ORIGINAL INVESTIGATION. Serum Ascorbic Acid and Gallbladder Disease Prevalence Among US Adults

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1 Serum Ascorbic Acid and Gallbladder Disease Prevalence Among US Adults The Third National Health and Nutrition Examination Survey (NHANES III) Joel A. Simon, MD, MPH; Esther S. Hudes, PhD, MPH ORIGINAL INVESTIGATION Background: Ascorbic acid deficient guinea pigs frequently develop gallstones, and ascorbic acid status may also affect the risk of gallbladder disease in humans. To examine the relationship of ascorbic acid, an antioxidant nutrient involved in cholesterol catabolism, to gallbladder disease, we analyzed data collected from a probability sample of US adults. Methods: Analyses of data from 7042 women and 6088 men enrolled in the Third National Health and Nutrition Examination Survey, , were performed. Multiple logistic regression models stratified by sex were examined, controlling for the effects of age, race, diet, body mass index, and other potential confounders. Results: A total of 761 women (11%) and 235 men (4%) reported a history of clinical gallbladder disease (symptomatic gallstones or cholecystectomy). Of the 9650 participants without a history of clinical gallbladder disease or abdominal pain consistent with gallbladder disease, and with valid abdominal ultrasonography, 408 (8%) of 4863 women and 274 (6%) of 4787 men had asymptomatic gallstones. Serum ascorbic acid level was inversely related to prevalence of clinical and asymptomatic gallbladder disease among women, but not among men. Among women, each SD (27 µmol/l) increase in serum ascorbic acid level was independently associated with a 13% lower prevalence of clinical gallbladder disease (P =.006) and asymptomatic gallstones (P =.048). Conclusion: Ascorbic acid, which affects the catabolism of cholesterol to bile acids and, in turn, the development of gallbladder disease in experimental animals, may affect the risk of gallbladder disease among women. Arch Intern Med. 2000;160: From the General Internal Medicine Section, Medical Service, Veterans Affairs Medical Center, San Francisco, Calif, and University of California, San Francisco (Dr Simon); and Department of Epidemiology and Biostatistics, University of California, San Francisco (Drs Simon and Hudes). GALLBLADDER disease is highly prevalent in the United States. 1 It has been estimated that as many as 20 million Americans have gallstones that are composed partially or entirely of cholesterol. 1-3 Gallstones form when bile that is supersaturated with cholesterol undergoes destabilization. 4 Ascorbic acid affects the rate-limiting step in the catabolism of cholesterol to bile acids in experimental animals, 5 and ascorbic acid deficient guinea pigs frequently develop cholesterol gallstones. 6-9 Because of the experimental animal evidence and the observation that many risk factors for gallbladder disease are associated with ascorbic acid status, we hypothesized that ascorbic acid status may be a risk factor for human gallbladder disease. 10 To ascertain whether ascorbic acid status is associated with gallbladder disease in humans, and particularly with the presence of asymptomatic gallstones, we analyzed data collected from the Third National Health and Nutrition Examination Survey (NHANES III) that included serum ascorbic acid levels and information on gallbladder disease among more than American adults. RESULTS The baseline characteristics of 7042 women and 6088 men, aged 20 to 90 years, enrolled in NHANES III with complete data available for these analyses are presented in Table 1. A total of 761 women (11%) and 235 men (4%) reported a history of clinical gallbladder disease. Among 9650 participants who had neither a history of symptomatic gallbladder disease nor a history of typical epigastric or right-upper-quadrant abdominal pain lasting 1 hour or more, 408 (8%) of 4863 women and 274 (6%) of 4787 men had gallstones detected by abdominal ultrasound examination. The age, race, BMI, and serum cholesterol levels of women and men were similar. A total of 62% of women reported ever 931

2 SUBJECTS AND METHODS SUBJECTS The NHANES III was a national probability survey of approximately Americans conducted between 1988 and 1994 that used a stratified, cluster sampling design to oversample populations of special interest. 11 Participants, aged 2 months to older than 90 years, were enrolled, interviewed, and examined by study personnel. 11 Complete data from 7042 women and 6088 men between the ages of 20 and 90 years were available for these analyses, representing approximately 83% of the total number of examined participants. The NHANES III recorded subjects older than 90 years as being 90 years old. Participants were excluded if there were missing data on variables judged to be potential predictors of gallbladder disease or if outcome data on gallbladder disease were unavailable. We also excluded as potentially unreliable historians an additional 4 participants who reported consuming more than 30 alcoholic beverages daily. Finally, 21 participants with extremely high serum ascorbic acid levels ( 170 µmol/l) were excluded because such values were of questionable validity. 