Effects of dietary oleic-rich oils (virgin olive and high-oleic-acid sunflower) on vascular reactivity in Wistar-Kyoto and spontaneously hypertensive rats

E ec s o die a y oleic- ich oils ( i gin oli e and high-oleic-acid sun lowe ) on ascula eac i i y in Wis a –Kyo o and spon aneously hype ensi e a s M. D. He e a 1 ,C.Pe ´ ez-Gue e o 1 , E. Ma huenda 1 and V. Ruiz-Gu ie ´ ez 2 * 1 Depa amen o de Fa macologı ´a, Facul ad de Fa macia, C/P o eso Ga cı ´a Gonza ´lez s/n, 41012 Se illa, Spain 2 Ins i u o de la G asa (CSIC), A da, Pad e Ga cı ´a Teje o no. 4, 41012 Se illa, Spain (Recei ed 18 Sep embe 2000 – Re ised 23 Feb ua y 2001 – Accep ed 3 Ap il 2001) The e ec s o wo monounsa u a ed a y acid (MUFA)- ich die s, con aining i gin oli e oil (OO) and high-oleic-acid sun lowe oil (HOSO), on de elopmen o ascula esponse om isola ed ho acic a ao a and lipid composi ion and a y acid composi ion we e s udied and compa ed wi h samples om a s ed on a con ol die . Die a y MUFA oils we e ed o 6 weeks o spon aneously hype ensi e (SHR) and Wis a –Kyo o (WKY) a s om 4 weeks o age. The maximum con ac ion o ao ic ing p epa a ions in esponse o phenyleph ine (10 26 m) was signi ican ly dec eased in SHR a s ed wi h OO (0:81 (sem 0:05) . 1:18 (sem 0:09) g,P,0:01Þ and ea men wi h HOSO did no al e he phenyleph ine-induced con ac ions. The elaxan esponses o ace ylcholine (10 25 m) we e signi ican ly enhanced (30:03 (sem 0:70) . 18:47 (sem 0:28) %, P,0:001Þin he ings om SHR a s ea ed wi h OO, and we e mo e p onounced han in WKY a s ðP,0:05Þ:In he same way, OO a enua ed he dose– esponse cu es induced by phenyleph ine (10 28 –10 25 m) om SHR a s, accompained wi h a slowe con ac ion. These esul s sugges ha only he ch onic eeding o OO die was able o a enua e he ascula esponse o a ao a. In addi ion, an inc ease in phospholipid con en (186:7(sd 3:2) . 159:1(sd 11:3) g/kg, P,0:01Þand changes in he a y acid composi ion o ao a (mainly a dec ease in a achidonic acid) could con ibu e o imp o ing endo helial unc ion. The e o e, he e ec s can no be a ibu ed exclusi ely o he con en o MUFA (mainly oleic acid). O he componen s o OO, such as polyphenols, no p esen in HOSO, may help o explain he ascula p o ec i e e ec o OO consump ion. Oli e oil: High-oleic-acid sun lowe oil: Vascula eac i i y: Ra ao a Hype ension is cha ac e ized by an inc eased sensi i i y o ascula smoo h muscle o asocons ic o s imuli (T iggle & Lahe , 1985; Boh & Webb, 1988). Hype ac i i y o asoac i e agonis s has been co ela ed wi h an enhanced p o ein kinase C ac i i y (Tu la & Webb, 1987; Sil e e al. 1992; Bazan e al. 1993), an augmen ed phosphoinosi ide me abolism (Tu la & Webb, 1990), and an inc eased Ca 2+ mobilisa ion (Tho in- T escases e al. 1994). The endo helium plays an impo an ole in con olling he a-agonis -induced con ac ion and he educed in luence o he endo helium in he s oke-p one spon aneously hype ensi e (SHR) a s ao a is mos likely o be due o a dec eased ac i i y o his endo helium (Osugi e al. 1990). The changes in endo helium-dependen elaxa ion may also a ec he con ac ile esponse o blood essels and, as a consequence, may be in ol ed in con olling blood p essu e (Shi asaki e al. 1988; Tes ama iam & Halpe n, 1988). In ac , he ascula endo helial unc ion has been shown o be impai ed in blood essels o hype ensi e animal models (Sunano e al. 1989), as well as in pa ien s wi h essen ial hype ension (Egashi a e al. 1995; Taddei e al. 1995). The Medi e anean egion has a e y low p e alence o ca dio ascula disease. The li e expec a ion in Medi e a- nean coun ies is cu en ly g ea e han ha in coun ies o no he n Eu ope and in he USA. Howe e , he mechanisms unde lying he ca dio ascula bene i s o Medi e anean- s yle die s and he e ec on endo helial dys unc ion a e no ully unde s ood (Massa o e al. 1999; Tsimikas e al. 1999). The die consumed by hese popula ions ha e a common cha ac e is ic, namely he high p opo ion o oli e oil (OO; *Co esponding au ho : D Ruiz-Gu ie ´ ez, ax +34 954 616790, email [email p o ec ed] Abb e ia ions: HOSO, high-oleic-acid sun lowe oil; MUFA, monounsa u a ed a y acids; OO, oli e oil. B i ish Jou nal o Nu i ion (2001), 86, 349–357 DOI: 10.1079/BJN2001397 qThe Au ho s 2001 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess ich in monounsa u a ed a y acids (MUFA), mainly oleic acid), whe eas he no he n Eu opean die includes a la ge p opo ion o sa u a ed animal a s (Keys, 1995). Despi e OO being he mos common highly monounsa- u a ed oil used o nu i ional s udies o e alua e he e ec s o MUFA-en iched die s (mainly oleic acid), o he sou ces o die a y MUFA (such as high-oleic-acid sun lowe oil; HOSO) a e now becoming a ailable. Howe e , he e is i ually no in o ma ion conce ning hei e ec s on endo helial unc ion. I is o in e es ha he bene icial e ec s o die a y OO a e no ound o be he same as o he HOSO- ich die , despi e he ac ha bo h ege able oils p o ide a simila concen a ion o MUFA (Ruiz-Gu ie ´ ez e al. 1996, 1999). The e o e, he weal h o mino componen s (including a ious polyphenols, such as oleu opein) con ained in he unsaponi icable ac ion o i gin OO (Mon eodo o e al. 1992) and no p esen in HOSO, appea s o be he key o he bene icial e ec s o i gin OO on ca dio ascula e en s (Visioli & Galli, 1994, 1995). The p esen wo k has been designed o compa e he e ec s o wo die s ich in oleic acid ( om i gin OO and om e ined HOSO) and a