olivas - PIFRECV

Transcription

olivas - PIFRECV

7° Curso Internacional del PIFRECV
"Enfermedades Coronaria y Aterosclerosis: Desde los mecanismos moleculares
a la prevención"
Jueves 30 de Septiembre - 1 de Octubre, Talca, Chile 2010
Aceites como Tratamiento Antioxidante
Montserrat Fitó Colomer
Cardiovascular Risk and Nutrition Research Group
CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN)
Institut Municipal d’Investigació Mèdica (IMIM-Hospital del Mar)
Barcelona, Spain
Mapa de las comarcas
del Estudi REGICOR
Hospital Comarcal
Hospital de Referencia
CONSUMO DE ALIMENTOS en el estudio REGICOR
(Mediana poblacional , n=2864)
*
g/dia
500
450
400
350
300
250
200
150
100
50
0
*
*
*
*
* P < 0.001
Verdura
Fruta
Legumbres
Lacteos
Frutos
Secos
Cereales
Pescado
Carne
Ingesta recomendada1
g/día
Raciones
Hombre
Mujer
>300
400
3d
3d
30
2-3 s
~250
5
~240
50g
50g
3d
1s
4-5 d
2-3 s
2-3 s
1. Sociedad Española de Nutrición Comunitaria. Aranceta J et al. Public Health Nutr 2001
La dieta mediterránea es rica en vegetales, fruta,
legumbres, otros alimentos procedentes de
plantas y se caracteriza por el consumo de
aceite de oliva virgen como principal aporte de
energía a través de la grasa
Consumo de grasa en el estudio REGICOR
(Media poblacional)
30
*
*
mL/día
25
20
Hombre
Mujer
15
10
* P < 0.005
5
*
*
0
Aceite de oliva:
Girasol
Oliva
Maiz
g/día
cucharadas soperas
30-50 mL
3-5 día
Soja
Margarina Mantequilla
La FDA (Food and Drug Administration) ha permitido un mensaje en las etiquetas de los aceites de oliva referente a los
beneficios sobre el riesgo cardiovascular del consumo de 2 cucharadas soperas (23g) de aceite de oliva al día,
beneficio atribuido al aporte de AGMI.
mL/día
Consumo de aceite de oliva en el estudio REGICOR
Media
Mediana
Aceite de oliva:
20
18
16
14
12
10
8
6
4
2
0
*
*
Hombre
Mujer
*
* P < 0.005
*
Oliva común Oliva virgen Oliva orujo
g/día
cucharadas soperas
30-50 mL
3-5 día
Oliva com Oliva virgen Oliva orujo
MUFA versus PUFA rich diets and
LDL oxidation
• Oleate-rich LDL is less susceptible against oxidative
modification than linoleate-rich LDL.1
• From 14 studies comparing the resistance of LDL to
oxidation only in 2 of them did MUFA-rich diets not
promote a higher resistance of LDL to oxidation than
PUFA-rich ones.2
1 Reaven
et al, J Clin Invest 1993; 2 Lapointe A et al. J Nutr Biochem 2006.
Polyunsaturated Fatty acids are key substrates
for lipid peroxidation
Propagation chain of lipid peroxidation via the
double bouds of the fatty acid
La FDA (Food and Drug Administration) permitió un mensaje en las
etiquetas de los aceites de oliva referente a los beneficios sobre el
riesgo cardiovascular del consumo de 2 cucharadas soperas (23g) de
aceite de oliva al día, beneficio atribuido al aporte de AGMI.
Si los efectes beneficiosos del aceite de oliva son sólo debidos a los
AGMI, el equilibrio gasto/beneficio no justificaría una recomendación a
la sociedad del consum de aceite de oliva virgen.
Tipos de aceite de oliva
OLIVAS
Separación de las hojas
ACEITES DE OLIVA
EXISTENTES EN EL MERCADO
Lavado
PRENSADO o
CENTRIFUGACION
REFINACIÓN
(>3,3º)
ACEITE DE ORUJO
REFINACIÓN
ACEITE DE OLIVA VIRGEN
Contenido alto en
compuestos fenólicos
(150-400 mg/Kg)
ACEITE DE OLIVA REFINADO
MEZCLA
MEZCLA
ACEITE DE ORUJO DE OLIVA
Contenido bajo en
ACEITE DE OLIVA COMUN
Contenido medio en
(10-70 mg/Kg)
Contenido nulo en
Composición del aceite de oliva
Fracción saponificable
 AG Saturados (8-14%)
 AG monoinsaturados (56-84%)
 AG poliinsaturados (4-20%)
Fracción insaponificable
 Vitamina E, carotenoides
 Squalene, Sterols, triterpenes
 Compuestos fenólicos en el aceite virgen de oliva
(tirosol, hidroxitirosol, oleuropeina, lignanos….)
EUROPEAN OLIVE OIL MEDICAL INFORMATION LIBRARY
Beneficios del consumo de aceite de oliva
sobre la salud
Antioxidantes
 Compuestos fenólicos
Consumo de ácidos grasos y perfil lipídico de riesgo
cardiovascular
Evidencia
científica
Ácidos grasos
 Saturados
 Isómeros trans
Adecuada
Efectos
Colesterol-LDL
Colesterol-LDL y Lp(a),
Colesterol-HDL
 AGPI
Colesterol-LDL
No efecto sobre colesterol-HDL
 AGMI
Colesterol-LDL
No efecto sobre colesterol-HDL
Katan MB, Am J Clin Nutr 1995; 61: 1368S-73S; Mensink RP, Arterioscler Thromb 1992; 12: 911-19; Gardner CD, ATVB 1995; 15: 1917-27
Beneficios del consumo de aceite de oliva
sobre la salud
Antioxidantes
 Compuestos fenólicos
Vissiers et al. J Nutr 2002.
Tyrosol, Hydroxytyrosol and their secoroids derivatives represents around 30% , and other conjugated
forms such as oleuropeine and ligstroside aglycone represents almost half, of the total phenolic content
of a virgin olive oil
Owen RM et al. Food Chem Toxicol 2000;38:647-659
MECANISMOS DE LOS EFECTOS ANTIOXIDANTES
DEL ACEITE DE OLIVA VIRGEN
Especie química definida que tiene en su estructura uno o
más electrones desapareados, lo que lo convierte en
altamente inestable y fugaz, con gran capacidad de formar
otros radicales libres por reacciones químicas en cadena
A concentraciones moderadas y dada su corta existencia, los
radicales libres pueden desempeñar un importante papel como
mediadores en la regulación de varios procesos fisiológicos
{Droge; Physiol Rev 2002}
ROS
(Radicales
libres)
Espacio
subendotelial
Fenoles
Fenoles
ApoB 100
ApoB100 ox.
ApoB 100
PUFA
MUFA
PUFA
MUFA
LDL
carotenoides
Vit. E
Fenoles
LDLox
Vit.E
ox.
carotenoides
PUFA ox.
MUFA
LDLox
Vit.E ox.
carotenoides
Receptores
scavenger, Cd36 y
Cd32
Macrófago/ Célula espumosa.
Covas MI. Int J Clin Pharmacol Res 2000;20:49-54.
Los radicales libres están controlados mediante un amplio
espectro de antioxidantes de origen:
ROS (Radicales
Endógeno: enzimas antioxidantes, glutation, albúmina, lactoferrina,
transferrina, ceruloplasmina, haptoglobulina, hemopexina, glucosa, ácido
úrico, bilirrubina, albúmina, coenzima Q.
libres)
Exógeno (a través de la dieta): vitaminas E y C,
carotenoides, selenio, compuestos fenólicos
Fenoles
Fenoles
Fenoles
ApoB 100
ApoB 100
PUFA ox.
PUFA
PUFA
MUFA
LDL
MUFA
LDLox
LDLox
MUFA
carotenoides
Vit. E
Vit.E
ox.
carotenoides
Vit.E
ox.
carotenoides
Vit.C
Vit. E
Vit.C ox
Receptores
scavenger,
Cd36 y Cd32
DNA
Macrófago/ Célula espumosa.
Codina O et al. Med Clín 1999; 112: 508-515; {Gutteridge; Clin Chem 1995}
Evidencia de los beneficios del consumo de los componentes del
aceite de oliva sobre el riesgo cardiovascular
Evidencia
científica
Adecuada
+
Adecuada
AGMI
(ácido oleico)
Incremento de
sensibilidad a la insulina
Reducción de la
oxidación lipídica
Alguna/adecuada
Compuestos
fenólicos del
aceite de oliva
Reducción de la
presión arterial
Insuficiente
Insuficiente
Mejora del perfil
lipídico
-
Efectos
antitrombótico
sv
Efectos
antiflamatorios
Estudios en
humanos
Modelos en
animales e in vitro
Hipòtesi oxidativa de la Arteriosclerosi:
Oxidació de la LDL
Propiedades aterogénicas de la LDL oxidada
 Inducen la expresión de la MCP-1 y de moléculas de adhesión como la VCAM-1 y la Pselectina en células endoteliales, lo que facilita la unión de monocitos circulantes al
endotelio.
 Promueven la diferenciación de monocitos a macrófagos.
 Provocan apoptosis de las células endoteliales y alteran la producción por las células
endoteliales de óxido nítrico.
 Estimulan la proliferación de células musculares lisas.
.
 Modulan la activación de factores como el factor nuclear kappa B (NF-kB), punto clave
en la activación de múltiples efectos ligados al proceso aterosclerótico.
 Promuevan la agregación plaquetar y la formación de trombos.
ATVB 2000; 20: 1572-79; J Biol Chem 1995; 270: 319-24; J Clin Invest 1996; 97: 1715-22; Clin Chem Lab Med 1999;
37: 777-87
Biodisponibilidad de los CF del aceite de oliva en humanos
Capacidad de unión de los compuestos fenólicos a las LDL humanas
Estudios efectos antioxidantes in vitro del aceite de oliva
Urinary levels of tyrosol and hydroxytyrosol at baseline,
after a single dose (50 mL)
g in 24h-urine
400
300
*
Tyrosol
200
24h
100
0
g in 24h-urine
baseline
1500
Hydroxytyrosol
single dose
*
1000
500
24h
0
baseline
single dose
* P < 0.05 versus baseline (Wilcoxon test)
Miró-Casas E, Covas MI, Fitó M y col. EJCN 2002; 56: 1-5.
Urinary levels of tyrosol and hydroxytyrosol at baseline, after a single dose
(50 mL) and after sustained doses (1 week, 25mL/day) of virgin olive oil
g in 24h-urine
400
300
*
Tyrosol
*
200
24h
100
0
g in 24h-urine
baseline
1500
Hydroxytyrosol
+
1w
single dose sustained doses
*
*
24h
1w
1000
500
0
baseline
single dose sustained doses
* P < 0.05 versus baseline (Wilcoxon test); + P < 0.05 versus single dose intervention
Miró-Casas E, Covas MI, Fitó M y col. EJCN 2002; 56: 1-5.
conc. ng/mL
Concentration vs Time curves for phenolic compounds in the 3 treatments (dose 25 mL)
Olive oils
20
HPC 460 mg/Kg
Tyrosol
MPC, 130 mg/Kg
10
LPC, 10 mg/Kg
0
0
4
8
12
16
20
24
conc. ng/mL
Time (hours)
20
0
conc. ng/mL
Hydroxytyrosol
10
6
5
4
3
2
1
0
0
4
8
12
Time (hours)
16
20
24
3-0-methyl-hydroxytyrosol
0
4
8
12
16
20
24
Time (hours)
Weinbrenner et al. J Nutr 2004
Estudios de biodisponibilidad
El tirosol y hidroxitirosol se absorben en humanos de una
forma dosi-dependiente, sin existir una concentración
umbral para su absorción.
Estudios ef. antiox in vivo del aceite de oliva
a corto plazo (en voluntarios sanos)
METHODS
Interventions: (dose 25 mL)
2 latin squares of 3 x 3 for six treatments were used to randomize olive oil administration
10 days
4 days
10 days
4 days
10 days
4 days
Order 1
wo
LPC
wo
MPC
wo
HPC
Order 2
wo
MPC
wo
HPC
wo
LPC
Order 3
wo
HPC
wo
LPC
wo
LPC
Order 4
wo
LPC
wo
HPC
wo
MPC
Order 5
wo
MPC
wo
LPC
wo
HPC
Order 6
wo
HPC
wo
MPC
wo
LPC
1
2
3
4
5
6
LPC: low phenolic content, 10 ppm; MPC: medium phenolic content, 130 ppm; HPC: high
phenolic content, 460 ppm, olive oils.
7
Week
-4 to –1
Period
Inclusion
Days
Wash-out
Days 1-7
Days 8-10
controlled*
low phenolic**
Day 1 (25ml)
Day 2/7+9+9 ml
Day 3/ 7+9+9 ml
Day 4/ 7+9+9 ml
low phenolic**
idem
idem
idem
Olive oil intervention
Diet
habitual
Controlled diet : with a moderate content of phenolic compounds: VLPC olive oil was given to the
participants for raw and cooking purposes. Vegetables (including pulses): one serve (little dish)/day.
Fruit (or juice): up to 2 per day Wine: up 2 glasses/day Tea/Coffee: up to 3 per day
**
** Low phenolic diet: without fruit (with the exception of banana), vegetables, coffee, tea, wine, and
coke.