12 Participants excluded because of missing data were compared with those analyzed; both groups were similar demographically regarding age and sex, but excluded participants were more likely to have attained a lower level of education and to be nonwhite. MEASUREMENTS The NHANES III questionnaire data included selfreported age; race (categorized as white, black, or other); sex; years of education completed; level of leisure-time physical activity; history of smoking; diabetes mellitus; level of alcohol intake; menopausal status; use of diuretic, cholesterol-lowering, and estrogencontaining medications; and dietary intake. 11 Quantitative nutrition data were collected by means of a 24-hour dietary recall. We calculated body mass index (BMI; in kilograms per meters squared) from weight and height data recorded during the physical examination. The questionnaires, dietary methods, and examination procedures used in NHANES III have been described elsewhere in detail. 11 We ascertained whether participants had a history of clinical gallbladder disease by a positive response to either of the following questions: Has a doctor ever told you that you had gallstones? and Have you ever had gallbladder surgery? 11 Both questions appeared twice in the NHANES III questionnaire; we used the response to the initial question for these analyses because of the greater number of responses. Participants who had gallstones on abdominal ultrasound examination, but reported no history of clinical gallbladder disease and also denied a history of right-upper-quadrant or epigastric abdominal pain lasting 1 hour or longer, were considered to have asymptomatic gallstones. Among the participants in our sample, a real-time abdominal ultrasound examination with valid results was performed on women and men aged 20 to 74 years by means of a standard protocol. 11 After exclusion of participants with known clinical gallbladder disease (n = 850) and with symptoms compatible with undiagnosed gallbladder disease (n = 682), there were 9650 participants available for the analysis of the relationship of acid level to asymptomatic gallstones. Serum ascorbic acid and serum vitamin E ( tocopherol) levels were measured at the Centers for Disease Control and Prevention, Atlanta, Ga, by isocratic highperformance liquid chromatography. 13 Ascorbic acid levels ranged from 0.0 µmol/l to the upper cutoff point of 170 µmol/l. Measurements of total and high-density lipoprotein cholesterol were performed by means of standardized protocols. 13 STATISTICAL METHODS We examined the distribution of ascorbic acid levels and other variables of interest using sample weights. We used age-adjusted and multivariable logistic regression to examine the associations of ascorbic acid level and other variables to the prevalence of clinical gallbladder disease and asymptomatic gallstones. Level of physical activity was analyzed as an ordinal variable. Because we detected a significant nonlinear relationship between age and gallbladder disease, and to improve the fit of the models, age adjustment was performed with the use of linear and quadratic terms for age. Multivariate models controlled for the effects of age, race, level of education, physical activity, BMI, diabetes mellitus, use of diuretic and lipidlowering medications, cigarette smoking, alcohol consumption, total serum cholesterol and high-density lipoprotein cholesterol levels, serum vitamin E level, and dietary intake of energy, fat, and fiber. For female participants, the multivariate models also controlled for the effects of oral contraceptive use, estrogen replacement therapy, history of pregnancy, number of live births, and menopausal status. Variables included in the multivariate models have been reported, in at least some studies, to be associated with gallbladder disease. 14 Serum vitamin E was included in the multivariate models because it is an important lipid-soluble antioxidant nutrient often correlated with ascorbic acid intake. We tested for nonlinear relationships by including both linear and quadratic terms for acid in the models. Analyses were performed with Stata software that included commands for the analysis of complex survey data. 15 For each predictor variable, we calculated an odds ratio (OR) and 95% confidence interval (CI). Because the probability of clinical and asymptomatic gallbladder disease in NHANES III is low, the ORs approximate the relative prevalence of disease. We considered 2-tailed P values of less than.05 to be statistically significant. Figures examining the independent association between acid level and gallbladder disease outcomes plotted the predicted prevalence of clinical gallbladder disease and asymptomatic gallstones as a function of acid level based on the multivariate models. For the figures, logistic coefficients were used to compute individual predicted probability of each outcome. These probabilities within each 0.06-µmol/L acid concentration were then smoothed twice by means of a locally weighted symmetric nearestneighbor linear smoother 16,17 that allows for the use of sample weights. 18,19 932

