AJCN. First published ahead of print September 16, 2009 as doi: 10.3945/ajcn.2009.27718.
Alcohol consumption is associated with high concentrations of urinary hydroxytyrosol1–4 Helmut Schro¨der, Rafael de la Torre, Ramo´n Estruch, Dolores Corella, Miguel Angel Martı´nez-Gonza´lez, Jordi Salas-Salvado´, Emilio Ros, Fernando Aro´s, Gemma Flores, Ester Civit, Magı´ Farre´, Miguel Fiol, Joan Vila, Joaquı´n Fernandez-Crehuet, Valentina Ruiz-Gutie´rrez, Jose Lapetra, Guillermo Sa´ez, and Marı´a-Isabel Covas for the PREDIMED Study Investigators ABSTRACT Background: Previously, we reported the presence of hydroxytyrosol in red wine and higher human urinary recovery of total hydroxytyrosol than that expected after a single red wine intake. We hypothesized that the alcohol present in wine could promote endogenous hydroxytyrosol generation. Objective: The objective was to assess the relation between alcohol consumption and urinary hydroxytyrosol concentrations. Design: This was a cross-sectional study with baseline data from a subsample of the PREvencio´n con DIeta MEDiterra´nea (PREDIMED) trial, an intervention study directed at testing the efficacy of the Mediterranean diet on the primary prevention of cardiovascular disease. Participants included 1045 subjects, aged 55–80 y, who were at high cardiovascular risk. Alcohol consumption was estimated through a validated food-frequency questionnaire. Urinary hydroxytyrosol and ethyl glucuronide, a biomarker of alcohol consumption, were measured. Results: Urinary ethyl glucuronide concentrations were directly related to alcohol and wine consumption (P , 0.001) as well as to urinary hydroxytyrosol in both sexes (P , 0.001). The degree of alcohol consumption was directly associated with urinary hydroxytyrosol in male alcohol consumers (P , 0.001). Multivariate logistic regression analyses showed a significant linear trend (P , 0.05) for elevated hydroxytyrosol concentrations with an increase in alcohol consumption. Intakes of .20 g (2 drinks)/d and .10 g (1 drink)/d alcohol in men and women, respectively, were associated (P , 0.05) with elevated concentrations of hydroxytyrosol. Conclusions: We report for the first time a direct association between urinary hydroxytyrosol and alcohol consumption at a population level. These findings reinforce previous work in human and animal models that examines wine as a source of hydroxytyrosol and alcohol as an indirect promoter of endogenous hydroxytyrosol generation. This trial was registered at controlled-trials.com/isrctn/ as ISRCTN 35739639. Am J Clin Nutr doi: 10.3945/ajcn.2009.27718.
INTRODUCTION
The health benefits of olive oil in humans have been attributed partially to olive oil’s unique composition of polyphenols (1). Polyphenols are found in olive fruits and leaves, mainly in the form of secoiridoids (2). A distinctive characteristic of olive oil is its enrichment in oleuropein, a member of the secoiridoid family, which hydrolyzes in the intestinal tract to catechol hydroxytyr-
osol, which has shown high antioxidant properties in experimental studies (3, 4). The presence of hydroxytyrosol in food was considered to be restricted to olive oil. Recently, we reported the presence of hydroxytyrosol in red wine (5). We also reported that the urinary recovery of total hydroxytyrosol in healthy volunteers after a single dose (250 mL) of red wine was higher than expected, taking into account the dose administered. This recovery was also higher than that observed after a single dose of virgin olive oil (25 mL), despite a 5-fold higher hydroxytyrosol dose in the olive oil than in the wine. Hydroxytyrosol is a minor dopamine metabolite also known in the neurochemistry nomenclature as 3,41 From the CIBER Fisiopatologı´a Obesidad y Nutricio´n (CIBEROBN), Instituto de Salud Carlos III, Santiago de Compostela, Spain (HS, RdlT, RE, DC, JS-S, ER, GF, MF, JL, and M-IC); the Cardiovascular Risk and Nutrition Research (HS, JV, and M-IC) and the Human Pharmacology and Neurosciences Research Group (RdlT, EC, and MF), Institut Municipal d´Investigacio´ Me`dica (IMIM-Hospital del Mar), Barcelona, Spain; the Department of Internal Medicine (RE) and Lipid Clinic, Endocrinology and Nutrition Service (ER), Hospital Clinic, Institut d´Investigacions Biome`diques August Pi Sunyer-IDIBAPS, Barcelona, Spain; the Departments of Preventive Medicine and Public Health (DC) and Biochemistry and Molecular Biology (GS), University of Valencia, Valencia, Spain; the Department of Preventive Medicine and Public Health, University of Navarra, Pamplona, Spain (MAM-G); the Human Nutrition Unit, School of Medicine, University Rovira i Virgili, Reus, Spain (JS-S); the Department of Cardiology, Hospital Txangorritxu, Vitoria, Spain (FA); the Primary Health Care Division, Barcelona, Spain (GF); the Institute for Health Sciences Investigation, Palma de Mallorca, Spain (MF); the Department of Epidemiology, School of Medicine of Malaga, Malaga, Spain (JF-C); the Instituto de la Grasa, CSIC, Seville, Spain (VR-G); and the Department of Family Medicine, Primary Care Division of Seville, San Pablo Health Center, Seville, Spain (JL). 2 HS and RdlT contributed equally to the study, and each can be regarded as first authors of this article. 3 Supported by the Spanish Network RD06/0045/0000 (ISCIII) and Sistema Nacional de Salud contracts (CP 03/00115 and CP06/00100) from Instituto de Salud Carlos III, and partially supported by the Generalitat of Catalunya (2005 SGR 00577). The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. 4 Address correspondence to M-I Covas, Cardiovascular Risk and Nutrition Research Group, Institut Municipal d’Investigacio´ Me`dica (IMIMHospital del Mar), Parc de Recerca Biome`dica de Barcelona (PRBB), Carrer Dr. Aiguader 88, 08003 Barcelona, Spain. E-mail:
[email protected]. Received March 5, 2009. Accepted for publication August 4, 2009. doi: 10.3945/ajcn.2009.27718.
