Liver International 2005: 25: 518526 Printed in Denmark.

All rights reserved

Copyright r Blackwell Munksgaard 2005

Clinical Studies

DOI: 10.1111/j.1478-3231.2005.01049.x

Antioxidant levels in peripheral blood, disease activity and brotic stage in chronic hepatitis C
                                          
Bandara P, George J, McCaughan G, Naidoo D, Lux O, Salonikas C, Kench J, Byth K, Farrell GC. Antioxidant levels in peripheral blood, disease activity and brotic stage in chronic hepatitis C. Liver International 2005: 25: 518526. r Blackwell Munksgaard 2005 Abstract: Background: This study addressed the suggested association between levels of the antioxidants glutathione (GSH), vitamin C and vitamin E in peripheral blood and the histological activity and brosis stage in chronic hepatitis C (CHC). We then determined whether regular antioxidant supplementation inuenced these antioxidant levels or disease severity. Methods: Clinical, biochemical, histological and demographic data were collected from 247 CHC patients at the time of liver biopsy. Whole blood total GSH, plasma vitamin C and E were assessed by high-performance liquid chromatography. Statistical analyses were performed to test for associations between the variables and to identify independent predictors for hepatic necroinammatory and brosis scores. Results: GSH and vitamin C, but not vitamin E correlated with both portal/periportal activity (r 5 0.19, P 5 0.004; r 5 0.19, P 5 0.009 respectively) and brosis stage (r 5 0.18, P 5 0.007; r 5 0.18, P 5 0.009 respectively). GSH was an independent negative predictor of portal/periportal inammation (P 5 0.02) and brosis (P 5 0.01). Vitamin C was an independent negative predictor of brosis stage (P 5 0.02). Antioxidant intake was associated with higher vitamin C (Po0.0001) and vitamin E (P 5 0.005) levels, but not GSH. Conclusions: Whole blood GSH and plasma vitamin C are negatively associated with hepatic portal/periportal inammation and brosis stage in CHC. Controlled intervention studies with vitamin C and agents that boost endogenous GSH levels are warranted.

Priyanka Bandara1, Jacob George1, Geoffrey McCaughan2, Daya Naidoo3, Ora Lux3, Chris Salonikas3, James Kench4, Karen Byth5 and Geoffrey C Farrell1
1 Storr Liver Unit, Westmead Millennium Institute, 2The Department of Medicine, A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, University of Sydney, 3Department of Clinical Chemistry, Prince of Wales Hospital, 4Department of Anatomical Pathology, 5Westmead Millennium Institute, Sydney, Australia

Key words: glutathione hepatitis C liver oxidative stress vitamin C vitamin E Geoffrey C Farrell, Storr Liver Unit, Westmead Hospital, Westmead, NSW 2145, Australia. Tel: 61-2-98459006 Fax: 61-2-9849 9100 e-mail: geoff_farrell@wmi.usyd.edu.au Received 28 May 2004, accepted 12 August 2004

Disease progression in chronic hepatitis C (CHC) depends on a range of host factors, the duration of infection, and exogenous inuences such as alcohol (15). Oxidative stress, an imbalance between pro-(mostly reactive oxygen species (ROS)) and antioxidants in favour of pro-oxidants is commonly associated with inammatory disorders (68), including acute and chronic liver diseases (915). A role for oxidative stress in the pathobiology of CHC has been suggested (16 21), but is not well characterised. Infection with the hepatitis C virus (HCV) results in a multispecic humoral and cellular immune response in the liver. Activated macrophages can generate ROS as can hepatocyte injury and cell death. Elevated ROS levels beyond that which can be neutralised by antioxidants can propagate cellular damage by oxidative modication of cellular components. ROS, as well as 518

oxidised cellular constituents such as lipid peroxides, are able to affect cellular signal transduction pathways leading to the activation of hepatic stellate cells (15, 22, 23), promoting hepatic brosis. Direct measurement of ROS to quantify oxidative stress is difcult because of their inherent high reactivity. Therefore, studies of oxidative stress rely on indirect measurements including changes in antioxidant levels and quantication of stable by-products of oxidative modication to cellular constituents. While previously published studies provide insights into associations between oxidative stress and the histological severity of CHC, most are limited by small patient cohorts, suboptimal matching and lack of multivariate data analysis. This study was designed to measure the blood levels of three key antioxidants (glutathione (GSH), vitamin C and vitamin E), in a large prospectively enrolled patient cohort in

