Serum β-Glucuronidase Activity in Response to Fruit and Vegetable Supplementation: A Controlled Feeding Study

Abstract

Background

Fruit and vegetable intake may lower the risk of some cancers. One hypothesized, but understudied, chemopreventive mechanism is that plant food constituents inhibit β-glucuronidase, an acid hydrolase that deconjugates glucuronides.

Methods

We conducted a cross-over feeding trial in 63 healthy women and men aged 20−40 years, to examine the effect of diet on serum β-glucuronidase activity. Participants were randomized to 2 two-week experimental diets with an intervening washout period: a diet high in selected citrus fruit, crucifers, and soy (F&V) and a diet devoid of fruits, vegetables, and soy (basal). Serum β-glucuronidase activity was measured during the pre-intervention, F&V, and basal periods. Linear mixed models were used to obtain effect estimates and 95% confidence intervals (CI).

Results

We observed statistically significantly higher β-glucuronidase activity during the F&V than the basal diet (ratio, F&V versus basal diet, 1.09; 95% CI, 1.05−1.13; P <0.01). These results were probably due to decreased β-glucuronidase activity during the basal diet (ratio, basal period versus pre-intervention, 0.93; 95% CI, 0.87−0.98; P=0.01), rather than increased enzyme activity during the F&V diet (ratio, F&V period versus pre-intervention, 1.01; 95% CI, 0.96−1.06; P=0.64). The diet-enzyme activity relation did not differ by sex (P interaction=0.30), but there was a suggestion of a short-term diet effect at 8 days versus 15 days (P interaction=0.06).

Conclusion

This intervention of selected fruits and vegetables did not lower β-glucuronidase activity. Further investigation is needed regarding what other foods and phytochemicals may influence β-glucuronidase activity and effect modifiers of this relation

INTRODUCTION

Fruit and vegetable intake may lower risk of some human cancers (1, 2). One hypothesized, but understudied, mechanism is the inhibition of the acid hydrolase, β-glucuronidase, found in most tissues, such as the liver, kidney, spleen, intestinal epithelium, and endocrine and reproductive organs (3). β-glucuronidase cleaves glucuronic acid from substrates (e.g. drug and non-drug xenobiotics, steroid hormones, and other endogenous compounds) making them less water-soluble and less able to be excreted. β-glucuronidase may increase cancer risk, because potential carcinogens and promoting agents, once deglucuronidated, have the ability to recirculate and interact with cells.

Plant food constituents, such as D-glucaric acid, may act as nontoxic β-glucuronidase inhibitors in humans (4) and thus, lower cancer risk. D-glucaric acid is converted into D-glucaro-1,4-lactone which competitively inhibits β-glucuronidase and has been shown to reduce chemical carcinogen-mediated mammary, liver, and skin tumors in animals (5-7). The effect of D-glucaric acid in preventing human cancer is unknown. Among commonly consumed plants, citrus and cruciferous foods are rich in D-glucaric acid (8).

Previously, we conducted a cross-sectional pilot study of 83 men and 120 women to examine dietary associations with serum β-glucuronidase activity (9). We used serum β-glucuronidase because it reflects tissue β-glucuronidase resulting from cell turnover, particularly from the liver which is the major source of the enzyme, and because serum collection requires minimally invasive methods. We found that β-glucuronidase activity was significantly inversely associated with intakes of plant protein, fruit, dietary fiber (r = −0.24 to −0.30; P < 0.01), the botanical groupings of Cucurbitaceae, Rosaceae, and Leguminosae (r = −0.16 to −0.19; P < 0.05), and serum alpha-carotene, beta-carotene and beta-cryptoxanthin (r = −0.18 to −0.26; P ≤ 0.01).

No human intervention study has examined this relation. We thus, conducted a cross-over feeding trial to compare the effects of a diet high in selected fruits and vegetables to a diet devoid of fruits and vegetables on serum β-glucuronidase activity. Based on earlier investigations of D-glucaric acid and our observational pilot study results , we hypothesized that a plant-rich diet would lower β-glucuronidase activity.

METHODS

The investigation was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board, Seattle, Washington. Informed written consent was obtained from all participants.