Am J Clin Nutr doi: 10.3945/ajcn.2009.27718. Printed in USA. Ó 2009 American Society for Nutrition
Copyright (C) 2009 by the American Society for Nutrition
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dihydroxyphenylethanol (DOPET). Dopamine can be formed from tyramine (6). A rise in dopamine body concentrations through its formation from the tyramine present in red wine was investigated, but urinary tyramine concentrations did not explain those of hydroxytyrosol obtained (5). However, the urinary concentration of homovanillic acid concentrations, a biomarker of the peripheral turnover of dopamine, was largely elevated. Because of this finding, we hypothesized that the alcohol present in red wine could promote endogenous hydroxytyrosol generation via an interaction with dopamine metabolism (5). Plasma and urinary hydroxytyrosol have been shown to increase in a dose-dependent manner with the phenolic content of the food (eg, olive oil) administered (7–10). Thus, if wine contains hydroxytyrosol, and alcohol could be capable of promoting an endogenous hydroxytyrosol generation, a direct relation between the degree of both alcohol and wine consumption and the concentrations of hydroxytyrosol in biological fluids would be expected. In the present work we tested this hypothesis at a population level in a large sample of individuals at high risk of coronary artery disease (CAD).
SUBJECTS AND METHODS
Study participants A cross-sectional study with baseline data from a subsample of participants in the PREDIMED (PREvencio´n con DIeta MEDiterra´nea) study was performed. The PREDIMED study is a large, parallel-group, multicenter, randomized, controlled 5-y clinical trial aimed at assessing the effects of the traditional Mediterranean diet on the primary prevention of cardiovascular disease (www.predimed.org). The detailed protocol of the study has been published previously (11). The Institutional Review Boards of the institutions involved approved the study protocol, and participants signed an informed consent. From October 2003 to January 2005, 1130 asymptomatic subjects at high risk of CAD, aged 55–80 y, were selected in 10 Spanish primary care centers. Inclusion criteria were the presence of diabetes or having 3 CAD risk factors [current smoking, systolic blood pressure .140 mm Hg, diastolic blood pressure .90 mm Hg, treatment with antihypertensive drugs, HDL cholesterol ,40 mg/dL in men and ,50 mg/dL in women, LDL cholesterol .160 mg/dL, treatment with hypolipidemic drugs, body mass index (BMI; in kg/m2) .25, family history of premature CAD]. Exclusion criteria were history of cardiovascular disease, any severe chronic illness, drug or alcohol addiction, history of allergy or intolerance to olive oil or nuts, or low predicted likelihood of changing dietary habits according to the stages of change model. Participants’ eligibility was based on a screening visit by the physician. Baseline assessments The baseline examination included the administration of the following: 1) a validated food-frequency questionnaire (12); energy and nutrient intake calculated from Spanish food composition tables (13); 2) the Minnesota Leisure Time Physical Activity Questionnaire, validated for its use in Spanish men and women (14, 15); and 3) a 47-item general questionnaire assessing lifestyle, health conditions, smoking habits, socio-
demographic variables, history of illness, and medication use. Food intake and alcoholic beverage consumption, including wine, beer, and spirits, were assessed through the food-frequency questionnaire (12). Alcohol intake (g/d) was calculated by multiplying the amount of the beverage (mL) by the respective grade (% alcohol) and the constant 0.80 to transform alcohol volumes into weight. We undertook all clinical procedures in accordance with the PREDIMED study manual of operations. Weight and height were measured with calibrated scales and a wall-mounted stadiometer, respectively. BMI was calculated as weight in kilograms divided by the square of height in meters. Waist circumference was measured with an anthropometric tape midway between the lowest rib and the iliac crest. Trained personnel measured blood pressure in triplicate with a validated semiautomatic sphygmomanometer (Omron HEM-705CP; Hoofddorp, Netherlands), with participants in a seated position after a 5-min rest. Urinary hydroxytyrosol and ethyl glucuronide analyses First spot morning urine was obtained at home, after an overnight fast, following