Dietary antioxidant lack, impaired hepatic glutathione reserve, and cholesterol gallstones

Clinica Chimica Acta 349 (2004) 157 – 165 www.elsevier.com/locate/clinchim

Dietary antioxidant lack, impaired hepatic glutathione reserve, and cholesterol gallstones Helen V. Worthingtona,1, Linda P. Huntb,2, Rory F. McCloyc, Jop B. Ubbinkd, Joan M. Braganzac,* a

b

Department of Dietetics, Manchester Royal Infirmary, Manchester, UK Faculty of Medicine Computational Group and the Pancreatobiliary Service, Manchester Royal Infirmary, Manchester, UK c Pancreatobiliary Service, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK d Department of Chemical Pathology, Pretoria University, South Africa Received 13 April 2004; received in revised form 16 June 2004; accepted 17 June 2004

Abstract Background: Theoretical considerations and experimental studies suggest a causal connection between micronutrient antioxidant insufficiency and the development of human gallstones. Methods: Fasting plasma/serum samples from 24 patients with cholesterol gallstones—on unchanged lifestyles—were analysed for the four main micronutrient antioxidants, glutathione and factors that impact or report upon glutathione homeostasis. The results were assessed by comparison with laboratory referent ranges. Results: The vitamin E:cholesterol ratio was lower in patients than controls ( P=0.021) as also concentrations of h-carotene ( P=0.001) and vitamin C ( P=0.001) but not selenium ( P=0.280). A fall in plasma glutathione ( P=0.001) was also accompanied by lower values of pyridoxyl-5-phosphate (the coenzyme that participates in vitamin B6-dependent enzyme reactions) which is involved in glutathione biosynthesis ( Pb0.001), and of folate ( P=0.012) but not vitamin B12 ( P=0.377) that participate in its regeneration via the methionine–homocysteine pathway. Despite these defects, values for plasma homocysteine were not significantly different from controls ( P=0.092)—an anomaly rationalised by poor levels of precursor methionine ( P=0.003) and cysteine ( P=0.046). Conclusions: Micronutrient antioxidant—including sulphur amino acid—lack, with disturbed glutathione homeostasis, are features of cholesterol gallstone disease. D 2004 Elsevier B.V. All rights reserved. Keywords: Cholesterol gallstones; Micronutrient antioxidants; Glutathione homeostasis; Homocysteine; Methionine

* Corresponding author. Tel.: +44 161 276 4534; fax: +44 161 273 3428. E-mail address: [email protected] (J.M. Braganza). 1 Current address: Community Dietitian, Manchester. 2 Current address: Department of Clinical Sciences, Bristol University, South Bristol. 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.06.022

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1. Introduction Macronutrient intake has been the focus of numerous investigations into the development of human

Fig. 1. Schematic representation of the intracellular pathway of methionine metabolism wherein the vertical rectangle indicates boundaries of the cell. Abbreviations: sulphadenosylmethionine (SAM), methyl thioadenosine (MTA), sulphadenosylhomocysteine (SAH), B6-dependent enzyme pyridoxal-5-phosphate (B6-PLP), glutathione in its reduced bioactive form (GSH), selenium-dependent glutathione peroxidase [GSH(Se)Px], riboflavin-dependent enzyme glutathione reductase [GSH(B2)Rx], oxidised form of glutathione involved in removal of hydrogen peroxide (H2O2) and lipid peroxide (LOOH) (GSSG), GSH-detoxified form when reactive xenobiotic metabolites are produced via cytochrome P450 (GSSR), gamma-glutamyl transpeptidase (g-GT ) involved in the recycling of constituent amino acids from GSH that finds its way into plasma.