Antioxidants in chronic hepatitis C order to investigate their associations to histologic markers of disease severity.
Patients and methods

Patient inclusion and data collection

Two hundred and forty-seven consecutive patients recruited into a longitudinal CHC natural history study at Westmead and Royal Prince Alfred hospitals were examined. The protocol was approved by the ethics committees of the Western and Central Sydney Area Health Services and the University of Sydney. All patients gave informed written consent. Participants were seropositive for anti-HCV antibodies (Monolisa anti-HCV; Sano Diagnostics Pasteur, Marnesla-Coquette, France) and HCV RNA (Amplicor HCV; Roche Diagnostics, Branchburg, NJ). Exclusion criteria were: evidence of hepatic malignancy or hepatic decompensation, co-infection with hepatitis B virus (HBV) or human immunodeciency virus (HIV), autoimmune hepatitis, Wilsons disease, haemochromatosis, a1-antitrypsin deciency or cholestatic liver disease. Demographic variables including age, gender and ethnicity were recorded. Patient height and weight were measured for determination of the body mass index (BMI) as weight (kg)/(height (m))2. Participants were interrogated in a structured interview for assessment of lifestyle and dietary factors. Average alcohol consumption (estimated and recorded in grams/day) was categorised as low (o10 g/day), medium (1040 g/ day) and high (440 g/day) and as current (intake in the preceding 6 months) or past. Those on regular (at least 3 days per week) supplements containing antioxidants (e.g. vitamin C, A and E, CoQ10 and herbals such as silymarin and CH100) or agents that boost antioxidative reactions (e.g. selenium, zinc) were labelled as antioxidants1 and others as antioxidants .
Clinical and laboratory tests

Liver tests (ALT, AST, GGT, bilirubin, albumin), ferritin and INR were performed using standard automated techniques in the clinical pathology laboratory. HCV genotyping was performed using a second-generation reverse hybridisation line probe assay (Inno-Lipa HCV II; Innogenetics, Zwijndrecht, Belgium). Viral load was measured by Roche Amplicor HCV Monitor version 2.0 kit (Roche Diagnostics, Raritan, NJ) and was categorised as low (o800,000 IU/ml) or high (4800,000 IU/ml). To estimate whole blood GSH, 800 ml of 5% metaphosphoric acid was added to 200 ml of freshly collected blood and incubated on ice for 10 min before centrifugation (2 min at 3300 g). The acidied supernatant was frozen at 70 1C. Total glutathione (GSH12 oxidised glutathione (GSSG)) was assayed by reverse phase high-performance liquid chromatography (HPLC) with uorometric detection (24). Vitamin E and C were measured in heparinised plasma, after collection of blood samples with light protection. Assays were performed on frozen ( 70 1C) plasma within 1 week, using HPLC with electrochemical detection for vitamin C (25) and UV detection for vitamin E (26). Liver biopsies were assessed blind by an experienced hepatopathologist (J. K.). Portal/periportal and lobular inammatory grade and brosis stage were graded from 0 to 4 according to Scheuer (27), and for purposes of data analysis were further categorised as mild (grade 1, 0 or stage 1, 0), moderate (grade 2 or stage 2) and severe (grade 3, 4 or stage 3, 4).
Statistical analysis of data

At the time of liver biopsy, venous blood was drawn following a 12-h overnight fast. The following parameters were assessed: whole blood total GSH, plasma vitamin C and vitamin E, vitamin E/total cholesterol, total cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), g-glutamyl transpeptidase (GGT), bilirubin, albumin, platelet count, international normalised ratio (INR), ferritin, HCV genotype and viral load. Previous biopsy data was used for 14 patients (within 3 months of study entry for four, 36 months for three, 612 months for four, 1224 months for three).