LPC olive oil was used for raw and cooking purposes during
washout periods and for cooking purposes during intervention
periods
10
0
-10
-20
10
0
-10
-20
-30
-40
-50
-60
-70
20
Oxidized LDL
20
-30
-40
% Change from Baseline
Linear trend:P = 0.036
14
12
10
8
6
4
2
0
HDL-cholesterol
8-Oxo-deoxyguanosine in mitochondrial DNA
Baseline
LPC
MPC
HPC
30
14
12
10
8
6
4
2
0
-2
Sustained effects of olive oil consunmption (4 days)
Glutathione peroxidase
-2
Malondialdehyde in urine
0
-20
-40
-60
-80
20
8-Oxo-deoxyguanosine in urine
0
-20
-40
-60
-80
Linear trend:P < 0.001
Baseline
LPC
MPC
HPC
Changes in the total phenolic content of the LDL are modulated by olive oil phenolic compounds
at 1h (A), and after 4 days (B) of 25 mL/day consumption of olive oils with high (HPC, 360 mg/Kg), medium
(164 mg/Kg), and low (2.7 mg/Kg) phenolic content
Change (%) from baseline
A
B
P = 0.032 for linear trend
80
a
60
P = 0.042 for linear trend
60
a
40
40
20
20
0
0
-20
-20
-40
LPC
MPC
a P< 0.05
Covas MI et al. Free Rad Biol Med 2006
versus LPC
HPC
-40
LPC
MPC
HPC
Estudios ef antioxidantes in vivo del aceite de oliva a corto plazo
Un consumo regular de aceite de oliva produce una disminución
del estrés oxidativo y un incremento del colesterol HDL y de
sus CF en la LDL, asociado al contenido en CF del aceite de
oliva (dosis depeniente)
Estudios ef. antioxidantes
in vivo del aceite de oliva a largo plazo
(RCT en voluntarios sanos)
DISEÑO DEL ENSAYO CLÍNICO RANDOMIZADO
30 voluntarios, miembros de un centro religioso
Edad: 23-91 años
Criterios de inclusión:
Hombres sanos no fumadores
Criterios de exclusión:
 Toma de fármacos con establecidas propiedades
antioxidantes
 Índice de masa corporal > 28 kg/m2
 Diabetes, enfermedades intestinales

Cualquier enfermedad o condición que pudiera
interferir el cumplimiento del estudio
Ensayo clínico randomizado, cruzado, controlado y a doble ciego
Orden 1
Orden 2
Orden 3
Extracción: 1
wo
Virgen
wo
Común
wo
Refinado
wo
Común
wo
Refinado
wo
Virgen
wo
Refinado
wo
Virgen
wo
Común
2
3
4
5
6
7
Tres intervención: 3 sem., 25 mL/día (aceite de oliva con distinto contenido fenólico, en crudo)
Período de lavado de aceite de oliva (wo): 2 sem., 25 mL/día (aceite de oliva refinado, en crudo)
Olive oil composition
Phenolic
comp.
Alfatocoferol
BetaCarotene
MUFA
PUFA
SAFA
(mg/kg CAE)
(mg/kg)
(mg/kg)
(%)
(%)
(%)
Refined
0
153
0
75%
11%
14%
Common
68
112
0,65
77%
8%
15%
Virgin
149
111
2,1
75%
10%
15%
Marrugat J, Covas MI, Fitó M y col. Sometido para publicación.
Order-adjusted levels of urinary tyrosol during the study
70
ug/L urine
60
Tyrosol
(ug/L urine)
50
40
*
*
*
30
20
Baseline
2 weeks
5 weeks
Post-WO
7 weeks
Post-WO
10 weeks 12 weeks
Post-WO
POST-INTERVENTION
* P < 0.05 versus previous period values; (MLG, Tukey’s correction)
Marrugat J, Covas MI, Fitó M y col. Sometido para publicación.
15 weeks
% of change in oxidative stress biomarkers after each intervention period
Oxidized LDL
30
A
% Change
20
10
0
*‡
-10
-20
-30
P for linear trend = 0.002
-40
-50
Refined
12
Common
Virgin
Resistance of LDL to oxidation
10
% Change
(Lag Time)
*
B
†
8
6
4
2
0
P for linear trend = 0.015
-2
-4
-6
Refined
Common
Virgin
Marrugat J, Covas MI, Fitó M et al. EJN 2004
Total phenolic content in LDL at the begining of the
study and after each intervention period
a
2
1,8
1,6
a
1,4
1,2
ug/mg apoB
1
0,8
0,6
0,4
0,2
0
Basal
Baseline
a P<0.05
a
PostPostCommon
refinado comercial
Refined
indican diferencias significativas (p=0,004)
PostVirgin
virgen
Estudios ef. antioxidantes in vivo del aceite de oliva
Después de un consumo regular de dosis moderadas
de aceite de oliva se observó, en relación directa al
contenido fenólico del aceite de oliva administrado:
1. Un incremento de los niveles urinarios de tirosol
e hidroxitirosol
2. Una protección de la LDL frente a la oxidación
Estudios
ef. antioxidantes
in vivo del aceite de oliva
(RCT en pacientes con enf
coronaria estable)
Estudios ef. antioxidantes
in vivo del aceite de oliva a largo plazo
Oxidative status markers in stable CHD patients after refined and virgin olive oil administration [mean (SD)]
n=40
Post
refined
olive oil
Post virgin
olive oil
Mean
difference
P
for olive oil
effect
P
for time
effect
(14.7 mg/Kg)
(161 mg/Kg)
(95% CI )
58.66
(23.05)
54.01
(19.89)
-4.66
(-7.08; -2.23)
< 0.001
<0.001
0.941
0.705
230
(122 - 495)
246
(140 - 487)
9.18
(-27.79; 9.42)
0.323
0.208
0.762
Lipid peroxides
(µmol/L)
1.47
(1.23)
1.23
(0.72)
-0.24
(-0.40; -0.09)
0.003
0.003
0.563
0.205
Glutathione Peroxidase
(U/L)
7308
(711)
7668
(854)
412
(35.98; 788)
0.033
0.033
0.346
0.258
Total antioxidant status
(mmol/L)
0.92
(0.12)
0.91
(0.11)
-0.01
(-0.03; 0.01)
0.301
0.715
0.172
Tyrosol (µg/L urine) *
23.68
(9.4– 53)
77.5
(75 – 81)
32.67
(3.2 – 62)
0.031
0.031
<0.001
0.459
Hydroxytyrosol
(µg/L urine) *
87.2
(74 – 156)
484
<0.001
< 0.001
<0.001
0.478
(439 – 531)
374
(310 – 438)
O-methylhydroxytyrosol
(µg/L urine) *
10.0
(2.93 – 17)
43.18
(31 – 64)
33.50
(4.7 – 62)
0.024
0.024
<0.001
0.651
Oxidized LDL
(µmol/L)
Antibodies against
oxidized LDL*
Adjusted by age, order of olive oil intervention and baseline vlues. * Median,
Fitó M, et al. Atherosclerosis (2005)
25-75 percentile
P for
interventi
on-period
effect
Changes in SBP after olive oil treatments in stable CHD patients (mean (SD))
Post
refined
olive oil
n=19
Post virgin
olive oil
Mean
difference
(161 mg/Kg)
(95% confidence
interval)
132.6
(5.6)
-2.53
(-3.78; -1.27)
(14.67 mg/Kg)
135.2
(6.58)
Systolic blood
pressure (mmHg)
P for olive
oil
effect
P for
time
effect
P for
interventi
on-period
effect
0.001
0.799
0.340
Changes in SBP after olive oil treatments according to SBP baseline values in stable CHD patients after
olive oil interventions
150
145
140
135
130
125
120
115
*
*+
Baseline values
Baseline
Fitó M, et al. Atherosclerosis (in press)
140 mmHg
Baseline values < 140 mmHg
Post-refined
olive oil
Post-virgin
olive oil
Estudios ef. antioxidantes in vivo del aceite de oliva
(RCT en pacientes con enfermedad coronária estable)
Después del consumo regular de dosis moderadas de aceite de
oliva se observó, en relación directa al contenido fenólico del
aceite de oliva administrado un incremento de los niveles urinarios
de tirosol e hidroxitirosol
Un consumo regular de aceite de oliva con un alto contenido
en CF, produce un mayor incremento del colesterol-HDL y una
mayor protección de la LDL frente a la oxidación que el
consumo de un aceite de oliva con un bajo contenido en CF
Randomized, crossover, controlled studies on the antioxidant effect of sustained olive oil phenolic compounds consumption on in vivo mark
of lípid and DNA oxidation. (Adapted from Fitó et al (2007) Mol Nutr Food Res 51, 1215-1224)
Daily olive
oil dose
Duration
3 weeks
High- vs Low-phenol
69 g
46 healthy
(31 women, 15
men)
2 weeks
without olives
and olive oil
3 weeks
High- vs Low-phenol
70 g raw
25 healthy
(14 women, 11
men)
2 weeks
without olives
and olive oil
3 weeks
Virgin vs Common vs
Refined
22 g (25 mL) raw
30 healthy men
2 weeks with refined
olive oil for
raw and cooking
purposes
4 days
High vs Medium vs
Low phenol
25 mL raw
12 healthy men
10 days: low phenol olive oil
for raw and
cooking; very-low
antioxidant diet
7 weeks
Virgin vs refined
40 mL raw
22 lipemic
(12 men, 10 women)
4 weeks only with refined
olive oil (low-phenol)
3 weeks
Virgin vs Refined
50 mL raw
40 coronary heart
disease men
2 weeks with refined
olive oil for all purposes
8 weeks
High- vs Low-phenol
ad libitum
in substitution of
other fats
10 postmenopausal women
2 weeks (usual diet)
3 weeks
Virgin vs Common vs
Refined
25 mL raw
200 healthy men
2 weeks w/o
olives and OO
•
•
•
•
•
•
•
Idem
Idem
Idem
Idem
Idem
• 8-oxo-dG
• 8-oxo-guanine
• 8-oxo-guanosine
a In vitro test. MDA,
Participants
Washout period
Oxidative markers
Effects
Reference
•
•
•
•
•
•
•
•
•
•
MDA
FRAP
LP
PC
LDL-resistance to oxidation a
MDA
FRAP
LP
PC
LDL resistance to oxidation a
None
Vissiers et al
(2001) (76)
None
Moschandreas
et al (2002) (77)
•
•
•
•
•
•
•
•
•
•
Plasma oxidized LDL
LDL resistance to oxidation a
Antibodies against oxLDL
HDL-C
Plasma oxidized LDL
MDA in urine
8oxodG in urine and lymphoctyes
F2-isoprostanes
GSH-Px
HDL-C
↓ with OO phenolics
None
↑ after VOO
Marrugat et al
(2004) (78)
None
↑ with OO phenolics
Weinbrenner et
al (2004) (79)
• Plasma antioxidant capacity
• F2-isoprostanes
None
Visioli et al
(2005) (80)
• Plasma oxLDL
• LP
• GSH-Px
Fitó et al
(2005) (81)
↓ with OO
Salvini et al
(2006) (82)
None
↑ non-related with OO
phenolics
None
Covas et al
(2006) (57)
↑ non-related with OO
phenolics
None
Machowetz
et al (2006) (30)
• Comet assay for DNA oxidation
Plasma oxLDL
Uninduced dienes
Hydroxy fatty acids
Antibodies against oxLDL
F2-isoprostans
GSH/GSSG
Antioxidant enzymes
malondialdehyde; FRAP, ferric reducing ability of plasma; LP, lipid peroxides; PC, protein carbonyl; 8oxodG, 8-position to
8-oxo-deoxyguanosine; GSH-Px, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; OO, olive oil; ↑, in crease; ↓, decrease
Antioxidant effect of olive oil phenolic compounds in randomized, crossover, controlled studies
in NON healthy individuals
Subjects (n)
( sex)
Intervention
Peripheral
Virgin vs
vascular
disease
for all purposes
Int
period
3 mo
refined
Washout
period
3 mo
Baseline
adjust.
No
Compliance
markers
No
usual diet
Markers
LPO in LDL
Macrophage plasma
oxidized LDL uptake
Effects
Reference
Decrease with
Ramírez-Tortosa
olive oil phenol
(all markers)
et al (1999)
(24, men)
Hyperlipemic Virgin vs
Patients (22)
refined (raw)
(12 men and
10 women)
(40 mL/day)
7w
4w
Yes
No
usual diet
3w
yes
yes
Increase with
antioxidant
phenol content
capacity
F2-isoprostanes
of olive oil
None
Stable CHD
Virgin vs
ox LDL, GSH-Px
Increase with
Patients
refined (raw)
inflammatory markers
olive oil phenol
(40, men)
(50 mL/day)
(IL6, CRP)
CHD, coronary heart disease
2w
Plasma total
(all markers)
Visioli et al
(2005)
Fitó et al
(2007)
Consensus report. Expert Panel
International Conference of Olive Oil and Health. Jaen, Spain October 2004
Olive oil phenolic compounds are bioavailable in humans
Data regarding the benefits of olive oil phenolic compounds in humans from real-life
daily doses of olive oil are controversial and scarce
The protective effects on lipid oxidation in these trials being better displayed in
oxidative stress conditions
Carefully controlled studies in appropriate populations (i.e. oxidative stress
conditions, or with a large sample size (in the case of healthy volunteers), are
required to definitively establish the health properties of olive oil phenolic compounds
in humans.
Expert Panel. Pérez-Jiménez, Coordinator, et al, Eur J Clin Invest 2005;35:421-4
THE EFFECT OF OLIVE OIL CONSUMPTION ON OXIDATIVE DAMAGE IN EUROPEAN
POPULATIONS. The EUROLIVE Study (QRLT-2001-00287)
Covas MI, Poulsen HE, Nyyssönen K, Zunft HFJ, Kiesewetter H, Gaddi A, López-Sabater C, Kaikkonen J,
on behalf of the EUROLIVE Investigators
Objectivo
UKU
JLB
Determinar el efecto de tres aceites de oliva
similares pero con diferente contenido en
RHK
compuestos fenólicos, en el perfil lipídico y el
DIfE
UBER
estado oxidativo/antioxidativo en voluntarios
UBGL
IMIM
UB
sanos
KEPKA
Protocol of management for Olive Oils
Characteristics of the olive oils
Determination of phenolic content in several virgin olive oils
(HPC)Virgin olive oil
Picual from Jaen (Andalucia, Spain, 366 ppm of PC)
Measurement of fatty acid profile and vitamin E
Determination of fatty acid profile and vitamin E of
several refined virgin olive oils from similar cultivar and soil
(VLPC) Refined olive oil
(similar characteristics to the virgin one)
Mixture
(MPC) Common olive oil
The EUROLIVE Study. Methods
Characteristics of the Olive Oils Administered
Type of olive oil
750
430
580
Free acidity (% oleic acid)
0.03
0.08
0.18
Peroxide value (mEq O2/kg)
4.12
5.89
11.28
C14:0
0.01
0.01
0.01
C16:0
10.63
10.50
10.63
C16:1
0.88
0.86
0.88
C17:0
0.05
0.05
0.04
C17:1
0.09
0.09
0.09
C18:0
3.27
3.13
2.84
C18:1
79.08
79.80
80.60
C18:2
4.64
4.21
3.35
C20:0
0.39
0.39
0.35
C18:3
0.58
0.58
0.58
C20:1
0.26
0.25
0.25
C22:0
0.11
0.10
0.10
C24:0
0.01
0.02
0.02
229
228
228
Phenolic compounds (mg/Kg)
2.7
2.7
164
164
366
366
Squalene (mg/g)
3.0
3.2
3.4
1.4
1.5
1.5
Fatty acids (%)
-Tocopherol (ppm)
-sitosterol (mg/g)
Study population: 200 healthy non-smoker males recruited between December 2002
and July 2003 in 6 Centers of 5 European Countries (Denmark, Finland, Germany (2
Centres), Italy, and Spain).