gallstones, among which the majority are cholesterolrich with a core of calcium bilirubinate. Yet animal models and theoretical considerations suggest that a reduction in micronutrient intake—of antioxidants in particular—may be more relevant across the gallstone spectrum [1]. Thus: guinea pigs, which like man cannot synthesise vitamin C, rapidly develop cholesterol stones when lithogenic diets are modified through vitamin C deprivation but not otherwise; the development of cholesterol gallstones is accelerated by vitamin E deficiency, and the stones dissolve when the vitamin is reintroduced; black pigment stones quickly form when dogs are reared on diets deficient in methionine [2]. These observations, and the recognized effect of antioxidant lack on the function of key enzymes that are involved in cholesterol and bilirubin metabolism [1], led us to examine micronutrient antioxidant intake in patients with cholesterol-rich stones. We found lower intakes by patients who had not changed their lifestyles than by matched controls of 10 among 16 antioxidants—methionine, a-tocopherol (vitamin E), vitamin D and manganese at the conventional 5% significance level; cysteine, h-carotene, vitamin C, selenium, zinc and phosphorous at the 10% level—when at most two differences might be expected by chance [3]. The aims of the present study of fasting blood samples in patients with cholesterol-rich stones were (i), to verify or refute the dietary findings in relation to the four best-known antioxidants (a-tocopherol, vitamin C, h-carotene, selenium); (ii) to analyse for the key endogenous antioxidant glutathione, and ancillary factors (Fig. 1) [i.e., vitamins B12 and folate, pyridoxyl-5-phosphate (PLP) which acts as a coenzyme in vitamin B6-dependent reactions, the sulphur amino acids methionine and cysteine, homocysteine, and the enzyme g-glutamyl transpeptidase (g-GT) located on the plasma membrane of hepatocytes] [4].

2. Participants and methods 2.1. Patients Recruitment was slow and only 24 patients could be investigated in the 18-month period of study. This was for two reasons. First, most patients with gallstones had already switched to a low-fat diet by

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the time of referral. Second, our study was restricted to patients with uncomplicated gallstone disease in whom this was the first medical problem. Thus, subclinical inflammation was excluded by normal values of plasma C-reactive protein, and subclinical biliary obstruction by ultrasound examination coupled with normal serum bilirubin and alkaline phosphatase. Furthermore, patients were automatically excluded if they had preexisting conditions that might limit antioxidant intake (e.g., eating disorders, prescribed low protein or low fat diets) and/or increase requirement (e.g., diabetes mellitus); we also excluded individuals on over-the-counter food supplements. Each participant had ultrasound-confirmed gallstones that were radiolucent on plain X-ray, which suggested the stones were rich in cholesterol. Table 1 gives

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social information including that on exposure to exogenous chemicals because these are potentially relevant to gallstone development via challenge to enzymes that are also involved in cholesterol and bilirubin metabolism [1]. The laboratory database at Manchester gave referent values for the antioxidant vitamins and glutathione. Hospital workers, their relatives and patients with minor surgical problems contributed to that database. It was stipulated that inclusion as a volunteer required that he/she did not smoke cigarettes or drink alcohol on a regular basis, had no dietary fads, did not take food supplements and had no previous or current medical problem. Residual deep-frozen samples from the patients, along with a subset of samples from controls who were age and

Table 1 Characteristics of patients

Cigarette and alcohol usage were extrapolated from amounts in a typical week aOccupations and chemical exposures were as stated by the patient.

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gender-matched, were analysed for the other variables at Pretoria, South Africa. 2.2. Procedure After an overnight fast, 30 ml of peripheral venous blood were drawn, of which 5 ml were placed into an EDTA tube (for assays of glutathione and haemoglobin in whole blood and plasma) and the remainder divided between plain tubes for serum-based measurements (selenium, vitamin E, h-carotene, vitamin B12, folate, cholesterol, g-GT) and lithium-heparin tubes for plasma analyates (vitamin C, its bioactive fraction as ascorbate, sulphur amino acids, homocysteine, PLP). All tubes were held in crushed ice during sample preparation. A portion of EDTA whole blood was processed immediately at the bedside to stabilise glutathione and minimise oxidation artifact: haemolysate and supernatant plasma obtained after centrifugation at 2900g for 6 min were prepared as detailed previously [5]. Heparinised tubes were spun down at 700g for 15 min at 4 8C. The separated plasma was deproteinised by the appropriate precipitant—sulphosalicylic acid for analysis of sulphur amino acids, trichloroacetic acid for homocysteine and total vitamin C, metaphosphoric acid for ascorbic acid—with vortexing to ensure thorough mixing before further centrifugation at 1500g for 20 min at 4 8C. Plain tubes were allowed to stand on ice until a clot formed, whereupon the samples were spun down at 1500g for 10 min at 4 8C and serum separated off. Aliquots of all prepared samples were stored at 70 8C and analysed within the known stability period for the various assays. The analysis for whole blood glutathione was done within 2 weeks and plasma analysis within a month. Plasma ascorbate and vitamin C were measured within 3 months and all the analyses were completed by 6 months.