Participants and Covariate Data

Participants were recruited from 193 individuals who had met the selection criteria and completed an initial cross-sectional study (10). Because of the aims of the parent study (10), participants were also recruited based on the UDP-glucuronosyltransferase (UGT). Of the 111 eligible for the feeding study, 72 consented and were randomized. Of these, 8 withdrew and 1 was noncompliant with the diet. Prior to the intervention, each participant provided demographic and health-related information and baseline data on dietary intake over the past 3 months.

Feeding Study Design

Participants were randomized and blocked on sex and UGT1A1 genotype. Using a crossover study design, participants consumed 2 two-week (14 days) experimental diets assigned in random order, with an intervening washout phase of at least 2 weeks when individuals resumed their habitual diet. The basal experimental (control) diet was low in phytochemicals, and contained low-fiber refined foods, without fruits, vegetables, herbs or spices (menu presented in (10)). The F&V experimental diet consisted of the basal diet supplemented with crucifers (i.e., broccoli, cabbage, and radish sprouts), citrus fruits (i.e., grapefruit/orange segments, grapefruit/orange juice and dried orange peel), and soy foods (i.e., tofu, soy nuts, and soy milk). Crucifers and citrus fruits contain D-glucaric acid, and therefore, were likely to decrease β-glucuronidase activity. We did not find literature suggesting that soy foods influence β-glucuronidase; soy was of interest to the parent study. For the F&V diet, individuals were dosed with fruits and vegetables according to body weight (10) and consumed ∼10 daily total servings. Both basal and F&V diets provided similar percent energy from carbohydrate (56%), protein (16%), and fat (28%). Nutrient intakes were calculated using the Nutrition Data System for Research software version V4.05_33 (Nutrition Coordinating Center, University of Minnesota; Minneapolis, MN).

Participants were instructed to eat only prescribed, study-provided beverages and foods. Compliance, as assessed by 24-h urinary excretion of isoflavones and isothiocyanates and by daily food check-off forms, was high (10).

Determination of Serum β-glucuronidase Activity

Prior to the intervention and at days 8 and 15 of each feeding period, morning blood (after 10 hrs of fasting) was collected for serum. β-glucuronidase activity from serum was determined using the following method modified from the Sigma β-glucuronidase kit (Sigma-Aldrich Company, St. Louis, MO), because the kit had been discontinued. Fifty μl of serum was used in the assay with a corresponding reduction in the volumes of the enzyme substrate, the acetate buffer, and water. The volume of the AMP buffer (stop reagent) was reduced by 20%. Serum was incubated with phenolphthalein glucuronic acid (Sigma), the β-glucuronidase substrate, at pH 4.5 for 4 hrs at 37° C. At exactly 4 hrs, the reaction was stopped using an alkaline buffer 0.1 M AMP buffer (Sigma) pH 11. Under standard conditions, β–glucuronidase cleaves phenolphthalein glucuronic acid liberating free phenolphthalein. A DU650 Spectrophotometer (Beckman Instruments, Fullerton CA) was used to monitor the intensity of the resulting pink color which is proportional to β–glucuronidase activity. We determined enzyme activity, expressed as μg phenolpthalein released /ml of serum /hr at 37° C, from standard curves. The intra-and inter- assay coefficient of variation (CV) were 2.9% and 5.7%, respectively.

Statistical Analysis

Before analyses, β-glucuronidase activity was log-transformed to improve normality. A linear mixed model was used to examine the effect of F&V on β-glucuronidase activity and whether the relation differs by subgroups of sex and days (8 or 15). We adjusted for sex, days on experimental diet, UGT1A1 genotype (one of the recruitment factors), order of assigned experimental diet, carryover effect, β-glucuronidase activity during the pre-intervention period, and 2-way interactions between sex, days on experimental diet, UGT1A1 genotype, and experimental diet. The log-back-transformed least-squared means and their associated 95% CIs were reported. Our main effect measure, the ratio of enzyme activity during each diet, was calculated by back-transforming (exponentiating) the difference of the log-transformed means from each diet. All statistical tests were 2-sided with P<0.05 considered statistically significant. Additional details are provided in Table footnotes. Analyses were conducted using SAS, version 9.0 (SAS Institute, Cary, NC).