Eligibility criteria: to be healthy on the basis of clinical exanimation and laboratory analyses;
willingness to provide written, informed consent; and to agree to the adherence to the protocol
Exclusion criteria:
-obesity (body mass index >30 kg/m2)
-smoking
-diabetes
-hypertension
-hyperlipidaemia
-aspirin, or drugs with established antioxidant properties
-intake of antioxidant supplements
-any condition limiting mobility
-celiac or other intestinal disease, life-threatening diseases, or any other disease or
condition that would impair compliance.
Flow-chart describing progress of participants through the EUROLIVE Study
Persons invited to be screened
n = 344
144 ineligible
98 Did Not Met Protocol criteria
46 Unwilling to Participate
200 Randomized
67 Assigned to Order 1,
(HPC, MPC, LPC)
6 Out of Follow-up
3 Unable to adhere
2 Moved away
1 Collateral event
61 Included in the Analysis
68 Assigned to Order 2,
(MPC, LPC, HPC)
5 Out of Follow-up
2 Unable to adhere
2 Moved away
1 Collateral event
LPC, MPC, and HPC, olive oils with low, medium and high phenolic content, respectively.
65 Assigned to Order 3
(LPC, HPC, MPC)
7 Out of Follow-up
4 Unable to adhere
1 Moved away
2 Collateral events
Latin Square for three treatments in the cross-over randomised trial
Examination Number:
Blood collection
Anthropometric measures
1
Order 1
Order 2
Order 3
3DDR
2
3
4
5
6
7
WO
HPC
WO
MPC
WO
LPC
WO
MPC
WO
LPC
WO
HPC
WO
LPC
WO
HPC
WO
MPC
3DDR
3DDR
FHD/PA
3DDR
PA
LPC, MPC, and HPC: olive oils with low, medium, and high phenolic content
WO: wash-out period of 2 weeks .Intervention periods 3 weeks (25 mL/day olive oil ingestion)
Examination number (general measurements and/or blood collection): 1, baseline; 2, post-first washout; 3, post-first intervention;
4, post-second washout; 5, post-second intervention; 6, post-third washout; 7, post-third intervention .
3DDR, 3 day dietary record questionaire; PA, physical activity questionaire;
Urinary tyrosol and hydroxytyrosol, the major olive oil phenolic compounds, as markers of
compliance
Outcome measurements in the Clinical Trials
Oxidative markers
Lipid oxidation
Antioxidative markers
DNA and RNA
oxidation
F2 isoprostanes (pl)
OH-Fatty acids (pl)
Conjugated dienes (LDL)
Oxidized LDL (pl)
8-oxo-dGuo (u)
8-oxo-Guo(u)
8-oxo-Gua (u)
Antibodies against oxidized
LDL (s))
Exogenous
Endogenous
AA (pl)
SOD (b)
Tocopherol (pl)
GSH-Px (pl)
-carotene (pl)
Lycopene (pl)
GR (pl)
GSH/GSSG (b)
Enterolactone (s)
PON (s)
3-O-methyl-hydroxytyrosol in urine as a marker of the individual bioavailability and metabolic capacity of the hydroxytyrosol
ingested.
Lipid status: Fatty acids in LDL; Serum Cholesterol, LDL, HDL
The EUROLIVE Study. Results
Mean daily energy consumption (SD) and selected nutrient intake (SD)
according olive oil intervention
Olive oil
Nutrient
Low phenolics
Medium phenolics
High phenolics
Energy (kcal)
2212
(690.6)
2228
(741.4)
2245
(650.0)
Carbohydrate (%)*
48.4
(27.6)
46.2
(9.5)
46.3
(9.3)
Protein (%)*
15.3
(3.3)
15.6
(3.7)
15.4
(3.6)
Total Fat (%)*
36.2
(8.1)
36.2
(8.1)
36.1
(8.2)
Saturated fat (%)*
12.6
(3.5)
12.6
(3.5)
12.8
(3.6)
Monounsaturated fat (%)*
14.6
(4.4)
14.7
(4.7)
14.7
(4.6)
Polyunsaturated fat (%)*
4.9
(2.0)
4.9
(1.9)
4.8
(1.9)
Vitamin C (mg)
102
(71.4)
104
(73.2)
115
(96.5)
Vitamin E (mg)
9.2
(4.8)
9.2
(5.6)
8.9
(4.9)
ß-carotene (mg)
2.4
(2.6)
2.6
(3.0)
2.2
(2.3)
*Expressed in percentage of total energy intake
Covas MI et al. Ann Intern Med. 2006; 145: 333-341.
Urinary tyrosol and hydroxytyrosol as markers of Compliance
Change (%)
3000
2500
Hydroxytyrosol
*†
2000
1500
1000
*
500
0
Change (%)
2000
1500
*†
Tyrosol
1000
*
500
0
LPC
MPC
HPC
Type of olive oil administered
LPC, MPC, and HPC, olive oils with low, medium and high phenolic content, respectively.
P < 0.001 for linear trend; * P< 0.05 versus LPC , † P< 0.001 versus MPC
Changes1 in Body Weight, Systolic and Diastolic Blood pressure, Glucose, and Blood Lipids after intervention
with olive oil with high (HPC), medium (MPC), and low ( LPC) phenolic content (mean (SD).
LPC
Post-int
MPC
P for linear
trend2
HPC
Change
Post-int
24.1 (2.8)
0.03 (0.31)
24.1 (2.8)
0.05 (0.33))
24.0 (0.21)
0.01 (0.30)
0.55
SBP( mmHg)
123 (0.88)
122 (0.95)
123 (0.89)
123 (0.85)
123 (0.82)
123 (0.93)
0.12
DBP (mmHg)
75 (0.65)
76 (0.64)
76 (0.68)
76 (0.60)
76 (0.62)
76 (0.65)
0.78
Glucose
86
-0.08 (11.2)
0.27
2
Body mass index
(k g/m )
(mg/dL)
Cholesterol
(6.6)
Change
Post-int
-0.29 (0.8)
86 (7.2)
-0.29 (8.5)
86 (5.9)
Ghange
(mg/dL)
Total
182 (37)
0.21 (21)
181 (36)
0.51 (24)
183 (37)
0.50 (17)
0.31
LDL
115 (34)
0.55 (19)
113 (33)
-0.88 (21)
2 115 (34)
-0.41 (18)
0.68
HDL
49.2 (10.8)
0.99(6.1)
(6.1)*
0.99
49.6 (10.3)
1.23 (6.5)*
91 (37)
-6.1(37)
(37)*
-6.1
92 (37)
-5.0(41)
(41)
-5.0
Total Cholesterol/HDL
3.88 (1.11)
-0.09 (0.58)
(0.58)†
-0.09
3.81 (1.06)
LDL/HDL ratio
2.46 (0.95)
-0.05 (0.49)
2.38 (0.88)
Triglycerides
(mg/dL)
1 General linear
*
*
50.4 (11.1)
‡
1.76
1.76(5.3)
(5.3)
‡
0.018
0.90
91 (32)
-4.7(32)*
(32)*
-4.7
)†
-0.10
-0.10(0.55
(0.55)
3.81 (1.07)
-0.012 (0.50)*
-0.12 (0.50)
0.005
0.005
-0.07(0.49)
(0.49)*
-0.07
2.41 (0.93)
-0-09
-0-09(0.45)
(0.45)†
0.052
0.052
model, * P < 0.05; P<0.01, ‡ P < 0.001 versus the corresponding baseline, Tukey´s test.
with post-intervention values adjusted by baseline values.
2 General linear model
*
Changes1 in oxidative stress biomarkers after intervention of olive oil with high (HPC), medium (MPC), and
low (LPC) phenolic content (mean (SD)
LPC
Post-int
(
F2 - isoprostanes
Antibodies against
oxidized LDL
(U/L)
Oxidized LDL
g/L)
3
(U/L)
Uninduced dienes
MPC
Change
28 (4.9)
0.15 (4.9)
590
(234 ; 1397)
-0.20
(-80 ; 85)
47 (18)
0.94 (17)
2.60 (0.96)
Post-int
28 (4.3)
636
(228 ; 1416)
46 (16)
-0.03 (0.83)
2.57 (0.92)
HPC
Change
Post-int
-0.31 (5.4)
28 (6.4)
9.8
(-61; 153)
557
(240 ; 1403)
-1.85 (14)
45 (16)
‡
-0.20(0.91)
(0.91)†
-0.20
2.50 (0.91)
P for linear Trend2
Change
0.23
0.08 (5.3)
8.3
(-67 ; 116)
†
-3.545.7
(15)(1.8)
0.60
*
0.014
0.014
†
†
-0.18
(0.78)
0.18
(0.78)
0.001
0.001
( mol/mol chol)
Hydroxyfatty acids
1.26 (0.18)
-0.03 (0.51)
1.25 (0.18)
-0.06 (0.49)
-0.08
1.22(0.49)
(0.026† ) *
0.004
18.1 (0.57)
†
-2.1
(0.55)
-2.1
(0.55)
0.16
1.21 (0.17)
( mol/L)
8 -oxo -deoxyguanosine
(nmol/24h
21.6 (0.74)
121 (6.5)
‡
‡
†
17.9 (0.53)
‡
-2.9
-2.9(0.50)
(0.50)
0.9(0.76)
21.0 (0.72)
0.4 (0.71)
21.5 (0.74)
0.3 (0.81)
0.23
28 (13)
160 (14.9)
25 (7.8)
152 (9.6)
24 (14.3)
0.39
-urine)
8 -oxo - guanine
(nmol/24h
-2.3(0.75)
(0.75)‡
-2.3
-urine)
8 -oxo - guanosine
(nmol/24h
18.6 (0.63)
-urine)
model, ‡ P<0.01, ‡ P < 0.001 versus the corresponding baseline, Tukey´s test.
with post-intervention values adjusted by baseline values.
3 median (25th-75th percentile)
1 General linear
-
Changes1 in antioxidative biomarkers after intervention of olive oil with high (HPC), medium
(MPC), and low (LPC) phenolic content (mean (SD)
LPC
Post-int
MPC
Change
Post-int
P for
linear
trend2
HPC
Change
Post-int
Change
Endogenous
Paraoxonase
(U/L)
Superoxide dismutase
(U/L)
165 (113)
0.27 (33)
1 64 (109)
0.13 (28)
165 (113)
-2.46 (25)
0.77
142 (20)
-0.47 (14)
141 (20)
-1.50 (13)
142 (20)
0.12 (14)
0.53
714 (162)
-3.7 (126)
0.31
63 (11)
0.78 (20)
0.02
Glutathione peroxidase
(U/L)
Glutathione reductase (
U/L)
62 (10)
-1.9 (18)
Reduced glutathione
( mol/L)
5.85 (0.64)
0.31
0.31 (0.40)
(0.40)‡
Oxidized glutathione
( mol/L)
Red/Ox glutathione ratio
7 08 (152)
-2.3 (119)
704 (134)
‡
0.84 (0.18)
-0.12(0.17)
(0.17)‡
-0.12
7.9 (2.2)
1.74 (2.9)
(2.9)‡
1.74
‡
‡
-1.1 (106)
63 (10)
-1.6 (19)
5.85 (0.67)
0.28(0.32)
(0.32)‡
0.28
0.82 (0.19)
-0.14 (0.16)‡
8.2 (2.6)
2.0
(3.4)‡
2.0 (3.4)
‡
‡
‡
1 General linear
model, ‡ P < 0.001 versus the corresponding baseline, Tukey´s test.
with post-intervention values adjusted by baseline values, order and. centre.
No changes in plasma exogenous antioxidants (vit C, vit E, -carot, Lycopene).
5.87 (0.56)
0.33(0.38)
(0.38)‡
0.33
0.83 (0.17)
-0.12 (0.18) ‡
8.1 (1.6)
‡
1.5
1.5(3.7)
(3.7)
‡
0.70
‡
‡
0.69
0.77
The EUROLIVE Study. Comments
Los cambios detectados en las variables analizadas fueron moderados, como se esperaba
por ser una intervención con dosis habituales de un único alimento durante tres semanas.
Los beneficios observados en los marcadores de estrés oxidativo fueron básicamente en tres
marcadores de oxidación de la partícula de LDL:
dienos conjugados y ácidos grasos hidroxilados (oxidación lipídica de la LDL) y LDL oxidada
(oxidación de la apolipoproteína B)
Los mecanismos implicados en los efectos beneficioses observados, pueden estar relacionados
con las propiedades antioxidantes de los compuestos fenólicos del aceite de oliva y/o el efecto
protector global de los compuestos fenólicos y el contenido en AGMI del aceite de oliva.
Limitatacions del estudi
No poder determinar la interacción entre los componentes del aceite de oliva y
otros compuestos de la dieta . Aunque la consistencia de los resultados entre
los distintos países, da solidez a las conclusiones.
El diseño de la randomización en cuadrado latín no permite revisar todos los
posibles efectos de arrastre
Los cuestionarios de consumo dietético son auto-suministrado y por tanto
pueden ser subjectvos
Aunque el ensayo era triple ciego los participantes podían haber identificado
los diferentes tipos ade aceite de oliva por el gusto y color
The EUROLIVE Study. Conclusions
 All olive oils increased HDL cholesterol and decreased triglycerides and DNA oxidation.
