ORIGINAL COMMUNICATION Reduced plasma homocysteine in ...

European Journal of Clinical Nutrition (2002) 56, 608–614 ß 2002 Nature Publishing Group All rights reserved 0954–3007/02 $25.00 www.nature.com/ejcn

ORIGINAL COMMUNICATION Reduced plasma homocysteine in obese red wine consumers: a potential contributor to reduced cardiovascular risk status JB Dixon1*, ME Dixon1 and PE O’Brien1 1

Monash University Department of Surgery, Alfred Hospital, Melbourne, Victoria, Australia

Background: Moderate alcohol consumption is associated with improved vascular risk profile and decreased mortality in the middle aged. An elevated homocysteine concentration is an independent risk factor for cardiovascular disease. Objective: To examine the relationship between alcohol consumption and homocysteine concentrations in severely obese patients (body mass index (BMI) > 35). Design: A careful alcohol history was obtained from 350 (male : female 1 : 5) consecutive patients as part of preoperative assessment for surgical treatment of obesity. Data were obtained concerning amount, frequency, timing and type of alcohol consumption. Fasting homocysteine, serum folate and vitamin B12 concentrations were measured. Differences between groups were assessed using Student t-test, and ANOVA. Linear regression was used to assess factors influencing homocysteine concentration. Results: There is a U-shaped relationship between alcohol consumption and homocysteine concentrations, with light to moderate consumption being associated with lower concentrations. Those consuming < 100 g=week (n ¼ 165) of alcohol had geometric mean (95% CI of mean) serum homocysteine concentrations of 8.5 (8.2 – 8.9) mmol=l compared with 9.5 (9.1 – 9.9) mmol=l for non or rare consumers (n ¼ 153; P ¼ 0.001). The lower concentrations of homocysteine in regular consumers were associated with higher folate concentrations of 9.4 (8.6 – 10.2) ng=ml when compared with non-consumers 7.5 (7.1 – 7.8) ng=ml (P ¼ 0.001). Red wine consumers (n ¼ 42) had lower fasting concentrations of homocysteine 7.8 (7.5 – 8.1) mmol=l compared with 153 non-consumers 9.4 (9.0 – 9.8) mmol=l (P < 0.001), 82 beer and spirit consumers 9.0 (8.4 – 9.7) mmol=l (P ¼ 0.005) and 73 white wine consumers 8.8 (8.2 – 9.4) mmol=l (P ¼ 0.013). Red wine consumption was an independent predictor for lower homocysteine concentrations. Conclusion: Mild to moderate alcohol consumption, especially red wine consumption, in obese subjects is associated with lower fasting homocysteine concentrations. This may reduce cardiovascular risk and help explain the ‘French paradox’. European Journal of Clinical Nutrition (2002) 56, 608 – 614. doi:10.1038=sj.ejcn.1601365 Keywords: red wine; alcohol; vascular risk; homocysteine; folate; vitamin B12; severe obesity

Introduction Elevated concentrations of homocysteine are associated with vascular and thromboembolic risk (den Heijer et al, 1996; Graham et al, 1997). Low to moderate alcohol consumption has been associated with reduced morbidity and mortality in

*Correspondence: JB Dixon, Monash University Department of Surgery, Alfred Hospital, Melbourne, Victoria, 3181, Australia. E-mail: [email protected] Guarantor: JB Dixon. Received 8 June 2001; revised 8 October 2001; accepted 17 October 2001

the middle aged to elderly (Gaziano et al, 2000) and epidemiological evidence suggests that consumption of wine may have added benefits (Gronbaek et al, 2000). There is also good epidemiological evidence of the ‘French Paradox’, a reduction in cardiovascular morbidity and mortality in areas where wine is commonly consumed, despite a diet high in saturated fat (Renaud & Gueguen, 1998; Renaud et al, 1999). It has recently been proposed that the presence of folate and vitamin B6 in beer may lead to a beverage-specific advantage through lowering of homocysteine concentration (van der Gaag et al, 2000). Current data indicate that very high alcohol consumption is associated with elevated homocysteine concentrations (Bleich et al, 2001). It is unclear