specific instructions for its collection (same as those given for routine laboratory analyses). Samples were coded, shipped to central laboratories, and frozen at 280°C until assay. Hydroxytyrosol was measured by gas chromatography–mass spectrometry. The analytic procedure has been published previously (16, 17). Control samples that were added to every analytic batch at concentrations of 10 and 35 ng/mL had CVs of 7.5% and 4.6%, respectively. Determination of urinary ethyl glucuronide (EtG) was made by using an enzyme immunochemical method for EtG (DRIEtG EIA; ThermoFisher/Microgenics, Fermont, CA) (18). The calibration concentration range of this methodology is between 100 and 2000 ng/mL. Samples with EtG concentrations outside the calibration range were considered either negative (if ,100 ng/mL) or diluted accordingly (if .2000 ng/mL). Control samples that were added to every analytic batch at concentrations of 375 ng/mL and 625 ng/mL had CVs of 5.0% and 3.6%, respectively. Inflammatory markers Serum interleukin-6 was measured by a standard enzymelinked immunosorbent assay, and high-sensitivity C-reactive protein by particle-enhanced immunonephelometry (11), in a subsample of 301 participants. Statistical analysis Differences in continuous variables were compared by using the Student’s t or Mann-Whitney U tests when indicated. The general linear modeling procedures (PROC GLM, version 9.1; SAS Institute Inc, Cary, N.C.) were used to estimate lifestyle, anthropometric, and socioeconomic variables according to the categories of alcohol consumption. Linear trend was tested by including the categorized variable as continuous in this model. The polynomial contrast was used to determine P values for linear trend for continuous variables. Spearman regression coefficients were calculated to assess the association between 1) self-reported alcohol consumption and the biomarker of alcohol consumption, namely, urinary EtG, and 2) urinary hydroxytyrosol concentrations and serum inflammatory markers. Weighted
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j analysis was performed to assess the agreement between categories of alcohol consumption and categories of urinary EtG concentrations. Linear regression models, adjusted for age, leisuretime physical activity, educational level, smoking, BMI, and olive oil consumption, were fitted (PROC REG; SAS Institute Inc) to determine the magnitude and strength of the association of alcohol consumption with urinary hydroxytyrosol concentrations in alcohol consumers. Multiple logistic regression analysis (PROC LOGISTIC procedure of SAS; SAS Institute Inc) was used to analyze the relation of elevated urinary hydroxytyrosol concentrations (defined as higher than the median value of the distribution values in each sex) with categories of alcohol consumption in the whole population. Linear trend was tested by including the categorized variable (categories of alcohol consumption) as continuous in this model. Differences were considered significant if P , 0.05. Values were expressed as means 6 SEMs or 95% CIs. RESULTS
Men were younger than women; spent more time in leisure-time physical activity; had a lower BMI, more education, and a higher dietary energy intake; and consumed more alcohol and olive oil than did women. Men also had higher urinary concentrations of hydroxytyrosol and EtG than did women (Table 1). Characteristics of the study participants according to alcohol consumption categories are provided in Table 2. Energy intake was positively associated with alcohol consumption in both sexes. The proportion of smokers and those with a higher educational level increased with higher alcohol consumption in women (Table 2). An inverse relation between urinary hydroxytyrosol and serum high-sensitivity C-reactive protein (r = 20.134, P = 0.020) and interleukin-6 (r = 20.128, P = 0.026) was observed. Urinary EtG concentration correlated significantly with the reported total alcohol consumption and alcohol from wine (Table 3). Weighted j analysis revealed good agreement (P = 0.435) between categories of alcohol consumption per day [1 = 0 g/d (0 drink/d), 2 = 0.1–20 g/d (1–2 drinks/d), and 3 = .20 g/d (.2 drinks/d)] and categories of urinary EtG (1 = 0–100, 2 = 101– 500, and 3 = 501–2499 ng/mL). These results indicate a valid
