Glutathione, glutathione S-transferase, and transmembrane transport of glutathione conjugate in human neutrophil leukocytes

Glutathione (GSH) conjugation is important in the detoxification of carcinogens and other exogenous electrophilic drugs and chemicals. The conjugation is catalyzed by GSH S-transferase. Neutrophil GSH content was 12.3 +/- 2.5 (mean +/- SD) nmol/10(7) cells, and transferase activity of cytosol preparations was 0.187 +/- 0.035 nmol/min/mg protein with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Maximal activity was found at a pH of 7.0 and a temperature of 40 degrees C. Apparent Km of transferase was 1.25 +/- 0.18 mmol/L CDNB, and apparent Vmax was 0.621 +/- 0.22 nmol/min/mg. GSH-CDNB conjugate was quantitated by HPLC in cells and in medium after CDNB exposure. Transport of conjugate from cells to medium increased with CDNB concentration to 50 mumol/L, and kinetic data showed two saturable transport mechanisms with apparent Km of 5.90 mumol/L and 0.265 mumol/L, respectively. Cellular GSH content fell rapidly with CDNB concentration greater than 2.5 mumol/L, and was depleted in a 10-minute incubation at a concentration of greater than 50 mumol/L CDNB. Neutrophils have a significant content of GSH and a significant amount of transferase activity, and transport of GSH conjugate involves two distinct saturable pathways. GSH depletion can be accomplished with a relatively low concentration of an exogenous chemical and could impair the ability of the cells to carry out their phagocytic functions.     

谷胱甘肽巯基转移酶基因多态性在原发性肝细胞癌人群中的研究

目的探讨原发性肝细胞癌人群中谷胱甘肽巯基转移酶基因多态性与肝癌发生、发展的关系。方法我院2008年10月-2010年4月收治的原发性肝癌患者与健康体检者各150名,提取血样,利用多重PCR和限制性片段长度多态性检测法分别检测肝癌人群与体检健康者的谷胱甘肽巯基转移酶基因多态性,并进行对比分析。结果该肝癌人群中:谷胱甘肽巯基转移酶基因多态性型别在病例和对照组研究对象中出现的频率无显著性差别,P>0.05;GSTM1-null、GSTT1-null、GST01-Ala140Asp、GSTP1-Ile105Val的多态性情况与该地区已有的报道一致。结论谷胱甘肽巯基转移酶基因多态性与肝癌的发生、发展无明确的相关关系。     

GSTM1基因多态性与子宫内膜异位症易感性的相关性研究

【目的】探讨谷胱甘肽S转移酶M1（GSTMI）基因多态性与子宫内膜异位症（EM）易感性的关系。【方法】应用内参照PCR技术检测96例Eros患者和88例对照妇女的GsTM1基因型。【结果】病例组GSTMI（-）频率为65．6％（63／96）,对照组为45．5％（40／88）,两组之间有非常显著性差异（P〈0．01）,其OR值为2．29（95％CI为1．27-4．15）,具有GsTM1（-）的个体患Eros的危险性增加1．29倍;病例组中Ⅰ-Ⅱ期与Ⅲ-Ⅳ期患者的GSTM1（-）频率无显著性差异（P〉0．05）,Ⅰ-Ⅱ期与对照组也无显著性差异（P〉0．05）,但Ⅲ-Ⅳ期患者与对照组有显著性差异（P〈0．01）。【结论】GSTM1基因多态性与Eros易感性有关,GSTM1（-）可能参与了Eros的致病过程。     

Glutathione S-transferase M1 gene polymorphism is related to COPD in patients with non-small-cell lung cancer

OBJECTIVE: Oxidative stress contributes to the development of both lung cancer and chronic obstructive pulmonary disease (COPD). Antioxidative enzymes may protect against such damage. We hypothesized that genetic variations in glutathione S-transferase M1 and/or T1 genes (GSTM1 and GSTT1, respectively) may influence susceptibility to COPD in patients with non-small-cell lung cancer. PATIENTS AND METHODS: The polymorphisms in GSTM1 and GSTT1 genes were examined in 110 patients (age: 63+/-1 years) with newly diagnosed non-small-cell lung cancer using the polymerase chain reaction. Respiratory function was assessed by bodyplethysmography. RESULTS: In the GSTM1 null (-/-) genotype, both FEV1 and FEV1/FVC were significantly lower than in the GSTM1 plus genotype (GSTM1 -/+ or +/+) (75.8+/-2.5 versus 86.6+/-3.6%, p<0.02; 69.1+/-1.6 versus 77.0+/-2.4, p<0.01, respectively). Among the patients with GSTM1 null genotype, 49% suffered from COPD as opposed to 21% of patients with GSTM1 plus genotype. In contrast, no differences were seen in FEV1 or FEV1/FVC when comparing patients with GSTT1 null genotype and GSTT1 plus genotype (81.4+/-4.9 versus 79.3+/-2.3, p=NS; 71.1+/-2.9 versus 72.2+/-1.6, p=NS). Multiple stepwise regression analysis identified the GSTM1 genotype (p<0.02) as a significant independent predictor of FEV1 in this group of patients. CONCLUSION: The present study suggests that in patients with non-small-cell lung cancer the presence of at least one active allele in GSTM1 has a protective effect against the development of COPD.     

