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Association Between Flavonoid-Rich Fruit and Vegetable Consumption and Total Serum Bilirubin Paul D. Loprinzi and Sara E. Mahoney ANGIOLOGY published online 27 May 2014 DOI: 10.1177/0003319714537111 The online version of this article can be found at: http://ang.sagepub.com/content/early/2014/05/27/0003319714537111
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Article
Association Between Flavonoid-Rich Fruit and Vegetable Consumption and Total Serum Bilirubin
Angiology 1-5 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003319714537111 ang.sagepub.com
Paul D. Loprinzi, PhD1, and Sara E. Mahoney, PhD1
Abstract Emerging work demonstrates that serum bilirubin is a novel biomarker implicated in cardiovascular and metabolic diseases. However, we have a limited understanding of the influence of flavonoid-rich fruit and vegetable consumption on bilirubin levels, which was the purpose of this study. Data from the 2003 to 2006 National Health and Nutrition Examination survey were used (n ¼ 1783; 18-85 years of age), with analyses performed in 2014. Total serum bilirubin was measured from a blood sample. Using a food frequency questionnaire (FFQ), a flavonoid index variable was created summing the frequency of consumption of flavonoid-rich foods. After adjustments, greater consumption of flavonoid-rich fruits and vegetables was positively associated with bilirubin levels. Our findings suggest an association between flavonoid-rich fruit and vegetable consumption and bilirubin levels. If confirmed by prospective and experimental studies, then regular consumption of flavonoid-rich fruits and vegetables should be promoted to increase levels of bilirubin. Keywords bilirubin, cardiovascular disease, epidemiology, flavonoid, nutrition
Background Bilirubin is derived from the breakdown of heme or hemoproteins.1 More specifically, bilirubin is the end product of heme catabolism in the systemic circulation and is formed by the action of hemeoxygenase, which catalyzes heme to biliverdin, subsequently converting to bilirubin by biliverdin reductase.2,3 Although once considered solely a toxic waste product due to neurological damage found in those with marked elevation,4 at levels elevated within normal circulating levels, bilirubin has now been shown to be associated with beneficial effects on the human body, including the scavenging of reactive oxygen species,5 anti-inflammatory actions,6 and influencing cell signaling.7 Bilirubin is almost 30 times more potent than other antioxidants such as Trolox, a vitamin E analog, in preventing low-density lipoprotein cholesterol oxidation.8 It has also been shown to play a key role in the antioxidant capacity of the blood plasma.9 Further, bilirubin inhibits tumor necrosis factor a-related adhesion molecules (eg, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1).10 Bilirubin has been implicated in several chronic diseases including diabetes, metabolic syndrome, stroke, cancer, and cardiovascular (CV) disease.11,12 With regard to the latter, individuals with lower bilirubin levels are at an increased risk of coronary atherosclerotic disease,11,13 likely through low bilirubin-induced coronary artery calcification14 and progression of the intima–media thickness of the carotid arteries.15,16
In contrast, individuals with higher levels of bilirubin are at a decreased risk of CV disease.17 Importantly, the potential beneficial effects of bilirubin in the atherosclerosis process may be secondary to a more beneficial CV risk profile. For example, in some studies, the association between bilirubin and CV disease is attenuated after controlling for CV risk factors.18-20 In addition to CV and metabolic disease, emerging work has also shown that bilirubin is linked with autoimmune21 and psychiatric disorders.22 As emerging evidence supports the role of bilirubin in CV disease, it is increasingly important to identify behaviors that may influence serum bilirubin. Although limited in examination, smoking is inversely associated with bilirubin,23,24 while physical activity is positively associated with bilirubin.24-27 Additionally, the effect of other health-enhancing behaviors (eg, diet) on bilirubin levels is unclear. It is plausible to suggest that adequate consumption of flavonoid-rich fruits and vegetables may be associated with bilirubin, given their influence on
1
Donna & Allan Lansing School of Nursing & Health Sciences, Bellarmine University, Louisville, KY, USA
Corresponding Author: Paul D. Loprinzi, Donna & Allan Lansing School of Nursing, Bellarmine University, Louisville, KY, USA. Email: [email protected]
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Angiology
inflammation,28 markers of CV disease,29 and more importantly, their ability to modulate enzymes (eg, uridine 50 -diphosphoglucuronosyltransferases) that influence bilirubin levels.30 However, at this point, the influence of diet on bilirubin is poorly understood. To our knowledge, only 3 studies have examined the association between dietary behavior and bilirubin.31-33 In general, these studies have observed an association between diet and bilirubin; however, these studies were conducted among a small sample, used a narrow age range of participants, isolated certain fruits or vegetables, and specifically focused on the effect of genotype or gender on the association between diet and bilirubin.31-33 As a result, our understanding of the association between fruit and vegetable consumption on bilirubin in the general population is limited. The purpose of this investigation was to examine the association between flavonoid-rich fruit and vegetable consumption and serum bilirubin levels in the broader population. We hypothesize that those with higher levels of fruit and vegetable consumption will have increased bilirubin levels, independent of other CV risk factors. To improve generalizability, data for the present study come from the National Health and Nutrition Examination Survey (NHANES), which employs a nationally representative sample of US adults up to 85 years of age.