estimation of alcohol consumption by means of the food-frequency questionnaire in the present population. A direct relation was observed between the urinary concentrations of EtG and hydroxytyrosol in both sexes. Multiple linear regression models were fitted to analyze the association between alcohol consumption and urinary concentrations of hydroxytyrosol (Table 4). Because of the skewed distribution of alcohol consumption, we excluded alcohol abstainers from these analyses. The average (median) amount of alcohol consumption among alcohol consumers was 15.9 g/d in men and 4.2 g/d in women. The age-adjusted models revealed a significant direct association (P , 0.001) between alcohol consumption and urinary hydroxytyrosol in men. The strength of the association was not attenuated when leisure-time physical activity, educational level, smoking, BMI, and olive oil consumption were controlled for. No associations were shown in women. There were no significant interactions between alcohol consumption and smoking in either sex. Multivariate logistic regression analysis was performed to assess the association between alcohol consumption and urinary hydroxytyrosol in the entire study population (Table 5). For this purpose, we categorized alcohol consumption as follows: 1) no alcohol consumption (reference category), 2) 10 g alcohol/d, 3) .10 g and 20 g alcohol/d, and 4) .20 g alcohol/d. Because only 3.7% of female participants reported alcohol consumption .20 g/d, we defined the highest category of alcohol consumption in women as .10 g/d. A significant linear trend for having elevated urinary hydroxytyrosol concentrations was observed in both sexes with the increase in alcohol consumption (P , 0.05). Alcohol consumption of .20 and .10 g/d was significantly associated with elevated urinary HT in men and women, respectively, even after potential confounders were controlled for. In women, the odds of having elevated hydroxytyrosol concentrations were 183% higher among the highest alcohol consumers than in alcohol abstainers. DISCUSSION
From a previous hypothesis concerning endogenous hydroxytyrosol generation after alcohol consumption via dopamine
TABLE 1 Characteristics of the study population1
Age (y) Current smokers (%) Body mass index (kg/m2) Educational level (%)3 Leisure-time physical activity (METs) Hydroxytyrosol (ng/mL) Ethyl glucuronide (ng/mL)5 Energy (kcal) Total alcohol consumption (g/d) Wine consumption (g alcohol/d) Other alcoholic beverages (g alcohol/d) Olive oil (mL) 1 2 3 4 5
Men (n = 506)
Women (n = 533)
P value
65.6 6 6.5 31.7 29.0 6 3.1 30.4 281 (152, 467)4 142.4 (67.1, 289.4) 239.5 (58.0, 828.0) 2479 6 647 11.8 (2.9, 28.7) 10.0 (0.7, 25.0) 2.4 (0.7, 6.1) 42.7 6 18.1
68.3 6 5.7 4.7 30.0 6 3.7 14.2 168 (76, 290) 83.7 (46.8, 161.1) 55.0 (17.0, 111.5) 2101 6 557 0.0 (0.0, 2.1) 0.0 (0.0, 0.7) 0.0 (0.0, 0.7) 40.3 6 16.6
,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.023
2
METs, metabolic equivalent tasks. Mean 6 SD (all such values). More than secondary school. Median; interquartile range in parentheses (all such values). Subsample of 350 men and 421 women.
0.016 0.841 0.005 0.001 0.970 ,0.001 0.008 (65.9, 68.6) (28.9, 30.7) (5.5, 15.7) (18.6, 35.2) (164, 243) (9.3, 10.4) (41.6, 49.5) (66.5, 68.2) (29.8, 31.0) (2.8, 9.2) (9.9, 20.7) (167, 217) (8.5, 9.2) (37.0, 42.1) 67.4 30.4 6.0 15.3 192 8.9 39.6 (68.5, 69.8) (29.5, 30.3) (0.3, 5.1) (6.7, 14.7) (186, 223) (8.2, 8.8) (37.7, 41.5) 69.1 29.9 2.7 10.7 205 8.5 39.6 0.049 0.235 0.094 0.337 0.950 ,0.001 0.158 (64.2, 66.0) (28.9, 29.3) (30.2, 42.9) (24.8, 37.4) (315, 389) (10.8, 11.5) (42.4, 47.3) 65.1 29.3 36.5 31.1 352 11.2 44.9 66.2 28.3 31.0 31.4 325 10.3 39.7 Age (y) BMI (kg/m2) Current smokers (%) Education3 (%) LTPA (METs min21 d21) Energy (MJ) Olive oil (mL)
67.3 29.8 30.5 17.5 348 9.1 40.1
(65.6, 69.0) (29.0, 30.5) (18.6, 42.4) (5.6, 29.5) (279, 418) (8.4, 9.7) (35.5, 44.7)
(64.5, 66.6) (28.2, 29.2) (19.1, 34.3) (26.8, 42.4) (293, 375) (9.3, 10.2) (39.5, 45.3) 65.6 28.7 26.7 34.6 328 9.8 42.4
(64.8, 67.5) (27.7, 29.0) (21.2, 40.9) (21.7, 41.1) (267, 382) (9.8, 10.9) (35.9, 43.4)
P value2 .20 g (n = 211) .10 and 20 g (n = 88) .0 and 10 g (n = 146) 0 g (n = 60)
Men
67.3 29.8 10.6 26.9 204 9.8 45.5
P value2 .10 g (n = 67) .0 and 10 g (n = 167) 0 g (n = 299)
Women Alcohol consumption TABLE 2 Characteristics of the study participants according to alcohol consumption categories1
1 All values are means; 95% CIs in parentheses. METs, metabolic equivalent tasks; LTPA, leisure-time physical activity. ANOVA was used to estimate variables according to categories of alcohol consumption. 2 For linear trend. 3 More than basic education.
