Analysis of nitrite/nitrate in biological fluids: Denitrification of 2-nitropropane in F344 rats

8-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats. To obtain evidence that denitrification of 2-NP also occurs in vivo we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction. However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na2CO3, and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H3P4. With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment. Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 mu M (PB-pretreated) and 940 mu M (vehicle-pretreated controls). The PB-pretreated and control rats, respectively, excreted 250 and 120 mu mol nitrate/nitrite in the 24-h urine post 2-NP treatment. The modifications described make the method more specific, reproducible, and more widely applicable. (C) 2000 Academic Press.