Methods Study Design and Participants Data from the 2003 to 2006 NHANES were used. All procedures for data collection were approved by the National Center for Health Statistics ethics review board, and all participants provided written informed consent prior to data collection. For the present analyses, 1783 adult participants (aged 18-85 years) provided data for all study variables.
Assessment of Flavonoid Index Based on the National Cancer Institute Diet History Questionnaire that is widely used in nutritional epidemiology research,34 participants completed the NHANES FFQ.35 Briefly, participants were asked to report the proportion of time certain types of foods were eaten over the past 12 months. For the present study, 15 foods rich in flavonoids (eg, apples, grapes, oranges, carrots, and broccoli) were identified using the US Department of Agriculture flavonoid content of foods.36 For each of the 15 food items, response options ranged from 1 to 11 and included never (1), 1 to 6 times/year (2), 7 to 11 times/year (3), 1 time/month (4), 2 to 3 times/month (5), 1 time/week (6), 2 times/week (7), 3 to 4 times/week (8), 5 to 6 times/week (9), 1 time/d (10), and 2 or more times/d (11). Responses were summed, with higher values indicating more frequent consumption of flavonoidrich foods. With 15 items, the possible range for the flavonoid index variable is 15 to 165.
Determination of Total Serum Bilirubin Total serum bilirubin was measured in mg/dL using the LX20 (Beckman Synchron), which uses a timed end point Diazo method to measure the total concentration of bilirubin. Further details can be found elsewhere.37
Covariates Covariates included age, gender, race ethnicity, socioeconomic status, body mass index (BMI), exercise, smoking status, hypertension, degree of insulin resistance/insulin sensitivity, and high sensitivity C-reactive protein (hsCRP). Information about age, gender, and race ethnicity were obtained from a questionnaire. As a measure of socioeconomic status, poverty to income ratio (PIR) was assessed, with a PIR value less than 1 considered below the poverty threshold. The PIR is calculated by dividing the family income by the poverty guidelines, which is specific to the family size, year assessed, and state of residence. The BMI was calculated from measured weight and height (weight in kg divided by the square of height in m). Based on the Global Physical Activity Questionnaire,38 participants were asked, ‘‘During the past 30 days, did you do moderate or vigorous activities for at least 10 minutes.’’ Responses were coded as yes or no. Serum cotinine was measured as a marker of active smoking status or environmental exposure to tobacco (ie, passive smoking). Serum cotinine was measured by an isotope dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Participants were considered to be hypertensive if they self-reported a physician diagnosis of hypertension or if their measured diastolic or systolic blood pressure was 90 or 140 mm Hg, respectively. The Homeostasis Model Assessment (HOMA) was used to evaluate insulin resistance using the following formula: fasting serum insulin (mU/mL) fasting plasma glucose (mmol/L)/22.5.39 Participants were classified as insulin sensitive if their HOMA score was 2.6, with a HOMA score >2.6 used to denote insulin resistance.40 The Tosoh AIA-PACK IRI, a 2-site immunoenzymometric assay, was used to measure blood insulin levels, with glucose measured spectrophotometrically. Further details of the assessment of insulin and glucose can be found elsewhere.41 The hsCRP concentration was quantified using latex-enhanced nephelometry.
