The association between coffee consumption and plasma total homocysteine levels: the ATTICA study

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1 Heart Vessels (2004) 19: Springer-Verlag 2004 DOI /s ORIGINAL ARTICLE Demosthenes B. Panagiotakos Christos Pitsavos Antonis Zampelas Akis Zeimbekis Christina Chrysohoou Lambros Papademetriou Christodoulos Stefanadis The association between coffee consumption and plasma total homocysteine levels: the ATTICA study Received: October 29, 2003 / Accepted: April 9, 2004 Abstract The aim of this work was to investigate the association between homocysteine levels and coffee consumption in a sample of cardiovascular disease-free men and women. From May 2001 to December 2002, we randomly enrolled 1514 men and 1528 women, stratified by age and gender, from the greater area of Athens. Blood samples were collected in the fasting state. Among other investigated factors, dietary habits (including coffee consumption in ml per day, adjusted for 28% caffeine containment) were evaluated using a validated food frequency questionnaire. Men consumed higher quantities of coffee compared with women ( vs ml/day, P 0.001), while homocysteine values were also higher in men than in women ( vs µmol/l, P 0.001). A dose response relationship of homocysteine levels with coffee consumption was observed (r 0.10, P 0.034). In particular, we found that homocysteine levels were µmol/ l for no consumption, µmol/l for 100ml/day, µmol/l for ml/day, and µmol/l for 500ml/day consumption (P 0.018). The observed trend remained significant even after controlling for the interactions between coffee consumption with gender, smoking habits, physical activity status, and eating habits. However, the sole effect of the consumption of filtered coffee on homocysteine levels was significant only in those who consumed more than 500ml/day (P 0.043). Although our findings cannot be evidence for causality, they can be the basis for hypotheses about the relation between homocysteine and coffee that can partially explain the mechanisms by which elevated homocysteine levels may influence coronary risk. Key words Homocysteine Cardiovascular Coffee Introduction During the past few years, elevated blood concentrations of total homocysteine have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. 1,2 Moreover, although homocysteine levels are lowered by the intake of folate, and vitamins B 6 and B 12, elevated levels of homocysteine as well have been related with lifestyle habits, such as smoking and alcohol consumption. 1 A randomized clinical trial 3 and some population studies have suggested that a high intake of coffee is associated with raised concentrations of plasma homocysteine. 4,5 On the other hand, no association between coffee consumption and plasma homocysteine levels was observed in participants of the Atherosclerosis Risk in Communities (ARIC) study. 6 These discrepant findings may be attributed to the quantity and type of coffee consumed. Thus, in this work we aimed to test the hypothesis of a dose response relationship between plasma total homocysteine levels and coffee consumption in a population-based sample of cardiovascular disease-free adults. D.B. Panagiotakos 1 (*) A. Zampelas Department of Nutrition and Dietetics, Harokopio University, Athens, Greece C. Pitsavos A. Zeimbekis C. Chrysohoou L. Papademetriou C. Stefanadis First Cardiology Clinic, School of Medicine, University of Athens, Greece Correspondence address: 1 46 Paleon Polemiston St. Glyfada, Attica , Greece Tel ; Fax d.b.panagiotakos@usa.net Subjects and methods Population of the study The ATTICA study 7,8 is a health and nutrition survey, which is being carried out in the province of Attica (including 78% urban and 22% rural areas), of which Athens is the metropolis. The sampling was random, multistage, and based on the age and sex distribution of the province of Attica provided by the National Statistical Service (census

2 281 of 2001). From May 2001 to December 2002, 4056 inhabitants from the this area were randomly selected to enter into the study. Among them, 1518 men (46 13 years old) and 1524 (45 13 years old) women agreed to participate (75% participation rate). Participants were interviewed by trained personnel (cardiologists, general practitioners, dietitians, and nurses) who used a standard questionnaire. The selected sample can be considered as representative since there were only minor, insignificant differences in sex and age distribution between the study and target populations. The number of enrolled participants was adequate to evaluate standardized differences between the investigated parameters greater than 0.5, achieving statistical power 0.08 at a 0.05 probability level. The study was approved by the Medical Research Ethics Committee of our institution, and was carried out in accordance with the Declaration of Helsinki (1989) of the World Health Organization. Signed informed consent was obtained from all participants. Dietary assessment Consumption of nonrefined cereals and products, vegetables, legumes, fruit, olive oil, dairy products, fish, pulses, nuts, potatoes, eggs, sweets, red meat, and meat products were measured as average per week during the year previous to the study through a validated food frequency questionnaire from the Unit of Nutritional Epidemiology of our Institute. 