con ol die , on he ascula eac i i y o Wis a –Kyo o (WKY) a s and SHR a s, in o de o p o ide he i s e idence o he e ec s and mechanisms o ac ion o bo h oils on endo helial dys unc ion p esen in blood essels o he hype ensi e animal model. In addi ion, we e alua ed he e ec s o bo h MUFA die s on lipid and a y acid composi ion phospholipids o he ascula wall, in o de o documen u he he bene icial e ec s on endo helial unc ion. Me hods Animals and composi ion o expe imen al die s Male SHR and WKY a s we e ob ained a he age o 4 weeks om Ha lan In e auna Ibe ´ ica, S.A. (Ba celona, Spain). They we e housed a 24 ^28C wi h 60 ^20 % ela i e humidi y, wi h a 12 h ligh –da k cycle. All expe imen s we e pe o med on 10–12-week-old a s. Bo h SHR and WKY a s we e di ided in o h ee g oups o six animals each. Each g oup was ed on one o he ollowing die s o 6 consecu i e weeks: a semisyn he ic die (basal die ) wi h 20 g unspeci ied lipid/kg, pu chased om Panlab SRL (Ba celona, Spain) (con ol g oup) o basal die modi ied by supplemen ion wi h 100 g i gin OO (Olea eu opea )/kg (OO g oup) o 100 g HOSO (Helian hus annus )/kg (HOSO g oup). The composi ion o he expe imen al die s is shown in Table 1. To minimise oxida ion, all die s we e p epa ed once pe week and s o ed a 48C unde an a mosphe e o N 2 un il needed. Changes in composi ion we e no de ec ed du ing s o age. The a y acid composi ion o he oils was analysed and is shown in Table 2. The non- a y acid componen s o he oils a e p esen ed in Table 3. Pha macological measu emen s The expe imen s we e pe o med on he ho acic ao a o male SHR and no mo ensi e WKY a s. A e 6 weeks o Table 1. Composi ion o expe imen al die s (g/kg) Ing edien s Con ol OO HOSO Casein 209 203 203 Suc ose 450 374 374 Co ns a ch 202 180 180 Lipid 20 20 20 OO – 100 – HOSO – – 100 Cellulose powde 52 56 56 Mine al mix* 57 57 57 Vi amin mix† 10 10 10 To al ene gy (MJ) 15:217 :117 :1 P o ein (% ene gy) 23:119 :719 :7 Lipid (% ene gy) 4:926 :326 :3 Ca bohyd a e (% ene gy) 71:953 :953 :9 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil. * Mine al mix (g/kg): NaCl 139:3, K 2 HPO 4 386:1, CaCO 3 381:4, Mg SO 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 (/kg): e inol 5:9 mg, cholecalci e ol 0:15 mg, hiamin 20 mg, ibo la in 15 mg, nico inic acid 70 mg, py idoxine 10 mg, inosi ol 150 mg, cyanocobalamin 50 mg, a- ocophe ol 170 mg, phylloquinone 40 mg, cho- line 1:36 g, olic acid 5 mg, p-aminobenzoic acid 50 mg, bio in 0:3 mg. Table 2. Fa y acid composi ion (g/kg o al a ) o he expe imen al oils Fa y acid Con ol OO HOSO 16:0 132 118 43 16:1 n-9 12 9 1 17:0 – 4 1 18:0 31 28 47 18:1 n-9 368 792 802 18:2 n-6 417 35 94 18:3 n-3 41 6 1 20:0 – 3 4 20:1 n-9) – 2 2 24:0 – 4 4 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil. Table 3. Composi ion o non- a y acid componen s o he expe imen al oils Non- a y acid OO HOSO To al unsaponi iable ac ion (g/kg) 15 10 To al s e ols (mg/kg) 1357 1534 Choles e ol (g/kg) 1:70 :1 B asicas e ol (g/kg) – 1:2 Campes e ol (g/kg) 3:8 102:5 S igmas e ol (g/kg) 8:1 115:9 b-Si os e ol (g/kg) 939:9 593:5 D-5-A enas e ol (g/kg) – 28:8 D-7-S igmas e ol (g/kg) 2:2 118:4 D-7-A enas e ol (g/kg) 1:532 :7 Squalene (mg/kg) 3000 90 Tocophe ols (mg/kg) 47 484 a-Tocophe ol (mg/kg) 34 484 g-Tocophe ol (mg/kg) 13 – F ac ion o polyphenols (mg/kg) 300 – To al polyphenols (mg/kg) 322 – O hodiphenol (mg/kg) 22 – OO, oli e oil; HOSO, high-oleic-acid sun lowe oil. M. D. He e a e al.350 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess ecei ing he espec i e die s (10-week-old animals), he a s we e killed by a blow on he head and he descending ho acic ao a was apidly dissec ed and placed in a modi ied K ebs–Henselei solu ion, con aining (mm): NaCl 118, KCl 4:75, NaHCO 3 25, MgSO 4 1:2, CaCl 2 1:8, KH 2 PO 4 1:2, glucose 11. A e excess a and connec i e issue we e emo ed, he ao a was cu in o 2–3 mm ings. Ao ic ings we e moun ed unde he basal ension o 1 gin 20 ml o gan ba hs con aining modi ied k ebs–Henselei solu ion and a ached o a o ce-displacemen ansduce (Pioden UF-1, Ha a d, Ken , UK) o isome ic con ac ion o ce as p e iously desc ibed (He e a e al. 1996). The signal was eco ded by a Powe lab w da a acquisi ion sys em (AD Ins umen s, Cas le Hill, Aus alia). The issue ba h was main ained a 378C and bubbled wi h a O 2 –CO 2 (95:5, / ). All expe imen s we e pe o med on ao ic ings wi h endo helium. The p esence o endo helium was con i med by he occu ence o elaxa ions induced by ace ylcholine (10 25 m) in ings con ac ed wi h no ad enaline (10 25 m). Each p epa a ion was allowed o equilib a e o a leas 90 min p io o ini ia ion o expe imen al p ocedu es and du ing his pe iod he incuba ion media we e changed e e y 20 min. A e equilib a ions, he ollowing expe imen s we e pe o med: (1) ao ic ings we e con ac ed by single submaximal concen a ions o 10 26 mphenyleph ine. When he con ac ile esponse o he agonis was s able, ace ylcholine was added (10 25 m) in o de o analyse he endo helium-dependen asodila ing esponse (Al a ez de So omayo e al. 1999). All esul s we e exp essed as a pe cen age o he maximal con ac ion o phenyleph ine induced esponses; (2) dose– esponse cu es o phenyl- eph ine-induced con ac ion in endo helium-in ac p ep- a a ions. The cumula i e concen a ion– esponse cu e was ob ained wi h phenyleph ine (10 28 –10 25 m) a 3 min in e als. Di e en ao ic ings we e used in each expe