Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems.
Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.
Biondi
R
R
Dipartimento di Medicina Clinica Sperimentale, University of Perugia School of Medicine, Perugia, Italy.
Xia
Y
Y
Rossi
R
R
Paolocci
N
N
Ambrosio
G
G
Zweier
J L
JL
D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005609:Free Radicals; D006801:Humans; D017665:Hydroxyl Radical / Q000032:analysis* / Q000378:metabolism; D006900:Hydroxylation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010649:Phenylalanine / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D005609:Free Radicals; D017665:Hydroxyl Radical; D014443:Tyrosine; D010649:Phenylalanine