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Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
3To whom correspondence should be addressed. E-mail: jlampe{at}fhcrc.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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2.5 or 
4.5 servings/d. Diet was assessed by 3-d food record and serum carotenoids were measured as biomarkers of V&F intake (e.g., servings V&F vs. 
-carotene; r = 0.47, P = 0.0001). Serum ß-glucuronidase activity (Modified Sigma Units/L), determined in blood samples collected on two consecutive days from fasting subjects, was higher in men than women (mean ± SEM: 20.4 x 103 ± 1.0 x103 and 17.0 x 103 ± 0.6 x 103, P = 0.002). ß-Glucuronidase activity (adjusted for sex) was inversely associated with intakes of plant protein, fruit, dietary fiber (r = -0.24 to -0.30; P < 0.001), botanical groupings Cucurbitaceae, Rosaceae, and Leguminosae (r = -0.16 to -0.19; P < 0.05), and serum - and ß-carotene and ß-cryptoxanthin (r = -0.18 to -0.26; P 0.01). Activity was not associated with overall vegetable intake. Although these associations are modest, the data suggest that plant foods, particularly constituents of fruits and fiber-containing foods, may influence human ß-glucuronidase activity in a potentially favorable direction.
KEY WORDS: • ß-glucuronidase • diet • serum • fruits and vegetables • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Hydrolysis of the glucuronide moiety can be carried out by ß-glucuronidase present in most tissues, particularly liver, kidney, spleen, intestinal epithelium, and endocrine and reproductive organs (1
). Thus, circulating glucuronyl conjugates of steroid hormones and other ligands, in the past generally considered inactive and destined for excretion, are now recognized as still having the potential to interact with target tissues [for review see (2
)]. ß-Glucuronidase is thought to play an important role in regulating glucuronidation of xenobiotics and endogenous compounds (3
). Furthermore, ß-glucuronidase inhibitors reduce the carcinogenic potential of certain compounds normally excreted in bile after glucuronidation (4
). Pronounced interindividual variation in ß-glucuronidase activity in human liver has been attributed to different levels of enzyme expression (5
).
Dietary modulation of ß-glucuronidase has not been investigated in humans; however, data suggest that certain foods have the potential to alter ß-glucuronidase activity. The dietary constituent D-glucaric acid is a precursor of the potent ß-glucuronidase inhibitor, D-glucaro-1,4-lactone. It is found in substantial amounts in plant foods, including a wide variety of fruits and vegetables. The D-glucaric acid content of commonly consumed plant foods ranges from 10 mg/100 g in lettuce and grapes to 
350 mg/100 g in bean sprouts, cruciferous vegetables, apples and grapefruit (6
). Oranges, apricots, cherries, and tomatoes also are significant sources of D-glucaric acid (6
,7
). In one observational study, urinary excretion of glucaric acid was higher in vegetarians than omnivores (8
), possibly due to differences in dietary exposure and/or glucuronidation. In mammals, glucaric acid is also an endogenous metabolite of glucuronic acid; thus, the contribution of exogenous sources to glucaric acid exposure remains to be established.
In vivo, the D-glucaric acid metabolite, D-glucaro-1,4-lactone, can increase detoxification of carcinogens and inhibit chemically induced carcinogenesis in animals, in part by inhibiting ß-glucuronidase. In rats, administration of supplemental calcium glucarate reduced ß-glucuronidase activity in liver microsomes and serum (7
). Also in rats, dietary D-glucaric acids reduced circulating levels of estrogens, possibly as a result of increased excretion as glucuronides (9
); however, direct inhibition of rat liver or blood ß-glucuronidase activity was not found (10
).