chromatography enabled measurement of methionine and cysteine [7,12]; total vitamin C was measured colorimetrically [13] and selenium by fluorometry [14]. Haemoglobin, cholesterol, g-GT, vitamins B12 and folate were determined in the routine hospital laboratory, using commercially available kits as required. Glutathione in erythrocytes is almost exclusively in the bioactive GSH form while that in plasma contains variable amounts of the oxidised GSSG form [5]. Plasma haemoglobin was measured to safeguard against spurious glutathione readings due to erythrocyte lysis invisible to the naked eye [5], and serum cholesterol to allow assessment of vitamin E status in relation to a key substrate in gallstone pathology. The assays for homocysteine and cysteine recorded the total of reduced, oxidised and proteinbound forms [7]. 2.4. Statistical methods Independent sample Student’s t-tests were used to compare mean blood concentrations for the patient and control groups. Where variances were found to differ significantly, separate (unpooled) variance ttests were used [15]. For variables found related to age (see below), age-adjusted differences in means were calculated using Analysis of Covariance. g-GT activity levels were positively skewed and logarithmic transformation was used prior to statistical analysis. No satisfactory transformation could be found for plasma haemoglobin, h-carotene and % oxidation in vitamin C and, for these, nonparametric Mann– Whitney U-tests were used. Mann–Whitney U-tests were also used for methionine and cysteine to accommodate single control measurements that were doutliersT but yet were considered valid results: the same applied to folate for which there was an outlier in the patient group. Two-tailed tests of significance were used throughout.

2.3. Chemical methods Full methodological details and performance characteristics have been published [5–8]. Briefly, a kinetic enzymatic method was used to measure erythrocyte and plasma glutathione [5]; HPLC was used to measure ascorbic acid [6], vitamins E and hcarotene [9], homocysteine [7,10] and PLP [11]; gas

3. Results 3.1. Age and gender The group of 54 controls in the Manchester database was younger than the gallstone group of 24

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(median 42, range 20–66 vs. 50, 24–80 years). However, analysis of control data showed that only retinol, vitamin E and vitamin E:cholesterol molar ratio correlated with age. Although these relationships were weak (r=0.336, P=0.021; r=0.374, P=0.010;

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r=0.304, P=0.038, respectively), age-adjusted differences are included in Table 2. Females were underrepresented among controls (female:male ratio, 56%:44% vs. 79%:21% in the gallstone group), but no gender differences could be found on statistical

Table 2 Summary of results N

Control meanFS.D.

N

Patients meanFS.D.

Difference in mean [95% confidence interval]

Main antioxidant nutrients Vitamin E (Amol/l)

47

27.25F6.08

18

25.49F11.15

(Amol:mol cholesterol)

47

5.24F0.98

18

4.41F1.30

1.76 2.56 0.83 0.92

47 47

279, 67–1153c 2.17F0.45

18 18

104, 7–672 1.98F0.69

45

1.14F0.20

24

54 41 39

68.80F26.86 54.21F24.18 16, 0–48c

29 29 16 13

h-carotene (nmol/l) Retinol (Amol/l) Selenium (Amol/l) Vitamin C (Amol/l) Ascorbate (Amol/l) Oxidation in vitamin C (%) Glutathione Whole blood (Amol/l) (Amol:g Hb) Male Female Plasma glutathione (Amol/l) Haemoglobin (mg/dl) gGT (u/l) Miscellaneous Methionine (Amol/l) Cysteine (Amol/l) Homocysteine (Amol/l) Homocysteine:methionine Vitamin B6-PLP (nmol/l) Folate (Ag/l) Vitamin B12 (ng/l)

