Short Communication In Vivo Disposition of 3-Nitro-L-Tyrosine in Rats: Implications on Tracking Systemic Peroxynitrite Exposure

In many pathological conditions such as inflammatory and neurodegenerative diseases, the in vivo toxicity of nitric oxide has been attributed to the toxic oxidant peroxynitrite. Interaction of peroxynitrite with biological molecules can modify tyrosine residues on the proteins at the ortho position resulting in the formation of the stable end-product, 3-nitro-L-tyrosine (3-NT). Recent investigations indicate that changes in the circulating concentrations of 3-NT in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. In the present study, we examined the in vivo disposition characteristics of 3-NT in rats after either a single i.v. bolus dose (10 mg/kg) or a loading and maintenance infusion at 10 or 30 mg/kg. Plasma concentrations of 3-NT were analyzed by a reversed-phase HPLC method. After a single bolus dose of 3-NT at 10 mg/kg, the average half-life of the elimination phase for the drug was 68.5 6 18.4 min (n 5 5). Infusions of 3-NT at two different doses (10 and 30 mg/kg) indicated that the pharmacokinetic properties of 3-NT below plasma concentrations of 100 mM were both linear and stationary. Urinary excretion of unchanged 3-NT was minimal, but two distinct metabolites of 3-NT were identified in the urine collected throughout the study. These findings may be useful in the interpretation of the plasma and urine 3-NT concentrations as possible indices of systemic peroxynitrite exposure. Peroxynitrite is an important toxic product of the reaction between nitric oxide and superoxide anion (O2 ). In many pathological conditions such as atherosclerosis, septic shock, cerebral ischemia, and Alzheimer’s disease, peroxynitrite has been implicated as a major contributing factor to the underlying pathology (Beckmann et al., 1994; Haddad et al., 1994; Smith et al., 1997; Tanaka et al., 1997). Peroxynitrite is a potent oxidant that reacts with major biological molecules. Nitration of tyrosine residues by peroxynitrite on the ortho position has been shown to result in formation of a stable product, 3-nitro-L-tyrosine (3-NT) (Crow and Beckman, 1995; Beckman, 1996). Nitrated tyrosine residues have been demonstrated to exist in proteins of damaged tissues (Beckmann et al., 1994; Haddad et al., 1994; Smith et al., 1997) and have been shown to be involved in regulating the functional activity of various proteins (Ischiropoulos et al., 1992). Recently, steady-state concentrations of free 3-NT in human plasma were determined and estimated to be about 0.05% of that observed for tyrosine (Kamisaki et al., 1996). In pathological conditions such as rheumatoid arthritis, septic shock, and amyotrophic lateral sclerosis, free 3-NT levels have been shown to be increased, possibly reflecting the degree of oxidative damage induced by peroxynitrite (Kaur and Halliwell, 1994; Bruijn et al., 1997; Fukuyama et al., 1997). Measurements of 3-NT levels in blood and synovial fluid samples obtained from patients with rheumatoid arthritis indicated an 80to 100-fold increase in the free 3-NT concentrations in comparison with those observed in normal volunteers (Kaur and Halliwell, 1994). These data indicate that the changes in the circulating 3-NT plasma concentrations in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. Because a basic understanding of the disposition properties of 3-NT can provide useful information in assessing the degree of peroxynitrite exposure, we have examined the in vivo disposition properties of 3-NT in rats after i.v. bolus administration. Furthermore, to simulate conditions under which a rapid surge in 3-NT concentrations is followed by a new steady-state and then a washout phase, we devised an infusion regimen that allowed us to examine both the effect of changes in dose and time on the in vivo disposition properties of 3-NT in rats. Experimental Procedures Materials. 3-NT, N-acetyl-tryptophan, heptane sulfonic acid (sodium salt), 3-amino-L-tyrosine, and p-hydroxyphenyllactic acid were purchased from Sigma Chemical Company (St. Louis, MO). 3-Nitro-4-hydroxyphenylacetic acid (NHPA) and tetranitromethane (TNM) were obtained from Aldrich Chemical Company (Milwaukee, WI). All solvents used were of HPLC grade and obtained from Fisher Scientific (Pittsburgh, PA). HPLC Assay. Plasma concentrations of 3-NT were analyzed by a reversedphase HPLC method. Briefly, 10 ml of the internal standard (N-acetyl-tryptophan, 0.5 mM) was added to 90 ml of plasma. Plasma proteins were then precipitated with 70 to 72% perchloric acid and samples were vortexed and centrifuged. The resultant supernatant was removed and a 50-ml aliquot was injected into a C8 reversed-phase Ultrasphere column (250 3 4.6 mm i.d., 5 mm) that was preceded by a guard column (45 3 4.6 mm i.d.), both from This work was supported in part by Grant HL22273 from the National Institutes of Health. 1 Abbreviations used are: 3-NT, 3-nitro-L-tyrosine; NHPA, 3-nitro-4-hydroxyphenylacetic acid; NHPL, 3-nitro-4-hydroxyphenyllactic acid; TNM, tetranitromethane; AUC, area under the plasma concentration-time curve; MRT, mean