Is glutathione reductase useful in hepatitis B virus-infected patients?

 Hepatitis B virus (HBV) infection There are about 257 million chronic HBV-infected people in the world[1] . In China, there are about 70 million cases of chronic HBV infection, of which about 20-30 million cases of Chronic Hepatitis B (CHB)[2] .

 

Current studies have shown that the imbalance between oxidative and antioxidative damage in CHB patients plays an important role in the progression of viral hepatitis to hepatocellular carcinoma [3]. Glutathione reductase (GR) is one of the most important enzymes in the redox system of the human body, and it is the main flavoprotein enzyme that maintains the level of reduced glutathione in cells. GR converts oxidized glutathione to reduced glutathione with the participation of NADPH. Reduced glutathione plays an important role in preventing the oxidative decomposition of hemoglobin, maintaining the activity of mercapto proteins, and ensuring the reducibility of mercapto proteins and cellular integrity [4].

 

In recent years, the significance of GR in patients with CHB has been gradually emphasized, and it has been regarded as one of the important complementary indicators for liver function tests. However, the trend of GR in viral hepatitis or in animal models of liver injury is controversial [5-8]. In this study, we investigated the differences in the distribution of serum GR activity between apparently healthy populations and HBV-infected diseases, as well as the correlation and differences with traditional liver function indices, in order to provide a basis for the clinical application of GR in the assessment of HBV-infected liver injury.

 

1 Information and methodology

1.1 General information Retrospective analysis From August 2019 to December 2019, 139 patients with liver diseases (112 patients with CHB, 12 patients with cirrhosis, and 15 patients with hepatocellular carcinoma), including 95 males and 44 females, with an age range of 19-76 years old, were seen in the Gastroenterology and Infection Departments of the Second People's Hospital of Jianli City, Hubei Province, because of HBV infection. In addition, 56 cases of apparent health checkups were selected from our medical checkup center, of which 30 cases were male and 26 cases were female, with an age range of 20-60 years old.

 

Inclusion criteria for healthy people: (1) age ≥ 18 years; (2) all HBV serological markers negative or only HBsAb positive, hepatitis C virus serum antibody negative; (3) traditional liver function tests within the normal range: aspartate aminotransferase (AST) ≤ 40 U/L for men and ≤ 32 U/L for women; alanine transaminase (ALT) ≤ 41 U/L for men and ≤ 33 U/L for women; total bilirubin (TBIL) ≤ 26 mmol/L for men and ≤ 21 mmol/L for women; direct bilirubin (DBIL) ≤ 8 mmol/L; and direct bilirubin (DBIL) ≤ 8 mmol/L for women. Total bilirubin (TBIL) ≤26 mmol/L for men and ≤21 mmol/L for women; direct bilirubin (DBIL) ≤8 mmol/L; alkaline phosphatase (ALP) 10-71 U/L for men and 6-42 U/L for women; and gamma-glutamyltranspeptidase (γ-GT) 10-71 U/L for men and 6-42 U/L for women. Exclusion criteria for healthy people: people with major organic lesions, history of tumors, history of liver transplantation, oral drug users, patients with HBV-infected liver diseases Inclusion criteria: (1) Age ≥ 18 years; (2) HBsAg-positive surface antigen of hepatitis B virus, negative serum antibody to hepatitis C virus. （Exclusion criteria for HBV-infected liver disease were: liver failure, combination of diseases other than liver, and history of liver transplantation.

 

1.2 Instruments and reagents   

GR kit (Jiangxi Lecheng Biological Co., Ltd., UV enzyme assay), reference range 33-73 U/L, detected by Beckman AU5800 automatic biochemical analyzer. AST, ALT, TBIL, DBIL, ALP, γ-GT were detected by Beckman original kit. Hepatitis B virus 5 and anti-hepatitis C virus antibody were detected by Abbott Automatic Immunoassay Analyzer Architect i200sr and its original reagents, and HBV-DNA was detected by AGS8830 real-time fluorescence quantitative PCR instrument produced by Hangzhou AnYu Technology Co.

