Abstract
During inflammation, leukocyte-derived eosinophil peroxidase catalyses the formation of hypobromous acid, which can brominate tyrosine residues in proteins to form bromotyrosine. Since eosinophils are involved in the pathogenesis of allergic reactions, such as asthma, urinary bromotyrosine level has been used for the assessment of children with asthma. However, little is known about the metabolism and disposition of bromotyrosine in vivo. The aim of this study was to identify the major urinary metabolites formed during bromotyrosine metabolism and to develop mass spectrometric methods for their quantitation. Deuterium-labeled bromotyrosine was synthesized by deuterium exchange. [D3]bromotyrosine (500nmole) was injected intraperitoneally into Sprague-Dawley rats and urine was collected for 24h in a metabolic cage. 13C-labeled derivatives of bromotyrosine and its major urinary metabolite were synthesized and used as internal standards for quantitation. Following solid phase extraction, urine samples were derivatized to the pentafluorobenzyl ester, and analyzed using isotope dilution gas chromatography and negative-ion chemical ionization mass spectrometry. A novel brominated metabolite, 3-bromo-4-hydroxyphenylacetic acid (bromo-HPA), was identified as the major brominated metabolite of bromotyrosine. Bromo-HPA only accounted for 0.43±0.04% of infused [D3]bromotyrosine and 0.12±0.02% of infused [D3]bromotyrosine was excreted in the urine unchanged. However, ~1.3% (6.66±1.33nmole) of infused [D3]bromotyrosine was excreted in the urine as the de-brominated metabolite, [D3]4-hydroxyphenylacetic acid, which is also a urinary metabolite of tyrosine in mammals. We also tested whether or not iodotyrosine dehalogenase can catalyse de-bromination of bromotyrosine and showed that iodotyrosine dehalogenase is able to de-brominate free bromotyrosine in vitro. We identified bromo-HPA as the main brominated urinary metabolite of bromotyrosine in rats. However, de-halogenation of bromotyrosine is the major metabolic pathway to eliminate free brominated tyrosine in vivo.
Highlights
Eosinophils play an important role in the mammalian immune system during allergic reactions, as well as in immune responses against extracellular parasites
For GC/MS analysis of bromotyrosine and tyrosine, the amine group was derivatized with ethyl heptafluorobutyrate, and the hydroxyl and carboxylic groups were silylated with tert-butyldimethylsilyl and the products analyzed by negative-ion chemical ionization mass spectrometry
Since eosinophil activation is involved in the pathogenesis of eosinophilic asthma, recent studies have focused on measuring urinary concentrations of bromotyrosine as a potential biomarker for assessment of patients with asthma [10,11]
Summary
Eosinophils play an important role in the mammalian immune system during allergic reactions, as well as in immune responses against extracellular parasites. Eosinophils release effectors, such as cationic proteins and eosinophil peroxidase, that lead to host defense and/or tissue damage [1]. Eosinophil peroxidase is a halide peroxidase that preferentially uses bromide to generate hypobromous acid, even at physiological halide concentrations where chloride is almost 1000-fold greater than bromide concentrations [1]. 3-bromotyrosine and 3,5-di-bromotyrosine are detectable following eosinophil peroxidase-induced protein oxidation [2]. Neutrophil-derived myeloperoxidase uses chloride, bromide and nitrite ions to generate halogenating and nitrating agents that lead to the formation of 3-chlorotyrosine, 3-bromotyrosine and 3-nitrotyrosine [4,5,6]. One of the major disadvantages of measuring bromotyrosine or chlorotyrosine is that oxidized proteins undergo proteolysis, and the resulting free halogenated amino acids are metabolized and excreted in the urine [9]
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