The statistical software package SPSS (Version 11) was used for data analysis. Two-sided tests with a signicance level of 5% were employed throughout. Spearmans rank correlation was used to quantify the pairwise associations between continuous or ordered categorical variables. MannWhitney tests and KruskalWallis nonparametric analysis of variance were used to test for associations between nominal categorical variables and continuous outcome variables. Multiple ordinal regression analysis with forward stepwise variable selection was used to identify the independent predictors of portal/periportal and lobular inammation and of brosis stage.
Results

Characteristics of the study cohort

The characteristics of the cohort are listed in Table 1. The mean age was 41 years (range 16 519

Bandara et al.
Table 1. Demographic, virologic and histopathologic characteristics in 247 patients with chronic hepatitis C
Variable Age Gender Male Female Country of birth Australia Outside Australia Mode of transmission Injecting drug use Blood transfusion Tattoo/body piercing Other/unknown Portal Inammation (n 5 242)n Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 On regular antioxidants (antiox1) Mean age Males No regular antioxidants (antiox ) Mean age Males Smokers BMI (kg/m2) Previous treatment status Treatment nave Unsuccessful treatment Ethnicity White Caucasian Other Caucasian East Asian (Chinese origin) Other HCV genotype 1 2 3 4 Other or untypable Fibrosis stage Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Past alcohol intake Low (o10 g/day) Medium (1040 g/day) High (440 g/day)
n

Mean SD [Range] or frequency (%) 41 10 [1667] 164 (66%) 83 (34%) 159 (64%) 88 (36%) 148 31 16 52 (60%) (13%) (6%) (21%)

3 (1%) 56 (23%) 135 (56%) 45 (19%) 3 (1%) 87 (35%) 43 9.3 50 (57%) 160 (65%) 41 9.6 114 (71%) 113 (46%) 26.3 4.9 [17.247.3] 217 (88%) 30 (12%) 180 46 15 6 123 13 90 15 6 46 83 65 30 23 (73%) (19%) (6%) (2%) (50%) (5%) (36%) (6%) (2%) (19%) (34%) (26%) (12%) (9%)

had never received interferon-based antiviral treatment, but 31 (12%) had had unsuccessful previous treatment. Current alcohol intake was low in 201 (81%), moderate in 33 (13%) and high in 13 (5%). Past alcohol consumption was evenly divided between the low, moderate and high groups (30%, 34%, 36% respectively). Smokers comprised nearly half of the study cohort (46%). Eighty-seven patients (35%) were taking at least one oral supplement with antioxidant properties on a regular basis (antioxidants1). Multivitamins and mineral preparations were the most commonly ingested form of antioxidants (64, 26%). Apart from multivitamins containing vitamin C and E, 11 (5%) and 21 (9%) were taking additional vitamin E and vitamin C respectively. Of herbal antioxidants, milk thistle (Silybum marianum) containing the active compound Silymarin, was consumed by 25 patients (10%). Most patients had mild to moderate CHC; 194/ 242 (80%) showed grade 02 portal/periportal inammation, 194/247 (79%) had brosis stages 02, 30 (12%) had stage 3 brosis while 23 (9%) had established cirrhosis (stage 4). Between the HCV genotype 1 (n 5 123) and genotype 3 (n 5 90) groups, disease severity (mean brosis score 1.6 for both), mean age (42 and 38 years, respectively) and the proportion of males (65% and 72%, respectively) were similar.
Univariate associations of antioxidant levels in peripheral blood

74 (30%) 83 (34%) 90 (36%)

Portal/periportal inammatory grade was not available for ve patients. BMI, body mass index; HCV, hepatitis C virus.