The animal literature suggests that the amounts of D-glucaric acid required to inhibit ß-glucuronidase may be greater than can be achieved by habitual diet; however, the relationship between dietary patterns and ß-glucuronidase activity in humans has not been explored. Serum ß-glucuronidase activity reflects tissue ß-glucuronidase activity resulting from cell turnover, particularly hepatic (11
), and provides a noninvasive surrogate measure of tissue ß-glucuronidase activity in humans. Thus, the objectives of this study were as follows: 1) to determine the within- and between-individual variation in serum ß-glucuronidase activity among free-living individuals consuming their usual diets and 2) to examine the relationship between habitual diet and serum ß-glucuronidase activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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) intakes of either 4.5 servings or 2.5 servings during an initial telephone interview. We have used this approach in several studies designed to recruit individuals with a wide range of V&F intakes. The participants were not informed of the exact purpose of the study to avoid biased reporting of V&F intake. In addition, because this study was designed to examine the effect of V&F intake on biotransformation enzymes, we established the following exclusion criteria to minimize effects of other exposures: history of gastrointestinal disorders; weight change > 4.5 kg within the past year; major changes in eating habits within the past year; exercise regimens requiring major short-term dietary changes; antibiotic use within the past 3 mo; body weight > 150% of ideal; current drug therapy for a diagnosed disease; chronic nonsteroidal anti-inflammatory drug use; alcohol intake > 2 drinks/d (2 drinks being equivalent to 720 mL of beer, 240 mL of wine, or 90 mL of spirits); occupational exposure to smoke (e.g., tobacco, wood) or organic solvents; chronic exposure to second-hand tobacco smoke; and intake of pharmacologic doses of dietary supplements. We also instructed participants to avoid all medication for 7 d before and during the study. The study activities were approved by the Fred Hutchinson Cancer Research Center Institutional Review Board and informed, written consent was obtained from all study participants.
Each participant completed a demographic and health questionnaire and recorded food intake for three consecutive days (d 1–3). Blood samples from subjects who had fasted for 12 h were obtained on d 3 and 4. Nutrient calculations, using the 3-d food records, were performed using the Nutrition Data System (NDS) software, developed by the Nutrition Coordinating Center (NCC, University of Minnesota, Minneapolis, MN, Food Database version 12A, release date: November 1996, Nutrient Database version 27, release date November 1996). Intakes of V&F and of subgroups of V&F were calculated from the food records using a V&F classification scheme developed by the Fred Hutchinson Cancer Research Center, Nutrition Assessment Shared Resource. Briefly, this classification scheme includes all edible plant tissues included in the NDS Food Database, excluding herbs, spices, and grains, except for sweet corn. Three main groups were used, i.e., "total vegetables," "total fruits" and V&F classified by botanical families. Standardized serving sizes of V&F were established, similar to those specified in the Dietary Guidelines for Americans (12
). Food-record data (3-d) were classified into 64 botanical groups. Comparisons with ß-glucuronidase were limited to botanical groups that were regularly consumed and that included previously identified D-glucaric acid–containing V&F, i.e., Cucurbitaceae, Brassicaceae, Roseaceae, Solanaceae, Leguminosae, Rutaceae, Liliaceae, Compositae and Umbelliferae.
ß-Glucuronidase was measured using Sigma Diagnostics kit (Procedure No. 325) according to the manufacturer’s instruction manual (Sigma Diagnostics, St. Louis, MO). Serum samples, previously stored at -70°C, and quality-control pooled samples were assayed in duplicate. The procedure was scaled down from 200 to 50 µL and validated for frozen samples. The assay, as described originally by Fishman et al. (13
), uses phenolphthalein glucuronidate as substrate. Under standardized conditions, ß-glucuronidase acts on phenolphthalein mono-ß-glucuronic acid liberating free phenolphthalein. The intensity of the resulting red color in alkali is measured at 550 nm and is proportional to the enzyme activity. ß-Glucuronidase activity is determined from the standard curves. The analytical interassay coefficients of variation (CV%) were 6.9% for lower level controls [11.1 x 103 ± 0.77 x 103 Modified Sigma Units (MSU)/L] and 6.2% for higher level controls (25.8 x 103 ± 1.61 x 103 MSU/L).
Serum carotenoids concentrations were measured in the blood sample drawn on d 4 as described previously (14
). The CV for the pooled quality-control samples was 10% for all analytes. Serum alanine aminotransferase (ALT) activity was determined on a Roche Cobas Mira Plus (Branchburg, NJ) centrifugal analyzer by measuring NAD+ formation at 340 nm. The intra- and interassay CV were < 5%.