Significance of difference P

[ 4.01 to 7.53] [ 1.27 to 6.39] [0.14 to 1.53] [0.36 to 1.48 ]

0.533a 0.187b 0.021a 0.002b

1.07F0.29

160 [76 to 268]d 0.19 [ 0.18 to 0.55] 0.22 [ 0.05 to 0.50] 0.07 [ 0.06 to 0.21]

0.001 0.298a 0.112b 0.280a

24 13 13

45.48F29.93 32.04F26.55 39, 0–70c

23.32 [9.72 to 36.92] 22.17 [6.37 to 37.98] 12 [ 32 to 4]d

0.001 0.007 0.150

1221F245 8.63F1.92 8.01F1.75 9.40F1.91

18 18 2 16

1244F270 9.13F2.05 7.40F1.36 9.35F2.05

23 [ 0.50 0.61 [ 0.05 [

0.763 0.401 0.644 0.947

30 30 26

6.08F1.46 1.60, 0–11.20c 14.3, 3–75e

17 17 20

4.62F1.28 1.40, 0–15.90c 41.6, 10–478e

1.47 [0.61 to 2.32] 0.00 [ 1.00 to 1.30]d 17.5 [ 45 to 9]d

14 14

23.42, 17.06–50.70c 280.8, 236–721.5c

12 12

17.61, 12.10–33.68c 250.1, 219.8–297.0c

5.79 [2.34 to 9.97]d 20.6 [0.40 to 43.7]d

14 13 14 14 14

7.20F1.33c 0.29, 0.133–0.450e 40.55F14.57 24.86, 16.67–38.66c 205.1F39.4

14 12 14 14 14

8.53F2.50 0.459, 0.283–1.080e 21.95F9.91 18.07, 13.61–66.50c 192.4F35.0

1.33 [ 2.88 to 0.23] 0.148 [ 0.287 to 0.034]d 18.60 [8.92 to 28.28] 5.97 [1.18 to 10.28]d 12.7 [ 16.0 to 41.6]

c

MeansFS.D. values compared by t-test (unless otherwise specified). a MeansFS.D. values compared by separate (dunpooledT) variance t-test. b MeansFS.D. values adjusted for age as a covariate. c Medians and ranges, with groups compared by Mann–Whitney U-test. d Median of pairwise differences with approximate 95% confidence interval. e Geometric mean and ranges compared by t-test on logged values.

177 to 131] [ 1.69 to 0.69] 2.13 to 3.52] 1.47 to 1.57]

0.001 0.920 b0.001

0.003 0.046 0.092 0.005 b0.001 0.012 0.377

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comparison for the study variables other than whole blood glutathione when expressed per g haemoglobin (control females 9.40F1.91 Amol/g Hb vs. males 8.01F1.75 Amol/g Hb, P=0.050) for which separate between-group comparisons were made for each gender (Table 2). 3.2. Main micronutrient antioxidants Whereas patients and controls had similar serum selenium concentrations, patients had lower levels of vitamin E relative to cholesterol (especially when adjusted for age), h-carotene and vitamin C (Table 2). Their higher levels of ascorbate oxidation-calculated as the difference between vitamin C and ascorbate values expressed as a percentage [8]—did not reach statistical significance, but raised the possibility of greater environmental exposure to oxidising agents. This aspect could not be properly probed because social details were available in relatively few of the controls—whose inclusion relied on their denial of regular alcohol or cigarette usage. Alcohol intakes were broadly similar overall in the group of 24 patients and a subgroup of 27 controls: zero intake in seven controls (26%) and nine patients (38%); 1–19 g per day in 17 (63%) and 8 (33%), respectively; z20 g per day in 3 (11%) and 7 (29%), respectively (Mann Whitney P=0.84). Scrutiny of the available information on cigarette usage in controls revealed that, in fact, one smoked 5 and another 40 cigarettes per day. When data on these two controls were omitted, along with that on eight cigarette users among the 24 with gallstones, the differences between the groups persisted in regard to total vitamin C (69.6F26.4 vs. 52.1F31 .8 Amol/l, P=0.031) and h-carotene (279, 67–1153 vs. 144, 6– 672 nmol/l, P=0.028) but was less marked for ascorbate (54.2F24.2 vs. 37.4F27.1 Amol/l, P=0.10) and vitamin E:cholesterol ratio (5.24F0.98 vs. 4.47F1.45, P=0.10) (statistical method as indicated in Table 2). Levels of vitamin C and vitamin E were highly correlated with each other, but only in the patient group (total vitamin C with vitamin E, r=0.755, Pb0.001 and with vitamin E:cholesterol ratio r=0.698, P=0.001 on 18 pairs; ascorbate, respectively, r=0.791, P=0.002 and r=0.614, P=0.034 on 12 pairs).

