Moderate alcohol consumption and lower levels of inflammatory markers in US men and women

Atherosclerosis 186 (2006) 113–120

Moderate alcohol consumption and lower levels of inflammatory markers in US men and women Jennifer K. Pai a,c,∗ , Susan E. Hankinson a,d , Ravi Thadhani e , Nader Rifai f , Tobias Pischon a,b,g , Eric B. Rimm a,b,d a

c

Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA b Department of Nutrition, Harvard School of Public Health, Boston, MA, USA Division of Preventive Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA d Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA, USA e Renal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA f Department of Pathology, Children’s Hospital Medical Center, Harvard Medical School, Boston, MA, USA g Department of Epidemiology, German Institute of Human Nutrition, Potsdam-Rehbruecke, Germany Received 27 April 2005; received in revised form 14 June 2005; accepted 23 June 2005 Available online 1 August 2005

Abstract Objective: Moderate alcohol consumption is associated with substantially lower risk of cardiovascular disease (CVD). We assessed the relationship between alcohol intake and inflammatory markers to partially explain this beneficial effect. Methods and results: From two large prospective studies, we sampled 959 healthy male and 473 healthy female health professionals with reported alcohol intake. Markers of inflammation were soluble tumor necrosis factor-alpha receptors 1 and 2 (sTNF-R1 and sTNF-R2), C-reactive protein (CRP), and interleukin-6 (IL-6). We found significant inverse linear trends for sTNF-R1 (p-trend < 0.001 men; 0.03 women) and sTNF-R2 (p-trend = 0.002 men; 0.08 women) with increasing alcohol intake. Compared to non-drinkers, men who consumed on average 1–2 drinks/day had 26% lower CRP (−0.66 mg/L, p = 0.13), and 36% lower IL-6 (−1.12 pg/ml, p = 0.02) levels. Among women, a similar though stronger association was observed at half drink per day. Compared to non-drinkers, both men and women who consumed 1–2 drinks/drinking day had significantly lower sTNF-R1 (−9% in men, −6% in women) and sTNF-R2 (−7% in men, −6% in women) levels as well as lower CRP (−10% in men, −32% in women) and IL-6 (−45% in men, −27% in women) levels. Conclusions: Alcohol in moderation is associated with lower levels of inflammatory markers and may lower risk of CVD through these mechanisms. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol; Inflammation; Epidemiology; Acute-phase proteins; Cytokines; Biological markers

1. Introduction Moderate alcohol consumption is associated with a decreased risk of cardiovascular disease (CVD) [1,2]. However, only half this benefit may be explained through beneficial effects on lipids [3–5]. Other potential mechanisms ∗ Corresponding author. Department of Epidemiology, Harvard School of Public Health, 665 Huntington Avenue, Bldg 2, Rm 315, Boston, MA 02115, USA. Tel.: +1 617 432 7722; fax: +1 617 566 7805. E-mail address: [email protected] (J.K. Pai).

0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.06.037

include the effects of alcohol on haemostatic factors, inflammatory processes and insulin sensitivity [6–10]. Several biological markers of inflammation are associated with risk of CVD. TNF-␣, a proinflammatory cytokine, has been associated with an increased risk for recurrent events after myocardial infarction [11] (MI), coronary heart disease [12] (CHD) and also with other established risk factors such as insulin resistance and obesity [13,14]. TNF-␣ activity is mediated by two TNF-␣ receptors, which in soluble forms—sTNF-R1 and sTNF-R2—can be used as proxies for TNF-␣ activity [15]. These soluble receptors bind to TNF-␣

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and may attenuate its bioactivity; however, they also serve as slow release reservoirs for TNF-␣, thereby prolonging TNF’s half life, and may be a better measure of total body TNF activity [16]. More specifically, TNF-receptors have been associated with cardiovascular events [17–19]. Additionally, C-reactive protein (CRP), an acute phase reactant, and IL-6, a proinflammatory cytokine that stimulates CRP production, have both been independently shown to predict cardiovascular events [11,17]. Several studies have assessed the association between alcohol consumption and inflammatory markers in welldefined populations [6,9,20–22], but frequency and consumption patterns have been described inconsistently. Furthermore, to our knowledge, no studies have examined the association of alcohol and sTNF-receptors, and these receptors may be important in predicting coronary risk. Therefore, we examined the association of sTNF-R1, sTNF-R2, CRP and IL-6 with alcohol consumption in men and women selected from two large ongoing cohort studies.