9 The frequency of consumption was then quantified approximately in terms of the number of times a month a food was consumed. All participants were asked to state their usual frequency of consumption of coffee over the previous year. Based on the distribution of coffee consumption, we categorized usual coffee consumption as: none, up to 100ml per day (rare consumption), ml per day (moderate consumption), or more than 500ml per day (heavy consumption). All reported types of coffee (instant coffee, Greek type, filtered, or cappuccino ) were adjusted for one cup (150ml) of coffee and concentration of 27.5% caffeine. 9 We also took into account the consumption of filtered coffee, tea, and caffeine-containing drinks (such as Coca-Cola) or chocolate consumption. Cessation of coffee consumption during the previous year (in months of abstinence) was recorded and taken into account as a covariate for the analysis which followed. Finally, alcohol consumption was measured by daily ethanol intake in wine glasses of 100ml (adjusted for 12 g ethanol concentration). Biochemical measurements During the enrollment, blood samples from the antecubital vein of all participants were collected between 08:00 and 10:00 h and after a 12-h overnight fast. Subjects lay supine for 10 min prior to the blood collection. Blood was collected into a glass tube without preservative (Vacutainer tube, Becton Dickinson, Franklin Lakes, NJ, USA) for serum lipids. All samples were collected without occlusion. The tubes for plasma were kept on ice until centrifugation at 3000rpm for 10min at 4 C within 2 4h of blood collection, while the tubes for serum were kept in room temperature for no more than 1h until centrifugation at 3000rpm for 10 min at room temperature (15 24 C). The biochemical evaluation was carried out in the same laboratory than followed the criteria of the World Health Organization Lipid Reference Laboratories. The concentration of total homocysteine in plasma was estimated based on the technology of fluorescence polarization immunoassay (using an automatic analyzer from Abott Axsym, Munich Germany), which is a modified version of the fully automated column-switching high-performance liquid chromatography (HPLC) method. The intra- and interassay coefficients of variation of total homocysteine levels did not exceed 5%. 10 Serum total cholesterol was measured using the chromatographic enzymic method in a Technicon automatic analyzer RA-1000 (Dade Behring, Marburg, Germany). An internal quality control was in place for assessing the validity of cholesterol methods (the intra- and interassay coefficients of variation of cholesterol levels did not exceed 3%). Demographic, clinical, and lifestyle characteristics The study questionnaire also included demographic characteristics, such as age, gender, financial status (average annual income during the past 3 years), and education level (in school-years). Moreover, current smokers were defined as those who smoked at least one cigarette per day, while former smokers were defined as those who had stopped smoking more than 1 year previously. The rest were classified as having never smoked. For the multivariate statistical analyses cigarette smoking was quantified in pack-years (cigarette packs per day years of smoking), adjusted for a nicotine content of 0.8 mg/cigarette. Physical activity was defined as leisure-time activity of a certain intensity and duration, at least once per week during the past year, and was graded in qualitative terms, such as light (expended calories 4 kcal/min), moderate (expended calories 4 7kacl/min), and vigorous (expended calories 7kcal/ min). 7 The rest of the subjects were defined as physically inactive. Body mass index (BMI) was calculated as weight (in kg) divided by standing height (in m 2 ). Obesity was defined as BMI 29.9kg/m 2. Arterial blood pressure was measured three times at the right arm (using an ELKA aneroid sphygmomanometer, Von Schlieben, Munich, Germany), at the end of the physical examination with the subject in a sitting position for at least 30 min. Patients whose average blood pressure levels were greater or equal to 149/90mmHg or who were under antihypertensive medication were classified as hypertensives. Hypercholesterolemia was defined as total serum cholesterol levels greater than 200 mg/dl, or the use of lipidlowering agents and diabetes mellitus as a blood sugar level 125mg/dl, or the use of antidiabetic medication.