67 years); 164 (66%) were male. The majority (180, 73%) were White Caucasians, and 159 (64%) were born in Australia. HCV genotypes 1 and 3 were most common, collectively representing 86% of the group. The most commonly identied route of HCV acquisition was injecting drug use (148, 60%), but no risk factor was identied in 52 (21%). Most patients (217, 88%) 520

Associations of GSH with host and viral factors, and with other antioxidants The median GSH in the study cohort, 1.67 mmol/ l (interquartile range: 1.421.92) RBC was below the reference range of the laboratory (1.88 2.58 mmol/l RBC). Univariate analysis (Table 2) indicated that GSH levels were higher in females (1.81 vs. 1.63 mmol/l RBC, P 5 0.002) and were negatively correlated with age (r 5 0.21, P 5 0.002) and BMI (r 5 0.23, P 5 0.001). Regular uncontrolled antioxidant intake, alcohol intake (current or past) or smoking did not signicantly inuence GSH levels. Whole blood GSH was not signicantly associated with either plasma vitamin C or E levels. GSH was not associated with serum ALT, but showed a negative correlation with hepatic portal/periportal activity (r 5 0.19, P 5 0.004) and brosis stage (r 5 0.18, P 5 0.007) (Table 2). The associations with hepatic portal/periportal inammation and brosis stage were further analysed, as these are key determinants of disease severity in CHC. Fig. 1 illustrates GSH levels

Antioxidants in chronic hepatitis C

Table 2. Associations of whole blood glutathione, plasma vitamin C and vitamin E with clinical parameters
Factor Median [IR]n

rw

P
0.002 0.08 0.3 0.002 0.1 0.001 0.004 0.2 0.007 0.5 0.4 0.04 0.2 o0.001 0.4 0.8 0.5 0.009 0.6 0.009 0.005 0.1 0.005 0.09 0.1 0.5 0.8 0.9 0.2

Glutathione (mmol/l RBC) Gender (Male, Female) Viral load (low, high) Antioxidant intake ( , 1) Age ALT BMI Portal/periportal activity grade Lobular activity grade Fibrosis stage Vitamin C Vitamin E Vitamin C (mmol/l) Gender (male, female) Viral load (low, high) Antioxidant intake ( , 1) Age ALT BMI Portal/periportal activity grade Lobular activity grade Fibrosis stage Vitamin E Vitamin E (mmol/l) Gender (male, female) Viral load (low, high) Antioxidant intake ( , 1) Age BMI ALT Portal/periporal Activity grade Lobular activity grade Fibrosis stage
n

1.63 [1.381.87], 1.81 [1.572.05] 1.70 [1.432.04], 1.63 [1.391.82] 1.64 [1.441.92], 1.67 [1.391.97] 0.21 0.11 0.23 0.19 0.09 0.18 0.04 0.05 31.5 [1548], 43.5 [1960] 39.8 [1655], 33.8 [1348] 24.0 [1244], 48.0 [3163] 0.06 0.02 0.05 0.19 0.04 0.18 0.19 20.5 [1626], 22.0 [1827] 22.1 [1726], 22.0 [1627] 20.0 [1625], 23.8 [1729] 0.12 0.11 0.04 0.02 0.01 0.09

Interquartile range, wSpearmans rank correlation. ALT, alanine aminotransferase; BMI, body mass index.

across increasing portal/periportal inammatory and brosis stage categories. GSH levels appeared to decrease with increasing portal/periportal activity, the change being statistically signicant only in males (Fig. 1A). Similarly, among men with severe hepatic brosis, GSH levels were signicantly lower (Fig. 1B). There was no difference in whole blood GSH between the HCV genotype groups. The high viral load group had a trend towards low GSH compared with the low group (P 5 0.08). Associations of plasma vitamin C levels Median plasma vitamin C in patients with CHC 36 mmol/l (interquartile range: 1650) was below the laboratory reference range (40100 mmol/l). As summarised in Table 2, higher vitamin C levels were detected in females than males (44 vs. 32 mmol/l, P 5 0.04). There was no association of vitamin C with age or BMI. Higher vitamin C levels were found in those taking oral antioxidant preparations (48 vs. 24 mmol/l, Po0.0001), but there was no association with alcohol intake or smoking. Vitamin C correlated with vitamin E (r 5 0.19, P 5 0.005). Vitamin C was not signi-