Glutathione synthetase deficient human fibroblasts in culture

Cultured skin fibroblasts from patients with 5-oxoprolinuria caused by hereditary deficiency of glutathione synthetase have decreased levels of the corresponding enzyme as well as of glutathione. Fibroblasts from the same patients accumulated gamma-glutamyl cysteine, but the levels were lower than those of glutathione in control fibroblasts. The uptake of [35S]cystine was equally rapid in control and patient fibroblasts. In the acid-soluble fraction gamma-glutamyl-[35S]cysteine accumulated in fibroblasts from patients but not from controls. Appreciable turnover of gamma-glutamyl cysteine and glutathione in the respective cell strains was observed, the half-lives of these pools being approximately 5 hours. The growth rate of mutant fibroblasts in culture was significantly slower than that of control fibroblasts. There was no significant accumulation of 5-oxoproline in the culture medium.     

Simultaneous determination of conjugated bile acids in human bile

We describe an optimized liquid chromatographic method for simultaneous analysis of 10 conjugated bile acids in gall bladder and ductal bile. A quick and effective one-step purification with Sep-pak C-18 was adopted. We used a reverse phase C18 stainless steel column and an isocratic mobile phase in a flow programme and monitored the column effluent at 205 nm. The within-day CV ranged from 0.3 to 1.8%, and the between-day CV from 1.2 to 7.2%. Absolute analytical recovery ranged from 89 to 107%. Linearity ranged from 0.3 to 3 mg/mL for most bile acids. The chromatographic analysis was completed in 18 min.     

Inorganic phosphorus in human bile.

The inorganic phosphorus concentration has been measured in fresh bile from patients undergoing cholecystectomy, usually because of cholelithiasis. The amount in common duct bile for patients with cholesterol on the stone surface was significantly higher than that for patients with some calcium carbonate in this area. For all patients with functioning gallbladders, inorganic phosphorus in gallbladder bile was always higher than that in the corresponding common duct bile. In bile from functioning gallbladders, inorganic phosphorus was linearly related to pH for cholesterol stone-formers, but values for calcium carbonate stone-formers were below the regression line and not related to pH. The results show that bile from calcium carbonate stone-formers is more dilute with respect to inorganic phosphorus.     

Selenium contents in human gallbladder bile

The selenium contents in human gallbladder bile were analyzed. Thirty-seven subjects were studied; 22 patients with cholelithiasis in Niigata Prefecture and 15 patients (13 with cholelithiasis and 2 with gallbladder polypus) in Kochi Prefecture. Five ml of bile was withdrawn with a syringe from the gallbladder during the operation and stored at -20 degrees C until analysis. For the analysis by gas chromatograph with an electron-capture detector, 0.2 ml of sample was used. The mean selenium contents in bile were 269 +/- 39.0 (mean +/- S.D.) ng/ml for the subjects in Niigata and 285 +/- 84.4 ng/ml in Kochi; without significant difference. Of 37 samples analyzed, the mean content was 276 +/- 61.0 ng/ml.     

Calcium in human gallbladder bile.

Biliary calcium is believed to be of great importance in gallstone pathogenesis. These studies were therefore performed to determine if quantitative and/or qualitative differences in calcium are present in gallbladder bile from patients with and without gallstones. Bile was obtained by direct gallbladder aspiration from 68 obese patients undergoing elective gastric bypass surgery. Forty-five patients had no evidence of gallstones or sludge, 18 had cholesterol gallstones, and five had black pigment stones. Gallbladder bile was also obtained from 27 nonobese patients undergoing elective cholecystectomy (19 cholesterol; eight black pigment gallstones). For all patients, total calcium ranged from 1.50 to 16.44 mmol/L (mean: 6.05 +/- 0.31 mmol/L); free Ca++ ion ranged from 0.53 to 2.83 mmol/L (mean: 1.28 +/- 0.05 mmol/L). Considerable overlap was observed between obese and nonobese subjects and between patients with and without gallstones. For all patient groups, calcium, Ca++, and bound calcium increased linearly with increasing concentrations of bile salt. No significant differences in the slopes of these relationships were observed with obesity or gallstones. In contrast, free Ca++ ion was greater in gallbladder bile from gallstone patient groups throughout the entire range of bile salt. We hypothesize that this observed increase in Ca++ resulted from increased Gibbs-Donnan forces and excess gallbladder mucin present within the gallbladder bile of patients with gallstones.     