Before statistical analysis, serum ß-glucuronidase activity, serum carotenoids and servings per day of the botanical groupings were log-transformed to normalize the data. Difference in race distribution between men and women was tested using the 
2 statistic. Differences in the means of continuous variables between men and women were examined using Student’s t test. Associations between dietary intakes and serum ß-glucuronidase activity, adjusted for sex, were tested using partial correlations. Values in the text are presented as means ± SD. P < 0.05 was set as the level of statistical significance.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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In Table 1
, we present characteristics and dietary intakes of the 203 participants and, because of the sex difference in ß-glucuronidase activity, of men and women separately. In Table 2
, we present correlation coefficients for nutrient intakes, V&F groupings and dietary biomarkers associated with serum ß-glucuronidase concentrations. With further adjustment of nutrient intakes for V&F intake, the correlation between ß-glucuronidase activity and animal protein intake was no longer significant (r = 0.11; P = 0.13), but the correlation between dietary fiber and ß-glucuronidase activity remained significant (P = 0.004). Thus, there was no indication that intake of animal products increased ß-glucuronidase activity independently of plant foods; however, other fiber-containing plant foods (e.g., whole grains and/or nuts), in addition to V&F, may contain ß-glucuronidase inhibitors.
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,7
); however, of these three, we found an association only with Rosaceae. Additional systematic analysis of the D-glucaric acid content of plant foods within botanical families, and more broadly beyond those in the V&F classes, would improve the ability to examine the relationship between diet and ß-glucuronidase activity.
The serum carotenoids, - and ß-carotene, which were most strongly associated with V&F intake in this cohort (r = 0.47 and 0.43, respectively; P = 0.0001), as well as ß-cryptoxanthin, were also inversely associated with serum ß-glucuronidase activity (Table 2)
. Serum lutein, lycopene, zeaxanthin and -tocopherol concentrations were not associated with ß-glucuronidase activity (data not shown). Conversely, serum 
-tocopherol concentrations were positively associated with ß-glucuronidase activity and inversely associated with serum carotenoids, i.e., higher serum -tocopherol concentrations likely reflect a dietary pattern with lower intakes of V&F and higher intakes of dietary fat (15
).
In this small observational study, we limited the scope of the work to determining short-term variability in serum ß-glucuronidase activity and the association between immediate diet and enzyme activity. The within-person variation in ß-glucuronidase activity between two consecutive days was very low; however, average values over longer periods remain undefined. Furthermore, the effect of long-term dietary pattern, compared with short-term dietary intake, on enzyme activity warrants evaluation. Thus, future research should establish long-term variability in ß-glucuronidase activity and the relationship between habitual diet and average ß-glucuronidase activity. Given the large interindividual variation in ß-glucuronidase activity and the modest associations between diet and enzyme activity, there are likely other factors and exposures that determine ß-glucuronidase activity. In addition, our results, based on averaged ß-glucuronidase activity and dietary intake data, are subject to random error, which acts to bias toward the null; thus, the true correlations between dietary intake and ß-glucuronidase activity are likely stronger than those presented (16
). Controlled dietary interventions will help to establish more accurately the relationship between diet and ß-glucuronidase activity and the rate of change in enzyme activity with dietary modification.
In conclusion, serum ß-glucuronidase activity was inversely associated with specific nutrients that characterize the intake of a more plant-based diet, namely, greater intakes of plant protein and dietary fiber. Similarly, ß-glucuronidase was inversely associated with biomarkers of V&F intake, including serum concentrations of - and ß-carotene, and ß-cryptoxanthin. Furthermore, several botanical groupings of V&F, e.g., Cucurbitaceae, Rosaceae and Leguminosae, were inversely associated with serum ß-glucuronidase activity and may thus be particularly good sources of D-glucaric acid or other ß-glucuronidase inhibitors. In our effort to understand the effect of endogenous ß-glucuronidase activity on carcinogenesis and modulation of risk factors for cancer, the capacity of a high plant food diet to reduce ß-glucuronidase in humans warrants further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Funded by National Institutes of Health grant R01 CA70913 and Fred Hutchinson Cancer Research Center. A portion of the work was also conducted through the Clinical Research Center facility at the University of Washington, supported by the National Institutes of Health, NCRR, Grant M01-N-00037.