Data Analysis All statistical analyses (STATA, version 12.0, College Station, Texas), which were performed in 2014, accounted for the complex survey design used in NHANES by using survey sample weights, clustering, and primary sampling units. The FFQ sample weights were used to provide nationally representative estimates. Means and standard errors were calculated for continuous variables and proportions were calculated for categorical variables. To examine the association between flavonoid consumption (index variable and independent variable) and total serum bilirubin (outcome variable), multivariable
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Table 1. Characteristics of the Analyzed Participants.a Variable
Mean/Proportion (95% CI)
Age, years % Female Race-ethnicity, % Non-Hispanic white Other Body mass index, kg/m2 Cotinine, ng/mL Poverty to income ratio Hypertensive, % Insulin resistance, % Insulin sensitive, % High-sensitivity C-reactive protein, mg/dL Exercise in past 30 days, % Bilirubin, mg/dL Bilirubin, mmol/Lb Flavonoid index
Table 2. Multivariable Linear Regression Analysis Examining the Association Between Flavonoid Consumption and Bilirubin (Outcome Variable).a
48.2 (46.8-49.6) 54.3 (51.7-56.9) 74.2 (69.4-79.0) 25.7 (20.9-30.5) 28.4 (27.9-28.9) 56.2 (45.3-67.2) 3.1 (3.0-3.2) 32.7 (29.3-36.0) 36.5 (33.8-39.3) 63.4 (60.6-66.1) 0.43 (0.38-0.48) 69.8 (67.1-72.6) 0.75 (0.73-0.78) 12.8 (12.5-13.3) 71.7 (70.6-72.9)
Abbreviations: CI, confidence interval; NHANES, National Health and Nutrition Examination Survey. a N ¼ 1783. NHANES 2003-2006. b To convert bilirubin from mg/dL to mmol/L, multiply mg/dL by 17.1.
linear regression was employed. Covariates included age, gender, race ethnicity, socioeconomic status, BMI, exercise, smoking status, hypertension, degree of insulin resistance/ insulin sensitivity, and hsCRP. All covariates were entered into the model at the same time, as there was no evidence of multicollinearity. Evidence of multicollinearity is likely to exist if there is a correlation of >0.8 between 2 covariates; if the mean variance inflation factor is >6, or if the highest individual variance inflation factor is >10, or if the tolerance statistic is 0.6. A P .05 (2-tailed) denoted statistical significance for all analyses.
Results Table 1 reports the study variable characteristics. Table 2 shows the multivariable linear regression analysis delineating the relationship between flavonoid consumption and bilirubin (outcome variable). After adjusting for age, gender, race ethnicity, socioeconomic status, BMI, exercise, smoking status, hypertension, degree of insulin resistance/insulin sensitivity, and hsCRP, greater consumption flavonoid-rich fruits and vegetables was positively associated with bilirubin (b ¼ .001; P ¼ .01). When expressed as a larger interval change, for every 50-unit increase in the flavonoid index variable, there was a 0.1 mg/dL increase in total serum bilirubin. Although there was no evidence of multicollinearity in the analytic model, we considered the possibility that the covariates may be taxing the regression model as a result of model overfitting. However, we ran minimally adjusted analyses (adjusting for age, gender, race ethnicity, and BMI), and the observed association was similar (b ¼ .001; P ¼ .004).
D (95% CI) in Bilirubin, mg/dL
P
0.001 (0.0001 to 0.001) .03 Flavonoid indexb Covariates Age, 1 year older 0.001 (0.0003 to 0.002) .01 Female vs male 0.20 (0.23 to 0.17) 1.78 ng/mL for men and >4.47 ng/mL for women was used to denote current smoker42), similar bilirubin levels were observed for smokers and nonsmokers (0.77 mg/dL vs 0.73 mg/dL; P ¼ .06). However, after adjustments, diet was not associated with bilirubin for current smokers (b ¼ .0004, P ¼ .35) but was for nonsmokers (b ¼ .002, P ¼ .005). With regard to insulin sensitivity/resistance, individuals with evidence of insulin sensitivity had higher bilirubin levels than those with insulin resistance (0.77 mg/dL vs 0.73 mg/dL; P ¼ .02). After adjustments, there was an association between diet and bilirubin levels for those with evidence of insulin sensitivity (b ¼ .001, P ¼ .05) but not for those with insulin resistance (b ¼ .0004, P ¼ .29).