cantly associated with serum ALT, but correlated negatively with hepatic portal/periportal inammatory grade (r 5 0.19, P 5 0.009) and brosis stage (r 5 0.18, P 5 0.009). Because gender and antioxidant intake appear to inuence the level of vitamin C, Fig. 2 illustrates vitamin C levels partitioned according to these variables. Apparently decreasing vitamin C levels were observed with increasing portal/periportal activity (Fig. 2A) in both men and women who did not admit to oral antioxidant intake, with signicant differences between the mild and severe groups (P 5 0.02 and 0.05 for males and females respectively). However, in those who did take antioxidant supplements (antioxidants1), vitamin C levels remained high, and unaffected by the severity of portal/periportal inammation. With regard to brosis stage, vitamin C levels tended to be lower with increasing brosis in antioxidants patients (P 5 0.06) (Fig. 2B), but antioxidants intake increased plasma vitamin C levels irrespective of the brosis stage. Plasma vitamin C levels were similar between the HCV genotype groups. Further, HCV viral load did not inuence the levels of vitamin C. 521

Bandara et al.
A
Glutathione (mmol/LRBC)
2.2 2 P=0.07 1.8 1.6 1.4 1.2 1 Female n = 19 44 18 Male 30 91 P=0.03 P=0.01 Fibrosis Mild Moderate Severe Portal Inflammation Mild Moderate Severe P=0.009

A
Vitamin C (mol/L)

90 80 70 60 50 40 30 20 10 0

P=0.05 P=0.02 Portal Inflammation Mild Moderate Severe

40

Female, Female, AO+ AO-

Male, AO+

Male, AO-

Glutathione (mmol/LRBC)

B
Vitamin C (mol/L)

2.2 2 1.8 1.6 1.4 1.2 1 Female n = 46 19 18

80 70 60 50 40 30 20 10 0 Female, Female, AO+ AOMale, AO+ Male, AO-

Fibrosis Mild Moderate Severe

83

Male 46

35

Fig. 1. Whole blood glutathione (GSH) against severity of portal/periportal inammation and brosis. GSH levels across the mild, moderate and severe portal/periportal inammation (A) and brosis (B) categories, separated based on gender. Error bars represent 95% condence intervals.

Fig. 2. Plasma vitamin C levels by portal/periportal inammation and brosis. Vitamin C levels across the mild, moderate and severe portal/periportal inammation (A) and brosis (B) categories separated based on oral antioxidant intake and gender. Patients on regular antioxidants are denoted AO1 and patients not taking antioxidants AO . Error bars represent 95% condence intervals.

Associations of plasma vitamin E levels Contrary to the two water-soluble antioxidants examined, median plasma vitamin E, 21 mmol/l (interquartile range: 1726) in the study cohort was well within the reference range (830 mmol/l). Patients taking antioxidant supplements showed higher plasma vitamin E levels than those not taking them (24 vs. 20 mmol/l, P 5 0.005). There was no signicant association between vitamin E and alcohol intake or smoking. Vitamin E levels in absolute terms (Table 2) or normalised to total plasma cholesterol (data not shown) were not signicantly associated with serum ALT, hepatic portal/periportal/lobular inammatory grade or brosis stage. Vitamin E levels were similar between the high and low viral load groups. Those infected with HCV genotype 3 had lower vitamin E levels compared with those with genotype 1 CHC (19 vs. 24 mmol/l, P 5 0.0002). Because vitamin E is associated with plasma lipoproteins (28), we investigated plasma cholesterol levels, which were found to be lower in patients infected with HCV genotype 3 compared with those with genotype 1 (4.7 vs. 3.9 mmol/l, Po0.001).
Antioxidant intake and CHC activity and severity

(antioxidants1) and the 160 patients not ingesting such supplements (antioxidants ) with respect to serum ALT (116 vs. 130 U/l), or the histologic grade (portal activity grade 1.93 vs. 1.97) or stage of CHC (1.53 vs. 1.65).
Multivariate analysis of predictors for hepatic inammation and brosis scores