 High- and medium-phenolic content olive oils decreased the in vivo lipid oxidative
damage.
 The increase in HDL cholesterol, and the decrease in lipid oxidative damage, were
observed in a direct dose-dependent manner with the phenolic content of the olive oil.
The EUROLIVE study provided the final degree of evidence required to recommend the
use of olive oil rich in phenolic compounds as a source of fat in order to achieve
additional benefits against cardiovascular risk factors.
Covas et al. Ann Int Med 2006; Machowetz et al. FASEB J 2007
The EUROLIVE Study. Recommendations
Recommendations which stem from the EUROLIVE study are:
• Among the olive oils with a taste that better suits personal
preferences, the best choice is that with the highest phenolic
content.
• For health policy makers, the phenolic content of an olive oil
should be present in the olive oil labels.
THE EFFECT OF OLIVE OIL CONSUMPTION ON OXIDATIVE DAMAGE IN EUROPEAN
POPULATIONS. The EUROLIVE Study
UKU
JLB
EUROLIVE Investigators:
Institut Municipal d´Investigació Mèdica (IMIM), Barcelona, Spain:
Lipids and Cardiovascular
Epidemiology Research Unit: Covas MI (Study
Coordinator), Marrugat J, Fitó M, Elosua R, Schröder H, Vila J
RHK
EUC
Pharmacology Research Unit, de la Torre R, Farré-Albaladejo M
DIfE
UBER
Associated Investigators. Sáez G, Tormos MC. Dpt. Of Biochemistry. Valencia
University. Ruiz-Gutierrez V, Perona J. Instituto de la Grasa. Sevilla, Spain
UBGL
IMIM
UB
KEPKA
Department of Clinical Pharmacology, Rigshospitalet,
Copenhagen, Denmark:
Poulsen H E (Centre Coordinator), Weimann A
University Hospital
Research Institute of Public Health, University of Kuopio, Finland: Salonen JT
(Centre Coordinator), Konttinen A, Nyyssönen K Mursu J; Rissanen T, Tuomainen T-P,
Valkonen V-P, Virtanen J
Department of Nutrition and Bromatology. University of
Barcelona, Spain:
López-Sabater C, Lamuela-Raventós R, de la Torre K,
Castellote AI
Oy Jurilab, Kuopio, Finland:. Kaikkonen J
KEPKA Consumers’ Protection Center. Thessaloniki,
Greece.Dragatsika A, Tsemperlidis N, Sidiropoulos I,
Koursioumi M. Grigoriadou K.
Centro per lo Studio dell'Arteriosclerosi e delle Malattie Dismetaboliche"GC
Descovich".
Dipartimento
di
Medicina
Clinica
e
Biotecnologia
Applicata.Policlinico S. Orsola-Malpighi, Bologna, Italy: Gaddi A (Centre
Coordinator), D’Addato S, Fiorito A, Grandi E, Linarello S, Nascetti S, Sangiorgi Z
German Institute of Human Nutrition Potsdam-Rehbruecke, Germany: Zunft, H-J
F (Centre Coordinator), Koebnick C, Machowetz A
Institute of Transfusion Medicine, Charité-University Medicine of Berlin,
Germany:
Kiesewetter H (Centre Coordinator), Bäumler H, Selmi H
U. de Lleida
EGEC Res.Group
Jaume Marrugat
Roberto Elosua
Susana Tello
Joan Vila
Isaac Subirana
Hector Sanz/Leny Franco
Marta Cabañero
Gemma Blanchart
María J. Motilva
URV
Rosa Solá
Dept. of Cardiology
Hospital del Mar
Mercedes Cladellas
CARIN Research Group
Julio Martí
Montserrat Fitó
Jordi Bruguera
Daniel Muñoz
Valentini Konstantinidou
Olga Castañer
Helmut Schröder
Human Neurociences R.G.
Saray Heredia
Rafael de la Torre
María Isabel Covas
Dept of Nutrition and
Bromatology.
Barcelona University
Carmen de la Torre
M. Carmen López-Sabater
Magí Farré
Karina de la Torre
Olha Khymenets
Rosa M. Lamuela-Raventós
M.Antonia Pujadas
ICAQ-CSIC
Esther Menoyo
Jesús Joglar
Bruno Almeida
Ana Isabel Castellote
Parc de Recerca Biomèdica de Barcelona (PRBB)
Gracias por su atención
Nutrigenomics
Olive Oil and the Mediterranean diet
regulates the expresion of inflammatory
genes in healthy subjects and CHD risk
individuals
Nutrigenomics or Nutritional Genomics
1) High-throughput genomics tools in nutrition research (Muller et al, Nature 2003)
2) Some diet-regulated genes (and their normal, common variants) are likely to play
a role in the onset, incidence, progression, and/or severity of chronic diseases.
(http://nutrigenomics.ucdavis.edu/nutrigenomics/)
3) Aims to define and characterize signaling pathways involved from the main
dietary signals (www.nugo.org)
4) Multidisciplinary science, not yet well defined but with high expectations
Barriers to overcome in Nutrigenomics studies
 Food is complex and variable mixture.
 Diet-related diseases are polygenic. Complex genotypes
 Specific function of genes still unclear
 Exact mechanisms still unclear
 Huge financial investment (analytical platforms, data ware housing, laboratory
information and management systems, databases structures, algorithms etc)
Hypothesis
• Nutrients can regulate the expression of genes at transcription, mRNA
processing, mRNA stability, and trans- and post-translational modification
stages (Salati, 2004).
• Changes in gene expression may support the beneficial changes observed in
phenotypic biomarkers for atherosclerotic processes after Mediterranean diet
and olive oil consumption.
• Mediterranean diet, and its main source of fat, the virgin olive oil, could
modify the human gene expression towards a protective mode for complex
diseases.
Objectives
a. Standardisation of the methodology used in the sample obtention and RNA extraction for
further gene expression analysis
b. To identify in vivo gene expression changes, related to a protective role towards
atherosclerotic processes, in healthy and high cardiovascular risk volunteers associated to:
a) olive oil consumption
b) Traditional Mediterranean diet consumption
c. To study if the gene expression changes are related with atherosclerosis-related systemic
markers
Task a. Standardisation of the methodology used in the sample obtention and RNA
extraction for further gene expression analysis :
The type of blood cells used in clinical studies is of great relevance in the
evaluation of gene expression profile.
Monocyte-macrophage cells are key cells in
the atherosclerotic plaque
development thus the target cell to study the expression of atherosclerosisrelated genes.
Exploratory Study Intervention
Volunteers: Six healthy male signed an informed consent and Ethical Committee’s approved the protocol
(74.1 ± 11.7 kg and BMI of 24.5 ± 3.55 kg/m2 )
Wash out period
1-4 days: habitual diet
controlling excess of
antioxidants, sunflower oil
for raw and cooking
purposes
•
Samples collection
5-7 days: diet with very
low phenolic content,
sunflower oil for raw and
cooking purposes
Intervention day
0h
1h
6h
50ml of VOO
ingestion
• 3 weeks (25ml/day, raw)
Protocol Summary
Six healthy male volunteers recruited (physical, biochemical and haematological tests
performed) and blood was collected (CPT tubes, Ethical Committee’s approval and
participants signed an informed consent)
Total RNA extraction from PBMNC
Ultraspec® RNA isolation procedure
RNA concentration, purity and integrity measured , A260/A280 and A260/A230 ≥ 1.8; and
8.5≤RIN≤9.5 (NanoDrop® ND-1000, Agilent 2100 Bioanalyzer)
Microarray experiment (CNIC, Madrid, Spain). Pool of 6 samples, at 3 time points,(technical
triplicates): baseline (0h), at 6 hours (postprandial state) and at 3 weeks after a regular olive oil
consumption.
Applied Biosystems, Human Genome Survey Microarray v2.0 and 1700 Chemiluminescent
Microarray Analyzer Software v.1.0.3
32,878 60-mer oligonucleotide probes representing 29,098 individual human genes (~97%
human genome)
GEO Accession Number GSE10590
Verification by Quantitative Real Time PCR (Applied Biosystems)
Microarray experiment
Candidate genes selection
29098 human
genes
1. Microarray data normalization
quantile normalization
S/N values >3 and/or
flags < 5,000 (1700 Chemiluminescent
Microarray Analyzer Software, AB)
15308 human genes
2. Panther Classification System
(Paul D et al, 2003, Genome Res)
3. NIH-DAVID Functional
Classification
log2ratio>0,5, fold change>1.41
log2ratio<-0,5, fold change <-1.41
p<0.05
Previously described, known genes
(Dennis G et al,2003, Genome Biology)
4. Gene Ontology Terms
(The Gene Ontology Consortium,2000,Nature Genetics)
•259 up regulated
•246 down regulated
5. Pubmed
(http://www.ncbi.nlm.nih.gov)
29,098 human genes
259
15308 human genes
246
Genes related with atherosclerosis regulated after olive oil ingestion (Fold change*, p<0.05)
Lipid Metabolism
Oxidative stress
related
Inflammation
PRKAG2 (2.09)
ABCA7 (1.47)
ABCB1 (1.46)
IL10 (1.66)
TXNL2 (1.62)
CD69 (-1.92)
NDUFA5 (-1.72)
IL8 (-1.80)
IFNG (-1.53)
DNA Repair
DCLRE1CARTEMIS (1.47)
Apoptosis
Insulin
sensibility
ATF7 (1.96)
OGT (1.68)
PDCD10 (-1.72)
POLK (1.44)
USP48 (2.16)
ADAM17 (1.42)
*Fold change formula at http://www.healthsystem.virginia.edu/internet/biomolec/genechipanalysismethods.cfm
Low Density Arrays-Quantitative Real Time qRT-PCR
 384 simultaneous, parallel reactions
 Pre-loaded TaqMan® Gene Expression Assay targets
 Completely customizable Array
http://www3.appliedbiosystems.com/AB_Home/index.htm
Figure 2. Assessment of gene expression levels by real-time PCR (RT-PCR). Log2ratio expresses the gene
expression changes in human mononuclear cells according to RT-PCR (black bars) and microarray (white bars).
Assessment of gene expression changes by RT-PCR and Microarray
3
2.5
2
1.5
Log2ratio
1
0.5
0
ADAM17
-0.5
IL10
OGT
USP48
AKAP13
-1
-1.5
-2
AKT3
DTTI4
IL8
IFNG
-2.5
Pool PCR
Pool MA
AKT3, v-akt murine thymoma viral oncogene; DDIT4, DNA-damage-inducible transcript 4; IFNG, interf eron gamma; IL8, interleukin 8; ADAM17, a disintegrin and
metalloproteinase domain 17; USP48, ubiquitin specif ic protease 48; OGT, O-linked N-acetylglucosamine transf erase; IL10, interleukin 10; AKAP13, A-kinase anchoring
protein 13.
Conclusions
• Changes in several genes related with oxidative stress associated diseases, such as cancer
and atherosclerosis, occur in human PBMNC of healthy volunteers at 6h postprandial
after 50ml olive oil ingestion.
• Changes were observed at a real-life dose of olive oil, as is daily consumed in some
Mediterranean areas.
• Our results support the hypothesis that the protective changes observed in secondary
markers for cardiovascular disease, related to olive oil consumption, could be mediated
through gene expression changes promoted by olive oil ingestion.
• No verification on the down-regulated expression of genes in general
TABLE 2. INSULIN, GLUCOSE AND LIPID PROFILE VALUES AT BASELINE, 1 HOUR AND 6 HOURS AFTER OLIVE OIL INGESTION
Parameters
Baseline
(0 hours)
1 hour
6 hours
P value for quadratic
trend
11 (4.0)
25 (12)*
7.5 (2.3)†
0.001
Glucose (mmol/L)
4.83 (3.1)
5.39 (0.9)
4.61 (0.3)†
0.045
Total cholesterol (mmol/L)
4.51 (0.80)
4.48 (0.80)
4.46 (0.83)
0.880
LDL cholesterol (mmol/L)
2.64 (0.62)
2.54 (0.62)
2.51 (0.67)
0.368
HDL cholesterol (mmol/L)
1.5 (0.37)
1.53 (0.36)
1.5 (0.34)
0.295
0.795 (0.57)
0.943 (0.49)
0.977 (0.64)
0.091a
Oxidized LDL (mU /mg LDL cholesterol)
66 (20)
60 (15)
75 (29)
0.042a
TBARS (μΜ/L )
4.6 (2.8)
3.4 (2.3)
9.03 (8.2)
0.044
Insulin (mU/L)
Triglycerides (mmol/L)*
Values are expressed as mean ± SD with exception of triglycerides and TBARS; * P<0.05 versus baseline; † P<0.05 versus 1h; a linear
trend
Figure 1. Time course changes (mean ± SEM) in the gene expression of ALOX5AP, arachidonate 5-lipoxygenase-activating protein;
CD36, CD36 molecule (thrombospondin receptor) and OGT, O-linked N-acetylglucosamine (O-GlcNAc) transferase after 50 mL olive oil
ingestion. *p<0.05 versus baseline; † p<0.05 versus 1h
Figure 2. Time course changes (mean ± SEM) in the gene expression of ADAM17, a disintegrin and metallopeptidase domain 17; ADRB2,
adrenergic beta-2- receptor; LIAS, lipoic acid synthetase and PPARBP, PPARγ binding protein after 50 mL olive oil ingestion.
Results
A down regulation at 1 hour and an up-regulation at 6h after olive oil ingestion were observed in OGT and ALOX5AP,
whereas CD36 was up regulated at 1 hour returning to basal values at 6 hours.
Because sustained insulin action would be detrimental to physiological homeostasis, several feedback mechanisms are
involved in attenuating the signalling of sustained insulin action (Saltiel and Pessin, 2002), (Zick, 2005).
ALOX5AP is required for the synthesis of leukotrienes, a protein family involved in inflammatory
responses and
contributes to CHD risk in patients with familial hypercholesterolemia (Van der Net et al, 2008, Atherosclerosis). The
expression of ALOX5AP, which has been associated with body weight and insulin resistance (Kaaman, 2006) followed
a similar pattern of OGT.