Discussion In a nationally representative sample of US adults, greater consumption of flavonoid-rich fruits and vegetables was associated
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with higher bilirubin levels. Secondary analyses demonstrated that this association may be moderated by gender, smoking status, and insulin sensitivity/resistance. Major strengths of this study include examining this underinvestigated topic and employing a nationally representative sample of US adults. Although these cross-sectional data do not allow for an assessment of the temporality of the association between fruit and vegetable consumption with bilirubin, it is plausible to suggest that adequate consumption of flavonoidrich fruits and vegetables may play a causal role in increasing bilirubin levels. The mechanism through which flavonoid-rich fruit and vegetable consumption may increase bilirubin levels is not clear; however, it is plausible through various biological pathways. Flavonoid-rich fruit and vegetable consumption may increase induction of hemeoxygenase through modulation of P-450-dependent metabolic activities,43,44 elimination of reactive oxygen species that may help to reduce the rate of decay in hemeoxygenase protein expression,45 and decreasing cellular Naþ and Ca2þ contents that can help to reduce edema formation46 and mitochondrial calcium-overload–induced cell death.45,47,48 Our findings are similar to the few other smaller scale convenience-based samples showing that fruit and vegetable consumption may help to increase bilirubin levels.31-33 These studies also show that the effects of fruit and vegetable consumption on bilirubin may be influenced by gender and UGT1A1 genotype. We were unable to control for UGT1A1 genotype, as this information was not collected in the NHANES; however, our secondary analyses support these previous findings indicating that the association between diet and bilirubin may be moderated by gender. In conclusion, these findings suggest an association between flavonoid-rich fruit and vegetable consumption and bilirubin levels. If confirmed by prospective and experimental studies, then regular consumption of flavonoid-rich fruits and vegetables should be promoted to increase levels of bilirubin.
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Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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20. Breimer LH, Mikhailidis DP. Is bilirubin a marker of vascular disease and/or cancer and is it a potential therapeutic target? Curr Pharm Des. 2011;17(33):3644-3655. 21. Yang Z, Liang Y, Li C, Xi W, Zhong R. Bilirubin levels in patients with systemic lupus erythematosus: increased or decreased? Rheumatol Int. 2012;32(8):2423-2430. 22. Radhakrishnan R, Kanigere M, Menon J, Calvin S, Janish A, Srinivasan K. Association between unconjugated bilirubin and schizophrenia. Psychiatry Res. 2011;189(3):480-482. 23. Van Hoydonck PG, Temme EH, Schouten EG. Serum bilirubin concentration in a Belgian population: the association with smoking status and type of cigarettes. Int J Epidemiol. 2001;30(6): 1465-1472. 24. Tanaka M, Budhathoki S, Hirata A, et al. Behavioral and clinical correlates of serum bilirubin concentrations in Japanese men and women. BMC Endocr Disord. 2013;13(1):39. 25. Devries MC, Samjoo IA, Hamadeh MJ, Tarnopolsky MA. Effect of endurance exercise on hepatic lipid content, enzymes, and adiposity in men and women. Obesity (Silver Spring). 2008; 16(10):2281-2288. 26. Swift DL, Johannsen NM, Earnest CP, Blair SN, Church TS. Effect of different doses of aerobic exercise training on total bilirubin levels. Med Sci Sports Exerc. 2012;44(4):569-574. 27. Loprinzi PD, Abbott K. Physical activity and total serum bilirubin levels among insulin sensitive and insulin resistant U.S. adults. J Diabetes Metab Disord. 2014;13(1):47. 28. Esmaillzadeh A, Kimiagar M, Mehrabi Y, Azadbakht L, Hu FB, Willett WC. Fruit and vegetable intakes, C-reactive protein, and the metabolic syndrome. Am J Clin Nutr. 2006;84(6): 1489-1497. 29. Hartley L, Igbinedion E, Holmes J, et al. Increased consumption of fruit and vegetables for the primary prevention of cardiovascular diseases. Cochrane Database Syst Rev. 2013;6:CD009874. 30. Mackenzie PI, Gregory PA, Lewinsky RH, et al. Polymorphic variations in the expression of the chemical detoxifying UDP glucuronosyltransferases. Toxicol Appl Pharmacol. 2005;207(2 suppl):77-83. 31. Saracino MR, Bigler J, Schwarz Y, et al. Citrus fruit intake is associated with lower serum bilirubin concentration among women with the UGT1A1*28 polymorphism. J Nutr. 2009; 139(3):555-560. 32. Peterson S, Bigler J, Horner NK, Potter JD, Lampe JW. Cruciferae interact with the UGT1A1*28 polymorphism to determine serum bilirubin levels in humans. J Nutr. 2005;135(5): 1051-1055. 33. Chang JL, Bigler J, Schwarz Y, et al. UGT1A1 polymorphism is associated with serum bilirubin concentrations in a randomized, controlled, fruit and vegetable feeding trial. J Nutr. 2007; 137(4):890-897. 34. Subar AF, Thompson FE, Kipnis V, et al. Comparative validation of the Block, Willett, and National Cancer Institute food frequency questionnaires : the eating at America’s table study. Am J Epidemiol. 2001;154(12):1089-1099.