There was no signicant difference between the 87 patients with uncontrolled antioxidant intake 522

In order to determine the independent predictors of hepatic inammation and brosis scores, we performed multiple ordinal regression analysis with the following input variables: ALT, AST, GGT, GSH, vitamin E, cholesterol, vitamin E/ cholesterol, ferritin, platelets, INR, age, BMI, past alcohol intake and smoking status. Vitamin C was not included as this was only available in 202 of the 247 subjects. The ordinal regression model for independent predictors of portal/periportal inammation (Table 3) included age, ALT, total cholesterol (negative association), GSH (negative association) and smoking status. These variables accounted for 25% of the variability in the grade of portal/periportal inammation. The ordinal regression model for lobular inammation included only ALT as an independent predictor, and accounted for just 8% of its variability. The ordinal regression model for independent predictive factors of hepatic brosis

Antioxidants in chronic hepatitis C

Table 3. Multiple ordinal regression analysis of factors associated with hepatic inammation and brosis stage in patients with CHC
Factor Portal/periportal activity Age (per year) ALT (per 10 U/l) Cholesterol GSH Non smoking status Lobular activity ALT (per 10 U/l) Fibrosis Age (per year) ALT (per 10 U/l) Cholesterol GSH Past alcohol Platelets (per 10 109 /l) Odds ratio 95% CI

Table 4. Multiple ordinal regression analysis for the subset of 202 patients with data on levels of glutathione, vitamin E and vitamin C
Factor Portal/periportal activity Age (per year) ALT (per 10 U/l) Cholesterol Non-smoking status Fibrosis Age (per year) ALT (per 10 U/l) Cholesterol GSH Vitamin C (per 10 lmol/l) Past alcohol Platelets (per 10 109 /l) Odds ratio 95% CI

P value
o0.001 0.002 0.001 0.02 0.015 o0.001 o0.001 0.008 o0.001 0.01 0.001 0.004

P value
o0.001 0.005 0.003 0.04 o0.001 0.006 o0.001 0.03 0.02 0.02 0.002

1.08 1.04 0.62 0.43 0.50 1.06 1.10 1.04 0.41 0.42 1.69 0.94

1.051.12 1.021.07 0.470.83 0.210.90 0.290.87 1.031.1 1.071.14 1.011.06 0.310.55 0.210.83 1.232.34 0.900.98

1.10 1.04 0.61 0.52 1.10 1.04 0.44 0.43 0.86 1.53 0.93

1.051.12 1.011.07 0.450.85 0.290.97 1.061.14 1.011.07 0.320.61 0.200.92 0.760.97 0.932.2 0.890.97

ALT, alanine aminotransferase; GSH, glutathione.

CHC,chronic hepatitis C; ALT, alanine aminotransferase; GSH, glutathione.

included age, ALT, total cholesterol (negative association), GSH (negative association), past alcohol intake and platelet levels (negative association). Together these variables accounted for 45% of the variability in brosis stage. A separate analysis was performed in the subgroup of 202 patients with available vitamin C data (Table 4). The ordinal regression model for portal/periportal activity included age, ALT, cholesterol (negative association) and smoking status, which accounted for 23% of the variability in portal/periportal activity grade. GSH and vitamin C levels failed to achieve statistical signicance after adjusting for these independent predictors (P 5 0.09 and 0.07 respectively). The ordinal regression model for brosis stage included age, ALT, cholesterol (negative association), past alcohol intake, platelets (negative association) as well as vitamin C and GSH (both negatively associated). These accounted for 43% of the variability in brosis score.
Discussion

Clinical experience and published data indicate that 3050% of persons with chronic liver diseases consume complementary and alternative medicines for their putative disease modifying antioxidant and antibrotic properties (29, 30). Despite evidence implicating oxidative stress in the pathology of CHC, data on the efcacy of antioxidants to modify CHC disease progression are lacking. In searching the literature, we found six studies (n 5 318 in total), one study (n 5 42) and two studies (n 5 42 and 20) respectively, that have examined, blood levels of GSH, vitamin C and vitamin E in CHC. All were in small cohorts