2. Methods

patterns and novel biomarkers of CVD, and the number of men and women were selected based upon power calculations and funding constraints. Aside from the exclusions above, characteristics between the 465 men and 473 women in this study, and the remaining men and women who gave blood, did not differ substantially [24]. Additionally, 519 HPFS participants were previously sampled as controls for a separate nested case-control study of plasma biomarkers and risk of MI [25], had alcohol intake and measures of inflammatory markers, and were not among the 465 men already selected. Diabetes mellitus was not an exclusion criterion for the controls, though less than 5% of controls reported history of diabetes. The 519 controls were slightly older, but were otherwise similar in characteristics. Thus, we pooled the two sub-samples of men for these analyses to increase the power, but included a flag variable in the multivariable analysis to adjust for this pooling. We further excluded all extreme drinkers from each sample (20 men in the biomarker sample, 5 men in the HPFS controls) who reported an average daily intake of >26 g of alcohol (>182 g/week), but consumed all alcohol on only 1–2 days/week (n = 25 men). The final analyses included 959 men and 473 women.

2.1. Study population 2.2. Alcohol assessment The Health Professionals Follow-up Study (HPFS) is a prospective cohort study of 51,529 US male health professionals aged 40–75 years in 1986, who completed detailed questionnaires assessing dietary intake, lifestyle factors and medical history at baseline [2]. The Nurses’ Health Study II (NHSII), established in 1989, is a prospective cohort study of 116,671 US female registered nurses aged 25–42 years at baseline, and who also completed detailed questionnaires assessing diet, lifestyle and medical history [23]. Follow-up questionnaires were mailed to participants of both studies every 2 years to update baseline information and to ascertain newly diagnosed disease. All participants gave written informed consent and the Harvard School of Public Health Human Subjects Committee Review Board approved the study protocol. All participants in both cohorts were invited to provide a blood sample. Between 1993 and 1995, we collected 18,225 samples in men, and between 1996 and 1999, 29,616 among women. Participants with a history of myocardial infarction, angina pectoris, stroke, type 2 diabetes, intermittent claudication, gastric or duodenal ulcers, gallbladder disease, liver disease, cancers (except non-melanoma skin cancer), or missing information on diet, smoking, alcohol consumption, or physical activity before blood collection were subsequently excluded [24]. From the remaining men and women, 465 men and 473 women were randomly selected based upon different self-reported patterns of alcohol intake and over-sampled to include a wide range of alcohol intakes determined by frequency, amount and alcohol intake with meals (i.e. none, light, moderate, heavy, binge, etc.). The purpose of this subset was to investigate the association between alcohol drinking

In both cohorts, alcohol consumption and other nutrient information was assessed every 4 years using a 131-item semiquantitative food frequency questionnaire (FFQ). Participants were asked to report their average intake of beer, white wine, red wine and liquor in the previous year. All alcohol and nutrient information for these analyses was based on the 1994 follow-up questionnaire for the HPFS, and 1995 follow-up questionnaire for the NHSII. Standard portions were specified as a glass, bottle or can of beer, a 4 oz glass of wine, and a shot of liquor. For drinking habits for each beverage type, there were nine possible response categories ranging from “never” or “less than once per month” to “six or more times per day”. To determine total grams of alcohol intake, we multiplied the frequency of each beverage type by the ethanol content in each portion (12.8 g for beer; 11.0 g for wine; 14.0 g for liquor) [26], and computed the sum of the beverage-specific intakes. Participants were also asked about the number of days per week on which they typically drank alcohol, with responses that ranged from 0 to 7 days per week. To calculate the usual quantity of alcohol consumed per drinking day, we divided the average weekly alcohol consumption (from the FFQ) by the number of drinking days per week. Previous work in this group has validated the assessment of alcohol consumption by the FFQ method. Among 136 men and 173 women, the alcohol assessment using FFQ was compared to multiple-week diet records over the same time period (gold standard), and were shown to be highly correlated- Spearman r = 0.86 in men and r = 0.90 in women [27].