imen . Ex ac ion and sepa a ion o lipids The in ima we e ca e ully sc aped wi h glass slides. Ex ac ion o o al lipids om in ima sc apings 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 0:001 ml bu yla ed hyd oxy oluene/l using an Ul aTu ax homogen- ise (model Type TP-18-1, Ul a Tu ax, Vineland, NJ, USA). To al lipids we e quan i ied by means o he Ia oscan TLC/FID echnique (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 Ia on Labo a o ies Inc., Tokyo, Japan ha ing a p ecoa ed hin ac i e laye o silica. Ch oma ods S we e ou inely s o ed in 4:5m-H 2 SO 4 . P io o use, he ods we e washed wi h dis illed wa e , d ied a 1108C o 15 min and ac i a ed by passing h ough he lame o he Ia oscan de ec o . To al lipids o phospholipids (3 ml) we e spo ed on each od using a 10 ml 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.). The phospholipids we e esol ed in wo s eps, s a ing wi h an ini ial de elopmen o ods in chlo o o m–me hanol–ace ic acid–wa e , (67:28:2:3, by ol.), d ying a 708C o 10 min, hen a second de elopmen 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 175 ml/min, ai low 1850 ml/ min, scanning speed 47 mm/s, cha speed 42 mm/min. An Ia oco de TC-11 in eg a o , New Technology Sys em, Rome, I aly, was used o eco ding and a ea in eg a ion (Ruiz-Gu ie ´ ez e al. 1995). Fa y acid analysis Lipids om he samples we e sepa a ed by TLC on pla es o silica gel 60 (Kieselgel 60 F254; Me ck, Ba celona, Spain) using a sol en sys em o hexane–die hyl e he –ace ic acid (80:20:1, by ol.). A e de elopmen o he pla e, he sol en was allowed o e apo a e. This sys em sepa a es phospholipids, choles e ol, iacylglyce ol and choles e yl es e s in inc easing o de o R (dis ance o spo cen e om s a poin /dis ance o sol en on om s a poin ). Indi idual lipid zones we e sc aped om TLC pla es and elu ed om he silica gel wi h chlo o o m–me hanol (2:1, / ) acco ding o he indi idual lipids. Fa y acid con en s we e de e mined by GC, as p e iously desc ibed (Ruiz- Gu ie ´ ez e al. 1992; Mu iana e al. 1995). Fa y acid me hyl es e analysis Phospholipids we e saponi ied by hea ing o 25 min wi h 5mlo 0 :2m-sodium me hyla e a 1208C and hea ed again a 808C o 25 min wi h 60 g H 2 SO/l anhyd ous me hanol. 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 (Hewle -Packa d Co., A ondale, PA, USA) equipped wi h a lame ioniza ion de ec o and using an Omegawax 320 used-silica capilla y column ð30 m 0:32 mm i.d., 0:25 mm ilm), ob ained om Supelco (Bella on e, PA, USA). The ini ial column empe a u e was 2008C, which was held o 10 min, hen p og ammed o change om 200 o 2308Ca 28C/min. Peak a eas we e calcula ed by a Hewle -Packa d 3390A eco ding in eg a o . Indi idual a y acid me hyl es e s we e iden i ied on iso he mal uns by compa ison o hei e en ion imes agains hose o s anda ds. Fa y acid me hyl es e s o which no s anda d was a ailable we e quan i ied using calib a ion ables o ela i e esponse a ios cons uc ed acco ding o C numbe using GC–mass spec ome y, pe o med on a Hewle -Packa d 5890 GC in e aced di ec ly o an AEJ MS30/70 VG mass spec ome e (VG Analy ical, Manches e , UK), using he elec on impac ioniza ion mode. The ion sou ce empe a- u e was main ained a 2008C, he mul iplie ol age was 4:0 kV, he emision cu en was 100 mA and he elec on ene gy was 70 eV. The da a we e p ocessed wi h a VG 11/ 250 da a sys em (VG Analy ical). Non- a y acid componen s Fo he ex ac ion o he unsaponi iable ma e , 20 g oils we e saponi ied o 30 min wi h 75 ml KOH (100 g/l e hanol). The solu ion was pu in a 5000 ml decan ing unnel, 100 ml dis illed wa e was added, and he mix u e was ex ac ed wi h 100 ml aliquo s o hexane. The hexane Die a y oils and ascula eac i i y 351 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess solu ion was e apo a ed o d yness in a o a y e apo a o a 308C unde 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 oils by column c oma og aphy on silica gel and analysed by capilla y GLC (Sulpioce & Fe ezou, 1984). The polyphenol ac ion was de e mined by capilla y GLC (A ce e al. 1998). D ugs Lipid and phospholipid s anda ds we e ob ained om Sigma Chemical Co, (S Louis, MO, USA). Fa y acid me hyl es e /s anda ds we e ob ained om La odan Fine Chemicals (Malmo, Sweden). The in e nal s anda d solu ion was p epa ed by dissol ing 200 mg icosanoic acid me hyl es e (23:0) in 100 ml hexane. The calib a ion solu ions we e p epa ed by dissol ing known amoun s o a y acid me hyl es e s anda ds in hexane con aining bu yla ed hyd oxy- oluene (2,6-di- e -bu yl-p-c esol) ob ained om Sigma (Poole, Do se , UK). Fo pha macological expe imen s, he ollowing d ugs we e used: no ad enaline bi a a e, ace ylcholine chlo ide and phenyleph ine chlo ide, all ob ained om Sigma Chemical Co. The d ugs we e dissol ed in dis illed wa e . S a is ical analysis Resul s a e exp essed as mean alues wi h hei s anda d e o s (pha macological expe imen s) o mean alues and s anda d de ia ions (lipid and a y acid composi ion) o p epa a ions ob ained om a leas six animals pe g oup. One-way ANOVA ollowed by Bon e oni es s we e used o s a is ical analysis. P alues ,0:05 we e conside ed as showing a signi ican di e ence. The analyses we e pe o med wi h he G aphPad P ism w s a is ical package ( e sion 2.01, 1996; G aphpad So wa e Inc., San Diego, USA). Resul s E ec s o i gin oli e oil and high-oleic-acid sun lowe oil on ascula eac i i y in Wis a –Kyo o