35. Subar AF, Dodd KW, Guenther PM, et al. The food propensity questionnaire: concept, development, and validation for use as a covariate in a model to estimate usual food intake. J Am Diet Assoc. 2006;106(10):1556-1563. 36. USDA. Database for the Flavonoid Content of Selected Foods. Release 2.1. Washington, DC: USDA; 2007. 37. Centers for Disease Control and Prevention. National Health and Nutrition Examination Survey. Standard Biochemistry Profile, 2008. http://www.cdc.gov/nchs/nhanes/nhanes2005-2006/BIOPRO_D.htm. Accessed February 20, 2014. 38. Bull FC, Maslin TS, Armstrong T. Global physical activity questionnaire (GPAQ): nine country reliability and validity study. J Phys Act Health. 2009;6(6):790-804. 39. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28(7):412-419. 40. Ascaso JF, Pardo S, Real JT, Lorente RI, Priego A, Carmena R. Diagnosing insulin resistance by simple quantitative methods in subjects with normal glucose metabolism. Diabetes Care. 2003; 26(12):3320-3325. 41. Centers for Disease Control and Prevention. National Health and Nutrition Examination Survey. Plasma Glucose and Insulin, 2009. http://www.cdc.gov/nchs/nhanes/nhanes2003-2004/L10AM_C. htm. Accessed February 20, 2014. 42. Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. Am J Epidemiol. 2009; 169(2):236-248. 43. Huang MT, Johnson EF, Muller-Eberhard U, Koop DR, Coon MJ, Conney AH. Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes. J Biol Chem. 1981;256(21): 10897-10901. 44. Lasker JM, Huang MT, Conney AH. In vivo activation of zoxazolamine metabolism by flavone. Science. 1982;216(4553): 1419-1421. 45. Szabo ME, Gallyas E, Bak I, et al. Heme oxygenase-1-related carbon monoxide and flavonoids in ischemic/reperfused rat retina. Invest Ophthalmol Vis Sci. 2004;45(10):3727-3732. 46. Szabo ME, Droy-Lefaix MT, Doly M, Carre C, Braquet P. Ischemia and reperfusion-induced histologic changes in the rat retina. Demonstration of a free radical-mediated mechanism. Invest Ophthalmol Vis Sci. 1991;32(5):1471-1478. 47. Kilic F, Handelman GJ, Traber K, Tsang K, Packer L, Trevithick JR. Modelling cortical cataractogenesis XX. In vitro effect of alpha-lipoic acid on glutathione concentrations in lens in model diabetic cataractogenesis. Biochem Mol Biol Int. 1998;46(3): 585-595. 48. Kilic F, Trevithick JR. Modelling cortical cataractogenesis. XXIX. Calpain proteolysis of lens fodrin in cataract. Biochem Mol Biol Int. 1998;45(5):963-978.
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Association Between Flavonoid-Rich Fruit and Vegetable Consumption and Total Serum Bilirubin Paul D. Loprinzi and Sara E. Mahoney ANGIOLOGY published online 27 May 2014 DOI: 10.1177/0003319714537111 The online version of this article can be found at: http://ang.sagepub.com/content/early/2014/05/27/0003319714537111
Published by: http://www.sagepublications.com
Additional services and information for Angiology can be found at: Email Alerts: http://ang.sagepub.com/cgi/alerts Subscriptions: http://ang.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - May 27, 2014 What is This?