RESULTS

Sixty-three healthy, non-smoking men and women, aged 20− 40 years, completed both feeding periods (Table 1). They were mostly Caucasian (68%) and Asian (25%). Their mean baseline BMI was within normal range, with men being taller (P<0.01) and heavier (P<0.01) than women. Men and women had statistically similar pre-intervention dietary intakes of fruits and vegetables (Table 1) even after adjusting for weight (data not shown), reporting, on average, approximately 4 daily servings. Men had higher pre-intervention β-glucuronidase activity than women (P<0.01) (Table 3).

Table 1.

Baseline characteristics of female and male participants^*

	Total (n=63)	Women (n=31)	Men (n=32)	P‡
Age, y	29.5 ± 5.57	28.8 ± 5.48	30.3 ± 5.65	0.29
Weight, kg	68.9 ± 12.0	62.2 ± 10.2	75.5 ± 9.98	<0.01
Height, m	1.72 ± 0.10	1.65 ± 0.08	1.78 ± 0.08	<0.01
BMI, kg/m2	23.3 ± 2.61	22.8 ± 2.82	23.7 ± 2.35	0.14
Race, n (%)
Caucasian	68	65	72	0.59
Asian	25	32	19	0.25
Other	6	3	9	0.61
Habitual servings/d†
Fruits	2.47 ± 2.08	2.45 ± 2.06	2.50 ± 2.13	0.87
Vegetables	1.87 ± 1.21	2.14 ± 1.30	1.61 ± 1.08	0.07
Citrus fruits	0.82 ± 1.10	0.85 ± 1.35	0.80 ± 0.82	0.82
Cruciferous vegetables	0.34 ± 0.40	0.30 ± 0.28	0.38 ± 0.50	0.67

^*Values for continuous variables are means ± SEM, and for categorical variables, percentages

^†Servings were calculated based on the standardized serving sizes from the Dietary Guidelines for Americans

^‡We tested whether the baseline data was statistically different by sex, by computing two-sample T-tests for continuous variables and the Fisher's exact test for the race variable; dietary intakes were log-transformed prior to hypothesis testing to improve normality. P<0.05 was considered statistically significant.

Table 3.

Serum β-glucuronidase activity during pre-intervention and experimental periods, stratified by sex and day on experimental diet

		Experimental Diets
	Pre-intervention diet* (μg/ml/hr)	Basal diet* (μg/ml/hr)	F&V diet* (μg/ml/hr)	Ratio of Basal and Pre-intervention diets † (95% CI)	Ratio of F&V and Pre-intervention diets † (95% CI)
All participants, averaged both days	5.89±0.34	5.46±0.06	5.96±0.06	0.93 (0.87−0.98)‡	1.01 (0.96−1.06)
Women §	5.02±0.40	4.50±0.40	5.01±0.46	0.90 (0.83−0.98)‡	1.00 (0.93−1.08)
Men §	6.89±0.51	6.58±0.49	7.05±0.51	0.96 (0.87−1.04)	1.02 (0.96−1.09)
Experimental day 8	5.89±0.34 ∥	5.49±0.07	6.22±0.06	0.93 (0.87−1.00)	1.06 (1.00−1.11)‡
Experimental day 15	5.89±0.34 ∥	5.45±0.06	5.72±0.06	0.93 (0.86−0.99)‡	0.97 (0.92−1.02)

Abbreviations: F&V, fruit and vegetable diet; CI, confidence interval

^*Geometric means ± SE of β-glucuronidase activity; means were unadjusted because paired t-tests were used to compare values

^†Experimental diet versus Pre-intervention ratios were calculated by exponentiating the difference of the log-transformed (unadjusted) geometric means. Paired t-tests, inherently adjusting for within-person characteristics, were used to calculate the significance of the ratios.

