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.