The postprandial lipid increase in triglycerides after the 50 mL olive oil ingestion could be involved in the CD36 upregulation observed, as a related mechanism to fatty acids uptake (Goldberg, 2008).
It has been reported that activation of the small-intestinal lipid messenger oleoylethanolamide, enabled by CD36mediated uptake of dietary oleic acid, serves as a molecular sensor linking fat ingestion to satiety (Schwartz, 2008).
At 1h post-prandial, the up-regulation of CD36 observed could be related with the oleic acid disposition after olive oil
ingestion and its relationship with the satiety feedback mechanism.
Results
Genes presenting a postprandial linear trend pattern: LIAS, PPARBP, ABRB2, and ADAM17, and also OGT with a quadratic
trend, were significantly up-regulated at 6 h after virgin olive oil ingestion.
Lipoic acid is a powerful antioxidant which can activate peroxisome proliferator-activated receptors (PPARα and PPARγ)
(Pershadsingh, 2007). Lipoic acid has shown to mimic insulin action via the insulin signalling pathway by several potential
intracellular mechanisms (Orasanu et al, 2008,J Am Coll Cardiol).
The improvement of insulin resistance by PPARγ agonists is primarily mediated by enhancing the PPARγ interaction with both
PPAR-binding protein and PPAR-interacting protein (Fujimura, 2006)
The up-regulation in the expression of LIAS, the gene which codifies the lipoic acid synthase, and that of PPARBP, a PPAR
coactivator, could be one of the feedback mechanisms for counteracting the postprandial oxidative stress involved in the
development of insulin resistance (Giugliano, 2008).
The increased expression of ADAM17 (TACE, a TNF-alpha-converting enzyme inhibitor), could also be a contributor for
improving the insulin resistance at 6h postprandial via a blockade of the TNF-alpha secretion, as has been described in fructose
fed rats (Togashi et al, 2002). ADAM17 partially protect from obesity and insulin resistance caused by a high-fat diet (Chen et al,
2007, Proc Natl Acad Sci USA)
The beta-2 adrenergic receptor mediates the insulin secretion, (Jalba et al, 2008, Obesity). A functional expression of
2
adrenergic receptors is considered to be related to a protection against oxidative stress through the promotion of glutathione
synthesis (Takahata, 2008).
The increase in OGT expression after 6 hour could be related with the feedback mechanisms developed to attenuate the
signalling of sustained insulin action (Yang, 2008).
Conclusions
Changes in the expression of insulin sensitivity-related genes occur in human
PBMNC after an oral load of olive oil.
These findings may be relevant concerning potential targets for controlling
insulin resistance, associated with metabolic dyslipidemia, which is largely a
postprandial phenomenon.
Table 1. Plasma lipid profile, oxidative stress, inflammation and glycemic homeostasis biomarkers
in volunteers during dietary intervention
Male group (n = 6)
Biomarkers
Values
Male and female combined group (n =
10)
Baseline
3 weeks diet
Baseline
3 weeks diet
Plasma lipid profile:
- total cholesterol
mg/dL
164.00
(17.45)
174.67
(30.08)
169.10
(27.98)
171.80
(27.26)
- LDL cholesterol
mg/dL
92.28
(20.48)
104.30
(20.03)
98.07
(21.37)
103.09
(20.46)
- HDL cholesterol
mg/dL
57.23
(16.35)
57.02
(12.44)
56.73
(14.50)
56.55
(11.11)
- triglycerides (TG)
mg/dL
68.45
(51.85 – 109.20)
62.80
(47.53 – 86.38)
68.45
(47.15 – 87.87)
61.00 *
(39.88 – 69.60)
U/L
66.96
(21.85)
80.37
(34.48)
63.33
(18.90)
71.14
(30.56)
mol/L
5.37
(2.94 – 8.46)
3.82
(2.92 – 9.70)
3.47
(2.76 – 7.27)
2.87
(2.59 – 4.72)
mg/L
0.025
(0.015 – 0.033)
0.020
(0.010 – 0.065)
0.020
(0.015 – 0.033)
0.020
(0.010 – 0.038)
mg/dL
89.67
(5.29)
91.65
(6.24)
87.01
(5.89)
87.5
(7.49)
Oxidative stress:
- oxidized LDL
- lipid peroxides[1]
Inflammation:
- C-reactive protein
Glucose homeostasis :
- Serum Glucose
[1] - the data are expressed as Median (Inter Quartile Range) for parameters with a non-normal distribution and as a Mean (Standard Deviation) for normally distributed ones. Non-parametrical tests were applied to all biochemical marker
comparisons because of the small number of subjects per group.
[1] – all samples for biochemical measurements were collected in the morning at a fasting stage.
* - changed value in comparison to its baseline at significance P < 0.05 by Wilcoxon test
[1] – this refers to a concentration of MDA (malondialdehyde), an end product of lipid peroxidation,measured in plasma using the TBARS test.
Selection of 23 candidate atherosclerosis-related genes based on
an exploratory study with virgin olive oil ingestion, in healthy human
Oxidative
stress
Apoptosis
LIAS
Inflammation
IL8RA
Cellular
differentation
IL23A
BIRC1
DHCR24
IL7R
IFNG
ADAM17(TACE)
Insuline
Sensibity
TNFSF10
CD36 ADRB2
RGS2
TRIB3
USP48
ADAMTS1
Lipid
Metabolism
GATA2
ALDH1A1
ALOX5AP
PPARBP (MED1)
DNA
Repair
Obesity
ERCC5
XRCC5
NMB
OGT
Selection of 23 candidate atherosclerosis-related genes based on
an exploratory study with virgin olive oil ingestion, in healthy human
Oxidative
stress
Apoptosis
LIAS
Inflammation
IL8RA
Cellular
differentation
IL23A
BIRC1
DHCR24
IL7R
IFNG
ADAM17(TACE)
Insuline
Sensibity
TNFSF10
CD36 ADRB2
RGS2
TRIB3
USP48
ADAMTS1
Lipid
Metabolism
GATA2
ALDH1A1
ALOX5AP
DNA
Repair
ERCC5
XRCC5
PPARBP (MED1)
Obesity
NMB
OGT
qPCR confirmation of microarrays detected expression changes for selected genes. Correspondence between qPCR analyses in pooled and individually performed samples.
Microarray
Pools qPCR
Individuals qPCR
Fold Changea
log2
(ratio)
Fold Changeb
log2
(RQ)
Fold
Changec
log2 (ratio)
(Mean (SD))
P-valued
1.855
0.89
2.250
1.170
1.991
0.993 (0.208)
< 0.001
-1.967
-0.98
1.201
0.264
1.504
0.589 (0.416)
0.018
-1.809
-0.85
1.037
0.052
1.175
0.233 (0.158)
0.015
1.980
0.99
2.244
1.166
1.793
0.843 (0.267)
< 0.001
-1.376
-0.46
1.004
0.006
1.038
0.054 (0.27)
0.645
1.803
0.85
2.306
1.205
2.191
1.132 (0.356)
< 0.001
1.455
0.54
1.668
0.738
1.419
0.505 (0.421)
0.032
-1.718
-0.78
1.038
0.053
1.084
0.116 (0.383)
0.491
1.543
0.63
2.086
1.061
1.978
0.984 (0.167)
< 0.001
-1.726
-0.79
-1.066
-0.093
1.243
0.314 (0.738)
0.345
-2.339
-1.23
-1.196
-0.259
-1.100
-0.138 (0.418)
0.456
-1.843
-0.88
-1.166
-0.221
-1.094
-0.130 (0.427)
0.486
1.898
0.92
2.218
1.149
1.928
0.947 (0.423)
0.003
-1.800
-0.85
-1.197
-0.259
-1.176
-0.234 (0.366)
0.179
1.633
0.71
2.276
1.187
2.091
1.064 (0.141)
< 0.001
-1.627
-0.70
-1.243
-0.314
-1.030
-0.042 (0.487)
0.840
2.200
1.14
2.947
1.559
3.037
1.603 (0.411)
< 0.001
1.520
0.60
2.610
1.384
2.293
1.197 (0.128)
< 0.001
1.923
0.94
1.580
0.660
1.494
0.579 (0.468)
0.029
1.802
0.85
2.086
1.061
1.912
0.935 (0.325)
< 0.001
-1.825
-0.87
-1.249
-0.320
-1.003
-0.004 (0.272)
0.973
3.110
1.64
2.059
1.042
2.066
1.047 (0.201)
< 0.001
1.811
0.86
2.151
1.105
1.752
0.809 (0.143)
< 0.001
a – Fold Chage in expression of genes estimated on the base microarray comparison between pooles of 6 individual tRNA correspon ding to basal and after 3-weeks virgin olive oil consumption
b – Fold Change in gene expression after 3-weeks virgin olive oil consumption estimated on the base of RQ values extracted from qPCR analysis in the same pooled samples used in microarrays. The baseline pool was taken as calibrator in relative-relative qPCR analysis.
c – Fold Change in gene expression provoked by 3-weeks virgin olive oil consumption estimated by qPCR analysis on the base of mean of individual log2 (ratios) in RQ values at baseline and after intervention periods corresponding to 6 males.
d – P-value calculated on the base of comparison of log2 (ratio) at baseline and after 3 -weeks virgin olive oil consumption using t-test for males. P-values < 0.001 are used as criteria for gene selection and are typed in bold.
Mononuclear Cell Transcriptome Response after Sustained
Virgin Olive Oil Consumption in Humans
qPCR verification
microarray pooled samples
individual volunteers samples
Cutoffs: P<0.05 & [log2(ratio)] i
n = 317
B
Generesponder
n = 10
n = 23
0.5
0.02
n = 1659
4
A
Additional cutoff: B-probability 20%
(B-statistics)
Data mining: Published reports
(PubMed database)
0.02
Microarray normalization and
filtering
Cutoffs: P < 0.05 (t-statistics)
[log2(ratio)] I 0.5 (M-statistics)
C
D
ADAM17
NMB
0.015
0.015
3
ALDH1A1
ALOX5AP
BIRC1
ERCC5
LIAS
0.01
p-value
0.01
p-value
2
-log10(p)
ADAMTS1
OGT
PPARBP
IL23A
IL8RA
IFNG
TRIB3
GATA2
0.005
1
0.005
PPARBP
CD36
DHCR24
ADAM17
-1
-0.5
0
0.5
1
log2(ratio)
Khymenets O et al. OMICS 2009; 13: 7-19
1.5
2
-2
-1.5
-1
-0.5
0
0.5
log2(ratio)
1
1.5
2
-2
-1.5
-1
-0.5
0
log2(ratio)
0.5
XRCC5
OGT
0
0
-1.5
USP48
LIAS
XRCC5
TNFSF10
IL7R
RGS2
ALDH1A1
ADRB2
0
-2
TNFSF10
USP48
ERCC5
BIRC1
1
1.5
2
Conclusions
Virgin olive oil may be involved in several molecular pathways involved in
antiatherogenic protection in humans in vivo, by both activating the cell death
pathway in certain cells and modulating the immune response.
Defining gene expression signatures induced by this dietary agent may point
towards potential targets for nutritional modulation of atherosclerosis risk in
populations.
Comparative qRT-PCR results between postprandial and sustained VOO consumption in gene
expression changes (log2ratio) for the insulin sensitivity related genes
6h Postprandial (n =11)
Sustained (3 weeks) (n =11)
Gene symbol
log2ratio SEM
P (t test)
log2ratio SEM
P (t test)
OGT
0,74 0,24
0,011
1,52 0,20
<0,001
ADAM17
0,35 0,16
0,049
1,01 0,12
0,001
LIAS
0,36 0,13
0,019
1,06 0,15
<0,001
PPARBP
0,38 0,14
0,001
1,19 0,18
<0,001
ADRB2
0,59 0,12
0,001
0,247 0,002
0,019
CD36
0,12 0,21
0,569
0,55 0,07
0,036
ALOX5AP
0,11 0,06
0,630
0,068 0,001
0,068
Changes in PPAR binding protein (PPARBP) gene expression in human PBMNC
at postprandial state after 50mL of virgin olive oil ingestion and
after sustained virgin olive oil consumption (25 mL/day, 3 weeks)
*
P<0.001 for linear trend;
*P <0.05 from baseline
*
Changes in n-acetylglucosamine(O-GlcNAc) transferase(OGT) gene expression in human PBMNC
*P <0.05 from baseline
P <0.001 for linear and cuadratic trend
*
*
*
*P <0.05 from
baseline
*
*
*
*
Changes in beta-2 adrenergic receptor (ADRB2) gene expression in human PBMNC
*P < 0.05 from baseline
*
P < 0.05 for linear and cubic trend
*
Conclusions
• Changes in human PBMNCs gene expression related with
atherosclerosis processes were observed after acute and sustained virgin
olive oil consumption.
• Sustained virgin olive oil consumption promoted the highest changes
observed.
Al comparar los dos intervenciones con aceite de oliva, en un caso con un alto contenido
en compuestos y en el otro con un nulo contenido, se ha obtenido una diferencia de una P<
0.050 en el caso de los siguientes genes: 1/ MED1 (PPARBP): PPAR binding protein y 2/
OSBP: oxysterol binding protein-like 8.
Y una P de <0.100 en los siguientes genes: 1. ABCG1: ATP-binding cassette, sub-family G
(WHITE), member 1 (p=0,076); 2. MSR1: macrophage scavenger receptor 1 (p=0,080); 3. OLR1:
oxidized low density lipoprotein (lectin-like) receptor 1 (p=0,067); 4. PPARA: peroxisome
proliferator-activated receptor alpha (p=0.067); 5. PPARD: peroxisome proliferator-activated
receptor delta (p=0,060);
Se aprecia una disminución de la expresión de ABCG1 y OSBP (p<0,082) y un incremento de la
expresión de LIPG (p<0,055) después del aceite de oliva virgen. Por otra parte se ha observado
una disminución de la expresión de LCAT, LIAS, MRS1, NR1H2, OLR1, PAFAH1B3, SAA1 y PLTP
(p<0,095) y un incremento de la expresión de ABCG4, LPL, MED1, PPARA y PPARD (p<0,099)
después del aceite de oliva virgen.
