Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine

Abstract

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

The Metabolism and Dechlorination of Chlorotyrosine in Vivo

“…Animal procedures were in accordance with the Home Office, UK guidelines. 4 ]Chlorotyrosine, [D 5 ]chloro-HPA, and [D 6 ]HPA were synthesized by deuterium exchange as described (12,17) using 50 mg of either chlorotyrosine, chloro-HPA, or HPA as starting material. The resulting products were dissolved in 0.1% (v/v) TFA/water (adjusted to pH 5.0 with ammonia solution) and extracted on an LC18 reverse-phase column, pre-washed with 2 ml of methanol and 5 ml of 0.1% (v/v) TFA/water (pH 5.0).…”

“…The products were washed with water, and the deuterated products eluted with 4 ml of 30% (v/v) methanol in water. The products were purified further by thin-layer chromatography (12), and their concentrations determined against known amounts of unlabeled standards by GC/MS (see below).…”

“…However, one of the major disadvantages of measuring chlorotyrosine or nitrotyrosine is that modified proteins may undergo rapid catabolism (10), and the resulting free chlorotyrosine or nitrotyrosine are taken up by cells, metabolized, and excreted. Thus, it is well established that nitrotyrosine is metabolized to 3-nitro-4-hydroxy-phenylacetic acid (nitro-HPA), which is the major urinary metabolite (11,12), and urinary levels increase during endotoxemia (12).…”

“…The products were purified using high pressure liquid chromatography as described (12). [ 13 C 8 ]HPA was synthesized following the deamination and decarboxylation of [ 13 C 9 ]tyrosine using Taiwan cobra venom as described previously (12).…”

“…The products were purified using high pressure liquid chromatography as described (12). [ 13 C 8 ]HPA was synthesized following the deamination and decarboxylation of [ 13 C 9 ]tyrosine using Taiwan cobra venom as described previously (12). [ 13 C 8 ]Chloro-HPA was synthesized in the same way as chlorotyrosine, and the concentrations of 13 C-labeled standards determined against known amounts of unlabeled standards by GC/MS.…”