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- MATERIALS AND METHODS
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.
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- MATERIALS AND METHODS
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.