Glutathione S-transferase levels in autoimmune chronic active hepatitis: a more sensitive index of hepatocellular damage than aspartate transaminase

Glutathione S-transferase (GST; EC 2.5.1.18), a sensitive marker of hepatocellular damage, was measured in patients on therapy for histologically proven, autoimmune chronic active hepatitis at various stages of the disease. GST levels were elevated in 65% of serum samples despite immuno-suppressive treatment compared with aspartate transaminase (AST) which was increased in only 23% of samples. In 55% of samples with normal AST concentrations, GST was elevated. No samples demonstrated abnormal transaminase with normal GST levels. It is concluded that continuing hepatocellular damage occurs in patients with autoimmune chronic active hepatitis on immuno-suppressive treatment.     

Glutathione status in liver and plasma during development of biliary cirrhosis after bile duct ligation

We do not know much about the changes that occur in reduced (GSH) and oxidized (GSSG) glutathione in the development of liver cirrhosis. Therefore, we investigated the glutathione redox system during development of liver cirrhosis after bile-duct ligation in rats. We compared the GSH and GSSG content of liver and plasma between bile-duct-ligated rats and sham-operated controls 6 and 24 h and 5, 15, 23, and 38 days after operation. Compared to controls (x±SD: 6.07±0.52 μmol/g wet wt.), liver GSH significantly increased 24 h (+37%) and 5 days (+53%) after bile-duct ligation. Thereafter, GSH continuously declined to 4.25±0.64 μmol/g (?31%; P<0.001) at the end of the observation period after 38 days. The GSH turnover in 5-day bile-duct-ligated rats with high GSH concentrations was not significantly different than in shamoperated controls (16 nmol/min per g after bile-duct ligation and 15 nmol/min per g in controls). GSSG (211±42 nmol/g wet wt. in controls) was significantly lower 6 and 24 h after bile-duct ligation (?34% and ?43%, respectively). Thereafter, GSSG increased and was about 100% higher than in controls after 23 and 38 days. The relation of GSSG to GSH in liver continuously increased from 3.4 to 20.5% after bile-duct ligation. The course of plasma GSH (9.57±0.79 μmol/l) paralleled hepatic GSH on a lower level: +14% at day 5, ?41% at day 15 and ?51% at the end of the observation period. Plasma GSSG (0.99±0.31 μmol/l) was inversely related to liver GSSG: there were increased concentrations early after bile duct ligation (day 5: +91%) and reduced concentrations (?44%) at the end of the observation period. Dynamic changes of the glutathione status occur in the development of liver cirrhosis after bile-duct ligation. These changes are consistent with increased oxidative stress in the liver and a deficit of transporting GSSG from the cells into plasma.     

Variability of glutathione S-transferase alpha in human liver and plasma

BACKGROUND: Glutathione S-transferases are a family of enzymes involved in the binding, transport, and detoxification of a wide variety of endogenous and exogenous compounds. Little information is available about the variability of class alpha glutathione S-transferases in human liver, where they are highly expressed, or in serum. METHODS: Both total class alpha glutathione S-transferase (GST-alpha, composed of GSTA1-1, GSTA1-2, and GSTA2-2) as well as GSTA1-1 concentrations were measured by specific and sensitive ELISA in liver cytosols of 35 organ donors and in plasma samples of 350 healthy controls. RESULTS: The mean total GST-alpha and GSTA1-1 in liver cytosols were 25.1 +/- 9.4 and 10.7 +/- 5.3 microg/mg protein, respectively, and did not correlate with activities of aspartate aminotransferase or alanine aminotransferase. The mean total GST-alpha in liver was significantly higher in females compared with males (28.8 +/- 10.0 vs 22.0 +/- 7.8 microg/mg protein; P <0.05). In contrast, the median total GST-alpha in plasma was lower in females compared with males (2.0 and 2.8 microg/L, respectively; P <0.0001). The median ratios for GSTA1-1/total GST-alpha in liver and plasma were 0.42 and 0.58, respectively. CONCLUSIONS: GSTA1-1 constitutes approximately one-half of the total amount of alpha class GSTs in human plasma and liver. Total GST-alpha values are higher in female liver but lower in plasma compared with the respective values in males.     