and spon aneously hype ensi e a s Du ing con ac ions caused by phenyleph ine (10 26 m) o 15 min, addi ion o ace ylcholine (10 25 m) e oked endo- helium-dependen elaxa ion in ao ic ing o WKY and SHR a s (Fig. 1). The maximum phasic con ac ion in esponse o phenyleph ine (10 26 m) was g ea e ðP,0:01Þin ao ic ings om SHR (1:18 (sem 0:09) g) han om WKY (0:88 (sem 0:14) g) a s. This con ac ion was signi ican ly dec eased in ao a om SHR a s ed wi h OO (0:81 (sem 0:05), g,P,0:01Þbu no in hose om WKY a s (0:89 Fig. 1. E alua ion o maximum ascula esponses o phenyleph ine (Phe)-induced con ac ion (g) (a, b) and maximum ascula esponses o ace ylcholine (ACh)- induced elaxa ion (%) (c, d) in a isola ed ao a p epa a ions ob ained om con ol a s, and om hose ecei ing an oli e oil (OO)- o a high-oleic-acid sun lowe (HOSO)- en iched die . SHR, Spon aneously hype ensi e a s; WKY, Wis a –Kyo o a s. Fo de ails o die s and p ocedu es, see Tables 1–3 and p. 350. Values a e means o six a s pe g oup wi h hei s anda d e o s ep esen ed by e ical ba s. Mean alues we e signi ican ly di e en om hose o he con ol g oup: * P,0:05;** P,0:01; *** P,0:001:Mean alue was signi ican ly di e en om ha o he HOSO die g oup; †P,0:01: M. D. He e a e al.352 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess (sem 0:10) g, NS.). The ea men wi h HOSO did no al e he phenyleph ine-induced con ac ions (0:96 (sem 0:06) g o SHR and 0:82 (sem 0:06) g o WKY a s) (Fig. 1). The elaxan esponses o ace ylcholine (10 25 m) we e signi ican ly enhanced by ea men wi h OO in bo h SHR (30:03 (sem 0:70), . 18:47 (sem 0:28) %, P,0:001Þand WKY (30:97 (sem 2:40), . 23:23 (sem 1:9) %, P,0:05Þ ings and he inc ease o elaxan esponses we e signi ican ly weake in he ings om SHR a s (23:47 (sem 0:36) %, P,0:01Þ ea ed wi h HOSO ðP,0:01 . OO ea men ) (Fig. 1). Dose- esponse cu es o phenyleph ine-induced con- ac ions (10 28 –10 25 m) in endo helium-in ac p epa a ions om SHR and WKY a isola ed ao a we e used as an indica o o Ca 2+ elease h ough he inosi ol 1,4,5- iphos- pha e ecep o pa hway. Phenyleph ine e oked simila concen a ion–con ac ion cu es in he SHR con ol ings and in he SHR ings om a s ea ed wi h HOSO, whe eas ea men wi h OO p oduced a p onounced dec ease o he con ac ion o ao ic ing in esponse o phenyleph ine a ec ing he a enua ion o he maximal esponse (Fig. 2). Mo eo e , he ime cou se o he con ac ion o his las p epa a ion was slowe , needing mo e ime o become s able (Fig. 2) Die s In o de o documen he in luence o he wo expe imen al oils on dis inc beha iou o he con ac ile and elaxan esponses o ho acic ao as, we examined he composi ion o wo oils exhaus i ely. The a y acid composi ion o OO and HOSO was cha ac e ized by a high con en o MUFA, mainly oleic acid (abou 800 g/kg o al a s) (Table 2). The majo di e ences we e g ea e concen a ions o palmi ic acid (16:0) in OO and linoleic acid (18:2) in HOSO. In ela ion o composi ion in non- a y acid componen s (mino componen s), signi i- can di e ences ðP,0:001Þwe e no ed o ocophe ols, squalene and polyphenols be ween he expe imen al oils (Table 3). The concen a ion o ocophe ols was g ea e (484 mg/kg) in HOSO han in OO (47 mg/kg) unlike squalene con en , which was 3000 mg/kg OO and 90 mg/kg HOSO. Howe e , polyphenols we e only ound in OO. E ec s o i gin oli e oil and high-oleic-acid sun lowe oil on he lipid composi ion o in ima ao a om Wis a –Kyo o and spon aneously hype ensi e a s Table 4 shows he e ec s o expe imen al die s on lipid composi ion o in ima ao a om WKY and SHR a s. The con en o memb ane phospholipid showed educed in animal model o hype ension (SHR) (159:1(sd 11:3) g/kg, P,0:001Þcompa ed wi h non-hype ensi e animals (WKY) (195:6(sd 6:0) g/kg). Phospholipid concen a ion was inc eased signi ican ly in SHR a s ed on he OO die (186:7(sd 3:2) g/kg, P,0:01Þapp oaching he alues o con ol WKY a s. Howe e , he HOSO die led o a signi ican dec ease in bo h g oups o animals ðP,0:001 . con ol g oup). The o al choles e ol le el emained Fig. 2. Dose– esponse cu es o phenyleph ine (Phe)-induced con ac ions (g) in a isola ed ao a p epa a ions ob ained om con ol a s (.), and om hose ecei ing an oli e oil (OO)- (W) o a high-oliec-acid sun lowe (HOSO)-en iched die (O) (a, b) and mean ime cou se (min) o Phe-induced con ac ions (c). No e he di e ence in he ime cou se in a isola ed ao a p epa a ions om con ol a s and hose ecei ing he OO die ((c), . . W). SHR, spon aneously hype ensi e a s; WKY, Wis a –Kyo o a s. Fo de ails o die s and p ocedu es, see Tables 1–3 and p. 350. Values a e means o six expe imen s wi h hei s anda d e o s ep esen ed by e ical ba s. Mean alue was sig- ni ican ly di e en om ha o he con ol g oup: * P,0:01: Die a y oils and ascula eac i i y 353 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess p ac ically unal e ed in HOSO- and OO- ed g oups, excep o SHR ea ed wi h OO, o which he alue was signi ican ly less han he con ol alue (11:2(sd 3:6) .18 :2 (sd 3:9) g/kg, P,0:05Þand iacylglyce ol le els inc eased wi h HOSO ea men (823:0(sd 19:8), P,0:05 o WKY and 865:6(sd 3:3) g/kg, P,0:01 o SHR a s). The HOSO- ed g oup showed a signi ican dec ease in he concen a ion o memb ane es e i ied choles e ol in SHR a s (1:1(sd 0:1) . 