Optimization studies of components in enzymatic cholesterol reagents containing cholesterol oxidase from Nocardia erythropolis,Streptomyces sp,or Pseudomonas fluorescens

Although enzymatic methods for serum cholesterol determination are widely used in clinical laboratories, little is known about the optimization of each component in enzymatic reagents. We investigated the optimal components in the reagents containing cholesterol oxidase isolated from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens. The optimal components in the reagents are: cholesterol oxidase 250 (Nocardia erythropolis), 250 (Streptomyces sp), or 300 (Pseudomonas fluorescens) U/L, cholesterol esterase 200 U/L, peroxidase 10,000 U/L, sodium cholate 3 mmol/L, 4-aminoantipyrine 0.5 mmol/L, phenol 20 mmol/L, Triton X-100 2 mL/L, and phosphate buffer, pH 7.0. Lower reaction sensitivity and lower cholesterol linearity, <18.1 mmol/L (700 mg/dL), could be obtained by using lower components than those suggested above. Pseudomonas fluorescens were an improper source for cholesterol oxidase; either Nocardia erythropolis or Streptomyces was suitable cholesterol oxidase. We prefer using Streptomyces sp cholesterol oxidase because of its economical cost and longest reagent stability. Sodium cholate must be included in the enzymatic reagent to prevent turbidity. However, sodium cholate of >5 mmol/L will suppress the reaction resulting in low cholesterol linearity. © 1996 Wiley-Liss, Inc.     

Opisthorchis viverrini infection causes liver and biliary cirrhosis in gerbils

Opisthorchis viverrini infection causes many hepatobiliary diseases, including cholangiocarcinoma. Hence, the study of O. viverrini infection in humans is subject to ethical limitations, so an animal model, the Syrian hamster, is often used. O. viverrini can develop into the adult stage not only in Syrian hamsters but also in other animals, including gerbils, but until now, there has been no report on pathology and susceptibility in gerbils. The present study revealed the pathology of O. viverrini infection in gerbils through gross appearance, histopathology, and worm recovery at various time points. Gerbils were infected with 50 O. viverrini metacercariae and then sacrificed at the time of observation. The gross appearance of the liver showed micronodules at the liver surface, suggesting liver and biliary cirrhosis. Light microscopic observation was correlated to the gross appearance with cholecystitis, fatty liver changes, fibrous septa, and generalized cirrhosis. The range of worm burden fluctuated from 1 to 25 worms with large body size, which was correlated with pathology. These novel findings indicate that O. viverrini infection can cause liver and biliary cirrhosis in gerbils, depending on the worm burden, worm size, and habitat.     

Anti-inflammatory, antioxidant and hepatoprotective effects of Thunbergia laurifolia Linn. on experimental opisthorchiasis

Thunbergia laurifolia Linn (Rang Chuet) possesses antioxidant and anti-inflammatory properties as well as anticancer activities. The aim of the present study was to evaluate the efficacy of T. laurifolia in reducing inflammation from pathological changes in Syrian hamsters infected with the human liver fluke Opisthorchis viverrini. Hamster groups were also administered N-nitrosodimethylamine (NDMA) and treated with T. laurifolia. Light microscopic observation of histopathological changes, liver function tests for alanine transaminase (ALT) and alkaline phosphatase (ALP) and kidney function tests for blood urea nitrogen (BUN) and creatinine were performed. Antioxidant effects of both fresh and dried Rang Chuet solutions were observed. Analysis of the histopathological changes showed anti-inflammatory properties, both in the case of O. viverrini infection or with NDMA administration, by reducing the aggregation of inflammatory cells surrounding the hepatic bile ducts as indicated by normal serum ALT, ALP, BUN and creatinine levels in treated Syrian hamsters. The present study found that fresh and dried Rang Chuet solutions clearly reduced the inflammatory cells in both O. viverrini-infected and NDMA-administered groups and was correlated with the total antioxidant capacity. These findings suggest that T. laurifolia possesses antioxidant and anti-inflammatory properties and that its application may be useful for prevention of the inflammatory process, one of the risk factors of O. viverrini-associated cholangiocarcinoma (CCA).     

A combination of praziquantel and the traditional medicinal plant Thunbergia laurifolia on Opisthorchis viverrini infection and cholangiocarcinoma in a hamster model

Cholangiocarcinoma (CCA) associated by Opisthorchis viverrini remains a health problem in Southeast Asia including Thailand. At present, there is still no efficient treatment for CCA. Thunbergia laurifolia is a traditionally used medicinal plant; its aqueous leave extract possesses the antioxidant activity and anti-inflammatory on hamster opisthorchiasis had been reported previously. Here, we demonstrate the combined effects of the T. laurifolia extract plus antihelminthic drug, praziquantel (PZ) on hamsters with opisthorchiasis and hamsters with opisthorchiasis related-cholangiocarcinoma through light microscopic observations of histopathological changes, as well as liver function tests for alanine transaminase (ALT) and alkaline phosphatase, and kidney function tests for blood urea nitrogen and creatinine. Results showed T. laurifolia extract combined with praziquantel reduced inflammatory cell aggregation and inhibiting CCA development, which were correlated to the serum ALT level. These present studies suggest that administration of T. laurifolia after praziquantel treatment clearly improve the hepatobiliary system and could reduce the risk of subsequent CCA development in human.     

Sex differences in opisthorchiosis and the development of cholangiocarcinoma in Syrian hamster model

Worldwide, the highest incidence of cholangiocarcinoma (CCA) is found in northeast Thailand, the endemic area of Opisthorchis viverrini infection. Cumulated clinical data revealed that the majority of CCA patients are men. However, many other types of cancers are more commonly found in women. In this study, we investigated the sex differences in the development of CCA, induced by O. viverrini infection and N-nitrosodimethylamine administration, in Syrian hamsters. Histopathology, liver function tests, and fecal egg counts were analyzed. The results showed that there are no sex differences in hamsters responses to O. viverrini infection and no prevalence of CCA development. Even though serum ALT level in O. viverrini-infected or CCA hamsters was significantly increased in female compared to male (p?<?0.05) and uninfected control (p?<?0.05), our results may imply that the higher prevalence of opisthorchiasis and CCA in men than in women in northeast Thailand may depend on behaviors of an individual exposed to risk factors rather than gender difference.