Bellido et al.
Butter and
walnuts, but not
olive oil elicit
NF B
postprandial
activation in
PBMC of healthy
volunteers
Am J Clin Nutr,
2004
Clinical, randomized, parallel, controlled, double blind trial with three dietary
interventions
Group
Baseline
sample
Intervention (3 months)
Post-intervention
sample
A
Traditional Mediterranean Diet +
Virgin Olive Oil
(1lt/week)
A1
B (TDM+OO
wo phenolic
compounds)
(n=30)
B
Traditional Mediterranean Diet +
Olive oil wo phenolic compounds
(1lt/week)
B1
C (HD)
(n=30)
C
Habitual Diet (Control)
C1
A (TDM+VOO)
(n=30)
GENERAL DIETARY RECOMMENDATIONS
 Med diet explanation
 Change the regular food consumption for the ones recommended.
 Involve in the diet 30 menus based on recommendations.
 Control body weight
 No specific advice for the control group
Exclusion criteria
1.
Previously documented cardiovascular disease: a)Isquemic cardiopathy,
infraction, b)Isquemic or hemorraic vascular-cerebral
angina,
myocardial
accident, c) Periferical vasculopathy
2.
Important medical disease which does not permit dietary interventions
3.
HIV pacients or patients with immunodeficiency
4.
Drug and/or alcohol addicted pacients (alcohol/day > 80 g)
5.
Patients with digestion problems
6.
Cronic pharmacological treatments (vitamins supplementation etc)
7.
Any type of physical or psycological limitations which could prevent dietary intervention.
8.
Two questions to check for volunteer’s follow up: a) Do you regularly eat
at work? and b) Do you always eat sitted?
home-cooked
food
Volunteers’ inclusion criteria
1.
Between 20 and 55 years
2.
Systolic arterial pressure < 140 mmHg and/or diastolic arterial
pressure < 90 mmHg without treatment
3.
BMI < 26 kg/m2
4.
Glucosa: 75-110 mg/dL
Triglicéridos: 40-150 mg/dL
Colesterol total: 129-238 mg/dL
Colesterol HDL: 41-59 mg/dL
Colesterol LDL: 80-149 mg/dL
CANDIDATE
GENES
AND THEIR
CATEGORIES(48)
CANDIDATE
GENES AND
THEIR
FUNCTION
(GO Terms)
4
1
1
3
1 1
3
4
8
3
4
6
6
3
Nucleic acid binding
Oxidoreductase
Protease
Receptor
Synthase and synthetase
Select regulatory molecule
Transcription factor
Signaling molecule
Transfer/carrier protein
Hydrolase
Select calcium binding protein
Kinase
Transferase
Molecular function unclassified
Cholesterol-Lipid Transport and metabolism: OLR1, ADRB2, LIAS, ARHGAP19, PPARA, CD36,
PPARG, PPARD, NR1H2, NR1H3, ABCA1, ARHGAP15, PLA2G4B, ABCG1,CETP, PPARBP, MSR1,
SCARB1, ANXA1
Inflammation: ADAM17, ADAMTS1, IL7R, IFNA1, IL6, TNFSF10, TNFSF13, TP53, IFNG, IL10,
CHUK, CCNG1
Oxidative Stress: NOX1, MPO, GAPDH, ALDH1A1, NOS2A, PTGS1, PTGS2, NFKB2, OSBP
DNA Repair protein: XRCC5, ERCC5, POLK, DCLRE1C
Atherosclerosis related: USP48, RGS2
Glucose Metabolism: OGT
Table 1. Volunteers’ baseline characteristics.
Parameter
Control
(N=29)
Age (y)
43 13
Men (%)
34.5
Weight (kg)
66
16
BMI (kg/m 2 )
25
4
SBP (mm/Hg)
117
DBP (mm/Hg)
69
Glucose (mg/dL)
85 15
12
10
TMD Global
(N=60)
45
10
TMD+WOO
(N=30)
44
25
68
10
TMD+VOO
(N=30)
45
27
13
69
10
23
13
68
14
25
4
26
5
25
4
116
15
117
14
114
16
72
10
72
10
84 12
84
14
72
10
84
9
Total Cholesterol (mg/dL)
202
54
207
50
200
47
214
54
LDL-C (mg/dL)
127
42
131
44
124
37
138
50
HDL-C (mg/dL)
58
13
60
14
58
13
61
15
Total Cholesterol /HDL-C
3.6
1.0
3.6
0.9
3.5
0.6
3.6
1.0
LDL-C / HDL-C
2.2
0.7
2.3
0.8
2.2
0.5
2.3
0.9
Triglycerides (mg/dL)
oxLDL (U/L)
Urinary F2a-isoprostanes
(pg/mmol creatinin)
IFNγ (ng/mL)
MCP-1 (pg/mL)
s-Pselectin (ng/mL)
67 (52, 83)
66
29
70 (57, 103)
63
21
67 (58, 102)
62
20
70 (57, 105)
64
22
42 (39, 79)
67 (39, 83)
54 (41, 79)
72 (39, 85)
0.086 (0.009, 0.124)
0.018 (0.001, 0.073)*
0.001 (0, 0.068)*
0.027 (0, 0.086)
282 (203, 369)
217 (170, 307)
240 (195, 349)
174 (143, 243)
935
741
743
493
768
496
710
498
s-CD40L (pg/mL)
937 (586, 2254)
1217 (602, 2354)
1389 (618, 2306)
1001 (558, 2449)
CRP (mg/dL)
Urinary 8-oxo-dG
(nmol /mmol creatinin)
0.02 (0.01, 0.09)
0.07 (0.03, 0.18)
0.11 (0.02, 0.25)
0.07 (0.03, 0.11)
EEPA (Kcal/day)
10.09
4.07
129 (25, 269)
11.32
4.01
130 (47, 224)
11.10
3.89
113 (49, 183)
11.55
4.19
139 (32, 229)
*p<0.05 vs control
Values are shown as mean SD for the normal variables and median (25 th, 75th) for the non parametric variables.
Univariate ANOVA was used to assess differences between groups for the normal variables and Kruskal -Walls test for non parametric variables
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL -C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; oxLDL, oxidized
low density lipoproteins; IFNγ, interferon gamma; MCP-1, monocyte chemoattractant protein 1; s-Pselectin, soluble P-selectin; s-CD40L, soluble CD40L CRP; C-reactive protein; 8-oxo-dG,
7,8-dihydro-8-oxo-2’-deoxyguanosine; EEPA, energy expenditure in physical activity in leisure time.
Table 3. Changes in biomarkers af ter 3 months of intervention. * p<0.05 versus baseline, † p<0.05 versus control, ‡p<0.05 ver sus TMD+WOO
CONTROL (N=29)
TMD Global (N=60)
TMD + WOO (N=30)
TMD + VOO (N=30)
Postintervention
Change
Post-intervention
Change
Post-intervention
Change
Post-intervention
Change
67 16
0.19 (-0.59 to 0.97)
68 14
-0.17 (-0.72 to 0.37)
69 14
-0.25 (-1.03 to 0.53)
67 14
-0.1 (-0.86 to 0.67)
25 4
0.081 (-0.2 to 0.36)
25 4
-0.068 (-0.26 to 0.13)
26 5
-0.1 (-0.38 to 0.18)
25 4
-0.04 (-0.31 to 0.24)
SBP (mmHg)
119 15
1.40 (-2.60 to 5.40)
115 15
-1.03 (-3.76 to 1.7)
116 14
-1.63 (-5.51 to 2.24)
114 15
-0.4 (-4.31 to 3.45)
DBP (mmHg)
71 10
1.67 (-1.23 to 4.58)
72 10
0.17 (-1.81 to 2.15)
71 9
-0.8 (-3.6 to 2.0)
73 10
1.12 (-1.69 to 3.93)
Glucose (mg/dL)
82 12
-2.55 (-5.4 to 0.31)
82 10*
-2.1 (-4.09 to -0.09)
82 11
-1.76 (-4.58 to 1.06)
82 9
-2.43 (-5.3 to 0.44)
Cholesterol
(mg/dL)
202 57
-0.12 (-8.56 to 8.33)
202 46
-4.85 (-10.75 to 1.04)
200 48
-0.2 (-8.1 to 7.7)
205 45 *
-10.5 (-19.1 to -1.84)
HDL-C (mg/dL)
57 13
-1.82 (-4.34 to 0.70)
57 13 *
-2.0 (-3.75 to -0.29)
57 12
-1.12 (-3.5 to 1.3)
58 15 *
-3.14 (-5.54 to -0.53)
LDL-C (mg/dL)
129 47
2.1 (-4.35 to 8.56)
128 40
-2.80 (-7.22 to 1.63)
126 40
1.4 (-4.6 to 7.4)
131 41 *
-7.5 (-13.8 to -1.2)† ‡
Cholesterol /
HDL-C
3.6
1.0
0.09 (-0.05 to 0.24)
3.6
0.8
0.04 (-0.06 to 0.14)
3.5
0.7
0.06 (-0.08 to 0.20)
3.6
0.8
0.02 (-0.13 to 0.17)
LDL-C / HDL-C
2.3
0.8
0.09 (-0.03 to 0.22)
2.3
0.7
0.03 (-0.06 to 0.12)
2.2
0.6
0.06 (-0.06 to 0.18)
2.3
0.8
-0.003 (-0.13 to 0.12)
Weight (kg)
BMI
(kg/m 2 )
Triglycerides
(mg/dL)
62 (49, 98)
-2.5 (-17, 17.3)
71 (59, 99)
4 (-14, 19)
73 (58, 100)
4.5 (-17.3, 18.5)
68 (60, 97)
4 (-10, 19)
OxLDL (U/L)
70 32
3.38 (-2.36 to 9.16)
64 23
2.3 (-1.69 to 6.19)
65 22
2.4 (-3.04 to 7.9)
63 24
2.1 (-3.68 to 7.83)
Isoprostanes
(pg/mmol urine
creatine)
39 (34, 65)
-2.8 (-14, 5.1)
49 (41, 66) *
-2.5 (-13.7, 6.6)
52 (43, 66)
-1.6 (-10.5, 7.4)
47 (35, 75)*
-4.3 (-18.2, 6.6)
8-oxo-GG (nmol
/mmol urine
creatine)
8.89 3.76
-1.1 (-2.47 to 0.26)
10.42 3.9
-0.95 (-1.89 to 0.003)
10.7 3.5
-0.41 (-1.75 to 0.93)
10.1 4.4*
-1.48 (-2.82 to -0.15)
IFNg (pg/mL)
61 (0, 113)
-11 (-52, 5)
0 (0, 46) *
0 (-45, 11)
16 (0, 51)
0 (-47, 33)
0 (0, 39) *
-2.5 (-47, 0)
MCP-1 (pg/mL)
247 (211, 317)
-36 (-119, 27)
202 (176, 305)
0.14 (-37, 35)
253 (175, 328)
8 (-60, 49)
194 (176, 250)
-6 (-25, 29)
s-Pselectin
(ng/mL)
696 (493, 1063)
-78 (-286, 323)
578 (346, 808) *
-30 (-383, 122) †
664 (368, 965)
19 (-375, 147)
549 (248, 634) *
-63 (-434, 75)
s-CD40L (pg/mL)
1267 (498, 2013)
-228 (-1109, 789)
943 (587, 2437)
-77 (-1077, 804)
1256 (706, 2773)
123 (-875, 915)
923 (455, 2467)
-81 (-1435, 602)
CPR (mg/dL)
0.04 (0.01-0.14)
0 (-0.01, 0.06)
0.04 (0.02 - 0.11)*
-0.02 (-0.07, 0) †
0.06 (0.02 – 0.12)*
-0.03 (-0.1, 0)
0.03 (0.02 – 0.11) *
-0.02 (-0.06, 0) †
129 (52, 226)
6.8 (-23.7 to 37.2)
117 (32, 206)
-1.8 (-23 to 19)
113 (61, 206)
6.7 (-23 to 37)
120 (23, 226)
-10 (-40 to 20)
EEPA (Kcal/day)
Univariate ANOVA, adjusted by sex and age, was used to assess differences between groups for the normal variables and Kruskal-Walls test for non parametric variables
Table 4. Changes in the expression of atherosclerosis-related genes after 3 months of intervention.
Gene Symbol
Symbols
Gene Name
Control
(n=20)
TMD-Global
(n=37)
P
between
groups
Cholesterol, Lipid Transport, and Metabolism
ABCA1
ATP-binding cassette, sub-family A,member 1
0.328
0.231
0.051
0.159
0.334
ABCG1
ATP-binding cassette, sub-family G,member 1
0.146
0.127
0.064
0.092
0.608
ANXA1
annexin A1
0.259
0.229
-0.444
0.161
0.160
ARHGAP15
Rho GTPase activating protein 15
0.448
0.175
-0.040
0.126
0.043
ARHGAP19
Rho GTPase activating protein 19
0.400
0.151
0.134
0.112
0.166
ARHGEF6
Rac/Cdc42 guanine nucleotide exchange factor 6
0.460
0.144
0.157
0.106
0.099
CD36
CD36 molecule (thrombospondin receptor)
0.197
0.170
-0.009
0.126
0.342
CETP
cholesteryl ester transfer protein, plasma
macrophage scavenger receptor 1
-0.262
0.331
-0.058
0.257
0.631
MSR1
0.542
0.222
0.253
0.157
0.301
PLA2G4B
phospholipase A2, group IVB
0.148
0.156
0.082
0.109
0.735
SCARB1
scavenger receptor class B, member 1
-0.025
0.078
0.085
0.056
0.261
IFNg
interferon, gamma
1.048
0.464
-0.109
0.330
0.049
IL10
interleukin 10
0.915
0.360
0.609
0.270
0.506
CHUK
conserved helix-loop-helix ubiquitous kinase
0.325
0.192
0.036
0.140
0.236
ADAM17
ADAM metallopeptidase domain 17
(tumor necrosis factor, alpha, converting enzyme)
ADAM metallopeptidase with thrombospondin
type 1 motif, 1
interferon, alpha 1
0.290
0.153
0.008
0.112
0.148
0.166
0.208
-0.120
0.150
0.277
0.726
0.356
0.001
0.258
0.117
0.195
0.219
-0.195
0.156
0.157
-0.021
0.102
0.133
0.075
0.235
Inflammation
ADAMTS1
IFNA1
TNFSF10
TNFSF12_13
tumor necrosis factor (ligand) superfamily, member 10
ttumor necrosis factor (ligand) superfamily,
member 12-member 13
IL6
interleukin 6
-0.017
0.588
0.356
0.401
0.612
IL7R
interleukin 7 receptor
0.580
0.182
0.095
0.132
0.037
USP48
ubiquitin specific peptidase 48
0.380
0.179
0.203
0.131
0.431
MPO
myeloperoxidase
-0.159
0.121
-0.013
0.090
0.343
RGS2
regulator of G-protein signalling 2, 24kDa
nuclear factor of kappa light polypeptide gene
enhancer in B-cells 2
0.439
0.268
0.289
0.196
0.656
-0.098
0.082
0.008
0.063
0.315
NFKB2
Gene expression changes, adjusted by age and sex, are presented as mean
SEM of the RQ log2 ratio (post-treatment versus basal values).