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TABLE 3 Spearman correlation coefficients for concentration of ethyl glucuronide (EtG; ng/mL) with alcohol consumption and urinary hydroxytyrosol in men and women1
Alcohol (g/d) Total P value Wine P value Other beverages P value Hydroxytyrosol (ng/mL) P value
Men (n = 350)
Women (n = 421)
0.600 ,0.001 0.585 ,0.001 0.442 ,0.001 0.202 ,0.001
0.348 ,0.001 0.358 ,0.001 0.300 ,0.001 0.111 0.023
1 Participants with .2500 ng/mL of urinary EtG were excluded from the analysis.
metabolism, we assessed in this study the association between alcohol consumption and urinary hydroxytyrosol concentrations at a population level. From our results, it appears that a direct relation exists between degree of alcohol consumption and urinary hydroxytyrosol concentrations in older men and women, independent of the amount of olive oil consumed. This relation was reinforced by the direct association observed between hydroxytyrosol and EtG urinary concentrations. Alcohol consumption was validated through urinary EtG, which is a reliable marker (19). Concerning alcohol drinkers, low alcohol consumption could explain the lack of association between alcohol intake and hydroxytyrosol in women. Also, we could not discard the possibility that a threshold of alcohol ingestion could exist for triggering the dopaminergic response leading to hydroxytyrosol formation. Hydroxytyrosol was, however, associated with EtG not only in men but also in women. This indicates that a relation between low alcohol intake and low concentrations of hydroxytyrosol in women could be detected by more precise measurements, such as biochemical determinations, rather than by self-reported alcohol consumption provided by questionnaires. The results of the present study are in agreement with our previous report (5) and support the hypothesis of alcohol, and particularly wine, as an indirect source of hydroxytyrosol. TABLE 4 Multiple linear regression analysis results showing the association of hydroxytyrosol with alcohol consumption in men (n = 446) and women (n = 234) who consume alcohol Hydroxytyrosol Regression coefficient1 Men Model Model Women Model Model 1
95% CI
P value
12 23
0.047 0.047
0.026, 0.069 0.023, 0.070
,0.001 ,0.001
12 23
0.005 0.005
20.063, 0.073 20.066, 0.077
0.889 0.887
Log transformed according to a 10-g increment of alcohol. Adjusted for age. 3 Adjusted for age, leisure-time physical activity, educational level, smoking, BMI, and olive oil consumption. 2
ALCOHOL CONSUMPTION AND URINARY HYDROXYTYROSOL TABLE 5 Odds ratios (ORs) and 95% CIs of elevated urinary hydroxytyrosol concentration according to categories of alcohol consumption1 OR (95% CI)2 Alcohol consumption Men 0 g/d .0 10 g/d .10 20 g/d .20 g/d Linear P for trend Women 0 g/d .0 10 g/d .10 g/d Linear P for trend
n
Model 13
Model 24
60 146 88 210
1 (Reference) 1.41 (0.75, 0.64) 2.01 (1.0, 3.96) 3.09 (1.6, 5.68) ,0.001
1 (Reference) 1.46 (0.7, 2.87) 1.84 (0.8, 3.81) 2.77 (1.4, 5.28) ,0.001
299 167 67
1 (Reference) 1.25 (0.8, 1.84) 1.93 (1.1, 3.34) 0.017
1 (Reference) 1.24 (0.8, 1.86) 1.83 (1.0, 3.25) 0.037
Hydroxytyrosol concentrations were .142.45 and .83.67 ng/mL in men and women, respectively. 2 ORs were determined by using logistic regression analysis. 3 Adjusted for age. 4 Adjusted for age, smoking, educational level, leisure-time physical activity, BMI, and olive oil consumption. 1
In addition to the hydroxytyrosol present in wine, a physiologic route for endogenous hydroxytyrosol production from alcohol consumption would be via dopamine metabolism. The deamination of biogenic amines to their corresponding aldehyde intermediates is catalyzed by the monoamine oxidase isoenzymes. Aldehyde intermediates are chemically very unstable and give rise metabolically to their corresponding acids or alcohols. In the case of dopamine, the aldehyde intermediate is known as 3,4-dihydroxyphenylacetaldehyde (DOPAL), whereas 3,4-dihydroxyphenylacetic acid (DOPAC) and DOPET (or hydroxytyrosol) are the corresponding acid and alcohol metabolites (20). Concentrations of DOPAC are relatively high in biological fluids and have been used as biomarkers of dopamine turnover, whereas those of DOPET are very low, and little attention has been paid to this minor compound of the oxidative metabolism of dopamine. The oxidation and reduction of aldehydes require a pyridine nucleotide coenzyme system. Alterations in the cellular ratio of NAD to NADH may result in alterations in the aldehyde metabolism and in the balance between acid (DOPAC) and alcohol (DOPET) metabolites of DOPAL. Ethanol is able to alter the NAD-to-NADH ratio, enhancing the formation of alcohol derivatives in humans. This has already been observed for serotonin metabolism, where the corresponding alcohol metabolite, 5-hydroxytryptophol, becomes more relevant than 5-hydroxyindolacetic acid (21) after ethanol intake, to the extent that 5-hydroxytryptophol has been considered as a biomarker of its consumption (22). In an experiment performed in rats, results for dopamine analogous to those seen for serotonin were reported (23). In short, ethanol alters dopamine metabolism, and the final product is no longer predominantly DOPAC but a mixture of DOPAC and DOPET. In the absence of ethanol, the ratio of DOPAC to DOPET is ’10, whereas in the presence of ethanol it is 0.25 (23). These observations in an animal model support the data obtained in the present study in humans, although the exact mechanism of the shift of the aldehyde metabolism in the presence of ethanol is not well understood and deserves further study. Therefore,
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hydroxytyrosol, in addition to being a natural antioxidant, is a metabolite of dopamine (DOPET), with an up-regulated synthesis in the presence of ethanol (5, 23). There is a large body of knowledge concerning the nutritional benefits derived from virgin olive oil ingestion that are attributable to both its monounsaturated fat content and its phenolic compounds, particularly hydroxytyrosol (24). Recent results of the Effect of Olive Oil Consumption on Oxidative Damage in European Populations (EUROLIVE) study, a randomized, crossover, controlled intervention trial in 200 European individuals, showed that olive oil is more than a monounsaturated fat and that olive oil polyphenols, including hydroxytyrosol, contribute to the virgin olive oil beneficial health effects (1). Endogenous production of hydroxytyrosol raises the question of its contribution to the human antioxidant defense pool, given that the amount of hydroxytyrosol generated by wine drinking (250 mL) appears to be higher than that of a standard dose of virgin olive oil (25 mL) (5). In this study, hydroxytyrosol concentrations were not related to classical cardiovascular risk factors but were inversely related to inflammatory status. Inflammation is a common feature in chronic degenerative oxidative stress–associated diseases such as cardiovascular and neurodegenerative diseases and cancer, and in the aging process (25). The in vivo antiinflammatory properties of hydroxytyrosol have been reported in animal models (26). A free-radical induction of mitochondrialpermeability transition has been implicated in the DOPALmediated death of dopamine neurons in Parkinson’s disease (27). Thus, alcohol-generated antioxidant hydroxytyrosol (DOPET) could be one mechanism for explaining the inverse association between alcohol consumption and the decreased risk of Parkinson’s disease observed in case-control and cohort studies (28–31). Moderate amounts of alcohol markedly reduce the risk of CAD (32) by means of an increase in plasma HDL cholesterol and an improvement of the clotting and thrombolytic processes (33). Alcohol, however, can generate reactive oxygen species in vivo because of a prooxidant effect on lipids, DNA, and proteins (34). In the current study, the association between alcohol consumption and urinary hydroxytyrosol was observed in a population at high risk of CAD, a situation in which a high oxidative status has been reported (35). The enhancement of hydroxytyrosol generation via dopamine and ethanol metabolism interaction may partially counteract the oxidative effect of alcohol. A contribution of hydroxytyrosol to the increase in HDL cholesterol associated with alcohol consumption cannot be discarded. Polyphenol-rich foods, including those rich in hydroxytyrosol, have been shown to elevate HDL-cholesterol concentrations in clinical intervention studies (1, 36, 37). At present, data on the in vivo oxidative damage linked to moderate alcohol consumption are controversial. In a cross-sectional study, alcohol consumption was directly related to the plasma concentration of in vivo oxidized LDL cholesterol (38). However, concentrations of DNA oxidative damage, which are related to both CAD and cancer processes (39), have been reported to be decreased with the amount of alcohol consumed (40). One limitation of the study is that, in agreement with previous reports (41), wine was the main source of alcohol in our Spanish participants. In our population, consumers of other alcoholic beverages were also wine consumers and only few of them consumed exclusively nonwine alcoholic beverages. Because of this, the relation between alcohol consumption from nonwine