^‡Serum β-glucuronidase activity was significantly different for the pre-intervention than the experimental diet, P<0.05

^§For experimental diets, data stratified by sex represent values averaged over days 8 and 15 of experimental diet

^∥Pre-intervention β-glucuronidase activity was sample at one time point

Table 2 shows the effect of F&V on mean β-glucuronidase activity. Contrary to our hypothesis, participants had significantly higher mean β-glucuronidase activity on F&V than on the basal diet (ratio, F&V versus basal diet, 1.09; 95% CI, 1.05−1.13; P <0.01). Seventy-six percent had higher β-glucuronidase activity on F&V than on the basal diet whereas 24% had lower levels. Response to diet appeared stronger for women than men (Table 2), but the test of interaction was not statistically significant (P interaction =0.30). There was also a suggestion of a short-term effect; the F&V diet was associated with a significant increase in β-glucuronidase activity half way through the intervention on Day 8 (ratio, F&V versus basal, 1.13; 95% CI, 1.07−1.20; P<0.01), but this relation was attenuated on Day 15 (ratio, 1.05; 95% CI, 0.99−1.11; P=0.12; P interaction=0.06). Order of diet assignment (F&V-basal or basal-F&V) had no effect on β-glucuronidase activity (P=0.70). Moreover, we did not observe specific responses among individuals that would allow for stratification of responder and nonresponder groups.

Table 2.

Effect of experimental diets on serum β-glucuronidase activity, stratified by sex and day on experimental diet

	Experimental Diets
	Basal diet* (μg/ml/hr)	F&V diet* (μg/ml/hr)	Ratio of F&V and Basal diets † (95% CI)
All participants, averaged both days	5.43±0.15	5.91 ± 0.17	1.09 (1.05−1.13)‡
Women §	5.19 ± 0.21	5.75 ± 0.23	1.11 (1.05−1.17)‡
Men §	5.69 ± 0.23	6.06 ± 0.25	1.07 (1.01−1.13)‡
Day 8	5.44 ± 0.17	6.15 ± 0.19	1.13 (1.07−1.20)‡
Day 15	5.42 ± 0.17	5.67 ± 0.18	1.05 (0.99−1.11)

Abbreviations: F&V, fruit and vegetable diet; CI, confidence interval

^*Back-log-transformed least-square means ±SE of serum β-glucuronidase activity. Means are adjusted for: sex, days on experimental diet, UGT1A1 genotype, order of assigned experimental diet, carryover effect, β-glucuronidase activity during the pre-intervention period, 2-way interactions between sex, days on experimental diet, UGT1A1 genotype, and experimental diet

^†Ratio was calculated by exponentiating the difference of the log-transformed adjusted, least-square means

^‡Serum β-glucuronidase activity was significantly higher during F&V than during basal diet, P<0.05

^§β-glucuronidase activity averaged over days 8 and 15 of experimental diet

Because the F&V results were unexpected, we subsequently examined the change in β-glucuronidase activity from the pre-intervention to each experimental diet period (Table 3). β-glucuronidase activity during the F&V period was not significantly different than the pre-intervention period (ratio, F&V period versus pre-intervention, 1.01; 95% CI, 0.96−1.06; P=0.64). However, during the basal diet, there was a statistically significant 7% decrease in β-glucuronidase activity compared with the pre-intervention diet (ratio, basal period versus pre-intervention, 0.93; 95% CI, 0.87−0.98; P=0.01), suggesting that our main finding was due to a decline in β-glucuronidase activity during the basal (control) diet. In subgroup analyses, this decline was slightly stronger for women (ratio, 0.90; 95% CI, 0.83−0.98) than men (ratio, 0.96; 95% CI, 0.87−1.04). The increase in β-glucuronidase activity from pre-intervention toDay 8 during the F&V diet was attenuated on Day 15.The magnitude of these changes was small.

DISCUSSION

In this randomized crossover study, we examined the effect of a diet rich in selected fruits and vegetables (F&V) on serum β-glucuronidase activity compared with a basal diet devoid of fruits and vegetables. Comparing F&V and basal diets, we observed an increase in β-glucuronidase activity. However, upon further analysis, we attribute this result to a decline in β-glucuronidase activity during the basal diet from the habitual, pre-intervention diet levels.