5:8(sd 0:3) g/kg, P,0:01Þ: To de e mine he phospholipid composi ion o in imal ao a, he majo phospholipid species we e quan i ied and he esul s o he analyses a e gi en in Table 4. In his able, we can obse e how he majo phospholipid species is sphingomyelin (SHR alues g ea e han hose om WKY, P,0:01Þ; ollowed by phospha idyl choline, phospha idly e hanolamine (WKY alues g ea e han hose om SHR, P,0:001Þ;phospha idyl se ine and lisophospha idyl e hanolamine ( hese la e wo had e y low alues). E ec s o i gin oli e oil and high-oleic-acid sun lowe oil on he phospholipid a y acid composi ion o in ima ao a om Wis a –Kyo o and spon aneously hype ensi e a s Table 5 shows he p opo ions o a y acids (g/100 g o al a y acids) in phospholipids o in ima ao a in a s ed on he di e en die s. In he p esen s udy, he mos signi ican Table 5. Fa y acid composi ion (g/100 g o al a y acids) o phospholipids o in ima ao a om no mo ensi e (Wis a –Kyo o; WKY) and spon- aneously hype ensi e (SHR) a s ed expe imen al die s‡ (Mean alues and s anda d de ia ions o six a s pe g oup) Die … Con ol OO HOSO WKY SHR WKY SHR WKY SHR Fa y acid Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 16:0 12:99 2:65 18:03††† 0:09 13:92 0:86 15:80 0:86 11:62 1:49 16:15††† 1:49 16:1 n-9 2:81 0:77 2:39 0:02 3:28 0:19 1:47**††† 0:19 1:63*** 0:17 1:10*** 0:17 18:0 22:76 3:76 22:05 1:32 21:54 1:50 17:81 1:50 22:88 2:69 22:69 2:69 18:1 n-9 17:95 2:33 17:53 1:66 27:59*** 0:42 32:58***††† 0:42 22:87*** 0:07 22:34*** 0:07 18:1 n-7 5:85 1:22 3:42††† 0:01 3:89*** 0:01 3:82 0:42 3:93*** 0:00 2:72†† 0:08 18:2 n-6 13:91 1:35 13:45 0:01 8:16*** 0:10 9:05*** 0:10 13:96 0:03 10:24****††† 0:03 20:0 0:38 0:01 0:1††† 0:01 0:38 0:03 0:14††† 0:03 0:39 0:02 0:85***††† 0:04 20:3 n-6 2:07 0:63 1:07† 0:63 1:03* 0:01 2:00* 0:63 0:38*** 0:44 0:32 0:04 20:4 n-6 18:33 2:86 17:39 1:09 18:49 1:60 13:68***††† 0:01 20:12 0:05 19:22* 0:05 22:5 n-3 1:04 0:27 0:69††† 0:02 0:68*** 0:01 0:60 :02 1:02 0:01 1:61***††† 0:01 22:6 n-3 1:44 0:33 1:70 0:58 1:10 0:33 0:78 0:33 1:20 0:78 1:45 0:78 SFA 36:13 6:42 40:18 1:42 35:84 2:39 33:75** 2:39 34:89 4:239 :69 4:22 MUFA 26:61 4:32 23:34 1:69 34:76*** 0:62 37:87*** 1:03 28:43 0:24 26:16 0:32 PUFA 36:79 5:44 34:30 2:33 29:46*** 2:05 26:11** 0:46 36:68 1:31 32:84 0:91 OO, oli e oil; HOSO, high-oleic-acid sun lowe 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. Mean alues we e signi ican ly di e en om hose o he con ol g oup * P,0:05;** P,0:01;*** P,0:001: Mean alues we e signi ican ly di e en om hose o he WKY g oup: † P,0:05;†† P,0:01;††† P,0:001: ‡ Fo de ails o die s and p ocedu es, see Tables 1–3 and p. 350. Table 4. Lipid composi ion and dis ibu ion (% w/w) o he majo phospholipid o in ima ao a om no mo ensi e (Wis a –Kyo o; WKY) and spon aneously hype ensi e (SHR) a s ed expe imen al die s‡ (Mean alues and s anda d de ia ions o six a s pe g oup) Die … Con ol OO HOSO WKY SHR WKY SHR WKY SHR Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD PL 19:56 0:60 15:91††† 1:13 21:40 1:59 18:67**†† 0:32 16:56*** 2:13 12:09***††† 0:59 PE 24:84 1:82 14:7††† 1:22 20:41* 3:511 :2††† 3:521 :32 0:311 :2††† 0:3 PS 1:90 :21 1:50 :21 2:03 0:71 :50 :92 :23 0:32 1:60 :3 PC 27:56 :227 :56 :227 :41 1:327 :41 1:328 :02 4:427 :02 4:4 SM 45:23 6:455 :23†† 5:449 :56 3:859 :56†† 3:848 :04 2:7 60††† 0:7 LysoPE 0:53 0:20 :63 0:20 :55 0:30 :32 0:30 :49 0:30 :42 0:3 CHOL 1:28 0:32 1:82 0:39 1:80 0:55 1:12* 0:36 1:02 0:28 1:27 0:29 TG 79:00 1:96 81:64 3:23 76:74 0:00 79:72 0:22 82:30* 1:98 86:56**†† 0:33 CE 0:16 0:09 0:58†† 0:03 0:16 0:06 0:52† 0:12 0:50* 0:41 0:11** 0:01 OO, oli e oil; HOSO, high-oleic-acid sun lowe oil; PL, phospholipid; PE, phospha idyl e hanolamine; PS, phospha idyl se ine; PC, phospha idyl choline; SM, sphingomyelin; LysoPE, lysophospha idyl e hanolamine; CHOL, ee choles e ol; TG, iacylglyce ol; CE, choles e yl es e . Mean alues we e signi ican ly di e en om hose o he con ol g oup: * P,0:05;** P,0:01;*** P,0:001: Mean alues we e signi ican ly di e en om hose o he WKY g oup † P,0:05;†† P,0:01;††† P,0:001: ‡ Fo de ails o die s and p ocedu es, see Tables 1–3 and p. 350. M. D. He e a e al.354 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess e ec s in he hype ensi e s a e we e on 16:0, 18:1 n-9, 20:0 and 22:5 n-3 con en . A educ ion in palmi ic acid and an inc ease in oleic acid, eicosanoic acid and eicosapen anoic acid in hype ensi e animals in ela ion o con ol animals (WKY). In he hype ensi e and no mo ensi e g oups, bo h die s inc eased he con en in he a ao a o oleic acid (18:1 n-9), whe eas he ise wi h he OO die was mo e signi ican . A signi ican dec ease in palmi oleic (16:1) and linoleic acid (18:2) was also e iden in SHR a s compa ed wi h baseline. On he o he hand, 20:4 n-6 was educed by 25 % only in hype ensi e animals ed he OO die . This educ ion was signi ican wi h espec o bo h WKY con ol and WKY ed OO. The di e ences be ween he wo MUFA die s in he s udy we e a dec ease in o al sa u a ed a y acids and polyunsa u a ed a y acids in SHR g oup ed on he OO die . Howe e , he o al MUFA inc eased in bo h WKY and SHR a s ea ed wi h his die . Discussion Two clea ends eme ge om he p esen s udy. Fi s , i is appa en ha he oil OO die a enua es phenyleph ine- induced con ac ions, and educed ascula eac i i y, which would con ibu e o dec easing ascula one. In addi ion, an inc ease in phospholipid con en and changes in a y acid composi ion o ao a could be he eason o he imp o emen o endo helial unc ion. The ascula smoo h muscle in gene ic hype ension is cha ac e ised by modi ica ions in he signal- ansduc ion sys em (Tho