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beverages and hydroxytyrosol urinary concentrations could not be evaluated independently. An open question, although unlikely, is whether other wine components besides alcohol and hydroxytyrosol could be responsible for the association observed. Although only 4% of the variability of hydroxytyrosol was explained by EtG, it should be taken into account that 99.2% of the participants consumed olive oil, the main food source of hydroxytyrosol. Also, a 24-h urine sample could have provided more information than a sample of first spot morning urine, despite the fact that hydroxytyrosol measured in the latter situation has been proven to be a useful marker of olive oil consumption (1, 16). Therefore, it is very likely that the degree of variability shown in the present study is underestimated because of the aforementioned settings of the present study. In summary, we report here a direct association between hydroxytyrosol urinary concentrations and alcohol consumption in a population at high risk of CAD. To our knowledge, this is the first time that such a relation has been reported. Our data reinforce previous work that examines wine as a source of hydroxytyrosol and alcohol as an indirect promoter of endogenous hydroxytyrosol generation. Further intervention studies are needed to explore the role of alcoholic beverages, other than wine, in the generation of endogenous hydroxytyrosol. We thank the participants for their enthusiastic collaboration, the PREDIMED personnel for excellent assistance, and the personnel of all primary care centers. The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. The authors’ responsibilities were as follows—HS and RdlT: preparation of the manuscript, with significant input and feedback from all coauthors; JV: analysis of data, with results interpreted by all coauthors; and RE, DC, JS-S, ER, GF, MF, JL, JV, M-IC, MF, EC, GS, MAM-G, JF-C, VR-G, and FA: design and execution of the study and critical revision of the manuscript for important intellectual content. None of the authors had potential conflicts of interest related to this manuscript, financial or otherwise.
REFERENCES 1. Covas MI, Nyysso¨nen K, Poulsen HE, et al.; EUROLIVE Study Group. The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial. Ann Intern Med 2006;145:333–41. 2. de la Torre K, Jauregui O, Gimeno E, Castellote AI, Lamuela-Ravento´s RM, Lo´pez-Sabater MC. Characterization and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and HPLC-MS/MS. J Agric Food Chem 2005;53:4331–40. 3. Vissers MN, Zock PL, Katan MB. Bioavailability and antioxidant effects of olive oil phenols in humans: a review. Eur J Clin Nutr 2004; 58:955–65. 4. Visioli F, Galli C, Plasmati E, et al. Olive phenol hydroxytyrosol prevents passive smoking-induced oxidative stress. Circulation 2000;102: 2169–71. 5. de la Torre R, Covas MI, Pujadas MI, Fito´ M, Farre´ M. Is dopamine behind the health benefits of red wine? Eur J Nutr 2006;45:307–10. 6. Hiroi T, Imaoka S, Funae Y. Dopamine formation from tyramine by CYP2D6. Biochem Biophys Res Commun 1998;249:838–43. 7. Pe´rez-Jimenez F, Alvarez de Cienfuegos G, Badimon L, et al. International conference on the healthy effect of virgin olive oil. Consensus report, Jaen (Spain) 2004. Eur J Clin Invest 2005;35:421–4. 8. Visioli F, Galli C, Bornet F, et al. Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett 2000;468:159–60. 9. Marrugat J, Covas MI, Fito´ M, et al. Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation. A randomized controlled trial. Eur J Nutr 2004;43:140–7. 10. Weinbrenner T, Fito´ M, de la Torre R, et al. Olive oils high in phenolic compounds modulate oxidative/antioxidative status in men. J Nutr 2004; 134:2314–21.