Downloaded from ang.sagepub.com at UNIV OF UTAH SALT LAKE CITY on November 28, 2014
Article
Association Between Flavonoid-Rich Fruit and Vegetable Consumption and Total Serum Bilirubin
Angiology 1-5 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003319714537111 ang.sagepub.com
Paul D. Loprinzi, PhD1, and Sara E. Mahoney, PhD1
Abstract Emerging work demonstrates that serum bilirubin is a novel biomarker implicated in cardiovascular and metabolic diseases. However, we have a limited understanding of the influence of flavonoid-rich fruit and vegetable consumption on bilirubin levels, which was the purpose of this study. Data from the 2003 to 2006 National Health and Nutrition Examination survey were used (n ¼ 1783; 18-85 years of age), with analyses performed in 2014. Total serum bilirubin was measured from a blood sample. Using a food frequency questionnaire (FFQ), a flavonoid index variable was created summing the frequency of consumption of flavonoid-rich foods. After adjustments, greater consumption of flavonoid-rich fruits and vegetables was positively associated with bilirubin levels. Our findings suggest an association between flavonoid-rich fruit and vegetable consumption and bilirubin levels. If confirmed by prospective and experimental studies, then regular consumption of flavonoid-rich fruits and vegetables should be promoted to increase levels of bilirubin. Keywords bilirubin, cardiovascular disease, epidemiology, flavonoid, nutrition
Background Bilirubin is derived from the breakdown of heme or hemoproteins.1 More specifically, bilirubin is the end product of heme catabolism in the systemic circulation and is formed by the action of hemeoxygenase, which catalyzes heme to biliverdin, subsequently converting to bilirubin by biliverdin reductase.2,3 Although once considered solely a toxic waste product due to neurological damage found in those with marked elevation,4 at levels elevated within normal circulating levels, bilirubin has now been shown to be associated with beneficial effects on the human body, including the scavenging of reactive oxygen species,5 anti-inflammatory actions,6 and influencing cell signaling.7 Bilirubin is almost 30 times more potent than other antioxidants such as Trolox, a vitamin E analog, in preventing low-density lipoprotein cholesterol oxidation.8 It has also been shown to play a key role in the antioxidant capacity of the blood plasma.9 Further, bilirubin inhibits tumor necrosis factor a-related adhesion molecules (eg, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1).10 Bilirubin has been implicated in several chronic diseases including diabetes, metabolic syndrome, stroke, cancer, and cardiovascular (CV) disease.11,12 With regard to the latter, individuals with lower bilirubin levels are at an increased risk of coronary atherosclerotic disease,11,13 likely through low bilirubin-induced coronary artery calcification14 and progression of the intima–media thickness of the carotid arteries.15,16
In contrast, individuals with higher levels of bilirubin are at a decreased risk of CV disease.17 Importantly, the potential beneficial effects of bilirubin in the atherosclerosis process may be secondary to a more beneficial CV risk profile. For example, in some studies, the association between bilirubin and CV disease is attenuated after controlling for CV risk factors.18-20 In addition to CV and metabolic disease, emerging work has also shown that bilirubin is linked with autoimmune21 and psychiatric disorders.22 As emerging evidence supports the role of bilirubin in CV disease, it is increasingly important to identify behaviors that may influence serum bilirubin. Although limited in examination, smoking is inversely associated with bilirubin,23,24 while physical activity is positively associated with bilirubin.24-27 Additionally, the effect of other health-enhancing behaviors (eg, diet) on bilirubin levels is unclear. It is plausible to suggest that adequate consumption of flavonoid-rich fruits and vegetables may be associated with bilirubin, given their influence on
1
Donna & Allan Lansing School of Nursing & Health Sciences, Bellarmine University, Louisville, KY, USA
Corresponding Author: Paul D. Loprinzi, Donna & Allan Lansing School of Nursing, Bellarmine University, Louisville, KY, USA. Email: [email protected]
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inflammation,28 markers of CV disease,29 and more importantly, their ability to modulate enzymes (eg, uridine 50 -diphosphoglucuronosyltransferases) that influence bilirubin levels.30 However, at this point, the influence of diet on bilirubin is poorly understood. To our knowledge, only 3 studies have examined the association between dietary behavior and bilirubin.31-33 In general, these studies have observed an association between diet and bilirubin; however, these studies were conducted among a small sample, used a narrow age range of participants, isolated certain fruits or vegetables, and specifically focused on the effect of genotype or gender on the association between diet and bilirubin.31-33 As a result, our understanding of the association between fruit and vegetable consumption on bilirubin in the general population is limited. The purpose of this investigation was to examine the association between flavonoid-rich fruit and vegetable consumption and serum bilirubin levels in the broader population. We hypothesize that those with higher levels of fruit and vegetable consumption will have increased bilirubin levels, independent of other CV risk factors. To improve generalizability, data for the present study come from the National Health and Nutrition Examination Survey (NHANES), which employs a nationally representative sample of US adults up to 85 years of age.
Methods Study Design and Participants Data from the 2003 to 2006 NHANES were used. All procedures for data collection were approved by the National Center for Health Statistics ethics review board, and all participants provided written informed consent prior to data collection. For the present analyses, 1783 adult participants (aged 18-85 years) provided data for all study variables.