in-T escases e al. 1994) and his is accoun ed o by medial hype ophy (Sudhi & Angus, 1990). Phasic con ac ions o a ao a induced by phenyleph ine we e used as an indica o o in acellula Ca 2+ elease h ough he inosi ol 1,4,5- iphospha e ecep o pa hway. NO selec- i ely inhibi s in acellula Ca 2+ elease s imula ed by inosi ol 1,4,5- iphospha e in ascula smoo h muscle (Ji e al. 1998). NO has been ex ensi ely shown o be a po en asodila o p oduced by ascula endo helial cells and NO elease is igge ed by pha macologically asoac i e subs ances such as ace ylcholine (Moncada e al. 1991; Moncada & Higgs, 1993). The in acellula NO in smoo h muscle cells in ol es he gene a ion o cGMP (Rapopo & Mu ad, 1983; Moncada e al. 1992), a dec ease in Ca mobiliza ion (Two & an B eemen, 1988; Komo i & Bol on, 1989) and inhibi ion o phosphoinosi ol b eakdown (Hi a a e al. 1990; Ru h e al. 1993). NO plays an ac i e ole in he balance be ween he con ac ion and dila ion o ascula smoo h muscle (Pe ´ ez-Gue e o e al. 2000). In he p esen s udy we desc ibe he impo an ole o endo helium in con olling he phenyleph ine-induced con ac ion, as he g ea e elaxa ion esponse o ace yl- choline is in ela ion o he weake esponse o phenyleph ine, in bo h p epa a ions om SHR ed wi h he OO die , indica ing an imp o emen o endo helial unc ion o his hype ensi e model. I is also clea ha he imp o emen o he endo helium plays an impo an ole in his educ ion, inhibi ing in acellula Ca 2+ elease by inosi ol 1,4,5- iphospha e pa hway. The ime cou se o he con ac ion by phenyleph ine was also a ec ed, especially in he p epa a ion om SHR ea ed wi h OO, he con ac ion becoming slowe han in SHR con ol, as well as ha ing a smalle esponse. I is known ha he deg ee o unsa u a ion o die a y oil in luences he a y acid composi ion o cells, undamen ally in he phospholipids o he cell memb ane (Ruiz-Gu ie ´ ez e al. 1990, 1998), and OO eeding leads o changes in lipid me abolism o he ascula compa men ha could be a ou able in he p e en ion o h ombosis and a he o- scle osis (Na a o e al. 1992). We wan ed o e i y ha he same changes occu ed wi h ega d o endo helial unc ion. We can p o e, a e analysis o lipid composi ion, ha he con en o memb ane phospholipid, which was ound o be educed signi ican ly ðP,0:001Þby abou 20 % in he animal model o hype ension (SHR) compa ed wi h non- hype ensi e animals (WKY), was inc eased signi ican ly ðP,0:01Þin SHR a s ed on he OO die app oaching he alues o hose o WKY a s. Howe e , he HOSO die led o a signi ican dec ease ðP,0:001Þ:I is well known ha phospholipids, in many cases, a e he main componen s o he cell memb anes in a li ing body. De ec s in phospholipid syn hesis, besides a ec ing he absolu e amoun s o phospholipids pe cell, lead o changes in he memb ane s uc u e and consequen ly he unc ion. Excep o a ew diseases, he in o ma ion a ailable is e y scan y. In ela ion o he ca dio ascula sys em, phospholipid changes ha e been obse ed in blood plasma o se um o pa ien s wi h hea disease (Ce c, 1993). The plasma le els o o al phospholipids a e dec eased compa ed wi h hose o no mal subjec s. Simila ly, a dec ease in phospholipids con en was seen in he p esen s udy in hype ensi e animals. Analysis o he a y acid composi ion in a ao a showed a signi ican inc ease ðP,0:001Þo 18:1 in ao a lipids ob ained om OO- and HOSO- ed a s and lowe sa u a ed a y acid le els in SHR ed wi h OO. 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 sup- plemen ed wi h oleic acid, an inc ease in i s con en is ound, accompained by a dec ease in he sa u a ed acid con en (Spec o & Yo ek, 1985). Con e sely, a lowe concen a ion o a achidonic (20:4) was de ec ed in he same g oup o a s. I is appa en ha he OO die supp essed he o ma ion o a achidonic acid in ao ic smoo h muscle phospholipids om SHR a s. In addi ion, phospholipids can ha e a ole as diagnos ic ma ke s. In gene al, such diagnos ic p ocedu es can be used o de ec ing changes in pa o he phospholipids, usually he a y-acid chain (Badley e al. 1993). To be exac , when sa u a ed and unsa u a ed a y acids o phospholipids ha e been examined in ela ion o hea disease (angina, hea a ack), a achidonic acid is signi ican ly aised, implying impo an links wi h p os aglandin ac i i y and me abolism (Skullado i e al. 1985). The e o e, he changes caused by he OO die , i.e. he con en o o al memb ane ao a phospholipids (ele a ion) and he con en o a achidonic acid (dec ease), may help o explain he ascula p o ec i e e ec o OO consump ion. The e ec s canno be a ibu ed exclusi ely o he con en o MUFA (mainly oleic acid) in he die , as high-oleic- sun lowe oil was unable o induce a ou able changes. Die a y oils and ascula eac i i y 355 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess Consequen ly, o he componen s o OO, like polyphenols, no p esen in HOSO, may be esponsible o he bene icial e ec s on he ca dio ascula sys em. The e a e e e ences o he p o ec o e ec o polyphenols ( om ed wine) on endo helial dys unc ion in human subjec s (Cue as e al. 2000). These polyphenols p oduce endo helium-dependen elaxa ion as a esul o enhanced NO syn hesis a he han enhanced biological ac i i y o NO o p o ec ion agains b eakdown by O 2 2 (And iambeloson e al. 1997; And ian - si ohaina 1999). We conclude ha HOSO and OO, p o iding a simila concen a ion o MUFA, ha e di e en e ec s on ascula eac i i y o isola ed a ao a and lipid composi ion