 

1.3 Methodology   

Three tubes of 4-6 mL of venous blood from each enrollee were centrifuged at 1912×g for 10 min to separate the serum. If it was too late for testing within 3 hours, the serum was frozen at -80 ℃ until testing. General clinical data were collected.

 

1.4 Statistical processing  

Statistics were performed using SPSS 16.0 software, and data were tested for normality using the Kol-mogorov-Smirnov test. Skewed measures were expressed as median (interquartile spacing), and comparisons between the two groups were made using the Mann-Whitney U test. Correlations were analyzed by Spearman's rank correlation, and a difference of P<0.05 was considered statistically significant.

 

2 Results

2.1 Comparison of liver function test results between hepatitis B virus-infected liver disease groups and the control group The liver function test indexes and age of the analyzed population were tested for normality, and only the age showed a normal distribution (age, P=0.163), and all the liver function indexes (GR, ALT, AST, ALP, GGT, TBil, DBIL) showed a skewed distribution, and all the data were expressed as the median (interquartile spacing), and the analysis method was non-parametric test. Therefore, all data were expressed as median (interquartile spacing) and analyzed by nonparametric test.

 

For the healthy subjects and the disease groups in this experiment, there was a significant difference in age composition (P<0.05). For traditional biochemical indicators, AST, ALT and γ -GT increased with the progression of liver disease after hepatitis B virus infection, and were significantly higher in the hepatocellular carcinoma group than in the cirrhosis group, and in the cirrhosis group than in the chronic hepatitis B group; they were significantly higher in the chronic hepatitis B group than in healthy subjects; TBIL, DBIL and ALP were significantly higher in the disease group than in healthy controls, and were significantly higher in the cirrhosis group than in the chronic hepatitis B group; but there was no significant difference between the chronic hepatitis B and cirrhosis groups; GR was significantly different in the chronic hepatitis group than in the healthy subjects. TBIL, DBIL and ALP were significantly higher in the disease group than in healthy controls, cirrhosis group and hepatocellular carcinoma group respectively, but there was no significant difference between the cirrhosis and hepatocellular carcinoma groups; GR was significantly higher in the chronic hepatitis group than in healthy controls at 59.4 (54.7-67.3) U/L (P < 0.001), and in the cirrhosis group at 73.1 (57.3-96.7) U/L (P < 0.001), while in the chronic hepatitis group, it was higher than that in the chronic hepatitis group (P < 0.001). In the cirrhosis group, 73.1 (57.3-96.7) U/L was significantly higher than that in the chronic hepatitis group (P<0.001), but in the hepatocellular carcinoma group, the concentration of GR was significantly lower than that in the other three groups (P<0.001), 26.5 (14.2-47.9) U/L (P<0.001). See Table 1.

 

2.2 Comparison of GR in various subgroups of chronic viral hepatitis B

Due to the small number of samples in the cirrhosis and hepatocellular carcinoma groups and the fact that the pattern of change of GR is different from other indexes, only the CHB group was further analyzed. The CHB patients were divided into different subgroups according to different criteria.

In patients with CHB, GR was significantly higher than in healthy controls when all other liver function tests were normal, and GR exceeded the upper limit of the reference interval in 6 cases, with a positivity rate of 8.2% (6/73). However, there was no significant change in GR in the group with any positive liver function tests compared to the group with normal liver function tests.

When patients with CHB were grouped according to whether they were HBeAg positive or negative, all indicators except TBIL were significantly higher in these two subgroups than in healthy controls, respectively. However, only ALP was higher in the HBeAg-positive CHB subgroup than in the HBeAg-negative subgroup.

The patients with CHB were categorized according to HBV-DNA positive or negative, and all the indexes were significantly higher in the DNA-positive group than in the healthy control group, while in the DNA-negative group, only GR, AST, and ALP were significantly higher than in the healthy control group, and GR was significantly higher in the DNA-positive group than in the DNA-negative group. See Table 2.

