Abstract
In the second World Health Organization MONItoring Trends and Determinants in CArdiovascular Disease (MONICA) Augsburg survey in 1989-1990 (#1=4,940), the association between nephelometric plasma fibrinogen level and lifestyle-related potential determinants was assessed in 4,434 subjects aged 25-74 years (89.8% of participants). Irrespective of pregnancy and the use of oral contraceptives, crude fibrinogen values were consistently higher in women than in men of all ages (age-standardized difference, 12.2 mg/dl; 95% confidence interval, 7.0-17.4 mg/dl). Fibrinogen concentrations were positively correlated (psO.0001) with age, body mass index, and waist-to-hip ratio in both sexes and with cigarette smoking In men and were negatively correlated with alcohol consumption in both sexes. In multiple linear regression analyses using categorized determinants as independent variables, a strongly J-shaped relation for body mass index in women and a linear association for waist-to-hip ratio in men were revealed. Smoking had a dose-dependent effect on fibrinogen concentration in men but a lesser effect in women. For alcohol consumption a U-shaped association was found, particularly in men. The curvilinear relations were confirmed in multiple polynomial regression models using continuous determinant variables. The potential epidemiological impact of a determinant was assessed by calculating differences in adjusted fibrinogen concentrations associated with the 10th and 90th percentile values of the determinant distributions actually observed among the study participants. This impact on the population fibrinogen level was most pronounced for age in both sexes, followed by body mass index, cigarette smoking, and alcohol consumption in women and by smoking, waist-to-bip ratio, and alcohol consumption in men. About 29% of the total variance in female and 26% in male fibrinogen values were explained by the three lifestyle factors plus age. Our findings are consistent with the hypothesis that fibrinogen may be an etiologic link between certain lifestyle characteristics and the course of cardiovascular disease. {Arterio- sclerosis and Thrombosis 1992;12:780-788) KEY WORDS • fibrinogen • cardiovascular disease risk factors • body mass index • waist-to-hip ratio • cigarette smoking • alcohol consumption T he evidence that clotting factors may be important in the evolution of atherosclerotic vascular disease was initially derived from case-control 1 " 3 and angiographic 4^ studies in which men with clinically manifest coronary heart disease showed elevated mean fibrinogen levels. Further evidence came from a number of prospective epidemiological studies indicating that plasma fibrinogen levels were strongly predictive of coronary heart disease and stroke incidence and mortality. 6 -12 An as-yet-undetermined part of the association between traditional risk factors and cardiovascular disease From the GSF-Institute of Epidemiology

T he evidence that clotting factors may be important in the evolution of atherosclerotic vascular disease was initially derived from case-control 1 " 3 and angiographic 4^ studies in which men with clinically manifest coronary heart disease showed elevated mean fibrinogen levels. Further evidence came from a number of prospective epidemiological studies indicating that plasma fibrinogen levels were strongly predictive of coronary heart disease and stroke incidence and mortality. 6 -is believed to be mediated through plasma fibrinogen (e.g., cigarette smoking 7 -10 and social factors 13 ' 14 ) . Thus, it is of importance to further investigate lifestyle-related, potentially modifiable determinants of fibrinogen concentration in the general population. We present cross-sectional analyses on the relation of the potential determinants body mass index (BMI), waist-to-hip ratio (WHR), cigarette smoking, and alcohol consumption and of age and sex with plasma fibrinogen concentrations. The analyses are based on a sample from a large population survey in southern Germany.
Methods

Study Population and Sampling Frame
The 1989-1990 MONItoring Trends and Determinants in CArdiovascular Disease (MONICA) Augsburg Survey was designed as a cross-sectional study to investigate the prevalence and the population distributions of major cardiovascular risk factors. The study area comprised the city of Augsburg and the two surrounding rural districts of Augsburg and Aichach-Friedberg in the south of the Federal Republic of Germany. From a target population of 349,050 inhabitants aged 25-74 years, 6,637 individuals were drawn applying two-stage, age/sex-stratified random sampling. The sampling methods are described in detail elsewhere. 15 Of the 6,420 eligible persons, 4,940 (76.9%) participated in the survey. 16 
Survey Methods
Participants were asked to submit to a standardized interview; three blood pressure measurements; recordings of height, weight, and waist and hip circumferences; a resting 12-lead electrocardiogram; the drawing of blood; and a self-administered questionnaire. The interview included questions on medical history, awareness of hypertension, socioeconomic factors, smoking and drinking habits, health behavior, and drug history.
Current regular cigarette smokers and occasional cigarette smokers (< 1 cigarette/day) were distinguished from former cigarette smokers and those who had never smoked cigarettes. The average intake of alcohol consumed per day was calculated from serf-reported consumption of alcoholic beverages over the last weekend and on the last workday by making the following conversions: 11 beer=40 g alcohol, 11 wine=100 g alcohol, and 1 shot (0.02 1) spirits=6.2 g alcohol. 17 Body weight and height were determined in light clothing without shoes, and BMI was calculated as kilograms per meter squared. The waist circumference was measured midway between the lower rib margin and the iliac crest, in a standing and slight expiratory position. The hip circumference was measured at the greatest circuit. The former divided by the latter is reported as the WHR.
Laboratory Methods
Blood sampling was performed under standardized conditions from sitting, nonfasting participants, with minimal tourniquet use and suction. Ten milliliters of blood was collected into Vio volume of 3.13% trisodium citrate in monovettes (Fa. Sarstedt, Numbrecht, FRG) and centrifuged for 10 minutes at 4°C. Plasma was immediately separated into three plastic cryotubes (500 til plasma in each), stored at 2-8°C for a maximum of 12 hours, and finally frozen at -70°C.
Measurements of fibrinogen concentration were performed by the Institute of Clinical Chemistry of the Ludwig-Maximilians-Universitat, Miinchen. Frozen samples were thawed for 10 minutes in a water bath maintained at 37°C, then removed, kept at room temperature, and analyzed within 20 minutes. The results of a pilot study carried out on 10 healthy volunteers to examine the stability of fibrinogen measurements according to different handling and storage procedures indicated that this procedure had no effect on the values of nephelometric fibrinogen determinations compared with analyses performed on samples immediately after drawing blood.
Fibrinogen concentration was determined by an immunologic nephelometric assay with a Behring nephelometer analyzer (Behringwerke, Marburg, FRG) using a monospecific antiserum against human fibrinogen (Behringwerke; lot 154424). The nephelometric assay was calibrated using standard plasma from Behringwerke (declared fibrinogen concentration, 345 mg/dl; lot 068019). The assay was calibrated regularly once a week and additionally if control values were out of the range of the control limits, as defined below.
Regular internal quality control measurements were performed before and during each series of analyses (after every 20 samples) and at the end of each run. For control samples we used Behring-N-Control Plasma (lot 048912) and Immuno Control Normal and Immuno Control Abnormal (lots KN26 and KP31, respectively; Immuno AG, Vienna, Austria). During an initial period of 30 days the mean values and SDs for each control sample were evaluated and compared with the declared fibrinogen concentration of the material. For the first control sample the mean±SD concentration was found to be 415.3±10 mg/dl (declared value, 415 mg/dl). For the second and third control samples the mean±SD concentrations were 335.4±10 mg/dl (declared value, 335 mg/dl) and 199.6 ±7 mg/dl (declared value, 200 mg/dl). Thus, the coefficient of variation of the method ranged between 2.4% and 3%, indicating an acceptable analytical precision for this nephelometric assay. Quality control limits were defined according to the results of the initial period as mean±2 SD of each control sample. The assay was considered invalid and requiring new calibration if one control value was out of the range of these limits. No lot number of any reagent, calibrator, or control was changed during the total measurement period.
Another day-to-day control was performed by comparing the median of the almost 100 values obtained daily with a reference median obtained from the approximately 500 values of the first 5 days of analysis. No value had to be excluded for reasons of fibrinogen measurement quality control. The results are reported in milligrams per deciliter.
