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).
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