Assessment of Flavonoid Index Based on the National Cancer Institute Diet History Questionnaire that is widely used in nutritional epidemiology research,34 participants completed the NHANES FFQ.35 Briefly, participants were asked to report the proportion of time certain types of foods were eaten over the past 12 months. For the present study, 15 foods rich in flavonoids (eg, apples, grapes, oranges, carrots, and broccoli) were identified using the US Department of Agriculture flavonoid content of foods.36 For each of the 15 food items, response options ranged from 1 to 11 and included never (1), 1 to 6 times/year (2), 7 to 11 times/year (3), 1 time/month (4), 2 to 3 times/month (5), 1 time/week (6), 2 times/week (7), 3 to 4 times/week (8), 5 to 6 times/week (9), 1 time/d (10), and 2 or more times/d (11). Responses were summed, with higher values indicating more frequent consumption of flavonoidrich foods. With 15 items, the possible range for the flavonoid index variable is 15 to 165.
Determination of Total Serum Bilirubin Total serum bilirubin was measured in mg/dL using the LX20 (Beckman Synchron), which uses a timed end point Diazo method to measure the total concentration of bilirubin. Further details can be found elsewhere.37
Covariates Covariates included age, gender, race ethnicity, socioeconomic status, body mass index (BMI), exercise, smoking status, hypertension, degree of insulin resistance/insulin sensitivity, and high sensitivity C-reactive protein (hsCRP). Information about age, gender, and race ethnicity were obtained from a questionnaire. As a measure of socioeconomic status, poverty to income ratio (PIR) was assessed, with a PIR value less than 1 considered below the poverty threshold. The PIR is calculated by dividing the family income by the poverty guidelines, which is specific to the family size, year assessed, and state of residence. The BMI was calculated from measured weight and height (weight in kg divided by the square of height in m). Based on the Global Physical Activity Questionnaire,38 participants were asked, ‘‘During the past 30 days, did you do moderate or vigorous activities for at least 10 minutes.’’ Responses were coded as yes or no. Serum cotinine was measured as a marker of active smoking status or environmental exposure to tobacco (ie, passive smoking). Serum cotinine was measured by an isotope dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Participants were considered to be hypertensive if they self-reported a physician diagnosis of hypertension or if their measured diastolic or systolic blood pressure was 90 or 140 mm Hg, respectively. The Homeostasis Model Assessment (HOMA) was used to evaluate insulin resistance using the following formula: fasting serum insulin (mU/mL) fasting plasma glucose (mmol/L)/22.5.39 Participants were classified as insulin sensitive if their HOMA score was 2.6, with a HOMA score >2.6 used to denote insulin resistance.40 The Tosoh AIA-PACK IRI, a 2-site immunoenzymometric assay, was used to measure blood insulin levels, with glucose measured spectrophotometrically. Further details of the assessment of insulin and glucose can be found elsewhere.41 The hsCRP concentration was quantified using latex-enhanced nephelometry.
Data Analysis All statistical analyses (STATA, version 12.0, College Station, Texas), which were performed in 2014, accounted for the complex survey design used in NHANES by using survey sample weights, clustering, and primary sampling units. The FFQ sample weights were used to provide nationally representative estimates. Means and standard errors were calculated for continuous variables and proportions were calculated for categorical variables. To examine the association between flavonoid consumption (index variable and independent variable) and total serum bilirubin (outcome variable), multivariable
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Table 1. Characteristics of the Analyzed Participants.a Variable
Mean/Proportion (95% CI)
Age, years % Female Race-ethnicity, % Non-Hispanic white Other Body mass index, kg/m2 Cotinine, ng/mL Poverty to income ratio Hypertensive, % Insulin resistance, % Insulin sensitive, % High-sensitivity C-reactive protein, mg/dL Exercise in past 30 days, % Bilirubin, mg/dL Bilirubin, mmol/Lb Flavonoid index
Table 2. Multivariable Linear Regression Analysis Examining the Association Between Flavonoid Consumption and Bilirubin (Outcome Variable).a
48.2 (46.8-49.6) 54.3 (51.7-56.9) 74.2 (69.4-79.0) 25.7 (20.9-30.5) 28.4 (27.9-28.9) 56.2 (45.3-67.2) 3.1 (3.0-3.2) 32.7 (29.3-36.0) 36.5 (33.8-39.3) 63.4 (60.6-66.1) 0.43 (0.38-0.48) 69.8 (67.1-72.6) 0.75 (0.73-0.78) 12.8 (12.5-13.3) 71.7 (70.6-72.9)
Abbreviations: CI, confidence interval; NHANES, National Health and Nutrition Examination Survey. a N ¼ 1783. NHANES 2003-2006. b To convert bilirubin from mg/dL to mmol/L, multiply mg/dL by 17.1.