(only one included4100 subjects), and none included multivariate data analysis to determine if antioxidant levels were independently associated with differences in liver pathology. The purpose of the present study was to establish, whether associations exist between blood levels of antioxidants and the severity of histopathological lesions in a large, prospectively enrolled cohort of CHC patients. It has previously been shown that CHC is associated with reductions in hepatic, plasma, lymphocyte, erythrocyte (16, 31, 32) and whole blood total GSH (18). In addition to conrming these data, we demonstrate that whole blood total GSH level negatively correlate with hepatic portal/periportal inammatory grade and brotic stage, and in multivariate analysis, is an independent predictor of the same. Vitamin C is the most oxidatively sensitive plasma antioxidant, functioning at the rst line of antioxidant defence (33). Low plasma vitamin C levels have been demonstrated in 42 CHC patients (18), and in a mixed group of patients with chronic hepatitis B and C (33), compared with healthy controls. Our study extends these ndings by demonstrating that plasma vitamin C correlates negatively with portal/periportal inammation grade and with brosis stage, and is an independent predictor of brosis stage in multivariate analysis. In contrast to the signicant associations observed for these two water-soluble antioxidants, we found no association between the levels of the lipid-soluble antioxidant vitamin E and the severity of CHC.
Whole blood GSH and CHC disease severity

Previous studies indicate that plasma GSH correlates best with hepatic GSH (r 5 0.895) (16), as it is almost exclusively of hepatocyte origin. 523

Bandara et al. However, in attempting to obtain a surrogate blood marker of hepatic oxidative stress, we chose to measure whole blood GSH, which is 499% of erythrocyte origin (34), for several cogent reasons. Firstly, over a time course, erythrocyte GSH parallels plasma GSH levels (35). Secondly, the 200-fold higher concentration of GSH in whole blood (34) is practical for sample preparation and assay in a clinical setting. This is crucial as it is well documented that plasma GSH measurements are highly susceptible to postblood collection artifacts from autooxidation and thioldisulphide exchange (36, 37). Thus, rapid disappearance of plasma GSH is evident, even when processed within 3 min after blood collection. GSH levels fall to 85% of total (from normal physiological levels of 9799%) with a concomitant rise in GSSG to about 15% (37). We did not separately estimate GSSG, because erythrocyte GSSG is minimally affected by cirrhosis because of chronic hepatitis C and B, and accounts for only 3% of total GSH (38). Jain et al. (18) have demonstrated no signicant difference in GSSG between noncirrhotic and cirrhotic CHC patients. Erythrocyte GSH synthesis depends on an adequate supply of the precursor L-cysteine (Cys) derived principally from hepatic GSH efux into plasma (36, 39). In turn, hepatic GSH synthesis depends on Cys derived from the transsulphuration pathway and exogenous (dietary) Cys (40, 41). The depletion of whole blood GSH that we observed with increasing portal/ periportal inammation and brosis suggests that hepatic GSH efux is reduced with advancing disease. This is likely because of decreased hepatic GSH synthesis as well as increased GSH consumption in the diseased liver. Reduced levels of hepatic GSH have been reported in patients with CHC (31). It has been reported that approximately half of hepatic GSH is synthesised incorporating Cys derived from the transsulphuration pathway (42), which has been demonstrated to be dysfunctional in patients with cirrhosis (43, 44). Thus, our data suggest that blood GSH depletion is reective of altered levels of hepatic GSH.
Plasma vitamin C and CHC disease severity

with a retardation of hepatic brosis progression in persons with CHC is unknown, but warrants further study. The reason for the observed higher levels of GSH and vitamin C in women than men is unclear; more detailed nutritional studies would be required to exclude dietary factors. It is known that males with CHC as well as other liver diseases appear to have an increased likelihood for advanced hepatic brosis (3, 4). Whether this could partly be explained by the observed differences in antioxidant defences is unknown.
Plasma vitamin E in CHC