J.K. Pai et al. / Atherosclerosis 186 (2006) 113–120

Other covariates, such as age, body mass index (BMI), smoking status, physical activity, medications and family history were assessed from the 1994 questionnaire for HPFS and 1997 questionnaire for NHSII. These represent the available data closest in time to the blood draws. 2.3. Laboratory methods Participants provided blood samples collected in three 10 mL blood tubes (containing EDTA for HPFS) and two 15 mL blood tubes (containing sodium heparin for NHSII samples), stored in styrofoam containers with ice packs, and returned back to the laboratory via overnight courier. Over 95% of the samples arrived within 24 h, and were centrifuged, aliquoted and stored in liquid nitrogen (−130 ◦ C or colder) until analysis. The stability within 36 h of transport of several of these plasma markers has been previously assessed, and shown to be stable [28]. Plasma sTNF-R1, sTNF-R2 and interleukin-6 were measured by means of enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN), which have a day-to-day variability of 3.5–9.0%. The levels of C-reactive protein were determined by means of a highly sensitive immunoturbidimetric assay with the use of reagents and calibrators from Denka Seiken (Niigata, Japan), which has a day-to-day variability of 1–2%. Total, HDL, and directly obtained LDL cholesterol were measured using standard methods with reagents from Roche Diagnostics (Indianapolis, IN) and Genzyme (Cambridge, MA). 2.4. Statistical analysis We conducted multivariable linear regression analyses to model the association between alcohol consumption and inflammatory markers. Robust variance methods were used to model non-normally distributed, continuous outcomes. We conducted separate analyses for men and women because the women were younger than the men. The main exposure of alcohol consumption was categorized into 0, 0.1–4.9, 5.0–14.9, 15.0–29.9 and ≥30 g/day. These categories were created to correspond approximately to 0, 0.5, 1, 2 and >2 drinks/day. Because not all participants consumed alcohol every day, we calculated and categorized the average grams of alcohol consumption per drinking day (g/dd), as abstainers, 0.1–14.9, 15.0–29.9, 30–49.9 and ≥50 g/dd. Due to limited numbers, we collapsed the categories in the analyses of beverage specific effects to be 0, 0.1–4.9, 5.0–14.9 and ≥15 g/day. Age was categorized in 5-year intervals and BMI was categorized as <20, 20–24.9, 25–29.9, 30–34.9 and ≥35 kg/m2 . Physical activity, total energy intake and nutrients were categorized into quintiles. Smoking status was characterized as never, former and current smoking 1–14 or ≥15 cigarettes per day. Current aspirin and ibuprofen use were dichotomous (yes/no). History of hypertension, diabetes, high cholesterol and parental history of MI before age 60 also were categorized as yes/no. Model testing for potential confounders was

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based on step-by-step inclusion of diet and lifestyle factors significantly associated with outcomes in univariate analyses. Final models included classic CVD risk factors and other predictors, which upon inclusion changed the main effects of alcohol by at least 10%. Tests for trend were calculated using the median value of each category, and modeled as a continuous variable. This value squared was used to model the quadratic trend. All p-values presented are two-tailed and p-values below 0.05 were considered statistically significant. All analyses were performed using SAS 8.2 (SAS Institute, Cary, NC).

