HT-2 Toxin 4-Glucuronide as New T-2 Toxin Metabolite: Enzymatic Synthesis, Analysis, and Species Specific Formation of T-2 and HT-2 Toxin Glucuronides by Rat, Mouse, Pig, and Human Liver Microsomes

Abstract

Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared.

Multi-mycotoxin analysis using dried blood spots and dried serum spots

“…2’ R -OTA was produced by thermal isomerization of OTA [25]. Glucuronic acid conjugates (DON-3-GlcA and HT-2-4-GlcA) were synthesized enzymatically using rat and pig liver microsomes [36]. ALT, AME, and AOH were isolated and purified according to a recently published procedure [37].…”

A comparative study of the human urinary mycotoxin excretion patterns in Bangladesh, Germany, and Haiti using a rapid and sensitive LC-MS/MS approach

“…ZEA, DON, FB 1 , OTA, OTα, T-2, and HT-2 were isolated and purified from fungal cultures (Beyer et al 2009;Cramer et al 2007Cramer et al , 2010Bittner et al 2013;Bretz et al 2006;Hübner et al 2012). Glucuronic acid conjugates (DON-3-GlcA, ZEA-14-GlcA, ZAN-14-GlcA, α-ZEL-14-GlcA, β-ZEL-14-GlcA, and HT-2-4-GlcA) were obtained from enzymatic synthesis using rat and pig liver microsomes (Welsch and Humpf 2012). Solid analytes were dissolved in ACN or ACN/H 2 O (DON-3-GlcA, FB 1 ).…”

Determination of mycotoxin exposure in Germany using an LC‐MS/MS multibiomarker approach

“…ZEN, DON, FB 1, FB 2 , OTA, OT␣, T-2, HT-2 were isolated and purified from fungal cultures [14][15][16][17][18][19][20]. Glucuronic acid adducts (DON-3-GlcA, ZEN-14-GlcA, zearalanone-14-O-glucuronide, ␣-zearalenol-14-Oglucuronide, ␤-zearalenol-14-O-glucuronide, and HT-2-GlcA were synthesized enzymatically using rat and pig liver microsomes according to a recently published procedure [13]. Solid analytes were dissolved in ACN or ACN/H 2 O (DON-3-GlcA, FB 1, FB 2 ).…”