Missing Data
Four hundred eighty-four participants were excluded from the analyses because of incomplete data, 426 of whom refused venipuncture. In addition, pregnant women (n=22) were removed from the data set. Thus, analyses were run with the data of 4,434 subjects (89.8% of all survey participants).
Statistical Methods
Statistical analyses were performed using SAS Release 5.2. Each potential determinant was categorized, and crude fibrinogen concentration means were calculated for each category. To detect differential associations over the wide age range of this survey, all data were analyzed after stratification for age (25-44 and 45-74 years) and sex. Next, covariance-adjusted estimates of fibrinogen concentration means were obtained in each determinant category of the four age/sex strata using linear regression techniques (SAS PROC GLM). Each regression model included as independent variables the categorized determinant of interest plus the other covariates as dummy variables and age as a continuous variable. The 95% confidence intervals (CIs) for the differences of adjusted fibrinogen concentration means x, were computed as where s, denotes the estimated standard error of x,, J = 1 or 2. This step served to assess the shape of the association for each determinant, including threshold effects. Last, polynomial regression techniques were employed involving each determinant as first-and secondorder continuous variables. The model of best fit was identified in a forward-selection manner running SAS PROC STEPWiSE with the maximum R 2 improvement option. For the sake of clearness and because only few age interactions were found by the categorized data analyses, age stratification was no longer maintained at this step. The results of the polynomial regression analyses served to estimate the epidemiological impact of each determinant on the population fibrinogen level.
Results

Fibrinogen Concentration in Categories of Determinants
Age and sex. The MONICA Augsburg population distribution of plasma fibrinogen concentration is given in Table 1 . Crude mean fibrinogen concentration increased with age in both sexes (see also Table 2 ). At all ages mean fibrinogen concentration was higher for women than for men (overall age-standardized difference, 12.2 mg/dl; 95% CI, 7.0-17.4 mg/dl). Figure 1 depicts the change in mean fibrinogen levels when including pregnant women (n=22, 6.9 mg/dl increase) or excluding women taking oral contraceptives (n = 134, 6.7 mg/dl decrease). Both conditions affected the estimate mainly in women aged 25-34 years.
Body mass index and waist-to-hip ratio. BMI and WHR were positively correlated with plasma fibrinogen concentrations in men and women (p^O.OOOl, Table 2 ). In men, however, the relation with BMI was less strong than that with WHR. Moreover, in the forward-selection stepwise procedure applied in the polynomial regression analyses (see below), WHR was consistently included before BMI in the models for men (Table 3) . Just the opposite was found for women. Evidently, WHR and BMI contributed differentially to the explanation of fibrinogen concentration variance in men and women. We therefore focused our further analyses on the associations of fibrinogen concentration with WHR in men and with BMI in women.
Crude and adjusted mean fibrinogen concentrations by categories of WHR and BMI are given in Table 4 and Smoking. In both age strata of men, fibrinogen levels increased consistently with the number of cigarettes smoked per day (Table 4, Figure 2 ). Adjustment for covariates did not markedly affect this relation. Mean fibrinogen concentrations in ex-smokers were intermediate between those of men who had never smoked and of men who were occasional or regular smokers. Among current smokers a clear dose-related association was seen. Thus, for example, at age 25-44 years, adjusted fibrinogen mean increased, relative to men who had never smoked, by 30.6 mg/dl (95% CI, 11.1-50.2 mg/dl) and 54.9 mg/dl (95% CI, 38.9-71.0 mg/dl) with the smoking of 1-10 and >20 cigarettes/day, respectively. In women, there was no correlation between fibrinogen levels and cigarette smoking (Table 2 ). Nevertheless, a smoking effect was detected between the adjusted fibrinogen concentration means of women who had never smoked and women who were current regular smokers, but no dose-related effects were found among the latter (Table 4, Figure 2 ). Particularly in current women smokers aged 45-74 years, covariance adjustment markedly contributed to a stronger association with fibrinogen concentration (Figure 2 ). Adjusted fibrinogen levels of current regular smokers (1-10 cigarettes/ day) and women who had never smoked differed by 18.9 mg/dl (95% CI, 3.4-34.4 mg/dl) and 29.7 mg/dl (95% CI, 11.9-473 mg/dl) in groups aged 25-44 and 45-74 years, respectively. Adjusted mean fibrinogen levels of older ex-smokers were almost identical to those of lifetime nonsmokers.
