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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Background:

Enhanced production of reactive oxygen species may play a pathogenic role in alcoholic liver injury.

Aims:

To investigate whether various antioxidant parameters in blood are affected in different stages of alcoholic liver disease and how specific the changes are relative to non-alcoholic cirrhosis.

Methods:

Patients with alcohol abuse without cirrhosis (n=14), with alcoholic cirrhosis [Child–Pugh scores A (n=9), B (n=5) and C (n=18)] and with non-alcoholic cirrhosis [Child–Pugh score C (n=6)] and healthy controls (n=13) were studied. Levels of reduced glutathione and glutathione peroxidase activity in blood, erythrocytic superoxide dismutase activity and carotenoids, α-tocopherol and malondialdehyde in plasma were measured.

Results:

Levels of reduced glutathione were significantly decreased in Child–Pugh score C cirrhotics, alcoholic or not in origin, whereas oxidized glutathione and glutathione peroxidase activity were not affected. Superoxide dismutase activity and α-tocopherol levels were not significantly different in the various groups. Carotenoid levels were significantly lower in alcoholic cirrhotics (Child–Pugh score C) vs. controls. Malondialdehyde levels were elevated only in cirrhotics Child–Pugh score C, alcoholic or non-alcoholic.

Conclusions:

Levels of reduced glutathione and malondialdehyde reflect the degree of liver impairment, more than the relation with alcohol intake. Decreases in several antioxidant levels are not specific to alcoholic liver injury.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

The production of reactive oxygen species is considered to be a major pathogenetic mechanism in alcoholic liver injury.1, 2 Most reactive oxygen species are formed in hepatocytes, in activated Kupffer cells and in inflammatory cells.2, 3 The increased amount of reactive oxygen species provokes damage to cellular lipids, proteins and DNA by peroxidation or oxidation;1–3 as a consequence of lipid peroxidation, malondialdehyde is formed.4, 5 Glutathione in its reduced form (GSH) represents an important substance for the protection of cells against oxidative injury.6–9 GSH depletion in alcoholic liver disease can be caused by acetaldehyde, a toxic degradation product of ethanol,2, 6, 7 but reduced GSH synthesis has also been documented in cirrhotic livers of non-alcoholic origin.6, 7, 10 In addition to GSH, antioxidant defence enzymes, such as glutathione peroxidase and superoxide dismutase, and antioxidant nutrients, such as α-tocopherol and carotenoids, are well-known antioxidants.1, 8, 11, 12 Reduced levels of carotenoids and α-tocopherol in cirrhotic livers have been proposed to result from both liver impairment and reduced dietary intake and absorption.5, 13

Antioxidant levels in plasma or red blood cells are attractive and easily accessible parameters to obtain information with regard to oxidative liver damage.1, 5, 8, 13 Complex defence systems cope with reactive oxygen species throughout the human body, and reactive oxygen species may understandably not be easily detected in peripheral blood, but the consumption of defence systems might be so. Therefore, we investigated blood/plasma levels of several antioxidants implicated in oxidative liver damage in order to select the most reliable ones for further clinical use, and to test whether some were more specifically disturbed in alcoholic liver injury.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Study population

All patients were examined between September 1998 and February 2001. They gave informed consent for blood sampling for the present study. This coincided with blood sampling for other diagnostic tests.

Alcoholic patients.

Blood samples were taken from patients with alcoholic hepatitis without cirrhosis (n=14) and from individuals with alcoholic cirrhosis (n=32). All had a history of continuous and excessive alcohol consumption for at least 4 years and were still actively drinking until 1 week or less before examination. The diagnosis of alcoholic hepatitis without cirrhosis was made by the appropriate clinical and biochemical tests to exclude other causes of liver disease (viral, autoimmune or metabolic) and by abdominal ultrasonography. In three of the 14 patients, a liver biopsy confirmed the diagnosis of alcoholic liver injury without cirrhosis. The patient characteristics are given in Table 1. The diagnosis of alcoholic cirrhosis was made by liver biopsy (23/32 patients) or by a combination of clinical presentation, ultrasound and/or computerized tomography scan and the presence of complications such as ascites and/or oesophageal varices (9/32 patients). The patient characteristics are given in Table 1. This group was subdivided according to the Child–Pugh score into class A (n=9), B (n=5) and C (n=18).