Table 4. Changes in the expression of atherosclerosis-related genes after 3 months of intervention.
Gene Symbol
Symbols
Gene Name
Control
(n=20)
TMD-Global
(n=37)
P
between
groups
Nuclear Receptors and Fatty acids Receptors
NR1H2
nuclear receptor subfamily 1, group H, member 2
-0.081
0.070
-0.003
0.050
0.369
NRIH3
nuclear receptor subfamily 1, group H, member 3
0.166
0.108
0.034
0.077
0.331
PPARA
peroxisome proliferator-activated receptor alpha
0.088
0.123
0.068
0.092
0.897
PPARBP
PPAR binding protein
0.341
0.160
0.022
0.105
0.084
PPARG
peroxisome proliferator-activated receptor gamma
0.002
0.242
0.235
0.175
0.463
PPARD
peroxisome proliferator-activated receptor delta
0.066
0.128
0.010
0.096
0.732
LIAS
lipoic acid synthetase
0.228
0.197
0.188
0.148
0.874
PTGS1
prostaglandin-endoperoxide synthase 1
-0.176
0.171
-0.170
0.117
0.978
PTGS2
prostaglandin-endoperoxide synthase 2
0.170
0.545
-0.231
0.379
0.557
0.521
0.948
0.113
0.580
0.724
Oxidative Stress
OLR1
oxidised low density lipoprotein (lectin-like) receptor 1
OSBP
oxysterol binding protein
0.219
0.130
0.035
0.093
0.260
ADRB2
adrenergic, beta-2-, receptor, surface
0.225
0.135
-0.138
0.098
0.036
OGT
O-linked N-acetylglucosamine (GlcNAc) transferase
0.373
0.235
0.014
0.162
0.218
ALDH1A1
aldehyde dehydrogenase 1 family, member A1
-0.101
0.187
-0.116
0.135
0.949
CCNG1
cyclin G1
0.396
0.192
0.004
0.139
0.106
POLK
polymerase (DNA directed) kappa
0.595
0.275
-0.115
0.204
0.045
TP53
tumor protein p53
-0.071
0.077
-0.048
0.056
0.812
DCLRE1C
DNA cross-link repair 1C
0.406
0.169
0.052
0.123
0.100
ERCC5
excision repair cross-complementing rodent repair deficiency,
complementation group 5
0.401
0.227
0.049
0.169
0.221
0.267
0.152
0.000
0.111
0.166
DNA Repair
XRCC5
X-ray repair complementing defective repair in Chinese hamster cells 5
(double-strand-break rejoining; Ku autoantigen, 80kDa)
Gene expression changes, adjusted by age and sex, are presented as mean
SEM of the RQ log ratio (post-treatment versus basal values).
Figure 2. Gene expression changes in adrenergic- -2 receptor (ADRB2), ), Rho-GTPase activating protein15 (ARHGAP15), interferon
gamma (IFN), and interleukin7 receptor genes before and after the 3-month interventions.
*(p<0.05) for linear trend, *p<0.05 versus control group
Human nutrigenomic studies concerning olive oil and MUFA-rich diets consumption
Type of Study
Participants
Intervention
Tissue
Up regulated Genes
Down regulated Genes
Accompanying
biomarkers
Reference
Postprandial randomized,
crossover, intervention-4
weeks
20 healthy males
Breakfasts rich in OO,
butter or walnut after
Westerm, Mediterranean
and CHO diets
PBMNCs
TNFa in butter, IL6 in butter and
OO, NS in MCP1
------------------------------------
↑ cholesterol and ↑ LDL-C,
and ↑ TG in Western Diet
↓ HDL-C in CHO diet
Ferrara et al
(2000)[43]
Parallel, controlled,
randomized, blind trial-3
months
49 individuals at
high-CVD-risk
TMD+VOO vs TMD+nuts
vs Control diet
WB Monocytes
CD36, TFPI, COX2, LRP1
in TMD+nuts
MCP1 and COX2 in
TMD+VOO
↓ SBP, ↓ plasma glucose, ↓
LDL ,
↓ cholesterol/ HDL
↑ HDL in TMD+VOO
Perona et al
(2004) [42]
Randomized, crossover
intervention-4 weeks
20 healthy young
males
Diets rich in SFAvs CHOrich vs Mediterranean diet
In vitro incubation of
HUVEC with
participants’ oxLDL
--------------------------------------
VCAM-1 and E-selectin
after Mediterranean and
CHO-diets
↑ LDL lag time after Med diet
Bellido et al
(2006) [61]
Postprandial linear
intervention
6 healthy individuals
Single dose VOO
consumption (50ml)
PBMNCs
Metabolism, primary metabolism,
regulation of biological process
genes etc
Biosynthesis, response to
biotic stimulus, defense
response, response to stress
etc
Postprandial linear
intervention
11 healthy
individuals
Single dose VOO
consumption (50ml)
PBMNCs
CD36, ADAM17, ADRB2, LIAS,
PPARBP at 1h postprandial
ALOX5AP,OGT at 6h
postprandial
OGT, ALOX5AP, CD36 at
6h postprandial
↑ insulin, glucose (1h)
↑ LDLox and TBARS (6h)
Konstantinidou
et al (2009)[66]
Parallel, controlled-3 weeks
10 healthy
individuals
VOO consumption
(25ml/day)
PBMNCs
ADAM17, ALDH1A1, BIRC1,
ERCC5, LIAS, OGT,
PPARBP,TNFSF10, USP48,
XRCC5
------------------------------------
--------------------------------
Khymenets et al
(2009)[67]
Parallel controlled-feeding
trial-8 weeks
20 individuals at risk
of metabolic
syndrome
SFA-rich diet vs MUFArich diet
Plasma and
adipose tissue
Inflammation-related genes in
SFA diet, anti- inflammatory in
MUFA diet
------------------------------------
↓ serum total cholesterol and
LDL-C, and
↑ in plasma oleic acid after
MUFA diet
van Dijk et al
(2009) [59]
Randomized, double-blind,
parallel intervention-16
weeks
81 healthy
postmenopausal
women
CLA-rich diet vs OO-rich
diet
Adipose tissue
TNFa in CLA-rich diet
GLUT4, LPL, and Leptin in
CLA-rich diet
↓ Body fat mass and
↑ Serum insulin in the CLArich diet
Raff et al
(2009)[60]
Randomized, double-blind
trial-26 weeks
111 elderly
individuals
EPA+DHA vs HOSF
capsule supplements
PBMNCs
Inflammation and atherogenicrelated genes after EPA+DHA
Inflammation and
atherogenic-related genes
after EPA+DHA
--------------------------------
Bouwens et al
(2009) [54]
Postprandial, randomized,
single-blind, crossover
21 healthy males
Consumption of a shake
enriched with PUFA,
MUFA or SFA
PBMNCs
--------------------------------------
LXR signaling genes after
PUFA
Cellular stress response
genes after PUFA
↑ triglycerides
↑ plasma DHA after PUFA
↓ cholesterol after MUFA
Bouwens et al
[53] (2010)
Randomized, parallel,
double-blind-3 months
90 healthy
individuals
TMD+VOO vs
TMD+WOO (25ml/day) vs
control
PBMNCs
--------------------------------------
ADRB2, ARHGAP15,
IFNγ and IL7R in
TMD+VOO
POLK in TMD
↓ in total cholesterol, HDL-C,
LDL-C, IFNγ, sP-selectin, F2aisoprostanes in TMD+VOO
Konstantinidou
et al 2010 [68]
Konstantinidou
et al (2009) [65]
CHO, low fat, high carbohydrates diet; CLA, conjugated linoleic acids; DHA, docosahexaenoic acid; HOSF, high-oleic acid sunflower oil; HUVECs, human umbilical endothelial cells; EPA, eicosapentaenoic acid; EVOO, extra virgin olive oil; FASN, fatty acid synthase; GLUT4, glucose transporter 4; LDL-C, low density lipoprotein cholesterol; LDL-ox, oxidized
LDL; LPL, lipoprotein lipase; OO, olive oil; PBMNCs, peripheral blood mononuclear cells; PC, phenolic compounds; SBP, systoli c blood pressure; TMD, Traditional Mediterrranean Diet; WB, whole blood; WOO, washed olive oil; TG, triglycerides; VOO, virgin o live oil; ↓ , decrease; ↑, increase
Olive oil and Inflammation
LDL induction of monocyte adhesion to endothelial cells was
lower after MUFA consumption than after those of SFA or PUFA in
healthy individuals1
Isolated human LDL enriched in oleic acid reduced monocyte
adhesion (77%), compared with linoleic-enriched LDL, when
exposed to oxidative stress 2
A decrease in the expression of ICAM-1 by PBMC was
observed in healthy subjects consuming an oleic acid-rich diet
during two months.3
1Mata P et al. Arterioscler Thromb Vasc Biol 1996; 2 Tsimikas et al. Arterioscler Thromb Vasc Biol 1999;
3Yaqoob
P et al Am J Clin Nutr 1998.
RADICAL LIBRE:
Especie química definida que tiene en su estructura uno o más
electrones desapareados
 inestable
 fugaz
 reacciones en cadena
Especies
reactivas
de oxígeno
Ballester M, Med Clin (Barc) 1996; 107: 509-15
RADICALES
LIBRES
Metales
de
transición
Compuestos antioxidantes según su modo de actuación:
Primarios: impiden la formación de radicales libres
(quelantes de metales de transición)
Secundarios: interrumpen la reacción de propagación por inactivación (como el alfa-tocoferol y
el ácido ascórbico) o desplazan a las especies reactivas de oxígeno (como el ácido ascórbico,
carotenoides, glutation y la mayoría de enzimas antioxidantes)
Terciarios: reparan el daño causado a las moléculas o eliminan aquellas que se han estropeado
Antioxidantes de membranas:
Vitamina E: inactiva la cadena de lipoperoxidación. Su protección antioxidante es esencial en las
membranas lipídicas. Al igual que la vitamina C, al actuar origina un radical de alta estabilidad y
baja agresividad y por tanto inocuo.
Beta-caroteno: desplaza las especies reactivas de oxígeno
Coenzima Q: desplaza radicales radicales peroxilo. Su forma reducida es la ubiquinona.
Compuestos fenólicos: quelantes de metales de transición y desplaza radicales libres
Gutteridge; Clin Chem 1995
Antioxidantes intracelulares:
Enzimas antioxidantes endógenas
Glutation: cuando se oxida el GSH se produce el radical tiilo (G-S.), que es muy estable y se dimerizan por un
enlace disulfuro (GSSG)
Antioxidantes extracelulares:
Enzimas antioxidantes endógenas y glutation
Ceruloplasmina: capta los iones de cobre
Transferrina: capta los iones de hierro
Lactoferrina: capta los iones de hierro a pH bajo
Haptoglobulina: capta hemoglobina libre, formando un complejo irreversible
Hemopexina:capta grupos hemo
Albumina: capta cobre, grupos hemo, y desplaza HOCl
Bilirrubina: desplaza radicales peróxilo
Ácido úrico: capta metales y desplaza radicales libres
Ácido ascórbico: desplaza radicales hidroxilo
{Gutteridge; Clin Chem 1995}
Presentación clínica de la enfermedad cardiovascular
(Angina de pecho, infarto de miocardio o cerebral, …)
Formas asintomáticas de enfermedad cardiovascular
(Estenosis, calcificación, poca capacidad
de dilatación de las arterias)
Factores de riesgo cardiovascular
(Colesterol, hipertensión, diabetes, obesidad,…)
Características genéticas
individuales
Estilo y forma de vida:
-Práctica de actividad física
-Dieta
-Consumo de tabaco
-Contaminación ambiental
conc. ng/mL
Concentration vs Time curves for phenolic compounds in the 3 treatments (dose 25 mL) (n=12)
conc. ng/mL
HPC, 486 mg/kg
Tyrosol
MPC, 133 mg/Kg
LPC, 10 mg/kg
10
0
0
4
8
12
Time (hours)
16
20
24
20
Hydroxytyrosol
10
0
conc. ng/mL
Olive oil
20
6
5
4
3
2
1
0
4
8
12
Time (hours)
16
20
24
3-0-methyl-hydroxytyrosol
0
4
8
12
Time (hours)
16
20
24
 No es van trobar diferències en l’estat basal entre els tres grups d’intervenció:
a/ Per valors d’energia, macronutrients, o els principals compostos antioxidants (vit
E, beta-carotè...) o pro-oxidants (ferro).
b/ Pels marcadors analitzats amb l’excepció dels anticosos anti-LDL oxidada en
l’ordre 1 i 8-dGuo en l’ordre 3 (P<0.05).
 No es va detectar efecte de arrossegament (carryover), avaluat com l’interacció
del tractament amb el període, en cap dels marcadors analitzats.
 No es va detectar canvis en el gast energètic en activitat física en el temps de
lleure, desde el principi de l’estudi fins al final (mitjanes, 282 vs 275 kcal/dia)
 La dieta va ésser similar entre els tres grups durant cada intervenció.