and a y acid composi ion o phospholipids o in ima ao a. Only he ch onic eeding o he OO die was able o a enua e ascula esponse o a ao a, p obably h ough an imp o emen o endo helial unc ion. Acknowledgemen s We wish o hank Acei es Toledo S.A. and Koipe S.A. o kindly p o iding he oils used in his s udy. We also g a e ully acknowledge he echnical assis ance o M . An onio Ma in Rod iguez. The s udy was suppo ed by g an s (ALI96-0456 and ALI99-0863) om Comision In e minis e ial de Ciencia y Tecnologia (CICYT). Re e ences And iambeloson E, Kleschyo AL, Mulle B, Be e z A, S ocle JC & And ian si ohaina R (1997) Ni ic oxide p oduc ion and endo helium-dependen aso elaxa ion induced by wine poly- phenols in a ao a. B i ish Jou nal o Pha macology 120, 1053–1058. And ian si ohaina R (1999) Regula ion o ascula one by plan polyphenols: ole o ni ic oxide. Gene ics, Physiology and Biophysics 18, Suppl. 1, 3–5. Al a ez de So omayo M, Pe ´ ez-Gue e o C, He e a MD & Ma huenda E (1999) E ec s o ch onic ea men wi h sim as a in on endo helial dys unc ion in spon aneously hype ensi e a s. Jou nal o Hype ension 17, 769–776. A ce L, Rios A & Valca cel M (1998) De e mina ion o an ica cinogenic polyphenols p esen in g een ea using capilla y elec opho esis coupled o a low injec ion sys em. Jou nal o Ch oma og aphy A 827, 113–120. Badley RA, Da is PJ & Tolley DM (1993) Phospholipids in diagnosis. In Phospholipids Handbook, pp. 833–853 [G Ce c, edi o ]. New Yo k, NY: Ma cel Dekke , Inc. Bazan E, Campbell AK & Rapopo RM (1993) E ec s o p o ein kinase C ac i a ion on no epineph ine-induced phospha idyl- inosi ide hyd olysis in in ac a ao a. Eu opean Jou nal o Pha macology 245, 173–177. Boh DF & Webb RC (1988) Vascula smoo h muscle memb ane in hype ension. Annual Re iew o Pha macology and Toxicology 28, 389–409. Ce c G (edi o ) (1993) Phospholipids Handbook, New Yo k, NY: Ma cel Dekke , Inc. Cue as AM, Guasch V, Cas illo O, I iba a V, Mizon C, San Ma in A, S obel P, Pe ez D, Ge main AM & Leigh on F (2000) A high- a die induces and ed wine coun e ac s endo helial dys unc ion in human olun ee s. Lipids 35, 143–148. De Sch ij e R & Ve meulen D (1991) Sepa a ion and quan i ica ion o phospholipids in animal issues by Ia oscan TLC/FID. Lipids 26, 74–75. Egashi a K, Suzuki S, Hi ooka Y, Kai H, Sugimachi M, Imaizumi T & Takeshi a A (1995) Impai ed endo helium-dependen asodila ion o la ge epica dial and esis ance co ona y a e ies in pa ien s wi h essen ial hype ension: di e en esponses o ace ylcholine and subs ance P. Hype ension 25, 201–206. Folch J, Lees M & Sloane-S anley GH (1957) A simple me hod o he isola ion and pu i ica ion o o al lipides om he animal issues. Jou nal o Biological Chemis y 26, 497–509. Ga cia Reguei o JA, Gilbe J & Diaz I (1994) De e mina ion o neu al lipids om subcu aneous a cu ed ham by capilla y gas ch oma og aphy and liquid ch oma og aphy. Jou nal o Ch oma og aphy A 667, 225–233. He e a MD, Za zuelo A, Jime ´nez J, Ma huenda E & Dua e J (1996) E ec s o la onoids on a ao ic smoo h muscle con ac ili y: S uc u e–ac i i y ela ionships. Gene ics and Pha macology 27, 273–277. Hi a a M, Kohse KP, Chang CH, Ikebe TJ & Mu ad F (1990) Mechanism o cyclic GMP inhibi ion o inosi ol phospha e o ma ion in a ao a segmen and cul u ed bo ine ao ic smoo h muscle cells. Jou nal o Biological Chemis y 265, 1268–1273. Ji J, Benishin CG & Pang PK (1998) Ni ic oxide selec i ely inhibi s in acellula Ca ++ elease elici ed by inosi ol isphos- pha e bu no ca eine in a ascula smoo h muscle. Jou nal o Pha macology and Expe imen al The apeu ics 285, 16–21. Keys A (1995) Medi e anean die and public heal h: pe sonal e lec ions. Ame ican Jou nal o Clinical Nu i ion 61, 1321S–1323S. Komo i S & Bol on TB (1989) Ac ions o guanine nucleo ides and cyclic nucleo ides on calcium s o es in single pa ch clamped smoo h muscle cells om abbi po al ein. B i ish Jou nal o Pha macology 97, 973–982. K ame JK, Blais L, Foucha d RC, Melnyk RA & Kalle y KM (1997) A apid me hod o he de e mina ion o i amin E o ms in issues and die by high-pe o mance liquid ch oma og aphy using a no mal-phase diol column. Lipids 32, 323–330. Massa o M, Ca luccio MA & De Ca e ina R (1999) Di ec ascula an ia he ogenic e ec s o oleic acid: a clue o he ca diop o- ec i e e ec s o he Medi e anean die . Ca diologia 44, 507–513. Moncada S & Higgs EA (1993) The l-a ginine–ni ic oxide pa hway. New England Jou nal o Medicine 329, 2002–2012. Moncada S, Palme RMJ & Higgs EA (1991) Ni ic oxide: physiology, pa hophysiology, and pha macology. Pha ma- cology Re iews 43, 109–142. Moncada S, Reed DD, Schulz R & Palme RMJ (1992) De elopmen and mechanism o a speci ic supe sensi i i y o ni o asodila o s a e inhibi ion o ascula ni ic oxide syn hesis in i o. P oceedings o he Na ional Academy o Science, USA 88, 2166–2170. Mon eodo o GF, Se ili M, Baldioli M & Minia i E (1992) Simple and hyd olyzable phenolic compounds in i gin oli e oil. Thei ex ac ion, sepa a ion and quan i a i e and semiquan i a i e e alua ion by HPLC. Jou nal o Ag icul u e and Food Chemis y 40, 1571–1576. Mu iana FJG, Ruiz-Gu ie ´ ez V, Blaya JA & Bolu e J (1995) Phospholipid a y acid composi ion o hepa opanc ea ic b ush- bo de memb ane esicles on he p awn Panaeus japonicus. Biochimie 77, 190–193. Na a o MD, Ho elano P, Pe iago JL & Pi a ML (1992) E ec o die a y oli e and sun lowe oils on he lipid composi ion o he ao a and pla