Table 1.  . Patient characteristics Thumbnail image of
Non-alcoholic patients.

A group of six patients with cirrhosis (Child–Pugh score C), not taking alcohol, was studied for comparison. The causes of their disease were hepatitis C (n=2), autoimmune hepatitis (n=1), Wilson's disease (n=1) and cryptogenic disease (n=2). Liver biopsies performed in all six patients confirmed these diagnoses. The patient characteristics are given in Table 1.

Controls.

A group of healthy controls (n=21), 10 male and 11 female, with no history of alcohol abuse or signs of liver disease, was also studied. Their age was 57 ± 4 years (mean ± S.E.M.).

Blood sampling

After an overnight fast, venous blood samples were taken. A dry tube for serum and a citrate-containing tube and an ethylenediaminetetra-acetate (EDTA)-containing tube for plasma were taken for clinical chemistry analyses. Blood from another EDTA-containing tube was used for all other tests. For glutathione determinations in particular, 300 μL of blood from the EDTA-containing tube was placed within 2 min into 1.2 mL of distilled water containing 5% metaphosphoric acid and 1 mM EDTA. Tubes were kept on ice for immediate processing.

Levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and protein-bound glutathione in blood

The blood specimen treated with metaphosphoric acid was centrifuged at 4 °C for 7 min at 6500 g, and the supernatant and precipitate were stored at − 80 °C until analysis. In the supernatant, the total glutathione content, considered to be the sum of GSH and GSSG, and GSSG were determined according to a recycling method, as described previously.14 In the pellet, protein-bound glutathione was determined by the same recycling method.14 Concentrations of total glutathione and protein-bound glutathione are expressed in μM. GSSG is expressed in μM and as the percentage of total glutathione: 2 × [GSSG]/[total glutathione]. GSH concentrations were calculated as [total glutathione] − 2 × [GSSG].

Glutathione peroxidase activity in blood

Twenty microlitres of blood was diluted with 400 μL diluting agent, provided in the Ransel kit (Randox Laboratories, Crumlin, UK), and stored at − 80 °C until analysis. Glutathione peroxidase activity was measured by the Ransel kit, based on the glutathione peroxidase-catalysed, cumene hydroperoxide-induced oxidation of added glutathione. The ensuing oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+ was photometrically measured at 340 nm.

Superoxide dismutase activity in erythrocytes

Erythrocytes from 500 μL of blood were washed three times in saline and haemolysed with distilled water (total volume, 2 mL). Forty microlitres of haemolysate was added to 960 μL KH2PO4 10 mM, pH 7.0, and stored at − 80 °C until analysis. Superoxide dismutase activity was measured by a Ransod kit (Randox Laboratories, Crumlin, UK) according to the manufacturer's instructions. This method is based on the photometric measurement of a red formazan dye. This dye is formed by the action of superoxide released from xanthine under the influence of added xanthine oxidase. The superoxide dismutase activity present in the haemolysate inhibits this dye formation because it catabolizes superoxide.

Levels of α-tocopherol, carotenoids and retinol in plasma

Plasma was stored at − 80 °C and used later for the determination of α-tocopherol, retinol and five carotenoids: α- and β-carotene, zeaxanthin/lutein, lycopene and cryptoxanthin. These five carotenoids are the main carotenoids in the plasma of subjects with a Western diet,5 and were measured by reversed-phase high-performance liquid chromatography (HPLC) as described previously.15 We used an HPLC apparatus type Waters 996 photodiode array detector with Millennium software (Waters Corporation, Milford, MA, USA). As internal standards, we used echinenone15 and, in addition, retinol acetate.

Levels of malondialdehyde in plasma

Plasma was stored at − 80 °C until analysis. The determination of thiobarbituric acid-reacting substances and the subsequent separation of malondialdehyde by reversed-phase HPLC (Waters 996 HPLC photodiode array detector with Millennium software; Milford, MA, USA) were carried out according to Knight et al.,4 modified by Jentzsch et al.16 As internal standard, we used 1,1,3,3-tetraethoxypropane.