3. Results The over-sampling of men and women across drinking patterns provided a broad range of alcohol intake (Tables 1 and 2). Among men in this sample, alcohol consumption was associated with smoking, aspirin or ibuprofen use, hypertension, parental history of MI, physical activity and omega-3 fatty acids DHA and EPA. Among women, alcohol consumption was associated with smoking, aspirin or ibuprofen use and greater caloric intake. Expected associations were observed with alcohol and HDL levels in both men and women. 3.1. Total average intake The age- and multivariable-adjusted associations between average alcohol intake and plasma marker concentrations are shown in Table 3. For sTNF-R1 and sTNF-R2, there was an inverse linear trend in both men (p-trend < 0.001 and 0.002, respectively) and women (p-trend = 0.03 and 0.08, respectively). Among men, we found a suggestion of a U-shaped association for CRP (p-quadratic = 0.06) and IL-6 (p-quadratic = 0.01). Men who consumed on average 15.0–29.9 g/day (1–2 drinks/day) had lower CRP (−0.66 mg/L, p = 0.13), and IL-6 (−1.12 pg/mL, p = 0.02) concentrations than non-drinkers. We observed a similar inverse association among women, but at lower concentrations of alcohol consumption (0.1–4.9 g/day). This trend was not statistically significant. Beverage-specific analyses were also performed but the sample size was limited. Beer, white wine, red wine and liquor (when controlling for other alcohol types) were not significantly different from each other, and were not independently significantly, inversely associated with inflammatory markers (data not shown). 3.2. Grams per drinking day We further calculated grams of alcohol per drinking day (g/dd) to assess whether total ethanol and frequency of intake may be related to plasma marker concentrations (Table 4). The number of non-drinkers differed from that in Table 3 because several light drinkers responded zero to the average

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Table 1 Characteristics by alcohol consumption of 959 US men in 1994: the Health Professionals Follow-up Study Alcohol consumption (g/day)a 0

0.1–4.9

5.0–14.9

15.0–29.9

30–105.92

Participants (n) Age (years) Body mass index (kg/m2 ) Alcohol intake in 1994 (g/day)

192 63.9 ± 8.8 25.3 ± 3.4 0.0 (0.0–0.0)

134 63.2 ± 8.9 26.2 ± 4.0 1.93 (1.02–3.56)

205 63.6 ± 8.7 24.9 ± 2.9 11.0 (7.88–13.0)

224 62.2 ± 8.7 25.7 ± 3.2 20.1 (16.9–24.4)

204 63.1 ± 8.0 25.6 ± 3.1 40.3 (35.4–48.5)

Plasma lipids Total cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL)

212 ± 44.6 45.0 ± 12.6 134 ± 36.6

205 ± 32.9 42.1 ± 9.42 129 ± 28.2

221 ± 42.5 55.3 ± 30.3 137 ± 33.7

240 ± 48.2 59.0 ± 16.9 144 ± 35.5

237 ± 52.2 59.6 ± 17.4 140 ± 41.1

Physical activity (METs/week)b

36.9 ± 42.1

35.8 ± 38.7

39.2 ± 37.8

43.1 ± 42.0

40.3 ± 39.7

Mean daily intakes Total energy intake (kcal/day) Total fat (g/day) Saturated fat (g/day) Omega-3 EPAc and DHAd (g/day)

1975 ± 656 67.7 ± 31.7 22.3 ± 11.3 0.27 ± 0.52

1976 ± 638 70.2 ± 33.0 23.6 ± 12.2 0.24 ± 0.26

1958 ± 586 66.7 ± 29.2 22.0 ± 10.8 0.32 ± 0.39

2049 ± 582 68.7 ± 28.8 22.2 ± 10.2 0.37 ± 0.39

2289 ± 615 75.2 ± 31.3 24.4 ± 11.3 0.31 ± 0.27

Cholesterol-lowering use (%) Current cigarette smokers (%) Current aspirin use (%) Current ibuprofen use (%) High cholesterol (%) Hypertension (%) Parental history of MIe before age 60 (%)

4.2 7.1 29.7 10.4 38.0 29.2 9.9

7.5 13.2 31.3 12.7 44.8 26.1 9.7

6.3 6.8 29.3 17.6 39.5 27.8 12.2

7.1 3.7 44.2 17.0 41.5 27.2 12.1

5.9 14.1 40.7 13.7 44.1 33.3 13.7

a b c d e

Values are means ± S.D., medians (interquartile range), unless otherwise indicated. Metabolic-equivalents. Eicosapentanoic-acid. Decosahexanoic-acid. Myocardial infarction.