4 Abbreviations used: ALT, alanine aminotransferase; MSU, Modified Sigma Units; NDS, Nutrition Data System; V&F, vegetable and fruit.
Manuscript received 1 January 2002. Initial review completed 14 February 2002. Revision accepted 13 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Dutton, G. J. (1980) Glucuronidation of Drugs and Other Compounds 1980 CRC Press Boca Raton, FL. .
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Belinsky, S. A., Kauffman, F. C., Sokolove, P. M., Tsukuda, T. & Thurman, R. G. (1984) Calcium-mediated inhibition of glucuronide production by epinephrine in the perfused rat liver. J. Biol. Chem. 259:7705-7711.
4.
Walaszek, Z., Hanausek-Walaszek, M. & Webb, T. E. (1984) Inhibition of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis by 2,5-di-O-acetyl-D-glucaro-1,4:6,3-dilactone, an in vivo ß-glucuronidase inhibitor. Carcinogenesis 5:767-772.
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Sperker, B., Mürdter, T. E., Schick, M., Eckhardt, K., Bosslet, K. & Kroemer, H. K. (1997) Interindividual variability in expression and activity of ß-glucuronidase in liver and kidney: consequences for metabolism. J. Pharmacol. Exp. Ther. 281:914-920.
6. Walaszek, Z., Szemraj, J., Hanausek, M., Adams, A. K. & Sherman, U. (1996) D-Glucaric acid content of various fruits and vegetables and cholesterol-lowering effects of dietary D-glucarate in the rat. Nutr. Res. 16:673-681.
7. Dwivedi, C., Heck, W. J., Downie, A. A., Larroya, S. & Webb, T. E. (1990) Effect of calcium glucarate on ß-glucuronidase activity and glucarate content of certain vegetables and fruits. Biochem. Med. Metab. Biol. 43:83-92.[Medline]
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9.
Walaszek, Z., Hanausek-Walaszek, M., Minton, J. P. & Webb, T. E. (1986) Dietary glucarate as anti-promoter of 1,7-dimethylbenz[a]anthracene-induced mammary tumorigenesis. Carcinogenesis 7:1463-1466.
10. Webb, T. E., Abou-Issa, H., Dwivedi, C. & Koolemans-Beynen, A. (1992) Synergism between glucarate and retinoids in the rat mammary system. Wittenberg, L. Lipkin, M. Boone, C. W. Kelloff, G. J. eds. Cancer Chemoprevention 1992:263-277 CRC Press Boca Raton, FL. .
11. Kim, D.-H., Shim, S.-B., Kim, N.-J. & Jang, I.-S. (1999) ß-Glucuronidase-inhibitory activity and hepatoprotective effect of Ganoderma lucidum. Biol. Pharm. Bull. 22:162-164.[Medline]
12. United States Department of Agriculture (1990) Nutrition, and Your Health. Dietary Guidelines for Americans 1990 United States Department of Agriculture, Department of Health and Human Services Washington, DC. .
13.
Fishman, W., Springer, S. & Brunetti, B. (1948) Application of an improved glucuronidase assay method to study of human blood (beta)-glucuronidase. J. Biol. Chem. 173:449.
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Zhang, C., Williams, M. A., Sanchez, S. E., King, I. B., Ware-Jauregui, S., Larrabure, G., Bazul, V. & Leisenring, W. M. (2001) Plasma concentrations of carotenoids, retinol, and tocopherols in preeclamptic and normotensive pregnant women. Am. J. Epidemiol. 153:572-580.
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White, E., Kristal, A. R., Shikany, J. M., Wilson, A. C., Chen, C., Mares-Perlman, J. A., Masaki, K. H. & Cann, B. J. (2001) Correlates of serum -tocopherol and -tocopherol in the Women’s Health Initiative. Ann. Epidemiol. 11:136-144.[Medline]
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