Combined detection of interleukin-6 and γ-glutamyltranspeptidase facilitates evaluation of therapeutic efficacy and prognosis of AFP-negative TACE-treated hepatocellular carcinoma patients / 解放军医学杂志

Objective To analyze the clinical value of combined detection of several serum markers in the treatment and prognosis of patients with alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). Methods A total of 120 patients (from September 2016 to March 2018) diagnosed with HCC followed by TACE are recruited in this study. Serum samples were collected and dynamic markers, serum AFP, carcinoembryonic antigen (CEA), carbohydrate antigen-199 (CA199), CA125, gamma-glutamyltranspeptidase (GGT), interleukin (IL)-2, IL-4, IL-6, IL-10, γ-interferon (IFN-γ), and tumor necrosis factor α (TNF-α) levels were detected in 44 AFP-negative patients and 76 AFP-positive patients prior, 1 and 3 months after TACE. During the follow-up period, the tumor activity of patients was monitored and the clinical value of different marker combinations on the efficacy and prognosis of AFP-negative HCC patients after TACE was analyzed until July 2018. Results There was no statistically significant difference in the pre-operative indicator levels between AFP-negative and AFP-positive patients (P>0.05). The sensitivity, specificity, negative predictive value, and positive predictive value of IL-6 combined with GGT in detecting the efficacy and prognosis of AFP-negative and AFP-positive patients were 95.2%, 87.0%, 87.0%, 95.2% and 85.7%, 59.6%, 70.1%, and 79.1%, respectively. The area under the ROC curve of IL-6 for the prognosis of AFP-negative and AFP-positive HCC patients was 0.839 and 0.666. Conclusion Serum IL-6 combined with GGT can be used as an effective indicator for predicting the efficacy and tumor activity of AFP-negative HCC patients after TACE.

Biochemical Predictors of Early Onset Non-Alcoholic Fatty Liver Disease in Young Children with Obesity

BACKGROUND: The prevalence of metabolic syndrome (MS) and non-alcoholic fatty liver disease (NAFLD) and their associated risk factors are not well-established in young children with obesity. The purpose of this study was to evaluate the prevalence of early onset NAFLD and identify its biochemical predictors in obese children aged less than 10 years. METHODS: Anthropometric measurements, blood pressure, laboratory tests, and abdominal ultrasonography (USG) were performed in all subjects. National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III) criteria for MS diagnosis and liver enzymes and USG for NAFLD diagnosis were assessed. RESULTS: A total of 356 children with obesity (233 boys, 123 girls) were included, with 172 children age ≤ 10 years and 184 adolescents. The prevalence of MS was 23.3% in young children and 35.3% in adolescents (P = 0.020); while the prevalence of NAFLD was 36.0% and 70.7%, respectively (P = 0.001). In obese children aged 10 years or less, there were significant differences in levels of serum γ-glutamyltranspeptidase (γGT) (P < 0.001), triglycerides (P = 0.042), and homeostatic model assessment of insulin resistance (P < 0.001) between the non-NAFLD and the NAFLD group. Multivariate logistic regression analysis revealed significant increase in serum γGT and uric acid levels in young children. CONCLUSION: Although MS and NAFLD were more prevalent in adolescents, young children also demonstrated MS and NAFLD as obesity-related complications. Elevated serum γGT and uric acid levels may serve as biochemical predictors in detecting NAFLD in young children with obesity before investigation with abdominal USG.

Detailed Distribution of Liver Enzymes according to Gender, Age, and Body Mass Index in Health Check-up Subjects / 대한소화기학회지

BACKGROUND/AIMS: The aim of this study was to examine the distribution of range of liver enzymes according to age and BMI in each gender using large-scale data. METHODS: Data were gathered from 65,715 subjects who underwent a routine health check-up and did not have HBsAg and anti-HCV. Boxplot analysis was used to examine the distribution of range of liver enzymes according to age and BMI in each gender. Multivariate linear regression analysis was performed for assessment of the association of liver enzymes with age and BMI, and to determine whether the range of liver enzymes was affected by risk factors for metabolic syndrome in each gender. RESULTS: ALT, AST, and GGT levels showed significant association with BMI in both male and female after adjusting for age. The range of ALT, AST, and GGT levels varied more widely according to the increase in BMI in males than in females, and this finding was more prominent in younger subjects than in older subjects. All risk factors for metabolic syndrome were shown to affect liver enzyme levels in male subjects. However, although most risk factors for metabolic syndrome affected liver enzyme levels, there might be weak or no effect of fasting hyperglycemia on AST, and low serum HDL-cholesterol level on GGT in female subjects. CONCLUSIONS: Age, BMI, and other risk factors for metabolic syndrome had a significant effect on the distribution of range of liver enzymes in each gender, even in this study conducted from Korean health checkup subjects.