We originally hypothesized that a plant-rich diet would lower β-glucuronidase activity. During the F&V diet, participants were fed citrus and cruciferous foods naturally rich in D-glucaric acid, a possible inhibitor of β-glucuronidase. Broccoli and grapefruit, for instance, contain ∼350mg/100g of D-glucaric acid and oranges have ∼129mg/100g of D-glucaric acid (8). However, not all D-glucaric acid may be bioavailable to lysosomes, where β-glucuronidase mostly resides (11). Additionally, other botanical groupings, not investigated here such as Cucurbitaceae (squash, melons), Rosaceae (stone-fruit), and Leguminosae (legumes) may be relevant, as suggested by our pilot results of habitual diet (9).

During the basal diet (averaged days 8 and 15), the decrease in β-glucuronidase activity from pre-intervention levels was interesting and needs further evaluation. One explanation may be that foods eaten habitually before the intervention contained constituents that maintain β-glucuronidase activity and the removal of these foods (basal diet) lowered activity. Our results though statistically significant, were small and the clinical relevance of these small changes on cancer risk is unknown.

In subgroup analyses, we observed a tendency for a stronger response on day 8 versus day 15, supporting an acute response to diet. This is consistent with the short-term effects of cruciferous vegetable feeding on acetaminophen conjugation (12) and oltipraz administration on the induction of detoxification enzymes (13). However, it is unknown whether the body adapts to elevated exposure over the long-term. There may be transient, acute responses to our 2-week dietary alterations that are different than responses to longer (>14 day) interventions.

This is the first human intervention study examining dietary supplementation on β-glucuronidase activity. Strengths of this investigation include: 1) controlled diets in which the plant foods were dosed according to body weight to minimize confounding by weight; 2) inherent adjustment for confounding and between-person differences as each person acted as his/her own control; and 3) high participant adherence to experimental diets. Lower β-glucuronidase activity (reviewed in (3)) has been associated with higher D-glucaric acid (4, 11), caloric restriction (14), silymarin (milk thistle extract) (15), Ganoderma lucidum (16), and the calcium modulating xenobiotics A23187 (17) and thapsigargin (17). Conversely, increased β-glucuronidase activity (reviewed in (3)) has been associated with male sex (9, 18), higher BMI (18), older age (3, 18), pathological conditions (i.e. cancer, liver disease, tuberculosis) (3), tobacco exposure (19), and spironolactone, a drug inducer of microsomal enzymes such as UGT (20).

Our restriction to healthy, nonsmoking, and nonmedicated participants minimized the impact of these and other non-intervention factors on β-glucuronidase activity. A study limitation is our reliance on serum β-glucuronidase, which are lower than tissue levels. Moreover, day-to-day, within-person variability in β-glucuronidase activity would attenuate our results. However, in our pilot study in which we had 2 measures of β-glucuronidase activity, the between-person CV (46%) was higher than the within-person CV (8%). While there is no established range of human β-glucuronidase activity, enzyme activity in this current study was similar to that of our pilot, after taking into account different assay temperatures.

The mechanisms underlying human β-glucuronidase induction are largely unknown. Analysis of the human β-glucuronidase gene suggests the presence of binding sites for 3 ubiquitous transcription factors with putative regulating roles: nuclear factor κB (NFκB), activating protein-2 (AP-2), and specificity protein 1 (Sp1). (cited in (21)). The possible β-glucuronidase-lowering effects of Silymarin (15), A23187 (17), and Thapsigargin (17), may be mediated by NFκB (22), whose expression is potentially induced by phytochemicals (23), and also, AP-2 (21). Moreover, Sp1 sites may prevent portions of the gene from becoming methylated (24). Three β-glucuronidase polymorphisms, whose frequencies in the general population still require quantification, have been associated with altered enzyme activity (18), and several isoforms of both microsomal and lysosomal origin have been identified (25), raising questions regarding whether the diet response varies by these factors.

In conclusion, we did not detect an effect of fruit and vegetable supplementation on β-glucuronidase activity, but rather, observed a small decrease in enzyme activity during the control diet devoid of fruits and vegetables. There are many unresolved questions and further investigation is needed regarding what other foods and phytochemicals may influence β-glucuronidase activity and the effect modifiers for this relation.