Proliferació de cèl.lules musculars llises
Inestabilitat de placa ateroscleròtica
Aterotrombosis:
un proceso generalizado y progresivo
fisura/ruptura
trombosis de
de la placa
normal
estrías
grasas
placa
fibrosa
placa
ateroasclerosa
Angina
inestable
IAM
Sd
coronario
agudo
AVC isquémico
/ ACVT
clínicamente silente
Angina estable
Claudicación intermitente
Edad creciente
Isquemia crítica
de la EEII
Muerte cardiovascular
Statistical analyses
•Normality of continuous variables was assessed by normal probability plots.
•One-factor ANOVA and Kruskal-Wallis test were used to determine differences in basal characteristics
and nutrient intake among the three olive oil interventions.
•A general linear model for repeated measurements was used, with multiple paired comparisons
corrected by Tukey s method, in order to assess differences among post-intervention values adjusted
by baseline values.
•The paired comparison of target concentrations post-intervention was carried out by a General Linear
Mixed Model (GLMM) with :
•Random effect: individual level of test subjects
•Fixed factor: the olive oil phenolic dose (high, medium, low) administered
•Covariates: basal values for each intervention period, olive oil administration order, age,
difference of fat and carbohydrate intake from baseline.
•An additional model with adjustment for 3-O-methyl, hydroxytyrosol levels was also fitted
Statistical significance was defined as P < 0.05 for a two-sided test. Analyses were performed using the
SAS System for Windows release 8.02.
Basal characteristics, glucose, lipid profile, and oxidative stress biomarkers at the beginning of the study by
subgroups of order of olive oil administration according to an intent-to treat analyis
Order 1
(n=67)
Order 2
(n=68)
Order 3
(n=65)
33.4 (11.2)
34.3 (11.0)
31.9 (10.8)
BMI (Kg/m2)
23.7 (2.8)
23.8 (2.5)
24.0 (3.2)
Physical activity (kcal/day)
312 (250)
294 (248)
288 (207)
Systolic blood pressure (mmHg)
125 (14.4)
125 (11.1)
123 (12.7)
77 (7.6)
78 (8.2)
76 (8.5)
Total cholesterol (mmol/L)
4.84 (0.96)
4.77 (1.06)
4.61 (1.09)
LDL cholesterol (mmol/L)
3.11(0.93)
3.08 (0.93)
2.95 (0.98)
HDL cholesterol (mmol/L)
46.9 (11.3)
46.4 (10.3)
48.5 (11.9)
Triglycerides (mmol/L)
102 (53)
1.2 (0.4)
1.0 (0.5)
Glucose (mmolL)
85 (9.4)
86 (10.4)
86 (9.4)
Oxidized LDL (U/L)
51 (26)
49 (20)
48 (22)
787 (120)*
1104 (153)
1092 (149)
Hydroxyfatty acids ( mol/L)
1.3 (0.33)
1.35 (0.46)
1.30 (0.57)
F2 -isoprostanes ( g/L)
30.6 (5.8)
Age (years)
Diastolic blood pressure
Antibodies against oxidized LDL (U/L)
Uninduced dienes ( mol/L)
*
11.3 (3.6)
29.1 (5.9)
11.4 (3.0)
20.9 (8.0)
31.6 (7.6)
12.1 (3.7)
17.6 (6.6)*
8-oxo-deoxyguanosine (nmol/24h)
22.4 (8.2)
8-oxo-guanosine (nmol/24h)
23.1 (9.5)
22.5 (8.4)
20.0 (8.6)
8-oxo-guanine (nmol/24h)
158 (100)
137 (84)
123 (92)
Values are men (SD). Order 1, High, medium , and low phenolic content olive oil; Order 2, medium, lo w, and high phenolic content olive o il; Order 3,low,
high, and medium phenolic content olive oil.. * P < 0.05 versus order 1 group.
Antioxidant status at the beginning of the study by subgroups of subjects depending on the order of olive oil
administration according to an intent-to treat analysis
Order 1
Order 2
Order 3
(n=61)
(n=63)
(n=58)
Superoxide dismutase (U/L)
142 (21)
144 (22)
140 (19)
Glutathione peroxidase (U/L)
719 (183)
686 (133)
692 (184)
64 (16)
64(16)
Endogenous
Glutathione reductase (U/L)
64 (17)
Reduced glutathione (GSH) ( mol/L)
4.71 (0.59)
4.53 (0.57)
4.61 (0.72)
Oxidized glutathione (GSSG) ( mol/L)
1.24 (0.12)
1.26 (0.12)
1.24 (0.12)
GSH/GSSG ratio
3.85 (0.62)
3.61 (0.57)
3.74 (0.71)
150 (95)
150 (90)
198 (142)
61 (26)
60 (23)
62 (23)
25.6 (5.7)
24.6 (6.3)
24.2 (6.8)
0.45 (0.39)
0.40 (0.28)
0.35 (0.25)
Lycopene ( mol/L)
0.46 (0.23)
0.42 (0.20)
0.43 (0.22)
Enterolactone (nmol/L)
16.8 (18.1)
22.6 (24.9)*
21.8 (36.6)
Enterodiol (nmol/L)
1.51 (5.30)
1.54 (4.13)
2.60 (9.6)
Paraoxonase
Exogenous
Ascorbic acid
-tocopherol ( mol/L)
-carotene ( mol/L)
Order 1, high, medium , and low phenolic content olive o il; Order 2, medium, lo w, and high phenolic content olive oil; Order 3,lo w, high, and medium phenolic
content olive oil.
Paired comparisons among values after olive oils interventions
Mean of Differences (SEM)
Variable
HDL Cholesterol (mg/dL)
HPC vs LPC
p
MPC vs LPC
p
HPC vs MPC
p
1.93 (0.77)
0.012
0.012
0.71 (0.66)
0.283
1.22 (0.64)
0.045
0.045
Uninduced dienes ( mol/L)
-0.060 (0.02)
0.013
-0.036 (0.02)
0.084
-0.024 (0.02)
0.218
Oxidized LDL (U/L)
-4.48 (1.73)
0.010
0.010
-2.96 (1.48)
0.046
0.046
-1.52 (1.43)
GLMM adjusted by basal values for each intervention period, olive oil administration order,center, and age
Results improved when difference of fat and carbohidrate from baseline are added to the model.
0.288
Table 4. Changes after olive oil interventions by centre
Variable
HDL cholesterol,
mg/dL
Low PC olive oil
Medium PC olive oil
High PC olive oil
Oxidized LDL, U/L
Low PC olive oil
Medium PC olive oil
High PC olive oil
Centre 1
(Barcelona)
Centre 2
(n =30)
(n =28)
(Copenhagen)
Centre 3
(Kuopio)
Center 4
(Bologna)
Center 5
(Postdam)
Center 6
(Berlin)
(n =30)
(n = 25)
(n = 38)
(n =32)
0.92
(-1.1 to 2.9)
1.38
(-0.08 to 2.8)
2.20
(0.58 to 3.8)
1.50
(-1.3 to 4.2)
3.63
(-0.7 to 7.9)
1.49
(-1.1 to 4.1)
0.93
(-1.1 to 2.9)
0.38
(-1.9 to 2.5)
1.73
(-0.2 to 3.7)
0.54
(-1.4 to 2.5)
0.11
(-2.2 to 2.4)
2.62
(0.2 to 5.0)
1.28
(-1.1 to 3.7)
1.38
(-0.6 to 3.3)
2.58
(0.6 to 4.5)
0.20
(-1.3 to 1.7)
0.52
(-1.3 to 2.3)
0.58
(-1.8 to 2.9)
3.38
(-2.6 to 9.4)
-1.62
(-6.2 to 3.0)
-3.10
(-6.9 to 0.8)
2.7
(-6.7 to 12)
-2.87
(-10.3to 4.6)
0.40
(-7.2 to 8.0)
-0.49
(-4.2 to 3.3)
-1.93
(-5.9 to 2.1)
-1.64
(-4.9 to 1.6)
2.30
(-4.6 to 8.7)
-0.84
(-10.0to 9.8)
-8.69
-5.53
(-11.8 to 0.7)
-2.12
(-6.6 to 2.4)
-8.75
(-15.8 to-2.2)
(-14.0 to –2.9)
2.26
(-2.7 to 7.2)
-2.0
(-5.7 to 1.6)
-2.28
(-5.7 to0.48 )
10
(109 to 106)
-3
(-127 to 134)
-22
(-116 to 71)
40
(-86 to 166)
-13
(-124to 150)
-114
(-247 to 19)
33
(-75 to 141)
-19
(-130 to 93)
-65
(-195 to 65)
-45
(-222to 132)
-165
-66
(-167 to 35)
14
(-87 to 114)
-14
(-85 to 56)
-130
(-273 to 13)
-69
(-189 to 30)
-76
(-174 to -4)
Hydroxy fatty
acids,* nmol/L
Low PC olive oil
Medium PC olive oil
High PC olive oil
(-299 to -31)
-109
(-223 to -9)
Difference in HDL (mmmol/L)
0.065
0.052
0.039
Threshold
0.026
0.01
0.00
LPC
MPC
HPC
Type of olive oil
A 0.026 mmol/L increase in circulating HDL cholesterol levels is associated with a decrease from 1 to
3.6% in cardiovascular mortality, and with a 3.7% reduction of the risk to develop acute myocardial
infarction (Stampfer MJ et al. JAMA 1996; 276: 882-8).
Olive oil and Inflammation
LDL induction of monocyte adhesion to endothelial cells was
lower after MUFA consumption than after those of SFA or PUFA in
healthy individuals1
Isolated human LDL enriched in oleic acid reduced monocyte
adhesion (77%), compared with linoleic-enriched LDL, when
exposed to oxidative stress 2
A decrease in the expression of ICAM-1 by PBMC was
observed in healthy subjects consuming an oleic acid-rich diet
during two months.3
1Mata P et al. Arterioscler Thromb Vasc Biol 1996; 2 Tsimikas et al. Arterioscler Thromb Vasc Biol 1999;
3Yaqoob
P et al Am J Clin Nutr 1998.
Inflammatory markers in CHD patients after olive oils administration (N=28, crossover)
Variable
CReactive
Protein
(mg/dL)
sIL-6
(pg/mL)
sICAM-1
(ng/mL)
sVCAM-1
(ng/mL)
Post ROO
Post VOO
Mean
(Low phenolic (High phenolic (95%CI)
content)
content)
difference
P for
diff.
P for
P
period intera
ction
0.329
(0.15 ;0.51)
0.279
(0.17 ;0.49)
-0.063
(-0.12 ; -0.007)
0.02
0.55 0.22
1.65
(0.92)
1.49
(0.41)
-0.166
(-0.26; -0.07)
0.002
0.75 0.98
423
(345 - 512)
402
(365 - 455)
-3.840
(-34; 26)
0.79
0.30 0.28
781
(441 - 1038)
711
(540 - 956)
-21.01
(-89; 47)
0.53
0.66 0.78
Fitó M et al. EJCN 2008
ROO, refined olive oil; VOO, virgin olive oil
Serum TXB2 ( g/mL) in hypercholesterolemic subjects administered
Virgin (EVOO, high phenolic content) or Refined (ROO, poor phenolic
content) Olive Oil for 49 days
EEVO
VOO
TXB2 ( g/ml serum)
0.27
0.25
a
ROO
ROO
0.23
0.21
0.19
0.17
0.15
T0
TXB2 ( g/ml serum)
0.29
T28
T49
WO
ROO
ROO
0.27
0.25
T 28
EVOO
VOO
T49
b
0.23
0.21
0.19
0.17
0.15
T0
T28
T49
WO
T28
T49
Serum TXB2 concentrations ( g/ml) in subjects administered EVOO or ROO for 49
days. Panel a, subjects were first given EVOO and then, after washout, ROO.
Panel b, subjects were first given ROO and then, after washout, EVOO. Data are
means ± S.D., n= 22. From Visioli et al, Eur J Nutr 2004 (on line)
Visioli F et al. Eur J Nutr 2004
La dieta mediterránea es rica en vegetales, fruta,
legumbres, otros alimentos procedentes de
plantas y se caracteriza por el consumo de
aceite de oliva virgen como principal aporte de
energía a través de la grasa
La susceptibilidad a la oxidación
de la LDL depende no solo del
contenido en ácidos grasos,
% of Total fat
Levels of oleic acid and antioxidants in LDL after sustained
(1 week, 25ml/day) doses of virgin olive oil
sino también del contenido en
vitamina E y compostos fenòlics
*
18
16
Oleic acid in LDL
14
12
10
g/mg protein
antioxidantes de la LDL com la
22
22
20
†
8.5
7.0
5.5
Vit E in LDL
4.0
(Fuller CJ, Jialal I. Am J Clin Nutr. 1994; 60:1010-3).
ng/mg protein
10
†
8.5
Phenolic compounds
in LDL
7.0
5.5
4.0
Baseline
Post-intervention
Gimeno et al. Eur J Clin Nutr 2002; 56:114-120
Chemical composition of olive oil
Unsaponifiable fraction
(about 2%)
Saponifiable fraction (98-99%)
(Main fatty acid present in triacylglicerols)
MUFA
• Oleic acid (18:1n-9) (55-83%)
• Lipophilic phenolics (tocopherols and tocotrienols)
• Hydrophilic phenolics (phenolic acids, phenolic alcohols,
secoiridoids, lignans and flavones)
PUFA
• Linolenic acid (18:3n-3) (0.0-1.5 %)
• Palmitoleic acid (18:3n-3) (7.5-20 %)
• Linoleic acid (18:2n-6) (3.5-21 %)
SFA
• Palmitic acid (16:0) (7.5-20 %)
• Miristic acid (14:0) (0-0.1 %)
• Estearic acid (18:0) (0.5-5 %)
Adapted from Escrich, E. et. al. (2007) Mol Nutr Food Res 51, 1279-1292
•
•
•
•
•
•
•
Volatile compounds
Pigments (chlorophylls)
Hydrocarbons (squalene, B-carotene, lycopene)
Sterols (B-sitosterol, campesterol, estigmasterol)
Triterpene alcohols
Aliphatic alcohols
Non-glyceride esters (alcoholic and sterol compounds, waxes)

olivas - PIFRECV

Transcription

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