11. Estruch R, Martı´nez-Gonza´lez MA, Corella D, et al; PREDIMED Study Investigators. Effects of a Mediterranean-style diet on cardiovascular risk factors: a randomized trial. Ann Intern Med 2006;145:1–11. 12. Martı´n-Moreno JM, Boyle P, Gorgojo L, et al. Development and validation of a food frequency questionnaire in Spain. Int J Epidemiol 1993; 22:512–9. 13. Mataix J. Tabla de composicio´n de alimentos. [Food composition tables.] Granada, Spain: University of Granada, 2003 (in Spanish). 14. Elosua R, Marrugat J, Molina L, Pons S, Pujol E. Validation of the Minnesota Leisure Time Physical Activity Questionnaire in Spanish men. The MARATHOM Investigators. Am J Epidemiol 1994;139: 1197–209. 15. Elosua R, Garcia M, Aguilar A, Molina L, Covas MI, Marrugat J. Validation of the Minnesota Leisure Time Physical Activity Questionnaire In Spanish Women. Investigators of the MARATDON Group. Med Sci Sports Exerc 2000;32:1431–7. 16. Miro´-Casas E, Farre´ Albaladejo M, Covas MI, et al. Capillary gas chromatography-mass spectrometry quantitative determination of hydroxytyrosol and tyrosol in human urine after olive oil intake. Anal Biochem 2001;294:63–72. 17. Miro´-Casas E, Covas MI, Farre´ M, et al. Hydroxytyrosol disposition in humans. Clin Chem 2003;49:945–52. 18. Bo¨ttcher M, Beck O, Helander A. Evaluation of a new immunoassay for urinary ethyl glucuronide testing. Alcohol Alcohol 2008;43:46–8. 19. Høiseth G, Bernard JP, Stephanson N, et al. Comparison between the urinary alcohol markers EtG, EtS, and GTOL/5-HIAA in a controlled drinking experiment. Alcohol Alcohol 2008;43:187–91. 20. Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Pharmacol Rev 2004;56:331–49. 21. Davis VE, Brown H, Huff JA, Cashaw JL. The alteration of serotonin metabolism to 5-hydroxytryptophol by ethanol ingestion in man. J Lab Clin Med 1967;69:132–40. 22. Helander A, Beck O, Borg S. The use of 5-hydroxytryptophol as an alcohol intake marker. Alcohol Alcohol Suppl 1994;2:497–502. 23. Tank AW, Weiner H. Ethanol-induced alteration of dopamine metabolism in rat liver. Biochem Pharmacol 1979;28:3139–47. 24. Covas MI. Olive oil and the cardiovascular system. Pharm Res 2007;55: 175–86. 25. Hensley K, Robinson KA, Gabbita P, Salsman S, Floyd RA. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med 2000;28:1456–62. 26. Bitler CM, Viale YM, Damaj B, Crea R. Hydrolyzed olive vegetation water in mice has anti-inflammatory activity. J Nutr 2005;135:1475–9. 27. Li SW, Lin TS, Minteer S, Burke WJ. 3,4-Dihydroxyphenyl acetaldehyde and hydrogen peroxide generate a hydroxyl radical: possible role in Parkinson’s disease pathogenesis. Brain Res Mol Brain Res 2001;93:1–7. 28. Ragonese P, Salemi G, Morgante L, et al. A case-control study on cigarette, alcohol, and coffee consumption preceding. Parkinson’s disease. Neuroepidemiology 2003;22:297–304. 29. Morano A, Jime´nez-Jime´nez FJ, Molina JA, Antolı´n MA. Risk-factors for Parkinson’s disease: case-control study in the province of Ca´ceres, Spain. Acta Neurol Scand 1994;89:164–70. 30. Gao X, Chen H, Fung TT, et al. Prospective study of dietary pattern and risk of Parkinson disease. Am J Clin Nutr 2007;86:1486–94. 31. Mukamal KJ, Conigrave KM, Mittleman MA, et al. Roles of drinking pattern and type of alcohol consumed in coronary heart disease in men. N Engl J Med 2003;348:109–18. 32. Suh I, Shaten J, Cutler JA, Kuller LH. Alcohol use and mortality from coronary heart disease: the role of high-density lipoprotein cholesterol. Ann Intern Med 1992;116:881–7. 33. Ruf JC. Alcohol, wine and platelet function. Biol Res 2004;37:209–15. 34. Wu D, Cerdebaum AI. Alcohol, oxidative stress and free radical damage. Alcohol Res Health 2003;27:277–84. 35. Weinbrenner T, Cladellas M, Isabel Covas M, et al. High oxidative stress in patients with stable coronary heart disease. Atherosclerosis 2003;168: 99–106. 36. Devaraj S, Vega-Lo´pez S, Kaul N, Sho¨nlau F, Rohdewald P, Jialal I. Supplementation with a pine bark extract rich in polyphenols increases plasma antioxidant capacity and alters the lipoprotein profile. Lipids 2002;37:931–4. 37. Mursu J, Voutilainen S, Nurmi T, et al. Dark chocolate consumption increases HDL cholesterol concentration and chocolate fatty acids may
ALCOHOL CONSUMPTION AND URINARY HYDROXYTYROSOL inhibit lipid peroxidation in healthy humans. Free Radic Biol Med 2004; 37:1351–9. 38. Schro¨der H, Marrugat J, Fito´ M, Weinbrenner T, Covas MI. Alcohol consumption is directly associated with circulating oxidized low-density lipoprotein. Free Radic Biol Med 2006;40:1474–81. 39. Andreassi MG. Coronary atherosclerosis and somatic mutations: an overview of the contributive factors for oxidative DNA damage. Mutat Res 2003;543:67–8.
7 of 7
40. Yoshida R, Shioji I, Kishida A, Ogawa Y. Moderate alcohol consumption reduces urinary 8-hydroxydeoxyguanosine by inducing of uric acid. Ind Health 2001;39:322–9. 41. Klipstein-Grobusch K, Slimani N, Krogh V, et al. Trends in self-reported past alcoholic beverage consumption and ethanol intake from 1950 to 1995 observed in eight European countries participating in the European Investigation into Cancer and Nutrition (EPIC). Public Health Nutr 2002;5:1297–310.