frequency question, but not zero for the beverage questions on average alcohol intake over the past year. Compared to non-drinkers, men consuming 15–29.9 g/dd had lower concentrations of sTNF-R1 (−116 pg/mL, p = 0.004), sTNF-R2 (−169 pg/mL, p = 0.01), CRP (−0.25 mg/L, p = 0.61) and IL-6 (−1.57 pg/mL, p = 0.04). Again, there was suggestion of a U-shaped association for CRP and IL-6, and significant inverse linear trends for the TNF receptors. This similar decrease was observed in women as well. Compared to nondrinkers, women consuming 15–29.9 g/dd had lower concentrations of sTNF-R1 (−65, p = 0.03), sTNF-R2 (−120, p = 0.04), CRP (−0.62, p = 0.12) and IL-6 (−0.41, p = 0.01) levels. Further adjustment for glycemic load, glycemic index, or dietary fiber did not appreciably affect these results.

4. Discussion In this study of US men and women, we found lower concentrations of sTNF-R1, sTNF-R2, CRP and IL-6 among participants who consumed moderate amounts of alcohol, 1–2 drinks/day for men, and half a drink per day for women, compared to non-drinkers. For grams per drinking day, we found the strongest inverse associations among men and women consuming 15–30 g of alcohol per drinking day. Overall, our results suggest a U-shaped association with increasing alcohol intake for CRP and IL-6. Additionally, we found a

strong inverse linear trend with increasing alcohol consumption for sTNF-R1 and sTNF-R2. Previous epidemiologic studies support our current findings on CRP and IL-6, but no studies have reported on the association between alcohol intake and soluble TNF receptors. In a national health survey of 781 German men and 995 women, Imhof et al. reported a significant U-shaped association between alcohol intake and CRP concentrations in men with the nadir at 20–40 g/day, and increased concentrations among those who drank >80 g/day. They reported a similar association among women with the nadir at 40–60 g/day [20]. We did not have sufficient power to examine these higher alcohol intake levels. In a cross-sectional analysis of 340 women in the Women’s Health Study, Bermudez et al. reported that women who reported alcohol at least weekly had lower concentrations of IL-6 compared to non-drinkers [6]. In a cross-sectional analysis of 1732 men and 1101 women in the Pravastatin Inflammation/CRP Evaluation Study (PRINCE) Study, Albert et al. found that CRP concentrations were lower in moderate drinkers who consumed 5–7 drinks weekly compared to light or occasional drinkers [9]. In the Health, Aging and Body Composition study of 2574 older men and women, Volpato et al. reported a significant J-shaped association between alcohol intake and IL-6 concentrations in both men and women, with the nadir at 1–7 drinks per week, and a similar trend for CRP, which was significant only among women [22]. In a random population sample of 198 men

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Table 2 Characteristics by alcohol consumption of 473 US women in 1997: the Nurses’ Health Study II Alcohol consumption (g/day)a 0

0.1–4.9

5.0–14.9

15.0–29.9

30–61.25

Participants (n) Age (years) Body mass index (kg/m2 ) Alcohol consumption in 1995 (g/day)

74 41.9 ± 4.0 25.6 ± 6.4 0.0 (0.0–0.0)

63 42.3 ± 3.3 24.8 ± 4.4 1.56 (0.77–3.23)

228 42.8 ± 3.9 23.5 ± 3.6 11.0 (9.46–12.6)

85 41.9 ± 4.1 24.5 ± 3.9 17.8 (16.4–24.1)

23 41.5 ± 3.3 24.8 ± 4.8 38.5 (32.8–50.4)

Plasma lipids Total cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL)

