe S.A. and Acei es Toledo. We a e g a e ul o Fe nanda Leo ´n and Manuel Rod iguez-Aguila o expe echnical assis ance. Re e ences Allain CC, Poon LS, Chang CSG, Richmond W & Fu PC (1974) Enzyma ic de e mina ion o o al plasma choles e ol. Clinical Chemis y 20, 470–475. A ce L, Rios A & Valca cel M (1998) De e mina ion o an i- ca cinogenic polyphenols p esen in g een ea using capilla y elec opho esis coupled o a low injec ion sys em. 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