3.3. Glutathione The concentration of glutathione in whole blood (essentially erythrocyte GSH) was similar in patients and controls. However, plasma glutathione (GSH plus some GSSG) was lower in patients and this was a genuine result in that plasma haemoglobin values were similar in patients and controls. This change was accompanied by an increase in serum activity of gGT (Table 2). 3.4. Miscellaneous factors Methionine, vitamin B6-PLP and folate values in patients were clearly lower than in controls (Table 2); their lower cysteine concentration just reached the conventional 5% significance level ( P=0.046); vitamin B12 and homocysteine values were not significantly different, but the homocysteine:methionine ratio was higher in the patients ( P=0.005) (Table 2).

4. Discussion There are three, largely novel, findings from our biochemical study in patients with cholesterol gallstones. First, micronutrient antioxidant deficiency is a feature. Second, this deficiency is accompanied by a fall in plasma glutathione but preservation of levels in the erythrocyte. Third, poor status of sulphur amino acids, folate and vitamin B6-PLP is revealed. Among the many micronutrients that are available in amounts large enough to fulfill an antioxidant function [16], the best known are vitamin E (primarily a-tocopherol), the carotenes (among which h-carotene is the most abundant), vitamin C (in its biologically active form of ascorbate) and selenium. Of these, the first two operate in the lipid phase within cells and extracellularly, and the latter two protect in the aqueous phase alongside the sulphur amino acids and their end-product GSH (Fig. 1). The various elements of the system interact in complex ways. Thus, ascorbate can compensate for low GSH status of tissues through redox and nonredox exchanges [17] and, so far as plasma is concerned, in vitro studies show that lipid peroxidation is postponed until all ascorbate is consumed, whereupon sulphydryls are

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rapidly degraded [18]. Furthermore, physiological levels of ascorbate have been shown to be as potent as is the drug probucol in protecting low density lipoprotein against oxidative modification and only the vitamin spared a-tocopherol and h-carotene from oxidation within this lipid fraction [18]. As to selenium, its antioxidant potential is generally equated with its presence at the active centre of the enzyme GSH-peroxidase (Fig. 1), but it also facilitates the metabolism of xenobiotics (e.g., occupational chemicals in Table 1) via cytochromes P450, monoxygenases that may inadvertently generate toxic metabolites and which are also involved in the metabolism of cholesterol and bilirubin [1]. With regard to the plasma/serum profiles of micronutrient antioxidants in patients with cholesterol gallstones, we are unaware of any previous report on atocopherol (vitamin E) and h-carotene (low) or selenium (normal). Our finding of low plasma ascorbate confirms and extends studies from the United Sates by Simon and Hudes [19,20] whose hypothesis of a pathogenetic association between ascorbate lack and cholesterol gallstones [21] was unfortunately not revealed by the literature search when we wrote our own [1] but which we cited in our subsequent dietary investigation [3]. The first US study identified an inverted U-shaped relationship between serum ascorbic acid and clinical gall bladder disease among women but not men [19]. The second showed each 27 Amol/l (standard deviation) increase in serum ascorbate to be independently associated with a 13% lower prevalence of both clinical disease and asymptomatic gallstones [20]. Total vitamin C—which includes two oxidised products of ascorbate—was not recorded in these studies. We cannot say for certain that low antioxidant intake by patients with cholesterol gallstones is the main explanation for our findings because we did not undertake detailed dietary studies in the laboratory controls nor did we measure blood urea as a rough and ready gauge of their protein intake. Nevertheless, our previous dietary investigation [3]—which fulfilled the prerequisite that controls and patients should be matched for age and gender—strongly supports this interpretation. When the new biochemical information in Table 2 is examined alongside data from that dietary study [3], it becomes clear that whereas the outcome in relation to vitamin E status relative to lipid