Clinical chemistry

Serum values of liver enzymes, albumin, cholesterol, triglycerides and bilirubin, in addition to the prothrombin time and haemoglobin levels, were measured by automated standardized procedures (Roche Hitachi 917/747, Mannheim, Germany) (Table 1).

Statistical analysis

Data are given as the mean ± S.E.M. or as the median [range] if they were not normally distributed. Differences between groups were analysed, respectively, with analysis of variance (ANOVA) or ANOVA-on-ranks. Multiple comparison testing was subsequently performed to find differences from the healthy control group. Significance levels were always taken at 0.05. Statistical analysis was carried out by SigmaSTAT 2.0 (Jandel Corporation, San Rafael, CA, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Blood levels of glutathione and glutathione peroxidase

Blood levels of GSH were significantly reduced in patients with cirrhosis (Child–Pugh score C), either alcoholic or non-alcoholic in origin (both P < 0.05 compared with healthy controls) (Figure 1 and Table 2). Blood levels of GSSG were not significantly different in the various groups, expressed in μM or as a percentage ratio (Table 2). Levels of protein-bound glutathione and glutathione peroxidase activity were not different in the various groups (Table 2).

Table 2.  . Concentrations of reduced, oxidized and protein-bound glutathione in blood and activities of erythrocytic superoxide dismutase and blood glutathione peroxidase Thumbnail image of
image

Figure 1. . Blood concentrations of reduced glutathione in the various groups. Individual data and the mean of each group are given. *P < 0.05 compared with the healthy control group.

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Erythrocyte superoxide dismutase activity

Erythrocyte superoxide dismutase activity was not significantly different in the various patient groups compared with the healthy controls (Table 2).

Plasma levels of malondialdehyde

Plasma malondialdehyde levels were significantly enhanced only in the cirrhotic Child–Pugh C groups, either alcoholic or non-alcoholic in origin (P < 0.05 vs. healthy controls) (Figure 2).

image

Figure 2. . Plasma concentrations of malondialdehyde as a reflection of lipid peroxidation in the various groups. Individual data and the median of each group are given. *P < 0.05 compared with the healthy control group.

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Plasma levels of α-tocopherol

Plasma levels of α-tocopherol were not different in the various groups (Table 3).

Table 3.  . Plasma concentrations of carotenoids, retinol and α-tocopherol in healthy controls, patients with alcoholic hepatitis without cirrhosis, alcoholic cirrhosis and non-alcoholic cirrhosis (Child–Pugh score C) Thumbnail image of

Plasma levels of β-carotene, other carotenoids and retinol

Plasma concentrations of β-carotene were significantly lower in patients with alcoholic hepatitis and alcoholic or non-alcoholic cirrhosis (Child–Pugh score C), compared with healthy controls (P < 0.05; Table 3). Concentrations of other carotenoids, determined in the same HPLC assay, are given in Table 3. Total carotenoid concentrations were significantly decreased in the alcoholic Child–Pugh C cirrhotic group (P=0.01; Table 3), but not in the non-alcoholic Child–Pugh C cirrhotic group (P=0.08), vs. healthy controls. Concentrations of retinol were significantly lower in both cirrhotic Child–Pugh C groups vs. healthy controls (P < 0.05; Table 3).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

GSH plays a crucial role in detoxification.7 Hepatic levels of GSH have been reported to be low in various liver diseases, including alcoholic cirrhosis.6, 10 Both reduced synthesis of cysteine, the central amino acid of GSH, and enhanced GSH consumption have been described in cirrhotic livers.2, 7, 10 Besides the liver, GSH is also present in erythrocytes.17 GSH, exported from hepatocytes to sinusoidal blood, is rapidly broken down to dipeptides and amino acids by γ-glutamyltransferase and dipeptidases.17, 18 After the uptake of precursor amino acids, erythrocytes can resynthesize GSH.19 Low GSH levels, as observed in the present study (Table 2), may therefore reflect both hepatic and erythrocytic GSH metabolism.19, 20