Red wine and lower homocysteine concentration JB Dixon et al

609 what effect, if any, low to moderate consumption has (Ayaori et al, 2000). Recently published studies have had variable results. A study examining the elderly (New Mexico Aging Process Study) found lower homocysteine concentrations in elderly subjects with light to moderate alcohol intake (Koehler et al, 2001), but another (Framingham offspring study) found a positive association with alcohol consumption (Jacques et al, 1999). Examination of their data, however, indicates no rise with an alcohol intake of less than 15 g=day. Severely obese patients are at increased risk of vascular morbidity and mortality (Bray, 1996). In this observational study, we examine obese subjects for any relationship between the amount and type of alcohol consumption and fasting plasma homocysteine concentrations.

Subjects and method The study group consisted of 350 of 353 consecutive severely obese patients presenting for gastric restrictive surgery. A history of chronic alcoholism or drug dependency is a relative contraindication to surgery. However, two patients with chronic alcoholism and one with binge drinking and recreational drug use were accepted for surgery and were therefore excluded from this study. A careful assessment of the pattern of alcohol consumption over the last 12 months was obtained at two separate medical consultations with physicians, separately in a questionnaire completed by the patient and finally in a structured interview. In this last interview the reason for any discrepancy in the pattern of reported alcohol consumption was sought from the patient or a relative and every effort was made to obtain accurate consumption information. Any reason for not consuming alcohol was also obtained. Alcohol consumption was classified into three groups: (1) nil (no alcohol or very rare consumption); (2) 100 g or less per week; and (3) greater than 100 g per week. Frequency of consumption was classified into four groups: (1) nil (including rare); (2) most months; (3) most weeks; and (4) most days or every day. Consumers were asked to nominate the most commonly consumed beverage: beer, wine or spirits. Wine consumers were asked to nominate the type of wine consumed, either white (including champagne-style) or red wine. The time of usual consumption was also recorded: social occasions, parties or weekend sessions, or with meals or immediately before or after meals. Any vitamin supplementation was noted. We did not further assess the micronutrient intake of the subjects. All patients gave informed consent to the gastric restrictive surgery. The study complied with the Helsinki Declaration. As a part of extensive pre-operative investigations, fasting plasma homocysteine concentrations, serum folate and vitamin B12 concentrations were measured in the month prior to surgery. Other than a 15 h fast immediately prior to collection, patients were not instructed to change their alcohol intake, diet or vitamin intake. Vitamin B6 levels were not measured. Patients were grouped in several ways: by amount,

frequency and most common type of alcohol consumed. All subjects gave written informed consent to surgery and preoperative assessments. The information obtained and the questionnaires used were approved by the hospital’s ethics committee as part of a larger study. A fasting venous blood sample for homocysteine assay was collected into EDTA tubes kept on ice and plasma separated off within four hours of collection. Specimens were stored at 7 20 C prior to batch analysis every 2 weeks. The reagent used was catalogue no. 3D39-20 Abbott IMX Homocysteine reagent and assay performed on the Abbott IMX analyser. The inter-assay and intra-assay coefficients for homocysteine level were 2.2 and 3%, respectively. Serum vitamin B12 and folate concentrations were measured using Chiron Diagnostics ACS-180 VB12 and Folate (catalogue no. 10481 and 672215) reagents, respectively, and assay performed on a Bayer ACS-180. The inter-assay and intraassay coefficients for vitamin B12 level are 5 and 6%, respectively, and for folate 5 and 10%, respectively. There are a number of genetic mutations, including MTHFR-mutations, that may adversely influence plasma homocysteine concentrations and the clinical effects of these are yet to be clarified. We have not sought to examine the genotype for patients in this study. The statistical software SPSS (1999) 10.00 was used for all analyses. Differences between groups were evaluated using Students t-test and analysis of variance with the Tukey method of post-hoc analysis. Linear regression was used to assess the effect of multiple variables on homocysteine concentration. The variables homocysteine, folate and vitamin B12 were not normally distributed, but right skewed, and required log transformation prior to parametric analysis and values are expressed as geometric mean and 95% confidence interval of the geometric mean. Chi-square method with Fisher exact method was used to test the significance of differences between proportions and categorical variables. A P-value of less than 0.05 was considered to be significant.