Purification and Characterization of Helicobacter pylori gamma-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) was purified to electrophoretic homogeneity from the cell extract of H. pylori. The purified enzyme consisted of heavy and light subunits with molecular weights of 38 kDa and 21 kDa, respectively. N-terminal amino acid sequence of heavy and light subunits revealed that H. pylori GGT was processed into 3 parts for a signal peptide of 27 amino acid residues, a heavy subunit of 352 residues, and a light subunit of 188 residues during translation. The reaction rate for hydrolysis of gamma-GpNA was 84.4 micromol/min per milligram of protein, and that for the gamma-glutamyl transfer from gamma-GpNA to gly-gly was 23.8 micromol/min per milligram of protein. The apparent Km values of H. pylori GGT for gamma-glutamyl compounds were on the order of 10-3 to 10-4 M and those for acceptor peptides and amino acids were on the order of 10-1 to 10-2 M. The GGT protein kept approximately 80% of the initial enzymatic activity on incubation at 60degrees C for 15 min. The optimum temperature and pH for reactions of both hydrolysis and transpeptidation were 40degrees C and 9.0, respectively. The transpeptidation and hydrolysis reactions catalyzed by H. pylori GGT were strongly inhibited by L-Gln and moderately inhibited by L-Ala, L-Ser, beta-chloro-L-Ala, and L-Glu. These results demonstrated that the biochemical properties of H. pylori GGT are different from those of other bacterial GGTs. Further, H. pylori GGT might degrade glutathione in the gastric mucous layer of humans if the enzyme could be secreted in the bacterial niches.

Alfa-fetoproteína y gamma-glutamiltranspeptidasa en pacientes con cirrosis hepática y carcinoma hepatocelular / Alpha-fetoprotein and gamma-glutamyltransferase in patients with liver cirrhosis and hepatocellular carcinoma

Introducción: El carcinoma hepatocelular (CHC) es una complicación de la cirrosis hepática (CH). El objetivo principal del estudio fue describir los niveles de los marcadores serológicos alfa-fetoproteína (AFP) y gamma-glutamiltranspeptidasa (GGT) en pacientes con CH y/o CHC. Métodos: Estudio de corte transversal con 99 pacientes con diagnóstico de CH y/o CHC. Resultados: Un total de 66 (66,7%) pacientes presentaban un diagnóstico de CH, 23 (23,2%) CHC asociado a CH y 10 (10,1%) CHC aislado. Los valores de AFP fueron mayores en individuos con CHC asociado a CH comparados con aquellos que solo tenían CH (20 y 2,93 ng/mL, p<0.05); los niveles de la GGT fueron también mayores en pacientes con CHC asociado a CH (208 y 109 UI/L, p<0,05). Ningún paciente con CHC tuvo en forma simultánea valores normales de AFP y GGT. Conclusiones: En pacientes con CHC asociado a CH los niveles de AFP y GGT son significativamente más altos que individuos con CH aislada.

Introduction: Hepatocellular carcinoma (HCC) is one of the complications associated with liver cirrhosis (LC). The main objective of the present study was to describe the levels of the serological markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase (GGT) in patients with LC and/or HCC. Methods: Cross sectional study that included 99 patients with a diagnosis of LC and/or HCC. Results: 66 (66.7%) patients had a diagnosis of LC, 23 (23.2%) had LC alongside with HCC and 10 (10.1%) had HCC without LC. AFP levels were higher in individuals with HCC associated with LC when compared with those with LC only (20 and 2.93 ng/mL, p <0.05), the levels of GGT were also higher in patients with HCC associated with LC (208 and 109 IU/L, p <0.05). Not a single patient with HCC had normal levels of AFP and GGT simultaneously. Conclusions: In patients with HCC associated with LC levels of AFP and GGT were significantly higher than those found in individuals with LC only.

Contribution of Ser463 residue to the enzymatic and autoprocessing activities of Escherichia coli -glutamyltranspeptidase.

A serine residue Ser463, required for proper function of E. coli -glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into - and -subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.

Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia / 中国生物工程杂志

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia / 中国生物工程杂志

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia / 中国生物工程杂志

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

Purification of ?-glutamyltranspeptidase strongly combined to Datura Stramonim (DSA) lectin from primary hepatic cancer tissue by affinity chromatography / 临床检验杂志

Objective To purify ?-glutamyltranspeptidase (GGT) strongly combined to datura stramonim (DSA) lectin from human liver cancer tissue,which will be used as the antigen to produce antibodies for early diagnosis of liver cancer.Methods The GGT strongly combined to DSA lectin in human liver cancer tissue was purified by lectin affinity chromatography from human liver cancer tissue, including rough extraction,dialysis,ion-exchange chromatography and lectin affinity chromatography.Results The catalysis units per milligram protein of GGT increased about 84.9 times.The yield of the GGT was 1.60%.Conclusion The protocol in this study is efficient to purity GGT from human liver cancer tissue.