3 282 Statistical analysis Continuous variables are presented as mean value standard deviation, while qualitative variables are presented as absolute and relative frequencies. Associations between categorical variables were tested by the used of contingency tables and calculation of the χ 2 test. Correlations between homocysteine levels and other cofactors were evaluated by the calculation of Pearson s correlation coefficient for the normally distributed variables and by the Spearman correlation coefficient for the skewed variables. Comparisons between normally distributed continuous variables and groups of coffee consumption were performed by multi-way analysis of covariance (ANCOVA), after testing for equality of variances (homoscedacity), and taking into account the effect of age, sex, BMI, smoking habits, physical activity, education status, food items consumed, and use of medication. In the case of asymmetric continuous variables the tested hypotheses were based on the calculations of the nonparametric test suggested by Kruskal and Wallis. Differences in homocysteine levels between subgroups of the participants according to coffee consumption were tested using post hoc analysis (after adjusting for multiple comparisons using the Bonferroni correction). Any interaction of coffee with the previously mentioned factors was assessed by calculation of the 2LogLikelihood ratio statistic between the model containing the interaction term and the main effects model. Since we have found in previous studies 7 that homocysteine levels are significantly higher in men, all the analyses applied in this work were stratified by gender. Finally, it has been suggested 3,4 that unfiltered coffee may have a different effect on homocysteine levels, as compared to filtered. Therefore, we also stratified our analysis by type of coffee consumed. All reported P values are based on two-sided tests and compared to a significance level of 5%. Statistical Package for Social Sciences software, version 11.0 (SPSS, Chicago, IL, USA) was used for all the statistical calculations. Results Demographic and clinical characteristics of the participants Ninety-one percent of men and 76% of women reported that they drank at least one cup of coffee per day. However, 12% of men and 8% of women reported that they drank decaffeinated coffee. Various demographic, clinical, and behavioral characteristics of men and women by coffee consumption status are presented in Table 1. A positive association was observed between coffee consumption and the prevalence of hypertension and hypercholesterolemia. An inverse association was found between coffee consumption and education status since coffee drinkers were less educated than nondrinkers (especially women). An inverse association was also observed between coffee drinking and obesity in women, but not in men (Table 1). A positive but nonsignificant trend was found as regards coffee drinking, smoking habits, and physical activity status of the participants. No significant associations were found regarding cof- Table 1. Demographic, lifestyle, and clinical characteristics of the participants Coffee consumption None 100 ml/day ml/day 500 ml/day P value Men No. of participants (%) 133 (9%) 758 (53%) 521 (36%) 27 (2%) Age (years) Education status (years of school) Currently smoking (%) Sedentary (%) Obesity (%) Hypertension (%) Hypercholesterolemia (%) Diabetes mellitus (%) Family history of CHD Women No. of participants (%) 366 (24%) 922 (61%) 211 (14%) 19 (1%) Age (years) Education status (years of school) Currently smoking (%) Sedentary (%) Obesity (%) Hypertension (%) Hypercholesterolemia (%) Diabetes mellitus (%) Family history of CHD (%) Data are expressed as mean standard deviation or percentages P values are from the analysis of variance test (for the comparisons between age and education status with homocysteine levels) and from the χ 2 test for the other comparisons CHD, coronary heart disease

4 283 Table 2. Association between total plasma homocysteine concentrations and various characteristics of the participants Men Women Standardized P value Standardized P value beta coefficient beta coefficient Age (years) Cigarette smoking (pack years) * * Body mass index (kg/m 2 ) * * Physical activity (yes/no) * * Year of school * * Annual income ( ) * * Fruit intake (servings/week) * * Vegetables (servings/week) * * Ethanol intake (g/day) * * * P values are from linear regression models after adjusting for the age of the participants Table 3. Homocysteine levels (µmol/l) and coffee consumption Coffee consumption None 100 ml/day ml/day 500 ml/day P value Men * 14 4* 14 5* Women * 12 4* P values are from the analysis of covariance test, after controlling for age, food items consumed, and smoking habits fee consumption, prevalence of diabetes mellitus, and family history of coronary heart disease (Table 1). Homocysteine and various characteristics of the participants Table 2 illustrates the associations observed between homocysteine levels and various characteristics of the participants. Homocysteine was positively associated with age in women, but not in men. The lack of association in men was confirmed even after adjusting for smoking habits (standardized β coefficient 0.002, P 0.23). Moreover, postmenopausal women had higher homocysteine levels compared with premenopausal women ( vs µmol/l, P 0.001), after taking into account the years of menstruation. Furthermore, we observed that age-adjusted homocysteine levels were positively associated with the daily number of cigarettes