Results There were 350 (58 male, 292 female) consecutive obese subjects, after the three exclusions. Characteristics of the group are shown in Table 1. Independent of the effect of alcohol consumption, four factors within the group had a significant impact on homocysteine concentrations. These were serum folate concentration (r ¼ 7 0.33, P < 0.001), serum vitamin B12 concentration (r ¼ 7 0.22, P < 0.001), male gender (9.9 (9.1 – 10.7) mmol=l vs 8.8 (8.5 – 9.1) mmol=l for women, P ¼ 0.006) and BMI (r ¼ 0.16, P ¼ 0.004). Subjects with higher BMI had significantly lower folate and vitamin B12 concentrations. Using linear regression analysis, independent predictors of fasting plasma homocysteine in the group were serum folate and male gender with a combined r2 ¼ 0.15 (P < 0.001). BMI and vitamin B12 levels did not European Journal of Clinical Nutrition


610 Table 1 Severely obese subjects (n ¼ 350) grouped by weekly alcohol consumption

Number Age (y) 2 BMI (kg=m ) Male (%) Smokers (%) Fasting plasma homocysteine (mmol=l)a Homocysteine > 10 mmol=l (%) d Serum folate (ng=ml) d Serum vitamin B12 (pg=ml) Taking multivitamin supplements (%)

Nil=rare

< 20 g

20 – 100 g

> 100 g

P-value

All groups

153 41.2 (11.3) 45.4 (8.3) 17 8 9.5 (9.1 – 9.9)a 32 a 7.5 (7.1 – 7.8) 379 (368 – 398) 20

58 a 38.6 (10.2) 42.3 (10.3) 10 7 7.9 (7.3 – 8.6)b 16 b 10.4 (9.6 – 11.2) 367 (347 – 406) 14

107 40.2 (8.9) 43.9 (8.1) 14 12 8.8 (8.3 – 9.2) 23 8.9 (8.5 – 9.4) 376 (358 – 395) 22

32 b 45.2 (8.6) 45.4 (6.7) 34 21 9.6 (8.4 – 10.9)a 25 a 7.4 (6.5 – 8.4) 391 (343 – 446) 15

0.027 NS c 0.023 NSc 0.001 0.073 < 0.001 NS c NS

350 40.8 (10.3) 44.6 (8.5) 16.6 10.2 9.0 (8.7 – 9.3) 24 8.7 (8.4 – 8.9) 376 (364 – 388) 19

a

Concentrations are significantly different from those marked. (ANOVA post hoc Tukey, P < 0.05). c 2 P-value obtained using w . d These variables log transformed prior to analysis: geometric mean ( 95% confidence interval of geometric mean). For other continuous variables: mean (standard deviation). b

contribute to the variance of homocysteine levels after controlling for serum folate concentration and gender. There was a U-shaped relationship between homocysteine concentrations and the amount of alcohol consumption each week (Table 1). Geometric mean homocysteine concentrations for 165 subjects consuming less than 100 g=week of alcohol were 8.5 (8.2 – 8.9) mmol=l compared with 9.5 (9.1 – 9.9) mmol=l for the non-consumers (P ¼ 0.001). The reduction in homocysteine concentration was associated with higher folate concentrations in consumers of less than 100 g=week, 9.4 (8.6 – 10.2) ng=ml compared with for non-consumers, 7.5 (7.1 – 7.8) ng=ml (P < 0.001). The lower homocysteine con-

centrations in those consuming less than 100 g=week were not significant after controlling for serum folate concentration. There was also a U-shaped relationship for homocysteine concentration when subjects were grouped on the basis of the frequency of alcohol consumption (Figure 1), with those consuming alcohol most weeks having lower homocysteine and higher folate concentrations than the nonconsumers. Geometric mean concentrations of fasting plasma homocysteine concentrations were significantly lower in red wine consumers than non-consumers and other alcohol drinkers (Figure 2). Red wine consumers (n ¼ 42) had concentrations

Figure 1 Geometric mean ( 95% CI of mean) fasting plasma homocysteine concentrations for groups based on frequency of alcohol consumption. Fasting plasma homocysteine concentrations are significantly higher and serum folate concentrations lower in those consuming most weeks compared with non-consumers with P-values of 0.026 and 0.025, respectively. (ANOVA using the Tukey method).

