| ObjectiveRat hepatocytes were cultured in vitro. The changes of Hcy metabolism weredetermined after methionine loading in order to explore the effects of methionineloading on some metabolic pathways of Hcy and the potential mechanisms involved.Methods1. The BRL rat hepatocytes were cultured in culture medium with10%fetalbovine serum.2. MTT method was used to investigate the effects of different concentrations ofMet on the survival rate of rat hepatocytes.3. The contents of Hcy and its metabolites in cell homogenates and culturemedium were determined by high performance liquid chromatography(HPLC).4. The contents of some amino acids related to the metabolism of Hcy in culturemedium were measured by an Amino Acid Analyzer and the activity ofbetaine homocysteine methyltransferase (BHMT) in cell homogenates wasassayed spectrophotometrically.5. The contents of S-adenosylmethionine (SAM) and S-adenosylhomoeysteine(SAH) and the activity of SAH hydrolase in cell homogenates were assayed byHPLC procedure.6. Spectrophotometrical methods were employed to measure the activities ofmethionine synthase (MS) and cystathionine β-synthase (CBS) in cellhomogenates.Results1. After BRL rat hepatocytes were exposed to Met loading (10,20,50,100and200mmol/L respectively) for1,2,3,4h, the changes of survival rate ofhepatocytes were measured. When the concentration of Met was less than100mmol/L, survival rate of hepatocytes was increased significantly. 2. After hepatocytes were exposed to Met loading (20,50and100mmol/Lrespectively), the content of Hcy in20and50mmol/L Met groups from1to4hwere not changed remarkably. However, the content of Hcy was increasedsignificantly in100mmol/L Met group.3. Compared to the control, the contents of Hcy, Cys, Ser and cystine wereincreased remarkably (P<0.05), and the contents of Glu, Gly and Taudecreased significantly (P<0.05) in the culture medium in20mmol/L Metgroup. The contents of Hcy, Cys and cystine were increased remarkably(P<0.05), whereas the contents of Glu, Gly decreased significantly (P<0.05) inthe culture medium of50mmol/L Met group. Compared to20mmol/L Metgroup, the contents of Hcy, Cys, GSH, Gly and Tau were increasedremarkably (P<0.05), and the content of Ser decreased significantly (P<0.05)in the culture medium of50mmol/L Met group.4. Compared to the control, the content of SAM, the ratio of SAM to SAH andthe hydrolytic and synthetic activity of SAH hydrolase were increasedsignificantly (P<0.05), and the activity of BHMT decreased remarkably(P<0.05) in the cell homogenates of20and50mmol/L Met groups;Theactivity of MS was decreased remarkably (P<0.05) in the cell homogenates of20mmol/L Met group; The content of SAH was decreased remarkably (P<0.05)in the cell homogenates of50mmol/L Met group. Compared to20mmol/L Metgroup, the contents of SAM and SAH were decreased remarkably (P<0.05),and the ratio of SAM to SAH increased significantly (P<0.05) in the cellhomogenates of50mmol/L Met group.Conclusion1. Met loading could increase the content of Hcy significantly in rat hepatocytes.2. The results of amino acids analysis indicated that the contents of Hcy, Cyswere increased significantly and the contents of Glu, Gly and Ser were alsochanged. The effect of20mmol/L Met was more obvious than that of50mmol/L Met.3. The changes of the contents of Hcy metabolites and enzymes related to Hcymetabolism demonstrated that the activity of SAH hydrolase was increasedsignificantly, while the activities of BHMT and MS decreased remarkably inMet loading groups. It was suggested that the remethylation pathway of Hcywas inhibited, and the effect of20mmol/L Met was more significant than that of50mmol/L Met. |
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