Cob(I)alamin for Trapping Butadiene Epoxides in Metabolism with Rat S9 and for Determining Associated Kinetic Parameters

Abstract

The reduced state of vitamin B(12), cob(I)alamin, acts as a supernucleophile that reacts ca. 10(5) times faster than standard nucleophiles, for example, thiols. Methods have been developed for trapping electrophilically reactive compounds by exploiting this property of cob(I)alamin. 1,3-Butadiene (BD) has recently been classified as a group 1 human carcinogen by the International Agency for Research on Cancer (IARC). The carcinogenicity of BD is considered to be dependent on the activation or deactivation of the reactive metabolites of BD, that is, the epoxides (oxiranes) 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol). Cytochrome P450 (P450) isozymes are involved in oxidation of BD to EB and further activation to DEB. EB and DEB are hydrolyzed by epoxide hydrolases (EH) to 3,4-dihydroxy-1-butene (BDdiol) and EBdiol, respectively. EBdiol can also be formed by oxidation of BDdiol. In the present study, cob(I)alamin was used for instant trapping of the BD epoxide metabolites generated in in vitro metabolism to study enzyme kinetics. The substrates EB, DEB, and BDdiol were incubated with rat S9 liver fraction, and apparent K(m) and apparent V(max), were determined. The ratio of conversion of EB to DEB (by P450) to the rate of deactivation of DEB by EH was 1.09. Formation of EBdiol from hydrolysis of DEB was ca. 10 times faster than that from oxidation of BDdiol. It was also found that the oxidation of EB to DEB was much faster than that of BDdiol to EBdiol. The study offers comparative enzyme kinetic data of different BD metabolic steps, which is useful for quantitative interspecies comparison. Furthermore, a new application of cob(I)alamin was demonstrated for the measurement of enzyme kinetics of compounds that form electophilically reactive metabolites.

Cob(I)alamin reacts with sucralose to afford an alkylcobalamin: Relevance to in vivo cobalamin and sucralose interaction

“…Presence of adventitious agents, promutagen activation and cytochrome P450 (3.34 nmoles/mL) content were provided by the supplier (Moltox). Epoxide hydrolase activity was assessed by hydrolysis of an oxirane (1,2-epoxy-3-butene) as in our earlier work (Motwani et al, 2009). …”

“…10 × excess of Cbl(I) (Scheme 3, Route A). A previously developed method in our earlier work for trapping electrophilic substances by Cbl(I) was employed (Haglund et al, 2006;Motwani et al, 2009). To obtain the Cbl(I)-species, OH-Cbl was reduced by sodium borohydride with cobalt(II) nitrate as reduction catalyst under an inert atmosphere using argon.…”

“…We considered the possibility that Cbl(I) generated from reduction of cobalamin species could scavenge the sucralose in vivo (Scheme 1), resembling the reactions with alkyl halides and oxiranes in aqueous solutions (Alsberg et al, 2001;Anderson et al, 1992;Dixon et al, 1983;Fred et al, 2004;Haglund et al, 2006;Haglund et al, 2003;Motwani et al, 2009;Schrauzer et al, 1969). To examine this we used an in vitro system made up of human liver S9 containing OH-Cbl.…”

“…hydroxocobalamin (OH-Cbl)}, following reduction to Cbl(I), is known to react with alkyl halides (Anderson et al, 1992;Schrauzer et al, 1969) and epoxides (Alsberg et al, 2001;Dixon et al, 1983;Fred et al, 2004;Haglund et al, 2006;Haglund et al, 2003;Motwani et al, 2009) to give alkyl-and 2-hydroxyalkyl-cobalamins, respectively, usually by a S N 2-type mechanism. This could be referred in vivo as scavenging of the alkylating substances by Cbl(I).…”

Quantitative analysis by liquid chromatography–tandem mass spectrometry of glycidamide using the cob(I)alamin trapping method: Validation and application to in vitro metabolism of acrylamide

“…Previously, we have introduced cob(I)alamin (Cbl(I)) as a reagent for rapid (ca. 10 5 times faster than standard nucelophiles [31]) trapping of butadiene epoxides formed during in vitro metabolism studies that result in formation of specific alkylcobalamins from each epoxide [32,33]. The alkylcobalamins are stable, have high molecular masses and are polar complexes, which makes them suitable for analysis by LC-UV and LC-MS [34,35].…”