European Journal of Clinical Nutrition


611 Table 2 Severely obese subjects (n ¼ 350) grouped by type of alcohol most commonly consumed

Number Age (y) 2 BMI (kg=m ) Male (%) Smokers (%) Fasting plasma homocysteine (mmol=l)d Homocysteine > 10 mmol=l (%) d Serum folate (ng=ml) d Serum vitamin B12 (pg=ml) Taking multivitamin supplements (%)

Nil

Beer and spirits

Wine

P-value

153 41.1 (11.2) 45.5 (8.3) 17 8 9.4 (9.0 – 9.8)a 32 a 7.5 (6.9 – 8.2) 379 (358 – 402) 20

82 a 38.2 (9.0) 43.65 (8.7) 20 14 9.0 (8.4 – 9.7) 26 8.6 (7.7 – 9.6) 372 (345 – 401) 19

115 b 42.2 (9.5) 43.6 (8.6) 13 9 8.3 (7.9 – 8.8)b 17 b 9.3 (8.5 – 10.2) 379 (358 – 402) 17

0.024 NS c NS NSc 0.007 0.030 0.007 NS NS

a

Concentrations are significantly different from those marked. (ANOVA post hoc Tukey). c 2 P-value obtained using w . d These variables log transformed prior to analysis: geometric mean ( 95% CI of mean). For other continuous variables: mean (standard deviation). b

17% lower than those of the nil group (P < 0.001) and 13% lower than the group of beer and spirits consumers (P ¼ 0.005). There were 22 regular beer consumers with geometric mean homocysteine concentrations of 8.7 (7.8 – 9.6) mmol=l which is 10% higher than red wine consumers (P ¼ 0.045). Wine consumers had higher folate levels than non-alcohol consumers (Table 2), but there was no difference between red and white wine consumers (Table 3). Linear regression analysis indicated that red wine consumption was an independent predictor of lower homocysteine concentrations when modelled with sex, age, weight, smoking, frequency and quantity of alcohol consumption, and serum folate and vitamin B12 concentrations. The consumption of red wine (b ¼ 7 0.16, P ¼ 0.002), high serum folate concentration (b ¼ 7 0.30, P < 0.001) and female gender (b ¼ 7 0.13, P ¼ 0.014) were independent predictors of lower homocysteine concentrations with a combined r2 of 18.2. Other modelled factors did not have significant influence on fasting plasma homocysteine levels. Only 7% of red wine consumers had a fasting homocysteine level greater than the AHA recommended risk threshold of 10 mmol=l (Malinow et al, 1999), compared with 32% for non-alcohol consumers (Table 3). The odds ratio for a fasting homocysteine level greater than 10 mmol=l for those nominating red wine as their usual beverage is 0.20 (95% confidence interval 0.06 – 0.65, P ¼ 0.003) compared with those not nominating red wine. Vitamin supplements were taken by 19% of patients and the proportion did not vary significantly between groups analysed. Those taking any supplementary vitamins (n ¼ 65) had higher folate (9.8 (8.8 – 10.8) vs 7.9 (7.1 – 8.8) ng=ml, P ¼ 0.005) and vitamin B12 (411 (381 – 444) vs 372 (349 – 397) pg=ml, P ¼ 0.032) concentrations than non-vitamin consumers but the geometric mean homocysteine concentrations were not significantly different (8.9 (8.3 – 9.6) vs 8.9 (8.4 – 9.5) mmol=l). Alcohol consumers (n ¼ 197) were grouped into those who usually consumed alcohol with or immediately before or

Table 3 Severely obese subjects who consume wine (n ¼ 115) grouped by the most commonly consumed type of wine (white or red)

