Aromatic hydroxylation of phenylalanine as an assay for hydroxyl radicals: Application to activated human neutrophils and to the heme protein leghemoglobin

Attack of hydroxyl radical (·OH), generated by a Fenton system at physiological pH, upon l-phenylalanine produces three isomeric tyrosines, o-tyrosine (2-hydroxyphenylalanine), m-tyrosine (3-hydroxyphenylalanine), and p-tyrosine (4-hydroxyphenylalanine). These may be separated by high-performance liquid chromatography and measured using an electrochemical detector. Since l-phenylalanine is relatively nontoxic, it is proposed that generation of these three tyrosines from phenylalanine can be used as an assay for ·OH in biological systems. The use of the assay to measure ·OH production by leghemoglobin (plus H2O2) and by activated human neutrophils is described. No ·OH production by activated human neutrophils was observed unless a source of iron ions was added to the reaction mixture, which suggests that these cells do not release an iron “promoter” of ·OH generation from superoxide and hydrogen peroxide.