Glutathione S-transferase gene polymorphisms are not major risks for susceptibility to posttransplantation diabetes mellitus in Taiwan renal transplant recipients

Glutathione S-transferase (GST) M1 null genotype has been reported playing a significant role in the diabetes mellitus (DM) susceptibility in Turkish population. We investigated whether the GSTM1, GSTA1, and GSTP1 gene polymorphisms are associated with posttransplantation diabetes mellitus (PTDM) in Taiwan. There were 283 renal transplant recipients (RTRs) enrolled. Polymerase chain reaction-restriction fragment length polymorphism was used for the measurement of GSTA1, M1, and P1 genetic polymorphisms. PTDM was diagnosed according to the American Diabetes Association guidelines. Eight-five patients (30%) were diagnosed with PTDM. The averaged posttransplant follow-up period was 77.9 ± 27.2 months. Duration from transplantat to diagnosis of PTDM ranged from 0.2 to 103.1 months (19.2 ± 26.3 months). There were significantly differences between non-DM and PTDM groups in age (50.6 ± 11.0 vs. 54.6 ± 9.36 years, P = 0.005), BMI (22.4 ± 3.6 vs. 24.3 ± 3.8, P<0.001). The distributions of GSTA1, GSTP1, and GSTM1 genotypes alleles were not significantly different between PTDM and non-DM group. Patients carrying the different GSTA1, GSTP1, and GSTM1 genetic and allelic polymorphisms had no differences for the development of PTDM. These overall results suggested a lack of strong association with GSTA1, GSTP1, and GSTM1 genetic polymorphisms to the susceptibility of PTDM in Taiwanese RTRs.     

Glutathione S-transferase Omega 2 DD genotype is associated with an increased risk of sporadic amyotrophic lateral sclerosis in Chinese men

ObjectiveTo investigate whether GSTA1, GSTO2, and GSTZ1 are relevant to an increased risk of amyotrophic lateral sclerosis (ALS) in a Chinese population.MethodsIn this study, 143 sporadic ALS (sALS) patients (83 men, 60 women) and 210 age- and sex-matched healthy subjects were enrolled. Blood samples were collected by venipuncture. Genomic DNA was isolated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) according to the manufacturer’s instructions. The potential associations between ALS and GSTA1, GSTO2, and GSTZ1 polymorphisms were estimated using chi-squared analysis and unconditional logistic regression.ResultsThe D allele and genotype frequencies of GSTO2 were increased in sALS patients compared with healthy subjects, indicating that the GSTO2 DD genotype was associated with an increased risk of sALS (odds ratio [OR] = 3.294, 95% confidence interval [CI] = 1.039–10.448). However, a significant association between the DD genotype and the risk of sALS was evident in men only (OR = 7.167, 95% CI = 1.381–37.202).ConclusionThis study revealed that the D allele and genotype frequencies of GSTO2 were increased in sALS patients. The GSTO2 DD genotype was associated with an increased risk of sALS in men in a Chinese population.     

Effects of bile and bile acids on cultured human fibroblasts

Impaired healing induced by leakage of bile has been postulated as one factor responsible for complications after reconstructive bile duct surgery. The cytotoxicity of human bile and its major bile acids on cultured human fibroblasts was therefore studied by evaluation of their effects on cell morphology and growth, on synthesis and secretion of 35SO4-mucopolysaccharides and on release of a lysosomal enzyme. Normal human fibroblasts derived from a standard culture strain (MRC-5) were grown to confluence and exposed to: (1) sterile human T-tube bile, (2) a mixture of bile acids resembling that of human bile, or (3) various concentrations of the glycine- and taurine conjugates of cholic, chenodeoxycholic or deoxycholic acid. Medium containing whole bile (total bile acid concentration 0.25, 0.75 or 1.6 mmol/l) exerted time and dose dependent cytotoxic effects on morphology and growth and release of lysosomal enzyme. Synthesis and secretion of 35SO4-mucopolysaccharides were markedly inhibited. The bile acid mixture exhibited the same time and dose dependent effects. The conjugates of deoxycholic acid were found to be the most toxic of the individual bile acids studied.     

Tetrahydroxylated bile acids in healthy human newborns

Urine was collected in four healthy infants, born after 35-39 weeks of gestation. The collections were made for 24 h starting immediately after birth. Bile acids were extracted, separated into conjugate groups, solvolysed, hydrolysed and quantified by gas-liquid chromatography as methyl ester trimethylsilyl ether derivatives. Identification was made by gas-liquid chromatography-mass spectrometry. Cholic acid was the predominating primary bile acid. Non-sulphated tetrahydroxylated bile acids, tentatively identified as 1,3,7,12- and 3,6,7,12-tetrahydroxycholanoic acids, were present in almost the same amounts as cholic acid. A previously unknown tetrahydroxylated cholanoic acid, which might be 2-hydroxylated hyocholic acid, was found in all infants in similar amounts. 3 beta-Hydroxy-5-cholenoic acid was present in the sulphate fraction in the urine from all infants. Lithocholic acid was not found. Small amounts of allo and 6-hydroxylated bile acids were mainly found in the sulphate fraction. Bile acids hydroxylated in the 1-position were found predominantly in the taurine fraction.