A method of measuring in vivo nitric oxide (NO) levels is required to detect pathological conditions in which endogenous production is decreased or to identify agents able to release this biomolecule. Unfortunately, nitric oxide has a very short biological half-life and is very difficult to measure. Assay of the oxidative products’ of NO levels, nitrite (NO2 -) and nitrate (NO3 -), measured as total amount, after the reduction of nitrate to nitrite, determined after conversion in an azo dye, is usually the used method, named NOx test. The NOx test is frequently used as a NO biomarker in human studies and also in animal experiments. The aim of this work is to evaluate the suitability of the NOx test for the detection of an instant release of nitric oxide.
Rabbits were used as experimental animals, a validated HPLC-UV/VIS method was used for speciation of nitrite and nitrate. The following substances were administered: blank; “negative blank”: phenyl-N-tert-butylnitrone (PBN); “positive blank” (nitroglycerin); nitrite.
PBN administration significantly increased nitrate and decreased nitrite levels, nitrite administration excessively increased nitrate levels, while nitroglycerin (1 mg/kg) significantly increased both nitrate and nitrite levels.
Results show that NOx test cannot be considered accurate in acute nitric oxide status testing. Nitrite alone should be used as an in vivo released nitric oxide marker.
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