Unlike the observations made for the two hydrophilic antioxidants, we found no association in this large cohort between plasma vitamin E and disease severity. Although a negative correlation with brosis has been reported in 33 CHC patients (18), and decreased levels compared with healthy controls were found in two separate mixed cohorts with chronic hepatitis B and C (45, 46), these studies lacked multivariate data analysis. In line with our observations, Yamamoto et al. (33) reported plasma vitamin E to be similar between a group of 28 chronic hepatitis B and C patients (with and without cirrhosis) and healthy individuals. They further reported that plasma vitamin E is resistant to early stage oxidative stress contrary to vitamin C.
Associations between blood antioxidants and HCV genotype

One explanation for the observed negative association between vitamin C and disease severity is that hepatic oxidative stress may increase consumption of this key antioxidant. In this regard, as vitamin C is entirely derived from the diet, the higher levels observed in those taking regular oral antioxidant preparations is not surprising. Whether intervention with controlled high dose vitamin C supplementation will be associated 524

We did not dene any association between the HCV genotype and whole blood total GSH or plasma vitamin C levels. In contrast, Barbaro et al. (16) reported a signicant depletion of plasma and lymphocyte GSH in patients infected with HCV genotype 1 compared with genotype 3. Although decreased GSH levels were clearly demonstrated, in the absence of multivariate analysis, we wonder if the reported genotype effect was inuenced by the presence of more males, increased age and more extensive brosis in their genotype 1 compared with their genotype 3 cohort. These confounders were similar between our genotype 1 and 3 groups. In contrast to GSH and vitamin C, we observed lower plasma vitamin E levels in association with HCV genotype 3 compared with genotype 1 infection. This may to be a consequence of the reduced plasma lipids in patients with genotype 3, observed by us and others (47). The mechanisms for HCVs genotype-specic perturbation of host lipid metabolism are not understood.

Antioxidants in chronic hepatitis C

Uncontrolled antioxidant intake and CHC pathogenesis
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This cross-sectional study also assessed uncontrolled antioxidant intake in patients with CHC at the time of liver biopsy. GSH levels were not inuenced by uncontrolled antioxidant ingestion (principally agents not known to have an effect on GSH) in this study, but reportedly can be boosted by supplementation with GSH (48), Nacetylcysteine (49), S-adenosyl-methionine (50) and Silymarin (51). In contrast, vitamin C levels were boosted by antioxidant intake, an effect undoubtedly because of the vitamin C contained in the supplements taken by 95% of patients in the antioxidants1group. The higher levels of plasma vitamin E in the antioxidants1group likewise may be explained by oral vitamin E intake and increased recycling of its oxidised radical form in the presence adequate vitamin C intake. Future studies should corroborate these ndings using known amounts of antioxidant supplements. This was not possible and is a limitation of the present study. In conclusion, data from this large prospective study and other reports indicate that oxidative stress is associated with CHC. The independent associations of brosis stage with endogenous levels of GSH and vitamin C, raise the possibility that these associations could have a pathogenic signicance. However, the associations in the present data are not strong enough to warrant the measurement of GSH and vitamin C as routine markers of the extent of liver disease in CHC. On the other hand, our data are sufciently encouraging to warrant consideration of randomised controlled trials with vitamin C and agents that can boost GSH in patients with CHC using histologic end points. Based on this study, there is less evidence to support the use of vitamin E to alter histologic end points in persons with CHC. Finally, our data do not support a role for uncontrolled antioxidant supplementation in clinical practice for persons with CHC.
Acknowledgements
This study was funded by the National Institutes of Health, USA grant DK56402. We thank Ms. Keshni Sharma, Ms. Barbara Charlton, Ms. Jasmin Canete, Ms. Seng Kee Teo, Mr. David Yau and Ms. Evelyn Crewe for their help with data collection and/or laboratory work. Dr. Calvin Lang (University of Louisville) and Dr. Jinah Choi (University of Southern California) are acknowledged for their technical advice and useful discussions respectively.

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