Number Age (y) 2 BMI (kg=m ) Male (%) Smokers (%) Fasting plasma homocysteine (mmol=l)b Homocysteine > 10 mmol=l (%) b Serum folate (ng=ml) b Serum vitamin B12 (pg=ml) Taking multivitamin supplements (%)

White wine

Red wine

P-value

73 42.3 (9.6) 43.2 (9.1) 14 8 8.8 (8.2 – 9.4)

42 42.1 (9.5) 44.3 (7.5) 12 12 7.8 (7.5 – 8.1)

NS NS a NS NSa 0.013

23 9.2 (8.3 – 10.2) 372 (345 – 401) 15

7 9.5 (8.0 – 11.2) 379 (358 – 402) 19

0.028 NS NS a NS

Unpaired Student t-test. a P-value obtained using w2. b These variables log transformed prior to analysis: geometric mean (95% CI of geometric mean). For other continuous variables: mean (standard deviation).

after meals (n ¼ 59) and those who usually consumed at social occasions, parties and weekend sessions (n ¼ 138). There were no differences in homocysteine, vitamin B12 or folate concentrations between these groups (not shown).

Discussion Light to moderate alcohol consumption is associated with lower fasting plasma homocysteine concentrations with the effect associated with higher serum folate concentrations in severely obese subjects, confirming the findings of a recent study in the elderly (Koehler et al, 2001). A sub-group of alcohol consumers, red wine consumers, have lower homocysteine concentrations and this appears to be an independent effect. Lower homocysteine concentrations seen in wine consumers, especially those drinking red wine, introduce another possible mechanism for the vascular protective effects seen European Journal of Clinical Nutrition


612

Figure 2 Geometric mean ( 95% confidence interval of mean) concentrations of fasting plasma homocysteine (mmol=l) for subjects grouped by the type of alcohol most commonly consumed. *Groups marked are a combination of the two subgroups following them in the figure. Red wine consumers had lower homocysteine levels when compared directly with any other group. (Student t-test, two tail).

in populations where wine consumption is common (Renaud & de Lorgeril, 1992). This possibly contributes to the ‘French paradox’. In contrast to the recent study by van der Gaag et al (2000) we have not confirmed that beer consumers have a specific advantage, when measuring fasting homocysteine concentrations. The possible mechanisms for the beneficial effect of red wine are unclear. The three micronutrients: folate, vitamin B12 and vitamin B6 have an important role in homocysteine metabolism (Selhub, 1999). However the effect of red wine on homocysteine concentrations appears to be independent of serum folate and vitamin B12 concentrations and, as red wine contains neglegible vitamin B6 (van der Gaag et al, 2000), it is unlikely these micronutrients alone explain the effect. There may be other lifestyle or dietary factors (Fung et al, 2001) that differ with the choice of beverage. Timing of alcohol consumption did not affect homocysteine concentrations in this study. It has been proposed that betaine that is found in some European wines may lower homocysteine concentrations as it is an alternative methyl donor for the methylation of homocysteine to methionine (Mar & Zeisel, 1999). The betaine in wine is a result of the addition of sugar beet. We have been advised that Australian conditions do not require the addition of sugar beet into local wines. To our knowledge betaine concentrations have not been measured in Australian wine, but concentrations would be expected to be low if any. We have recently demonstrated that homocysteine concentrations rise with weight loss following gastric restrictive surgery, and that higher serum concentrations of micronutrients folate and vitamin B12 are required to maintain homocysteine concentrations (Dixon et al, 2001). There is European Journal of Clinical Nutrition