188 ± 32.7 60.6 ± 11.5 108 ± 29.3

190 ± 27.0 62.7 ± 13.0 108 ± 20.6

195 ± 31.5 70.1 ± 15.8 107 ± 27.8

200 ± 35.8 68.6 ± 16.6 112 ± 30.2

207 ± 37.9 79.7 ± 19.2 112 ± 34.8

Physical activity (METs/week)b

17.1 ± 18.5

19.4 ± 24.4

24.1 ± 28.6

23.8 ± 22.5

20.3 ± 17.0

Mean daily intakes Total energy intake (kcal/day) Total fat (g/day) Saturated fat (g/day) Omega-3 EPAc and DHAd (g/day)

1770 ± 618 58.5 ± 29.0 20.7 ± 11.7 0.17 ± 0.13

1847 ± 482 58.7 ± 19.9 20.2 ± 7.7 0.15 ± 0.10

1836 ± 488 58.7 ± 21.9 19.7 ± 8.0 0.20 ± 0.18

1981 ± 570 63.2 ± 23.6 21.5 ± 8.7 0.21 ± 0.23

2018 ± 380 60.0 ± 18.5 20.5 ± 7.1 0.16 ± 0.10

Never oral contraceptive use (%) Current cigarette smoker (%) Current aspirin use (%) Current ibuprofen use (%) High cholesterol (%) Hypertension (%) Parental history of MIe before age 60 (%)

25.7 2.7 5.4 28.4 12.2 6.8 13.5

17.5 4.8 25.4 49.2 17.5 6.4 12.7

14.9 6.1 12.7 44.3 14.5 5.7 17.5

12.9 12.9 18.8 51.8 21.2 5.9 15.3

8.7 26.1 17.4 56.5 17.4 0.0 17.4

a b c d e

Values are means ± S.D., medians (interquartile range), unless otherwise indicated. Metabolic-equivalents. Eicosapentanoic-acid. Decosahexanoic-acid. Myocardial infarction.

from south London, Mendall et al. reported an inverse association between alcohol intake and TNF-␣ concentrations but a positive association between alcohol intake and IL-6 concentrations comparing men with more than one drink weekly to those consuming one or fewer drinks weekly [21]. However, the dichotomous assessment of alcohol may limit the interpretation of these results across a larger range of alcohol consumption. Finally, in a 12-week intervention trial of wine and gin among 42 healthy men, Estruch et al. reported lower levels of CRP, but no change in TNF-␣ concentrations after wine and gin intake [29]. Nonetheless, though mostly consistent, our study further identified a strong inverse association between alcohol consumption and soluble TNF receptors, which have been associated with cardiovascular risk, as well as describing the usual quantity of alcohol consumed per drinking day as a better measure of overall alcohol intake. The association between alcohol and inflammation has strong biological plausibility. On one hand, alcohol in high quantities and its metabolites may exert direct inflammatory effects on the liver. Acetaldehyde, in particular, may induce free radical production and subsequently increase lipid peroxidation and tissue inflammation [30]. While excessive alcohol has also been associated with increased IL-6 production, lower concentrations, on the other hand, may inhibit IL-6 secretion from adipocytes [31]. Additionally, alcohol may suppress macrophage-induced TNF-␣ production, and lead

to downstream anti-inflammatory effects on CRP and IL-6 [32]. Our findings that alcohol intake is inversely linearly associated with soluble TNF receptors are novel among humans but supported by animal models. With increasing blood alcohol levels, there was a dose–response depression of serum TNF levels among endotoxin-induced rats [35], and also less TNF-␣ was observed bound to cell-surface receptors among alcohol-treated rats compared to respective controls [34]. Moreover, our findings support the hypothesis that alcohol may have health effects beyond lipids and acute phase inflammatory markers, possibly through insulin sensitivity. It has been shown in animal studies that TNF-␣ and IL-6 impair insulin sensitivity [15,33]. Thus, the beneficial effects of alcohol on insulin levels shown in human metabolic studies [36] may be mediated through changes in TNF-␣ activity and insulin sensitivity. The U-shaped association for CRP concentrations may be partially caused by the upstream effects of moderate alcohol consumption on TNF-␣ activity and partially through the response of separate hepatotoxic effects of alcohol at higher intake levels. Further research that can differentiate the effects of alcohol on the inflammatory cascade is necessary. Despite these results, several limitations should be addressed. The blood samples were collected over a period of 2–3 years. For consistency, we used data from the single questionnaire cycle closest to blood collection, and recognize