 

2.3 Correlation between GR and other indicators in chronic viral hepatitis B

Spearman's correlation analysis showed that in patients with CHB, GR was significantly and positively correlated with other indices of conventional liver function (ALT, AST, TBIL, ALP, GGT) and DNA content (after conversion from DBIL), with the exception of DBIL.  In patients with CHB, except for DBIL, GR showed a significant positive correlation with other indicators of conventional liver function (ALT, AST, TBIL, ALP, GGT) and DNA content (after lg conversion). See Table 3

 

3 Discussion

Hepatocyte mitochondria, hepatic blast cells and inflammatory cells all produce reactive oxygen species (ROS) and free radicals, and the liver also exerts antioxidant effects through protein synthesis. In the pathogenesis of hepatitis B, there is a broad spectrum of oxidative stress that occurs at all levels, including lipids, DNA and proteins[3] . Glutathione peroxidase (GPX) and GR are important components of the glutathione (GSH) antioxidant system, which can scavenge ROS and repair oxidative damage[4] . In this study, GR was elevated in viral hepatitis B before conventional liver enzymes and bilirubin, and was further elevated in patients with cirrhosis of viral hepatitis B and decreased in hepatocellular carcinoma of hepatitis B virus infection. In the available literature, the pattern of changes in GR in hepatitis B virus-infected liver injury varies. Li Shuli et al. showed that GR levels were elevated in patients with chronic hepatitis B virus[7] , and Fan Yuchen et al. showed that GR was decreased in patients with CHB[8] .

 

In patients with CHB, the results of this study were consistent with those of Li Shuli et al [7]. Through the statistics of traditional liver function tests, it can be concluded that the patients with CHB enrolled in this study were in the state of mild liver insufficiency, and the liver function indicators such as aminotransferases were much lower than those in the study by Fan Yuchen et al[8] . A possible mechanism is that the liver maintains the balance of ROS and antioxidants in the body under normal conditions. When HBV virus infects the human body, oxidative stress is triggered by proteins such as HBx protein, HBsAg and HBcAg [9-11], and the body recycles oxidized glutathione by increasing GR levels to improve the harmful environment in the body [12]. Therefore, the elevation of GR precedes the conventional changes in liver enzyme profile and bilirubin. However, when the liver is subjected to successive oxidative damage, ROS damage is further increased, leading to inflammation and fibrosis, regenerative nodules and loss of liver function[13- 14] . Moreover, when liver injury is further increased, too much ROS are produced and the compensatory increase in GR is less than the amount consumed, leading to a decrease in GR. In patients with hepatocellular carcinoma, the GSH microcirculatory system may be disrupted in cancer cells, resulting in a compensatory increase in GR. However, the number of hepatocellular carcinoma patients in this study was small, and further expansion of the sample size is needed to verify this conclusion.

In this study, GR was not associated with the negativity or positivity of HBeAg, but with the negativity or positivity of HBV-DNA. Further analysis showed a positive correlation between GR and HBV-DNA (r=0.396, P<0.001), suggesting that the oxidative damage increased further with the increase of viral load[15] . This finding is consistent with the study of Tianlu Zhou et al [16].The relationship between GR and HBV-DNA suggests that GR may be used as one of the indicators for antiviral monitoring of hepatitis B virus.

 

There are some limitations to this study: (1) The statistical results of this study showed a positive correlation between GR and age. The control group in this study was not matched by age and gender. (2) The number of cases of cirrhosis and hepatocellular carcinoma in this study is relatively small, which needs to be verified by enlarging the sample size.

 

4 Conclusion

GR is elevated in patients with hepatitis B virus infection in the early stages of liver injury and can be used as an important supplement to traditional liver function tests and as a monitor of the effectiveness of antiviral therapy for hepatitis B virus.

 

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