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is similar ( P=0.021 vs. P=0.032), the biochemical study shows vitamin C, h-carotene and vitamin B6PLP levels to be disproportionately lower (plasma/ serum P=0.001 vs. diet P=0.100 for the first two, Pb0.001 vs. PN0.100 for the third). Since these three vitamins are highly susceptible to oxidative attack, the implication seems to be that heightened oxidation contributes to the subnormal blood levels in patients with gallstones. There was no suggestion from our previous dietary work that in vitro losses during food preparation might have been responsible. Hypothetically [1], oxidising agents in cigarette smoke, occupational chemicals or alcohol (Table 1)—individually and collectively—could be responsible. The identification of these requires painstaking studies, as we found in our investigation of chronic pancreatitis, a calcifying process in an adjacent organ [22–24]. The corollary of heightened in vivo oxidation, oxidative stress, was supported by glutathione measurements—with the liver implicated as the site of the problem. Thus, it is known that surplus GSH generated in the liver is the source of the tripeptide in plasma and that, when hepatic oxidative strain compromises the delivery of GSH into plasma, constituent amino acids from the existing plasma GSH pool are made reavailable to hepatocytes and erythrocytes via increased activity of plasma g-GT (without increase in bilirubin or alkaline phosphatase) [4]. Against this background, the low total plasma glutathione but excellent level in erythrocytes—which facilitate the interorgan transfer of GSH—seems to indicate that hepatic GSH metabolism is under particular strain in patients with cholesterol gallstones. The Pretorian limb of our study focused on homocysteine and related metabolic pathways (Fig. 1). Basically, homocysteine may undergo remethylation, or it may be driven by transsulphuration reactions towards GSH synthesis. The former requires vitamin B12 and methyltetrahydrofolate, as coenzyme and cosubstrate, respectively [25]; the overall NADPH-linked reduction of CH2–H4 folate to CH3– H4 folate is irreversible and commits folate-bound one-carbon units to the remethylation of homocysteine [26]. The transsulphuration of homocysteine involves two coenzymes that participate in B6dependent reactions. Thus, hyperhomocysteinemia develops easily whenever folate, vitamin B6 or vitamin B12 are deficient [27]. In our patients with

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cholesterol gallstones, levels of both folate and vitamin B6-PLP were subnormal, while the homocysteine:methionine ratio was higher than in controls indicating lower remethylation. Yet, homocysteine levels were not elevated (Table 2). This paradox is rationalised by the finding of subnormal plasma concentrations of both methionine and cysteine which, in turn, point to poor intakes. Of interest, subnormal intakes of methionine and cysteine were identified in our earlier dietary study [3] and are conducive to experimental gallstones (citations in Refs. [1–3]). In conclusion, our study brings antioxidant deficiency—and, by implication, free radical pathology— into the field of cholesterol gallstones, as has been recognised for some time in relation to black pigment stones (citations in Ref. [1]) and underlined by a recent report [28]. However, it does not help to determine whether antioxidant lack facilitates a particular stage in gallstone formation—where possibilities include the inception stage in the hepatocyte [1,21], destabilisation of lipids in secreted bile [29], or cholesterol crystallisation in the gall bladder [30]—or by a combination of adverse effects. It would also be premature to conclude that antioxidant deficiency underlies the full spectrum of gallstones, from pure cholesterol through to pure pigment, although other strands of evidence raise this intriguing possibility [1].

Acknowledgements We thank the North West Regional Health Authority for a grant to H V Worthington to facilitate the study, which was approved by the Ethical Committee. We appreciate the expertise of chemists in the laboratories at Manchester and Pretoria, and extraordinary secretarial assistance by Jenny Parr.

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