Alcohol consumption. There existed a moderate negative correlation between alcohol consumption and plasma fibrinogen concentration (Table 2) . Categorizing dairy alcohol intake revealed, however, a rather U-shaped relation in each age/sex stratum ( Figure 2 ). The U shape was less marked in men older than 45 years and was absent in young women. It was distinctly attenuated by covariance adjustment in the heavy alcohol consumption Figure 2 ). Independent of age and sex, nondrinkers consistently showed the highest fibrinogen values. The lowest mean fibrinogen concentrations were mostly seen with an alcohol consumption of 40.0-59.9 g/day. Table 3 contains the results of polynomial regression analyses. In this table, the partial regression coefficients indicate the alteration of fibrinogen levels per unit change of a determinant; thus, product terms of defined rises or drops in a determinant with the respective coefficients provide statistical estimates of the related change in plasma fibrinogen concentration. For both sexes, all regression coefficients in Table 3 were highly significant. Essentially, polynomial regression analyses confirmed the shape of associations detected by the categorized data analyses above. Apart from WHR in men, all other determinant variables were included in Based on regression estimates of Table 3. the models as second-order terms. The explanation of total variance of the fibrinogen concentration was slightly higher in women (7? 2 =28.5%) than in men (i? 2 =25.9%).
Polynomial Regressions of Fibrinogen Concentration on Continuous Determinant Variables
Based on the models in Table 3 , specific adjusted fibrinogen concentration means were calculated for the 10th and 90th percentile values of the study participants' determinant distributions. The difference of these two means relates the observed range in a given determinant to its expected impact on fibrinogen concentrations in the study population (Table 5) . Given the wide age range in our study, the strongest impact on population fibrinogen levels was observed for age (101.9 mg/dl in men and 87.8 mg/dl in women). In the male population, cigarette smoking (47.4 mg/dl) and WHR (41.2 mg/dl) had similarly substantial impacts. In women, the effect of BMI (52.4 mg/dl) was more pronounced than that of cigarette smoking (25.3 mg/dl). Due to high fibrinogen concentrations in nondrinkers, regular alcohol use appears to have a moderately decreasing impact on population fibrinogen levels.
Discussion
In confirmation of results from other groups, 18 -22 the quantitatively most relevant determinant of plasma fibrinogen concentration in our study population was age. In addition, we found significant sex differences in plasma fibrinogen levels independent of the prevalence of hormone-related conditions. The use of oral contraceptives 18 -23 and pregnancy are known to induce temporary rises in fibrinogen concentration and to contribute to higher fibrinogen levels in females. In a recent report, however, sex differences in the concentration of thrombin-clottable fibrinogen could not be confirmed when using a heat precipitation technique, and the authors hypothesized an increased fibrinogen clottability in females. 19 Results obtained in our study by nephelometric assay clearly indicate concentration differences between men and women that are independent of the hemostatic action of fibrinogen. A moderate acceleration of the increases in fibrinogen concentration in women's sixth decade of life (Figure 1) Although the biological processes involved in aging of the human body appear to account for the largest differences in fibrinogen levels, we were able to identify effects of sizable magnitude for further potentially modifiable determinants. Consistent with other reports, 18 -19 the association of fibrinogen concentration with BMI was substantially less striking in men than in women. WHR in men, however, was as powerful a predictor (Tables 2 and 3 ) and as considerable a population impact estimator (Table 5) for fibrinogen concentration as was BMI in women. The relevance of abdominal (WHR) versus general (BMI) adiposity for male cardiovascular risk has been corroborated by results of a Swedish cohort study in which plasma fibrinogen concentration and WHR in men were each independently and significantly related to stroke, but BMI was not. 27 The effect of WHR on fibrinogen concentration disappeared, however, in another study when including the maximal oxygen uptake in multiple regression analyses, indicating that physical activity may be closely involved in this relation. 28 Cigarette smoking and plasma fibrinogen concentration were consistently found to be positively associated in male populations Direct and/or indirect pathophysiological pathways have been hypothesized, 34 but the precise mechanisms by which nicotine and presumably other constituents of cigarette smoke may adversely affect the hemostatic system are still unclear. Likewise, the effect of smoking on plasma fibrinogen concentration in our male population was strongly dose related, with fibrinogen concentrations of ex-smokers intermediate between those of current smokers and lifetime nonsmokers, thus supporting a direct and at least partly reversible effect. Assuming that the full effect of quitting smoking on lowering plasma fibrinogen levels takes as long as 10 years, 12 -19 -35 the attenuation of the difference in fibrinogen levels between those who have never smoked and ex-smokers at older ages (Table 4, Figure 2 ) probably reflects a longer period since giving up smoking. Thus, fibrinogen levels may provide an etiologic link between the number of years since giving up smoking and the decline of risk for ischemic heart disease. 19 For women only a weak dose-dependent relation with smoking was found, in accordance with the few publications presently available.
