Characterization of in vitro metabolites of toad venom using high-performance liquid chromatography and liquid chromatography-mass spectrometry.
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 5beta-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.
Shimada
Kazutake
K
Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. shimada@p.kanazawa-u.ac.jp
Miyashiro
Yoshimichi
Y
Nishio
Tadashi
T
D000664:Amphibian Venoms / Q000737:chemistry*; D000818:Animals; D002018:Bufanolides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D020723:Glucuronic Acid / Q000378:metabolism; D006801:Humans; D008297:Male; D053858:Metabolic Networks and Pathways; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013463:Sulfuric Acid Esters / Q000378:metabolism
D000664:Amphibian Venoms; D002018:Bufanolides; D013463:Sulfuric Acid Esters; C005734:bufogenin; D020723:Glucuronic Acid; C026738:bufotoxin