an alteration in the dose – response relationship between homocysteine concentration, and folate and vitamin B12 concentrations. In addition, the influence of folate and B12 on homocysteine concentrations is increased in subjects who have lost weight. Perhaps components of red wine such as the polyphenols alter the dose – response curve in the other direction with lower homocysteine concentrations achieved with similar micronutrient concentrations. There are many influences on plasma homocysteine levels that may confound our findings (Jacques et al, 2001). All of our patients are obese and therefore relatively insulin resistant and this may have an impact on levels (Emoto et al, 2001). Certainly light to moderate alcohol consumption reduces insulin resistance and the risk of type 2 diabetes (Bell, 1996). The findings in our group may not necessarily be applicable to non-obese subjects. Our finding that red wine consumers have lower homocysteine levels contrasts with data from the Framingham Offspring Study that showed a rise in homocysteine levels in regular red wine consumers (Jacques et al, 2001), however data was collected and analysed in a different manner. In our study subjects were grouped by their nominated most frequently consumed beverage, thus a spirit consumer having an occasional glass of red wine is analysed as a spirit consumer. An intervention study with subjects given 30 g of alcohol=day found raised homocysteine levels after consumption of all alcoholic beverages (Bleich et al, 2001), but the level of consumption of 210 g=week is above our threshold for lower fasting homocysteine concentrations. Chronic alcoholism, acute alcohol intoxication and heavy alcohol consumption are associated with high homocysteine concentrations that may well be related to relative or absolute folate or vitamin B6 deficiency (Bleich et al, 2000b;


Cravo et al, 1996). Hyperhomocysteinemia has been found in rats after ethanol feeding (Stickel et al, 2000). In addition, it has been shown that long-term ingestion of large quantities of ethanol causes inhibition of methionine synthase activity due to its breakdown product acetaldehyde (Kenyon et al, 1998) and it has been proposed that elevated blood levels of ethanol, rather than nutritional factors are responsible for changes in homocysteine metabolism (Bleich et al, 2000c). With high alcohol intake beer, rich in folate and vitamin B6, may reduce the risks of raised homocysteine concentrations (Cravo et al, 1996; van der Gaag et al, 2000). Elevated homocysteine concentrations in chronic alcoholics predict the risk of fitting with alcohol withdrawal (Bleich et al, 2000a) and may be an important factor related to the increased risk of direct neuronal damage (Bleich et al, 2000b) or stroke (Hultberg et al, 1993). This group of severely obese subjects has relatively normal homocysteine concentrations and one may question the clinical relevance of the apparently small variations seen in this study. As for other cardiovascular risk factors there appears to be a graded effect over a wide range of homocysteine concentrations, with lower concentrations associated with lower risk (Malinow et al, 1996; Perry et al, 1995). Relatively small falls in the population mean homocysteine levels may be highly relevant (Jacques et al, 1999) and we have demonstrated fewer alcohol consumers have homocysteine levels of higher risk (Malinow et al, 1999). Obese subjects are certainly a group at high risk of cardiovascular disease (Bray, 1996). Moderate alcohol consumption is associated with greater insulin sensitivity (Facchini et al, 1994), reduced risk of type 2 diabetes (Bell, 1996), favourable higher HDL-cholesterol concentrations (Gaziano et al, 1993) and less thrombogenic platelet function (Renaud & de Lorgeril, 1992). Lower homocysteine concentrations, in addition to the antioxidant effects of polyphenols (Brouillard et al, 1997), may confer the red wine consumer a beverage-specific advantage. Severely obese patients are at considerable risk of cardiovascular morbidity and mortality. It has recently been shown that light to moderate alcohol consumption is of benefit to the obese in terms of type 2 diabetes (Bell, 1996). Our data would indicate a similar benefit for this group in the reduction of cardiovascular risk. References Ayaori M, Hisada T, Yoshira H, Shige H, Ito T, Nakajima K, Higashi K, Yonemura A, Ishikawa T, Ohsuzu F, Saionji K, Tamai S & Nakamura H (2000): Effect of alcohol intake on the levels of plasma homocysteine in healthy males. J. Nutr. Sci. Vitaminol. (Tokyo) 46, 171 – 174. Bell DS (1996): Alcohol and the NIDDM patient. Diabetes Care 19, 509 – 513. Bleich S, Degner D, Bandelow B, von Ahsen N, Ruther E & Kornhuber J (2000a): Plasma homocysteine is a predictor of alcohol withdrawal seizures. Neuroreport 11, 2749 – 2752. Bleich S, Degner D, Javaheripour K, Kurth C & Kornhuber J (2000b): Homocysteine and alcoholism. J. Neural Transm. 60(Suppl), 187 – 196.

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