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Table 3 Mean plasma inflammatory markers between non-drinkers and drinkers among men and women Grams of alcohol per day 0

0.1–4.9

5.0–14.9

15.0–29.9

≥30

0 192

1.93 134

11.0 205

20.1 224

40.3 204

sTNF-R1 (pg/mL) Crudea Adjustedb

1339 1341

1245e 1249e

1263 1271

1199c 1200c

sTNF-R2 (pg/mL) Crudea Adjustedb

2396 2391

2301 2307

2232e 2231e

CRP (mg/L) Crudea Adjustedb

2.28 2.55

2.38 2.35

IL-6 (pg/mL) Crudea Adjustedb

2.83 3.12

p-Trend linear

p-Trend quad

1189c 1176c

<0.001 <0.001

0.11 0.22

2185d 2192d

2165c 2160d

0.001 0.002

0.05 0.09

2.12 2.05

1.94 1.89

2.92 2.80

0.33 0.49

0.06 0.06

3.47 3.53

2.35 2.18

2.04 2.00e

3.30 3.18

0.80 0.99

0.01 0.01

0 74

1.56 63

11.0 228

17.8 85

38.5 23

sTNF-R1 (pg/mL) Crudea Adjustedb

1073 1048

1008 992

1006e 1019

957d 958e

958e 954e

0.003 0.03

0.19 0.86

sTNF-R2 (pg/mL) Crudea Adjustedb

2253 2216

2065e 2037e

2105e 2132

2105e 2097

1978d 1940e

0.02 0.08

0.82 0.39

CRP (mg/L) Crudea Adjustedb

2.70 2.25

1.03d 0.79d

1.63 1.87

1.73 1.81

1.75 1.21

0.48 0.86

0.38 0.43

IL-6 (pg/mL) Crudea Adjustedb

1.66 1.61

1.19e 1.16e

1.59 1.62

1.46 1.45

1.30 1.33

0.63 0.96

0.45 0.31

Men Median n

Women Median n

sTNF-R1, soluble tumor necrosis factor-receptor 1; sTNF-R2, soluble tumor necrosis factor-receptor 2; CRP, C-reactive protein; IL-6, interleukin-6. a Adjusted for age only. b Adjusted for age, bmi, smoking, physical activity, ibuprofen use, high cholesterol, parental history of myocardial infarction before age 60, omega-3 fatty acids (DHA + EPA only), and total caloric intake. c p < 0.001 compared to zero intake. d p < 0.01. e p ≤ 0.05.

that alcohol and covariate data at the time of venipuncture was estimated. However, bias from residual confounding is unlikely because the age-adjusted and multivariable-adjusted results were similar. We collected only one sample per subject, whereas multiple collections are ideal to reduce variability from acute infection or other short-term insult. However, we have previously reported good overall 4-year intra-class correlations for these biomarkers (sTNF-R1, 0.81; sTNF-R2, 0.78; CRP, 0.67 and IL-6, 0.47) [24]. Previous work also assessed the effect of transport conditions and processing of the samples, and found the inflammatory marker concentrations to be stable [28]. Reported inflammatory marker concentrations, specifically for CRP and IL-6, were lower than those presented in other studies. However, despite reduced variability in biomarkers, our findings still are in agreement

with a direct beneficial effect of moderate alcohol consumption on these inflammatory markers. Both populations were health professionals, and reported alcohol consumption was not heterogeneous enough to examine inflammatory markers at the extremes of drinking pattern. Only 6% of men consumed >50 g/day and 5% of women consumed >30 g/day. We assessed alcohol intake and potential confounders with a self-administered questionnaire which may limit our ability to study the associations at the extremes where the validity of the questionnaire may be lower. Nonetheless, this method of alcohol assessment was previously validated and found strongly correlated with the gold standard [27]. However, despite the careful adjustment in multivariable models, alcohol use is associated with other life-style and health-related factors, and residual confound-

