BALB-DTNB法の改良によるラットの各種リパーゼ活性の測定

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In this study, we developed a method of assaying rat lipase activities, such as pancreatic lipase ,(PL, EC3.1.1.3), lipoprotein lipase (LPL, EC3.1.1.34), hepatic triglyceride lipase (HTGL), hormone-sensitive lipase (HSL) and adipose tissue total lipase (ATTL) using BALB-DTNB method with modifications. A 0.0335% BALE (2,3-dimercaptopropan-l-ol tributyreta) water solution was used as a substrate, that of 0.01% DTNB (5,5'-dithiobis (2-nitrobenzonic acid)) as a chromogenic reagent and that of 0.0287% SDS (sodium dodecyl sulfate) as a lipase activator to revitalize each lipase. Especially, 0.15 mole of Nad Tris-HCl buffer was used to revitalize LPL activity, while 1 mole of the same buffer was used to inhibit. A water solution of 0.1 mole of SDS and 0.2 mole of Tris-HCl (pH8.2) was used to inhibit HTGL activity. The activity value of LPL or HTGL was calculated by subtracting the inhibited value from the revitalized value. PMSF (phenylmethylsulfonyl fluoride) ethanol solution was used as an esterase inhibitor. A solution of 0.74% PMSF was used for LPL and HTGL assay, that of 1.8% for HSL and ATTL and that of 3.6% for pancreatic tissue PL. One mole of Tris-HCl was used ,as a reaction buffer (at pH8.8 for HTGL assay, pH8.5 for PL, pH8.2 for LPL and ATTL and pH7.0 for HSL). A one percent SDS water solution was used as a reaction stopper (at pH2.8 to stop HTGL reaction, pH3.0 for PL, pH3.2 for LPL and ATTL and pH5.8 for HSL). PL and HSL activity reaction was carried out at 30 ± 1 ℃ and for the other lipases at 28 ± 1 ℃ for 30 minutes. Under these conditions, two linear standard curves of each lipase were obtained using sample values 'of 20 to 80 μl 1 and a reactive time of 5 to 50minutes. However, HSL showed stable values after reaction for 20 to 60 minutes. Each lipase showed a CV value less than 5% in ten tests. These results suggest that the present method is possible to assay rat PL, LPL, HTGL, HSL and ATTL activity.
東海大学の論文
2004-03-31

BALB-DTNB法の改良によるラットの各種リパーゼ活性の測定

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