3 Table 1. Characteristics of Participants Aged 20 to 90 Years Enrolled in the Third National Health and Nutrition Examination Survey, * Women (n = 7042) Men (n = 6088) Characteristic Serum ascorbic acid level, 47±27 38±24 µmol/l Dietary ascorbic acid intake, 98 ± ± 119 mg/d Age, y 45 ± ± 16 Level of education, y 12 ± 3 13 ± 3 Body mass index, kg/m ± ± 4.7 Energy intake, kj/d 7443 ± ± 4845 Dietary fat intake, g/d 68 ± ± 57 Dietary fiber intake, g/d 14 ± 8 20 ± 12 Alcohol intake, drinks/wk 1 ± 5 5 ± 11 Serum cholesterol level, mmol/l (mg/dl) 5.33 ± 1.14 (206 ± 44) 5.22 ± 1.06 (202 ± 41) HDL cholesterol level, mmol/l (mg/dl) 1.42 ± 0.41 (55 ± 16) 1.19 ± 0.36 (46 ± 14) Number of live births 2.1 ± 1.8 NA Postmenopausal, % 41.9 NA Use of estrogen medications, % 14.3 NA History of birth control pills, % 61.5 NA Ever pregnant, % 83.8 NA Diabetes mellitus, % History of cigarette smoking, % Current cigarette smoker, % White race, % Exercise level, % More active Similar Less active Diuretic use, % Use of lipid-lowering medications Clinical gallbladder disease, % Asymptomatic gallstones, % *Values are mean ± SD unless otherwise specified. HDL indicates high-density lipoprotein; NA, not applicable. Self-reported level of physical activity compared with others of the same age. Detected by abdominal ultrasound examination among 4863 women and 4787 men between ages 20 and 74 years. having used oral contraceptives, and 3% of both women and men reported the current use of lipid-lowering medications. Among women, acid level was associated with prevalence of gallbladder disease. Ageadjusted models demonstrated that acid levels were inversely associated with clinical and asymptomatic gallbladder disease; each SD (27 µmol/l) increase in acid level was associated with an approximately 23% lower prevalence of clinical gallbladder disease and a 22% lower prevalence of asymptomatic gallstones (both P.001). Further adjustment for potential confounders including race, BMI, diabetes, diet, menopausal status, use of estrogen-containing and lipid-lowering medications, and other variables attenuated these associations; an SD increase in acid levels was independently associated with an approximately 13% lower prevalence of clinical gallbladder disease (P =.006) and asymptomatic gallstones (P =.048) (Table 2, Figure 1, and Figure 2). The acid Table 2. Relation of Serum Ascorbic Acid Level (per SD Increase) to Clinical Gallbladder Disease and Asymptomatic Gallstones Among Men and Women Aged 20 to 90 Years Enrolled in the Third National Health and Nutrition Examination Survey, Clinical Gallbladder Disease Asymptomatic Gallstones* Women No. of subjects No. of prevalent cases Odds ratio (95% confidence interval) Age-adjusted 0.77 ( ) 0.78 ( ) Multivariate-adjusted 0.87 ( ) 0.87 ( ) Men No. of subjects No. of prevalent cases Odds ratio (95% confidence interval) Age-adjusted 0.94 ( ) 0.93 ( ) Multivariate-adjusted# 0.97 ( ) 0.91 ( ) *Detected by abdominal ultrasound examination performed on subjects between the ages of 20 and 74 years. Participants with clinical gallbladder disease or a history of right-upper-quadrant abdominal pain lasting 1 hour or longer were excluded; hence, the 2 outcomes, clinical gallbladder disease and asymptomatic gallstones, are mutually exclusive. Adjusted for age and age squared. P.001. Adjusted for age; age squared; race; level of education; physical activity; body mass index; use of oral contraceptive and estrogen-containing medications; history of pregnancy and number of live births; menopausal status; dietary intake of energy, fat, fiber, and alcohol; history of smoking; history of diabetes; use of diuretic and cholesterol-lowering medications; serum level of total and high-density lipoprotein cholesterol; and serum vitamin E level. P.01. P.05. #Adjusted for age; race; age squared; level of education; physical activity; body mass index; dietary intake of energy, fat, fiber, and alcohol; history of smoking; history of diabetes; use of diuretic and cholesterol-lowering medications; serum level of total and high-density lipoprotein cholesterol; and serum vitamin E level. percentiles for Figures 1 and 2 are presented in the following tabulation: Percentile, % Serum Ascorbic Acid, µmol/l Among men, there were no significant age-adjusted or multivariate-adjusted associations detected between serum ascorbic acid level and gallbladder disease (Table 2). Additional analyses that excluded 607 participants whose 933