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Table 4 Mean plasma inflammatory markers among men and women, according to grams of alcohol per drinking day Grams of alcohol per drinking day 0

<15

15–29.9

30–49.9

≥50

224

136

221

190

156

sTNF-R1 (pg/mL) Crudea Adjustedb

1321 1324

1306 1312

1202 1208d

1184e 1187c

sTNF-R2 (pg/mL) Crudea Adjustedb

2351 2349

2358 2345

2174d 2180e

CRP (mg/L) Crudea Adjustedb

2.32 2.48

2.20 2.19

IL-6 (pg/mL) Crudea Adjustedb

3.26 3.47

p-Trend linear

p-Trend quad

1193c 1174c

<0.001 <0.001

0.007 0.03

2118c 2131d

2194e 2184e

0.003 0.005

0.003 0.01

2.23 2.23

2.15 2.19

2.71 2.42

0.58 0.99

0.42 0.52

2.47 2.48

1.96 1.90e

2.60 2.63

3.12 2.84

0.94 0.79

0.14 0.12

101

143

130

64

35

sTNF-R1 (pg/mL) Crudea Adjustedb

1058 1034

1025 1034

961d 969e

978e 980

993 990

0.02 0.08

0.02 0.19

sTNF-R2 (pg/mL) Crudea Adjustedb

2220 2183

2133 2151

2051d 2063e

2029d 2025e

2153 2148

0.06 0.17

0.004 0.05

CRP (mg/L) Crudea Adjustedb

2.33 1.99

1.71d 1.86

1.21 1.37

1.81 1.79

2.03 1.83

0.54 0.64

0.02 0.24

IL-6 (pg/mL) Crudea Adjustedb

1.54 1.50

1.62 1.63

1.10d 1.09e

1.66 1.64

2.27 2.39

0.32 0.25

0.10 0.11

Men n

Women n

sTNF-R1, soluble tumor necrosis factor-receptor 1; sTNF-R2, soluble tumor necrosis factor-receptor 2; CRP, C-reactive protein; IL-6, interleukin-6. a Adjusted for age only. b Adjusted for age, bmi, smoking, physical activity, ibuprofen use, high cholesterol, parental history of myocardial infarction before age 60, omega-3 fatty acids (DHA + EPA only), and total caloric intake. c p < 0.001 compared to zero intake. d p < 0.01. e p < 0.05.

ing may still exist. Because this study was cross-sectional, we are limited in our ability to show causality. However, it seems unlikely that concentrations of inflammatory markers among these generally healthy individuals would affect self-reported alcohol intake (i.e. reverse causality). Finally, our questionnaires did not include questions on the use of hydroxymethylglutarylcoenzyme A reductase inhibitors (statins) because these drugs were not widely used at time of blood sampling. However, the reported use of cholesterol-lowering drugs was generally low in the men. In summary, we found an inverse association between moderate alcohol consumption and the inflammatory markers sTNF-R1, sTNF-R2, CRP and IL-6 in both men and women. To our knowledge, this study is the first to describe an inverse linear association between alcohol and sTNF-R1 and sTNF-R2. Although moderate alcohol intake may elicit favorable anti-inflammatory effects, the well-known side effects

of excessive alcohol consumption, as well as specific contraindications, should be considered carefully when providing recommendations to the general population. Our findings support the hypothesis that moderate alcohol consumption and drinking patterns may be important in the inflammatory pathway. These results further suggest a possible mechanism for the beneficial effect of moderate alcohol intake on insulin sensitivity and its protective role in CHD.

Acknowledgments The authors thank Dr. Meir Stampfer for his invaluable comments and Lydia Liu for programming assistance. Supported by research grants from the National Institutes of Health (CA55075, CA67262, HL35464 and AA11181), and partial funding from Merck Research Laboratories for labo-

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