Detection of hydroxyl radicals by D-phenylalanine hydroxylation: A specific assay for hydroxyl radical generation in biological systems

Roberto Biondi, Yong Xia, Ruggero Rossi, Nazareno Paolocci, Giuseppe Ambrosio, Jay L. Zweier

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)138-145
Number of pages8
JournalAnalytical Biochemistry
Volume290
Issue number1
DOIs
StatePublished - Mar 1 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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