ele s and on blood eicosanoids in a s. A e ioscle osis and Th ombosis 12, 830–835. Osugi S, Shimamu a K & Sunano S (1990) Dec eased modula ion by endo helium o no ad enaline-induced con ac ions in ao a om s oke-p one spon aneously hype ensi e a s. A chi es o In e nal Pha macodynamics 305, 86–99. Pe ´ ez-Gue e o C, Al a ez de So omayo M, He e a MD & M. D. He e a e al.356 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess Ma huenda E (2000) Endo helium modula es con ac ile esponse o sim as a in in a ao a. Zei sch i u ¨ Na u - o schung 55, 121–124. Rapopo RM & Mu ad F (1983) Agonis -induced endo helium- dependen elaxa ion in a ho acic ao a may be media ed h ough cyclic GMP. Ci cula ion Resea ch 62, 961–964. Ruiz-Gu ie ´ ez V, Molina MT & Va ´zquez CM (1990) Compa a- i e e ec s o eeding di e en a s on a y acid composi ion o majo indi idual phospholipids o a hea s. Annals o Nu i ion and Me abolism 34, 350–358. Ruiz-Gu ie ´ ez V, Mon e o E & Villa J (1992) De e mina ion o a y acid and iacylglyce ol composi ion o human adipose issue. Jou nal o Ch oma og aphy B 581, 171–178. Ruiz-Gu ie ´ ez V, Pe ona JS, Pacheco YM, Mu iana FJ & Villa J (1999) Inco po a ion o die a y iacylglyce ols om oli e oil and high-oleic sun lowe oil in o VLDL iacylglyce ols o hype ensi e pa ien s. Eu opean Jou nal o Clinical Nu i ion 53, 687–693. Ruiz-Gu ie ´ ez V, Mu iana FJG, Gue e o A, Ce AM & Villa J (1996) Plasma lipids, e y h ocy e memb ane lipids and blood p essu e o hype ensi e women a e die a y oleic acid om wo di e en sou ces. Jou nal o Hype ension 14, 1483–1490. Ruiz-Gu ie ´ ez V, Mu iana FJG, Maes o R & G aciani E (1995) Oleu opein and lipid and a y acid composi ion o a hea . Nu i ion Resea ch 15, 37–51. Ruiz-Gu ie ´ ez V, Mu iana FJG & Villa J (1998) El acei e de oli a i gen en la p e encio ´n de en e medades ca dio ascula es pe il lipı ´dico en plasma y composicio ´n lipı ´dica de la memb ana de e i oci o humano. G asas y Acei es 49, 9–29. Ru h P, Wang GX, Boekho I, May B, P ei e A, Penne R, Ko h M, B ee M & Ho mann F (1993) T ans ec ed cGMP- dependen p o ein kinase supp esses calcium ansien s by inhibi ion o inosi ol 1,4,5- iphospha e p oduc ion. P oceedings o he Na ional Academy o Science, USA 90, 2623–2627. Shi asaki Y, Kolm P, Nickols GA & Lee TJ-F (1988) Endo helial egula ion o cyclic GMP and ascula esponses in hype en- sion. Jou nal o Pha macology and Expe imen al The apeu ics 245, 53–58. Sil e PJ, Cumiskey WR & Ha is AL (1992) Vascula p o ein kinase C in Wis a –Kyo o and spon aneously hype ensi e a s. Eu opean Jou nal o Pha macology 212, 143–149. Skullado i G, Ha danson T & Sig usson N (1985) A achidonic acid le els in se um phospholipids o pa ien s wi h angina pec o is o a al myoca dial in a c ion. Ac a Medica Scandina- ica 218, 55–58. Spec o AA & Yo ek MA (1985) Memb ane lipid composi ion and cellula unc ion. Jou nal o Lipid Resea ch 26, 1015–1035. Sudhi K & Angus JA (1990) Con ac ile esponses o alpha 1-ad enocep o s imula ion du ing ma u a ion in he ao a o he no mo ensi e and spon aneously hype ensi e a : ela ion o s uc u e. Clinical and Expe imen al Pha macology and Physiology 17, 69–82. Sulpioce JC & Fe ezou J (1984) Squalene isola ion by HPLC and quan i a i e compa ison by HPLC and GLC. Lipids 19, 631–635. Sunano S, Osugi O & Shimamu a K (1989) Blood p essu e and impai men o endo helium-dependen elaxa ion in spon- aneously hype ensi e a s. Expe ien ia 45, 705–708. Taddei S, Vi dis A, Ma ei P, Ghiadoni L, Genna i A, Fasolo CB, Sudano I & Sal e i A (1995) Aging and endo helial unc ion in no mo ensi e subjec s and pa ien s wi h essen ial hype ension. Ci cula ion 91, 1981–1987. Tes ama iam B & Halpe n W (1988) Endo helium-dependen and endo helium-independen asodila ion in esis ance a e ies om hype ensi e a s. Hype ension 11, 440–444. Tho in-T escases N, Hamil on CA, Ja dine E & Reid JL (1994) Signal ansduc ion mechanisms o he asocons ic ion in hype ension. Eu opean Jou nal o Pha macology 268, 199–207. T iggle CR & Lahe I (1985) A e iew o changes in ascula smoo h muscle unc ions in hype ension: isola ed issue e sus in i o s udies. Canadian Jou nal o Physiology and Pha macology 63, 355–365. Tsimikas S, Philis-Tsimikas A, Alexopoulos S, Siga i F, Lee C & Rea en PD (1999) LDL isola ed om G eek subjec s on a ypical die o om Ame ican subjec s on an olea e- supplemen ed die induces less monocy e chemo axis and adhesion when exposed o oxida i e s ess. A e ioscle osis Th ombosis and Vascula Biology 19, 122–130. Tu la MB & Webb RC (1987) Enhanced ascula eac i i y o p o ein kinase C ac i a o s in gene ically hype ensi e a s. Hype ension 9, III150–III154. Tu la MB & Webb RC (1990) Augmen ed phosphoinosi ide me abolism in ao as om gene ically hype ensi e a s. Ame ican Jou nal o Physiology 258, H173–H178. Two CHC & an B eemen C (1988) Cyclic guanosine monophospha e-enhanced seques a ion o Ca 2+ by sa coplas- mic e iculum in ascula smoo h muscle. Ci cula ion Resea ch 62, 961–964. Visioli F & Galli C (1994) Oleu opein p o ec s low densi y lipop o ein om oxida ion. Li e Sciences 55, 1965–1971. Visioli F & Galli C (1995) Na u al an ioxidan s and p e en ion o co ona y hea disease: he po en ial ole o oli e oil and i s mino cons i uen s. Nu i ion and Me abolism in Ca dio ascula Disease 5, 306–314. Die a y oils and ascula eac i i y 357 h ps://doi.o g/10.1079/BJN2001397 Published online by Camb idge Uni e si y P ess