4 Predicted Prevalence of Clinical Gallbladder Disease Serum Ascorbic Acid Level, µmol/l Figure 1. The relationship between acid concentration and the predicted prevalence of clinical gallbladder disease among 7042 women enrolled in the Third National Health and Nutrition Examination Survey, , based on the multivariate model noted in Table 2. Predicted Prevalence of Asymptomatic Gallstones Serum Ascorbic Acid Level, µmol/l Figure 2. The relationship between acid concentration and the predicted prevalence of asymptomatic gallstones detected by abdominal ultrasound among 4863 women enrolled in the Third National Health and Nutrition Examination Survey, , based on the multivariate model noted in Table 2. self-reported race was other than white or black produced virtually identical results. We also analyzed the relationship of acid, categorized by quartile, to prevalence of clinical and asymptomatic gallbladder disease. When compared with women in the lowest acid quartile, multivariate analyses showed that women in the highest quartile had a lower prevalence of clinical gallbladder disease (OR, 0.61; 95% CI, ; P.01) and asymptomatic gallstones (OR, 0.67; 95% CI, ; P =.06). In similar comparisons, men in the highest serum ascorbic acid quartile, however, did not have a significantly lower prevalence of either clinical gallbladder disease (OR, 0.85; 95% CI, ; P =.53) or asymptomatic gallstones (OR, 0.82; 95% CI, ; P =.58) when compared with men in the lowest quartile. We explored factors that might confound the relationship between acid and gallbladder disease by examining the effect of potential confounders when added singly to age-adjusted models. Among women, BMI was the only variable that substantially affected the OR estimates. Inclusion of BMI in the ageadjusted models resulted in an approximately 8% attenuation in the magnitude of the association of acid to clinical and asymptomatic gallbladder disease. For example, a 1-SD increase in acid level (27 µmol/l) was associated with a 23% lower prevalence of clinical gallbladder disease before adjustment for BMI, but only a 17% lower prevalence after adjustment (ageadjusted OR, 0.77; age- and BMI-adjusted OR, 0.83; both P.001). There were no variables that significantly confounded the relationship between acid and gallbladder disease among men. We also examined the relationship of ascorbic acid supplement use (or use of multiple vitamins containing additional ascorbic acid) to gallbladder disease by substituting supplement use for acid in the multivariate models presented in Table 2. Among women, supplement use was independently associated with a 34% lower prevalence of clinical gallbladder disease (P =.02) but was not associated with prevalence of asymptomatic gallstones (P =.86). Among men, ascorbic acid supplement use was associated with a trend toward a higher prevalence of clinical gallbladder disease (P =.07), but was not associated with asymptomatic gallstones (P =.54). Smoking, which lowers acid levels, was also analyzed as a separate predictor of gallbladder disease. In analyses that excluded acid, smoking among women was nonsignificantly associated with clinical gallbladder disease (OR, 1.10; 95% CI, ; P =.43) and asymptomatic gallstones (OR, 1.29; 95% CI, ; P =.18). Because ascorbic acid may have important effects on the metabolism of alcohol, we examined whether alcohol consumption may modify the association between serum ascorbic acid level and prevalence of gallbladder disease. We found no evidence of such an interaction. COMMENT Our principal finding was a consistent inverse relationship between serum level of ascorbic acid and prevalence of gallbladder disease among women. We also observed among women that ascorbic acid supplement use was associated with a lower prevalence of clinical gallbladder disease. We found no significant relationship, however, between serum level of ascorbic acid (or ascorbic acid supplement use) and prevalence of gallbladder disease among men. Three reports have examined the relationship of ascorbic acid to gallbladder disease among human populations, and our findings are, in general, similar to those in these reports In a small case-control study among 23 men and 77 women, Ortega and colleagues 20 found an association between lower dietary intake of ascorbic acid and gallbladder disease among women, but not men. In another study, Simon et al 21 examined ascorbic acid supplement use as a correlate of clinical gallbladder disease among postmenopausal women with coronary heart disease. Among women who consumed alcohol, ascorbic acid supplement use was independently associated with a 50% reduction in prevalence of self-reported gallstones and a 62% reduction in cholecystectomy. 21 This study, however, examined a somewhat atypical group of women with a high (23%) prevalence of self-reported gallbladder disease. Further- 934

5 more, this study was limited by lack of data on dietary ascorbic acid intake or blood ascorbic acid levels. We also analyzed data from the Second National Health and Nutrition Examination Survey and reported a significant nonlinear relationship between acid level and prevalence of clinical gallbladder disease among women; self-reported prevalence of gallstones and cholecystectomy was lower among women with both low and high serum levels of ascorbic acid. 22 We found no relationship, either linear or nonlinear, between acid level and gallbladder disease prevalence among men enrolled in the Second National Health and Nutrition Examination Survey. Thus, the association between ascorbic acid status and gallbladder disease has been reported only among women and may be the result of a biological interaction between ascorbic acid status and sex. However, these findings may also reflect the lower prevalence of gallbladder disease among men and reduced statistical power to detect such an