Application of Raney nickel to measure adducts of styrene oxide with hemoglobin and albumin

Abstract

Adducts of styrene 7,8-oxide (SO) with the blood proteins, Hb and albumin (Alb), were measured following treatment of the proteins with Raney nickel (Ra-Ni) to cleave 2-phenylethanol, which was subsequently assayed. Internal standards were prepared by modifying Hb and Alb with 4-methyl-SO to produce similar adducts. In a preliminary experiment with human blood, which had been modified with [14C]SO in vitro, it was determined that Ra-Ni released 6% of the total binding to globin and 76% of the total binding to Alb. Since Ra-Ni primarily cleaves covalently-bound sulfur species, this suggests that much more binding to human serum Alb occurred at a free cysteine residue than to human Hb. The overall rate of reaction of SO at 37 degrees C in vitro had a half-time of 0.70 h in human blood and of 0.36 h in blood from Sprague-Dawley rats. Adducts of SO with Hb and Alb were measured following SO modification of blood from humans and rats in vitro at 37 degrees C. Slopes of the linear relationships between adduct level and SO concentration allowed the following second-order rate constants [(L/mol of protein)/h] to be estimated: rat Hb, 72; human Hb, 2.4; rat Alb, 63; human Alb, 32. These rate constants show that intrinsic reactivity of SO toward the cysteine residue of human Hb is much lower than that of rat Hb or of Alb from either species. Adducts of Hb and Alb were also measured following administration of both SO and styrene to rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin and hemoglobin adducts as biomarkers of exposure to styrene in fiberglass-reinforced-plastics workers

“…The reagents used for the protein-adduct analyses were the same as those reported by Rappaport et al [17]. The protein-bound internal standards, 4-methyl-SO-Alb and 4-methyl-SO-Hb, were formed by reaction of an aqueous solution of either Alb or globin with 4-methyl-SO.…”

“…In animal experiments it has been demonstrated that both styrene and SO produce cysteine adducts in albumin (Alb) and hemoglobin (Hb) [17,22], N-terminal valine adducts in Hb [11], and carboxylic acid adducts in Hb [19]. However, applications of these assays to styrene-exposed workers in the reinforced-plastics industry have produced inconsistent results [2,3,19,20,25].…”

“…Adducts of Hb and serum Alb were quanti®ed using the Raney-nickel assay to cleave adducts formed by nucleophilic attack of cysteine on either the a-or the b-carbon of SO [17,22]. Thus, two dierent adducts were measured, namely, 2-phenylethanol (2-PE, arising from acarbon attack) and 1-phenylethanol (1-PE, arising from b-carbon attack).…”

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

“…Over the last few years, modified Edman degradation and Raney Nickel procedures have been widely used for the biological monitoring of exposed workers (27,(51)(52)(53)(54)(55). Methods based on modified Edman degradation allow for the determination of adducts on the N-terminal Valine of the globin chains.…”

Fractionation of protein adducts in rats and mice dosed with [ 14 C]pentachlorophenol

“…Several studies have successfully applied Raney Ni to cleave cysteinyl adducts in various tissues, i.e., those of styrene 7,8-oxide styrene in blood proteins (Ting et al 1990;Rappaport et al 1993;Yeowell-O'Connell et al 1996), of benzoquinone in blood and bone-marrow proteins Rappaport et al 1996), of chlorinated quinones from PCP in blood and liver proteins (Lin et al 1999;Tsai et al 2001;Waidyanatha et al 1996), and of polychlorinated biphenyl quinones in liver and brain proteins (Lin et al 2000). By administering [ 14 C/ 12 C 6 ]PCP to animals along with the sequential counting scheme shown in Fig.…”