101118 Different smoking behaviors (light smoking, 36 reduced depth of inhalation) or hormone-related intima protection mechanisms may account for this observation. An essentially U-shaped relation between alcohol consumption and plasma fibrinogen concentration was observed in men and presumably also existed in women, although in the latter the low frequency of heavy drinking reduced the precision of our estimates. Other studies in men found either no association 18 -28 or a threshold effect between drinkers and nondrinkers and an inverse relation in women. 19 Nondrinkers 37 -38 and alcoholics 39 are reported to have higher total and cardiovascular mortality rates than light or moderate drinkers, and a number of mechanisms have been invoked to support the concept of a "protective" value of moderate alcohol intake. 40 - 42 Misinterpretation, particularly in cross-sectional studies, may, however, originate from movement of men with preexisting cardiovascular or related illnesses into the nondrinking or occasional-drinking categories. 43 Altogether, our findings of a U-shaped alcohol/fibrinogen relation are compatible with these results, although specific and biologically plausible explanations are still missing.
The joint predictive power of the determinants included in our polynomial regression models, assessed as the portion of total variance explained (Table 3) , was sizable in men (25.9%) and women (28.5%). This portion is substantially higher than that obtained in the Scottish Heart Health Study (SHHS) population. 19 Even when using models similar to the ones used in the SHHS (first-order terms only, age 40-59 years), the marked differences persisted (men, 14.7% versus 6.5%; women, 19.7% versus 10.8%, MONICA Augsburg versus SHHS, respectively). The major reason for these interstudy differences may be found in divergent methods for determining plasma fibrinogen concentration. In the MONICA Augsburg study, determinations of fibrinogen concentration were based on a fully automated nephelometric assay, whereas in the SHHS, thrombin clotting and the relatively imprecise heat precipitation techniques were employed. Although clottable and nephelometric fibrinogen concentrations are highly correlated, the absolute values of nephelometric fibrinogen concentrations are about 40-50% higher than those obtained by thrombin clotting assays (our pilot study comparisons 44 ). Recently, the immunologic determinations of fibrinogen concentration have been proven to be superior to clotting techniques in predicting the incidence of ischemic heart disease 12 and in separating patients attending a lipid clinic from healthy controls. 45 Altogether, these observations might reflect a greater precision of nephelometric determinations of fibrinogen concentrations, different effects of and determinants for the hemostatic function of plasma fibrinogen as opposed to its rheological impact, or possibly an as-yetundetermined additional role of degradation products of fibrin and fibrinogen in the atherosclerotic process.
The results of our cross-sectional study do not necessarily imply causal relations. We suppose, however, that a sizable portion of the impact of Lifestyle factors on cardiovascular diseases may be mediated through plasma fibrinogen. Thus, modification of lifestyle-related factors may exert its beneficial influence on cardiovascular risk partly through lowering of plasma fibrinogen concentrations. This conclusion awaits, however, further confirmation.

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