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Characterization of in vitro metabolites of toad venom using high‐performance liquid chromatography and liquid chromatography–mass spectrometry
Author(s) -
Shimada Kazutake,
Miyashiro Yoshimichi,
Nishio Tadashi
Publication year - 2006
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.699
Subject(s) - chemistry , chromatography , microsome , s9 fraction , hydroxylation , glucuronide , mass spectrometry , toad , high performance liquid chromatography , in vitro , enzyme , metabolite , biochemistry , medicine
The characterization of the in vitro metabolites of toad venom, which has been widely used as a traditional Chinese drug, Ch'an Su, has been completed. Toad venom contains bufotoxins (such as marinobufotoxin; marinobufagin 3‐suberoylarginine ester) and bufogenins (such as marinobufagin and bufalin) as the main cardiac steroids. An in vitro experiment using the rat or human liver cytosolic fraction disclosed that marinobufotoxin produced marinobufagin, but not its 3‐hemisuberate. Marinobufagin was subjected to the enzyme reaction using the rat or human liver microsomal fraction together with NADPH and NAD, which produced 3‐dehydromarinobufagin and 3‐epimarinobufagin. Marinobufagin produced its 3‐sulfate upon treatment with the rat or human liver cytosolic fraction and 3′‐phosphoadenosine 5′‐phosphosulfate. Bufalin was also subjected to the above enzyme reactions and showed almost the same results except for the result that the hydroxylation occurred at the 5 β ‐position. On the other hand, small amounts of marinobufagin 3‐glucuronide were obtained only by treatment with the human liver microsomal fraction and uridine 5′‐diphosphoglucuronic acid. The structures of these metabolites were confirmed using authentic samples regarding their high‐performance liquid chromatographic behavior and/or liquid chromatography–mass spectrometry analysis. Copyright © 2006 John Wiley & Sons, Ltd.