title: New method for the quantitative determination of major protein carbonyls, alpha-aminoadipic and gamma-glutamic semialdehydes: investigation of the formation mechanism and chemical nature in vitro and in vivo.
abstract: Alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) are identified as the major carbonyl products in oxidized proteins. To elucidate the formation pathway of AAS and GGS in vivo, we developed and validated a new quantification method. AAS and GGS in proteins were derivatized by reductive amination with NaCNBH(3) and p-aminobenzoic acid, a fluorescent reagent, followed by acid hydrolysis. It is noteworthy that the fluorescent derivatives were completely stable during acid hydrolysis. The present method permitted the specific, accurate, and sensitive quantification of both semialdehydes by fluorometric high-performance liquid chromatography. Analysis of proteins oxidized by various oxidation systems revealed that AAS and GGS are notably generated by the reaction of proteins with (*)OH, which is produced by metal-catalyzed oxidation (MCO). Furthermore, exposure of transferrin and human plasma to ascorbic acid and H(2)O(2) significantly promoted the formation of AAS and GGS in vitro, suggesting that both semialdehydes can be generated by MCO in vivo. We also demonstrated their generation through oxidative stress induced by acute iron overload in vivo. In this paper, we describe this analytical technique for simple and precise measurement of AAS and GGS and discuss their formation mechanism in vivo.
authors.affiliation: Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599-8531, Japan. akagawa@biochem.osakafu-u.ac.jp
authors.forename: Mitsugu
authors.initials: M
authors.lastname: Akagawa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sasaki
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Ishii
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kurota
authors.forename: Mari
authors.initials: M
authors.lastname: Yotsu-Yamashita
authors.forename: Koji
authors.initials: K
authors.lastname: Uchida
authors.forename: Kyozo
authors.initials: K
authors.lastname: Suyama
mesh_terms: D015074:2-Aminoadipic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000737:chemistry; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001798:Blood Proteins / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005971:Glutamates / Q000032:analysis / Q000097:blood / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010084:Oxidation-Reduction; D050050:Protein Carbonylation / Q000187:drug effects; D051381:Rats; D014168:Transferrin / Q000494:pharmacology
chemical_list: D001798:Blood Proteins; D005971:Glutamates; D014168:Transferrin; D015074:2-Aminoadipic Acid; C000061:allysine; C021199:glutamic acid gamma-semialdehyde; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid