Hydrolysis of Urine 17-OS Conjugates by Ampullaria Hydrolases, and Effect of Pi Removal on Sulfatase Activity

概要

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Glucuronidase and sulfatase which hydrolyzed various conjugates in urine were found to occur in the extracts of digestive juice from Ampullaria and to be useful rapid hydrolysis of steroid conjugates. Ampullaria hydrolases had a good thermal stability, at 60°C, were not inactivated for 3 hours in urine. More than 90% activity of the sulfatase activity was inhibited by Pi in urine, but β-glucuronidase activity was slightly inhibited by urine. Pi could be removed by treating with the magnesium and ammonium solution at pH 11.5. Incubation for 2hr at 60°C of the Pi free urine with 800 U β-glucuronidase and 400 Roy U sulfatase from Ampullaria per milliliter at pH 5.0 resulted in complete hydrolysis of 17-OS conjugates in the sample. This hydrolysis method showed a good recovery of dehydroepiandrosterone (DHEA) sulfate by gas-chromatography. A good correlation between this rapidly hydrolysis method and the long time incubation with Helix pomatia enzyme was obtained expect the value of DHEA.(y=2.806x-0.068 y: Ampullaria x: Helix pomatia) Normal range of DHEA on male was obtained 0.12-5.20 mg/day, this value was higher than that by the other hydrolysis methods.
Japan Society of Clinical Chemistryの論文