linear regression was employed. Covariates included age, gender, race ethnicity, socioeconomic status, BMI, exercise, smoking status, hypertension, degree of insulin resistance/ insulin sensitivity, and hsCRP. All covariates were entered into the model at the same time, as there was no evidence of multicollinearity. Evidence of multicollinearity is likely to exist if there is a correlation of >0.8 between 2 covariates; if the mean variance inflation factor is >6, or if the highest individual variance inflation factor is >10, or if the tolerance statistic is 0.6. A P .05 (2-tailed) denoted statistical significance for all analyses.
Results Table 1 reports the study variable characteristics. Table 2 shows the multivariable linear regression analysis delineating the relationship between flavonoid consumption and bilirubin (outcome variable). After adjusting for age, gender, race ethnicity, socioeconomic status, BMI, exercise, smoking status, hypertension, degree of insulin resistance/insulin sensitivity, and hsCRP, greater consumption flavonoid-rich fruits and vegetables was positively associated with bilirubin (b ¼ .001; P ¼ .01). When expressed as a larger interval change, for every 50-unit increase in the flavonoid index variable, there was a 0.1 mg/dL increase in total serum bilirubin. Although there was no evidence of multicollinearity in the analytic model, we considered the possibility that the covariates may be taxing the regression model as a result of model overfitting. However, we ran minimally adjusted analyses (adjusting for age, gender, race ethnicity, and BMI), and the observed association was similar (b ¼ .001; P ¼ .004).
D (95% CI) in Bilirubin, mg/dL
P
0.001 (0.0001 to 0.001) .03 Flavonoid indexb Covariates Age, 1 year older 0.001 (0.0003 to 0.002) .01 Female vs male 0.20 (0.23 to 0.17) 1.78 ng/mL for men and >4.47 ng/mL for women was used to denote current smoker42), similar bilirubin levels were observed for smokers and nonsmokers (0.77 mg/dL vs 0.73 mg/dL; P ¼ .06). However, after adjustments, diet was not associated with bilirubin for current smokers (b ¼ .0004, P ¼ .35) but was for nonsmokers (b ¼ .002, P ¼ .005). With regard to insulin sensitivity/resistance, individuals with evidence of insulin sensitivity had higher bilirubin levels than those with insulin resistance (0.77 mg/dL vs 0.73 mg/dL; P ¼ .02). After adjustments, there was an association between diet and bilirubin levels for those with evidence of insulin sensitivity (b ¼ .001, P ¼ .05) but not for those with insulin resistance (b ¼ .0004, P ¼ .29).
Discussion In a nationally representative sample of US adults, greater consumption of flavonoid-rich fruits and vegetables was associated
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Angiology
with higher bilirubin levels. Secondary analyses demonstrated that this association may be moderated by gender, smoking status, and insulin sensitivity/resistance. Major strengths of this study include examining this underinvestigated topic and employing a nationally representative sample of US adults. Although these cross-sectional data do not allow for an assessment of the temporality of the association between fruit and vegetable consumption with bilirubin, it is plausible to suggest that adequate consumption of flavonoidrich fruits and vegetables may play a causal role in increasing bilirubin levels. The mechanism through which flavonoid-rich fruit and vegetable consumption may increase bilirubin levels is not clear; however, it is plausible through various biological pathways. Flavonoid-rich fruit and vegetable consumption may increase induction of hemeoxygenase through modulation of P-450-dependent metabolic activities,43,44 elimination of reactive oxygen species that may help to reduce the rate of decay in hemeoxygenase protein expression,45 and decreasing cellular Naþ and Ca2þ contents that can help to reduce edema formation46 and mitochondrial calcium-overload–induced cell death.45,47,48 Our findings are similar to the few other smaller scale convenience-based samples showing that fruit and vegetable consumption may help to increase bilirubin levels.31-33 These studies also show that the effects of fruit and vegetable consumption on bilirubin may be influenced by gender and UGT1A1 genotype. We were unable to control for UGT1A1 genotype, as this information was not collected in the NHANES; however, our secondary analyses support these previous findings indicating that the association between diet and bilirubin may be moderated by gender. In conclusion, these findings suggest an association between flavonoid-rich fruit and vegetable consumption and bilirubin levels. If confirmed by prospective and experimental studies, then regular consumption of flavonoid-rich fruits and vegetables should be promoted to increase levels of bilirubin.
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Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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