smoked, as well as with the BMI of the participants. No associations were found between homocysteine levels and physical activity status, years of school, and annual income of the participants. Regarding the associations between homocysteine levels and food items consumed, we observed an inverse relationship of homocysteine with fruit and vegetable intake, in both genders, while no associations were observed regarding the other foods consumed. Moreover, a dose response relationship was observed between homocysteine and alcohol drinking. Homocysteine levels and coffee consumption A dose response relationship of homocysteine levels with quantities of coffee consumed was observed. Since homocysteine values were higher in men compared with women ( vs µmol/l, P 0.001), we stratified our analysis by gender. Table 3 presents the mean homocysteine levels by coffee consumption. After controlling for age, food items consumed, and smoking habits, we observed that men who consumed even low quantities of coffee ( 100ml/day) had significantly higher homocysteine levels compared with non-coffee drinkers. The effect of coffee consumption on homocysteine levels among women was observed only when larger quantities were consumed ( 200ml/day). Although homocysteine concentrations were higher in men than in women, and the quantity of coffee consumed was also higher, no gender coffee interaction was observed regarding homocysteine levels (P 0.675). Since the associations presented in Table 2 could alter the significance of the investigated relationship between homocysteine and coffee drinking, we performed a multivariate analysis. Thus, after controlling for age, gender, smoking habits, BMI, physical activity status, presence of hypertension and hypercholesterolemia, fruit and vegetable consumption, quantity of alcohol drinking, menopausal status, intake of caffeine-containing drinks, and months of abstinence from coffee drinking during the previous year, we observed that an intake of 200ml/day of coffee was associated with a significant increase in homocysteine levels (β coefficient standard error for ml per day, P 0.023; and for 500 per day, P

5 284 Table 4. Homocysteine levels (µmol/l) and coffee consumption by fruit intake Coffee consumption None 100ml/day ml/day 500ml/day P value a Fruit consumption None * 16 4* 18 5* items/day * 13 3* 15 4* items/day * 13 5* items/day * * P 0.05 for the comparisons between coffee consumption and no coffee drinking a P values for linear trend between coffee drinking and homocysteine levels more than 5 fruits per day we observed that the adverse effect of heavy coffee drinking on homocysteine levels was statistically significant only when large quantities of coffee were drunk (i.e., 500ml per day). Discussion Fig. 1. Homocysteine levels by coffee consumption and smoking status. *P 0.05 for comparisons between coffee and non-coffee drinkers, derived from multi-way analysis of covariance 0.001). However, when we focused our interest on filtered coffee drinking we observed that the adverse effect of coffee consumption on homocysteine levels was significant only in those who consumed more than 500ml per day (P 0.043). A significant synergistic effect was observed between coffee consumption and smoking on homocysteine levels (P 0.001). Specifically, the effect of even low levels of coffee drinking ( 100ml per day) in men who smoked than 20 cigarettes per day resulted a 4 1µmol/l increase in homocysteine levels compared with nondrinkers who smoked similar quantities of cigarettes (P 0.02). A significant increase was also observed among women (3 1µmol/l, P 0.03). As expected, the synergistic effect of smoking and coffee consumption on homocysteine levels was more prominent when participants consumed higher quantities of coffee and smoked more cigarettes per day. Figure 1 illustrates the effect of coffee drinking on homocysteine concentrations by smoking status. Finally, we stratified our analysis by quantities of fruit consumed. We observed that increased fruit intake attenuates the effect of coffee on homocysteine levels (Table 4). In particular, when we focused on participants who consumed We revealed a positive relationship between coffee consumption and plasma homocysteine levels, after taking into account several demographic, clinical, biochemical, and lifestyle-related characteristics of 3042 randomly selected men and women form the Attica region. The increase was significant in men who consumed more than 100ml per day and in women who consumed more than 200ml per day, as compared to those who did not drink coffee. Moreover, consumption of filtered coffee seems to moderate the effect of caffeine on homocysteine levels, since only when high quantities of coffee were consumed (i.e., 500ml per day) was there an association with a significant increase in homocysteine levels. In the Hordaland Homocysteine study, 11 daily use of coffee was reported by 89% of the participants of whom 95% used filtered coffee. The investigators reported a positive association between high ( 9 cups per day) coffee consumption and total homocysteine, even after adjusting for smoking habits, intake of vitamin supplements, and fruit and vegetables consumption. In the present study, we found that the increase in homocysteine levels was significant even with low levels of consumption (see Table 1). On the contrary, when we focused on filtered coffee drinkers we observed that the effect of coffee consumption on homocysteine levels was significant only in those who consumed high quantities (i.e., 500ml per day). The different dose response effect of coffee on homocysteine levels observed in our study as compared to the Hordaland Study may be attributed to the much higher frequency of unfiltered coffee consumed in our population. It has been suggested that unfiltered coffee increases plasma homocysteine concentrations in people with normal baseline concentrations. 2,4,5,11,12 Randomized clinical trials suggest that there is a homocysteine-raising effect of both filtered 13 and unfiltered coffee. 2 However, coffee intake in these studies was high (1l per day) and at these levels of consumption, our results agree with the results of the above studies. It is