association. Unlike the previous studies examining the relationship of ascorbic acid status to gallbladder disease, NHANES III collected data on the sonographic presence of gallstones. These data are important because previous studies could not exclude the possibility that participants changed their diets after being diagnosed as having gallbladder disease. Although it seems implausible that women with gallbladder disease would change their diets and consume less ascorbic acid containing fruits, vegetables, and dietary supplements, this possibility cannot be excluded. However, because this study also examined participants with gallstones present on abdominal ultrasound who denied any previous symptomatic gallbladder disease and reported no history of abdominal pain consistent with undiagnosed gallbladder disease, it strengthens the hypothesis that ascorbic acid status may be an important risk factor for gallstone formation among women. We hypothesized that an inverse relationship between acid levels and gallbladder disease in humans might be observed on the basis of the results of animal experiments. 10 In guinea pigs, ascorbic acid affects the activity of cholesterol 7 -hydroxylase, the enzyme that regulates the ratelimiting step in the catabolism of cholesterol to bile acids. 12 Ascorbic acid supplementation increases cholesterol 7 -hydroxylase activity by as much as 15-fold compared with ascorbic acid deficient guinea pigs, 23 and ascorbic acid deficient guinea pigs frequently develop cholesterol gallstones. 7,8,24 Additionally, hypersecretion of mucin, a glycoprotein that is secreted by the epithelium of the gallbladder, has been found to precede cholesterol destabilization and gallstone formation. 25,26 Because hydroxyl and oxygen radicals stimulate the hypersecretion of mucin, 27 the inhibition of oxidative changes within the gallbladder by ascorbic acid may also decrease mucoprotein production and gallstone formation. Our study has a number of strengths and limitations. Because NHANES III surveyed a large probability sample of Americans, our findings, which are derived from analyses of data from approximately 83% of examined NHANES III participants, should be generalizable to the US population. Although participants excluded from the analyses were similar to included participants in regard to age and sex, they were more likely to be nonwhite and to have achieved a lower level of education. Nevertheless, we believe these differences are unlikely to substantially affect the generalizability of our findings. Furthermore, the measurement of acid levels on a large sample of the population allows a more reliable assessment of ascorbic acid status as a correlate of gallbladder disease compared with studies using dietary intake estimations only. Plasma ascorbic acid levels are strongly correlated with leukocyte ascorbic acid levels, an indicator of tissue levels, 28 and reflect at least the previous several months of dietary intake, even during periods of seasonal variation. 29 It is also unlikely that recall bias affected our findings, since participants obtained blood levels of ascorbic acid and completed a comprehensive questionnaire that included information on many health outcomes. Most importantly, we have data on asymptomatic gallbladder disease that are not subject to interviewer or recall bias and are not available from previous studies. However, for the outcome of clinical gallbladder disease, we relied on self-reported history and may, therefore, have misclassified some outcomes. Such misclassification would likely have decreased the power of our study to detect associations. It is also possible that unknown or unmeasured confounders may have affected our results and that acid may simply be a healthy lifestyle marker. Furthermore, we cannot exclude the possibility that a systemic oxidative stress may both lower acid levels and result in gallstone formation. Finally, because of the cross-sectional nature of the study, we cannot be certain that differences in acid level preceded gallbladder disease, and inferences regarding causality should be made cautiously. Serum ascorbic acid level is an important correlate of symptomatic and asymptomatic gallbladder disease among American women. Our study is the first to report an association between acid levels and asymptomatic gallstones. Examination of this relationship among other populations is indicated, and clinical trials of ascorbic acid for the primary or secondary prevention of gallbladder disease among women should be considered. Accepted for publication July 19, This study was supported by Roche Vitamins, Inc, Parsippany, NJ, and grant HL53479 from the Public Health Service, Bethesda, Md. Reprints: JoelA. Simon, MD, MPH, GeneralInternalMedicine (111A1), San Francisco VA Medical Center, 4150 ClementSt,SanFrancisco,CA94121( jasimon@itsa.ucsf.edu). REFERENCES 1. Gallstones and laparoscopic cholecystectomy. NIH Consens Statement. September 14-16, 1992;10(3): Trotman BW, Ostrow JD, Soloway RD. Pigment vs cholesterol cholelithiasis: comparison of stone and bile composition. Am J Dig Dis. 1974;19:

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