Construction Fermentation of Engineered Strain and Properties of Recombinant ?-glutamyltranspeptidase / 中国生物工程杂志

The ?glutamyltranspeptidase encoding gene(ggt) from Bacillus subtilis SYU 20016 was amplified by PCR. The ggt gene was inserted in pBV220 to yield the recombinant expression vector pBV220ggt. Overexpression of ggt in E.coli JM109 was achieved with pBV220ggt. SDSPAGE analysis showed an overexpressed recombinant product at about 65kDa,consistent with the molecular weight predicted from gene sequence. The ferment conditions of r-glutamyltranspeptidase were also discussed. The optimum temperature and pH for the enzyme were determined as 30℃ and 7.2 respectively.The cultures were incubated at 42℃ for 4h with broth volume 20ml/250ml flask and the yield of 6U/ml was obtained, enzyme activity of B. subtilis NX2 was only 3.2 U/ml.

Níveis séricos de aminotransferases, bilirrubinas e gama-glutamil transpeptidase após a admininstração de óleo de copaíba em ratos

Copaiba oil is a oil-resin used as a phytotherapic in Amazon region in a folkloric way. This use is empirical and little is known about its effect in the organic systems. Objective: To determine serum levels of aminotransferases, bilirrubins and gama-glutamyl transpeptidase after copaiba oil administration. Methods: Male Wistar rats (n=20) divided in two groups of 10 animals: Group Cop which received a daily oral administration of copaiba oil, 0.63ml/kg, during 5 days; Group P in which any substance was administrated. In the sixth day the animals of both groups were submitted to inalatory anesthesia, laparotomy and collection of blood samples (3ml) from inferior vena cava for the determination of serum aminotransferases, bilirrubins and gama-glutamyl transpeptidase levels. Results: The results were compared between groups and analyzed by t-Student test. Group Cop showed AST, ALT and GGT levels significantly decreased when compared with Gruop P (p 0.01), while total bilirrubins levels increased by its direct fraction. Conclusion: Copaiba oil can alter serum levels of aminotransferases, bilirrubins and gama-glutamyl transpeptidase, showing a possible interaction between this substance and liver, but this considerations require posterior evalutaion.

O óleo de copaíba é um óleo-resina empregado como fitoterápico na região Amazônica. Sua utilização se faz de forma empírica e pouco se conhece à respeito de seus efeitos sobre os sistemas orgânicos. Objetivo: Verificar os níveis séricos de aminotransferases, bilirrubinas e gama-glutamil transpeptidase após a administração do óleo. Métodos: Foram utilizados 20 ratos Wistar, machos, pesando entre 250 e 300g, distribuídos em 2 grupos: Grupo Cop (n=10) submetidos diariamente à gavagem com administração de 0,63ml/kg do óleo, por 5 dias; Grupo P (n=10), animais nos quais nenhuma substância foi administrada. Ao sexto dia, em ambos os grupos, procedeu-se anestesia inalatória e obtenção de 3ml de sangue da VCI para dosagem dos níveis séricos de ALT, AST, bilirrubinas e GGT. Resultados: Os resultados obtidos com o GCop foram comparados com os do GP e analisados estatisticamente pelo teste t -Student. O GCop apresentou níveis de ALT, AST e GGT significantemente mais baixos que o GP (p 0,01), enquanto os níveis de BT elevaram-se às custas da fração direta. Conclusão: O óleo de copaíba altera os níveis das aminotransferases, bilirrubinas e GGT, sem alterar os níveis da fração indireta.

Purification of r-Glutamyltranspeptidase from Rat Primary Hepatoma Tissue and Preparation of a Tumor Associated Antigen

r-Glutamyltranspeptidase (r-GT) from a rat hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me DAB) was purified 833 fold. The purified enzyme had a specific activity of 15.0 U/mg protein with an overall yield of 3.8%. The molecular weight of native r-GT was estimated as about 350,000 daltons, whichs a multicomplex of a single polypetide having a M W of 59,000. Anti r-GT rabbit antiserum cross-reacted with kidney r-GT as well as liver r-GT. Tryptic digestion of r-GT followed by separation with Con A sepharose column chromatography resulted in two major glycopeptides. A tumor associated antigen was prepared by the conjugation of a tryptic glycopeptide of r-GT to keyhole limpets hemocyanin and an antibody against this antigen cross-reacted preferentially with r-GT in rat hepatoma tissue.