6 285 therefore unclear whether the differences exist in lower levels of consumption. However, it has to be mentioned that results from a recent study suggest that abstention from 4 cups of filtered coffee per day could decrease homocysteine levels and reduce the homocysteine-attributed ischemic heart disease risk by 10%. 14 On the other hand, it should be mentioned that the association between coffee consumption and plasma homocysteine was not confirmed in participants in the Atherosclerosis Risk in Communities Study. 6 However, the majority of the participants probably consumed filtered coffee. The synergistic effect of cigarette smoking and coffee drinking on homocysteine concentrations has already been reported in the Framingham Offspring cohort 5 and the Hordaland Homocysteine study. 11 A coffee smoking interaction was observed in our study, too. In particular, coffee drinking was associated with a much higher increase in homocysteine levels, especially when we focused only on people who smoked more than 20 cigarettes per day (Fig. 1). Since both coffee and smoking seem to be associated with homocysteine levels, coexistence of these two habits (which is very common in the population) could be a plausible explanation of the increased homocysteine levels observed in the present as well as previous studies. We also observed that consumption of fruit might moderate the effect of coffee intake on homocysteine levels. In particular, we found that a statistically significant increase in homocysteine levels among participants who consumed more than 5 fruits per day was observed only in the high coffee-drinking group ( 500ml per day). However, as fruit consumption decreased, the effect of coffee on homocysteine became significant even when lower quantities of coffee were consumed (Table 4). It has been suggested that at least 5 servings of fruits and vegetables a day are necessary in order to observe a decrease in homocysteine levels, 15,16 and this agrees with our findings. It has been suggested that this protective effect of fruit against the homocysteine-raising effect of coffee could be due to folic acid that may affect the homocysteine concentration. 10,17 Limitations This was a cross sectional study that could not provide causal relationships, but only generate hypotheses. However, the large and representative sample as well as the random selection may strengthen the importance of our findings. Although fruit and vegetable intake was taken into account, based on a validated food frequency questionnaire, the lack of serum or plasma folate measurement and data on other vitamins are a major limitation of our findings. Moreover, since coffee drinking as well as other lifestyle habits (e.g., smoking, physical activity) were evaluated by self-reporting, the information retrieved could be biased due to several psychosocial or other unknown factors. Conclusion The results regarding the effect of coffee consumption on the cardiovascular system are conflicting. Some reports showed a positive association between coffee intake and coronary heart disease, while other reported no relationship. 21,22 Several investigators suggested that the increase in the risk of coronary heart disease may be the result of elevated blood pressure and cholesterol levels due to unhealthy dietary habits or increased smoking habits associated with coffee consumption In this work we revealed that unfiltered coffee drinking was associated with increased homocysteine levels, a potential maker of cardiovascular disease. This effect was enhanced when heavy cigarette smoking existed. However, the association between filtered coffee drinking and homocysteine levels was significant only when large quantities of coffee were consumed, which is in accordance with several other similar studies Moreover, fruit consumption seems to moderate the effect of coffee drinking on homocysteine levels. Acknowledgment The ATTICA study is supported by research grants from the Hellenic Cardiological Society (HCS2002) and the Hellenic Atherosclerosis Society (HAS2003). The authors thank the field investigators of the ATTICA study: Dr. Natasa Katinioti (physical examination), Dr. Akis Zeimbekis (physical examination), Dr. Spiros Vellas (physical examination), Dr. Efi Tsetsekou (physical/psychological evaluation), Dr. Dina Masoura (physical examination), and Dr. Lambros Papadimitriou (physical examination), as well as the technical team: Dr. Marina Toutouza (senior investigator/biochemical analysis), Ms. Carmen Vasiliadou (genetic analysis), Mr. Manolis Kambaxis (nutritional evaluation), Ms. Konstadina Paliou (nutritional evaluation), Ms. Constadina Tselika (biochemical evaluation), Ms. Sia Poulopoulou (biochemical evaluation), and Ms. Maria Toutouza (database management). References 1. Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. 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