The exact relation between hepatic and erythrocytic GSH content needs to be further clarified. We found that blood levels of GSH were significantly lower only in advanced cirrhosis: Child–Pugh score C (Table 2). This implies that the synthesis and consumption of GSH are adequate in erythrocytes of alcoholic patients with a moderate degree of oxidative stress. Advanced liver impairment, however, is characterized by an imbalance between the synthesis and consumption of GSH. This observation of reduced blood levels of GSH is in agreement with the observation that the administration of S-adenosylmethionine, a precursor in GSH synthesis, to severe alcoholic cirrhotics can improve the blood content of glutathione, but not to normal levels.20 Our observations suggest that decreased GSH levels are not due to an enhanced conversion to GSSG, but rather due to a production failure in the liver, because GSSG, as well as the enzyme glutathione peroxidase involved in the oxidation of GSH, are unaffected (Table 2). The glutathione peroxidase defence mechanism, needed to remove hydrogen peroxide, does not seem to fail in the different stages of alcoholic liver injury (Table 2), which has also been reported by others.8

Data on superoxide dismutase activity, an antioxidant defence enzyme that reduces superoxide to hydrogen peroxide,21 in the blood of alcoholics are contradictory: increased8, 22, 23 and decreased23, 24 activity have been reported. In the present study, differences between the superoxide dismutase activities of alcoholic patients, who continuously consumed excess alcohol, patients with end-stage non-alcoholic liver disease and healthy controls were small (Table 2) and probably not important.

We observed low levels of carotenoids and vitamin A in advanced cirrhotics only, and this reached significance in the alcoholic Child–Pugh C group alone (Table 3). It is not clear whether reduced levels of carotenoids and vitamin A are due to the severity of the hepatic process or to malnutrition or malabsorption, often present in patients with cirrhosis.5, 13, 25, 26 Lower plasma levels were also observed in patients with chronic cholestasis.27 The latter study demonstrated the role of bile in the absorption of these liposoluble nutrients in the gut.27 A reduction in plasma carotenoid levels is therefore not specific to alcoholic cirrhosis, because plasma and liver concentrations are involved in complex interactions.13, 28 The measurement of low plasma carotenoid levels in cirrhotic patients, as in the present study, cannot allow a distinction to be made between a disease-related low nutritional intake or absorption and a disease-related impaired metabolism.29, 30 Therefore, these tests are not attractive as parameters of oxidative stress for further studies.

α-Tocopherol is mainly stored in adipose tissue, in addition to smaller amounts in liver, muscle and other tissues.13, 31 Plasma tocopherol was found to correlate with the content of adipose tissue in healthy subjects.32 We observed normal plasma levels in our alcoholic patient groups (Table 3), which is in agreement with previous studies.13, 33 Nevertheless, the hepatic content of α-tocopherol was reported to be low in alcoholic cirrhosis.13 In addition to changes in adipose tissue, the variation in plasma α-tocopherol levels, if any, can be partly explained by altered lipoprotein levels,34 also in alcoholics.5, 8 Therefore, this parameter is not suitable for use as a measure of oxidative stress in alcoholic liver damage.

Malondialdehyde levels were reported to be elevated in the plasma of alcoholics8 and in hepatitis C patients with elevated transaminases.35 In the present study, we confirmed that elevated malondialdehyde levels were not specific to alcoholic liver injury (Figure 2). Our observation that plasma malondialdehyde levels were only significantly elevated in Child–Pugh C cirrhotics, alcoholic or non-alcoholic (Figure 2), might suggest that they are not only due to enhanced hepatic lipid peroxidation, but possibly also to impaired removal in the case of severe cirrhosis.

In conclusion, GSH and malondialdehyde levels reflected the severity of cirrhosis, rather than the relation with alcohol. Production failure of GSH, rather than a deficit of glutathione peroxidase activity in red blood cells, seemed to be responsible for this decrease in blood GSH levels; increased malondialdehyde levels might also be due to impaired removal in end-stage liver disease. Decreases in blood levels of several antioxidants were not specific to alcoholic liver injury. Protective mechanisms against oxidative stress due to alcohol or other insults may be measured in blood, but their relationships to hepatic levels often remain difficult or doubtful.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

The authors thank Professor T. Roskams for histochemical work on all liver biopsies and Professor J. van Pelt for helpful suggestions. This study was supported by a grant from the Foundation for Scientific Research, FWO-Vlaanderen (Belgium), n° b.0111.98, to FN.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References
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