Quantifying nanomolar levels of nitrite in biological samples by HPLC-Griess method: special reference to arterio-venous difference in vivo.
Nitrite (NO(2)(-)) is assumed to play an important role in regulation of vascular tone as a reservoir of nitric oxide (NO). To examine its physiological contribution, however, a sensitive method is required for determination of the true level of NO(2)(-) in biological samples. To this end, practical consideration to avoid NO(2)(-) contamination through the quantification procedure is important. We present here a highly sensitive and accurate method for determining NO(2)(-) in plasma by improving the HPLC-Griess system with minimal NO(2)(-) contamination in the samples. The system achieved high sensitivity (detection limit of 2 nM and sensitivity to 1 nM) and complete separation of the NO(2)(-) signal peak by modifying the system setup and mobile phase. Using this method, we achieved acceptable quantification of low NO(2)(-) levels in plasma. Deproteinization by ultrafiltration and exposure to atmosphere before measurement were identified as the major sources of NO(2)(-) contamination during sample processing. We addressed these issues by the use of methanol for deproteinization and gas-tight caps. These countermeasures allowed us to detect small arterio-venous NO(2)(-) differences in rabbit plasma that may indicate kinetic difference of NO(2)(-) in a small number of samples (n = 6). This difference became prominent when NO(2)(-) or a NO releasing agent, NOR1, was intravenously applied. Our results indicate that application of a sensitive method with careful handling is important for accurate determination of NO(2)(-) and that our method is applicable for further examination of the kinetic features of NO(2)(-) in vivo.
Ishibashi
Takaharu
T
Department of Pharmacology, School of Nursing, Kanazawa Medical University, Ishikawa, Japan. ishitaka@kanazawa-med.ac.jp
Miwa
Tomoko
T
Shinkawa
Ikumi
I
Nishizawa
Naoki
N
Nomura
Mihoko
M
Yoshida
Junko
J
Kawada
Tomie
T
Nishio
Matomo
M
D000818:Animals; D002339:Carotid Arteries / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005029:Ethylenediamines; D005263:Femoral Artery / Q000378:metabolism*; D005268:Femoral Vein / Q000378:metabolism*; D006801:Humans; D007601:Jugular Veins / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000032:analysis* / Q000097:blood*; D011817:Rabbits; D013424:Sulfanilamides
D005029:Ethylenediamines; C095000:Griess reagent; D009573:Nitrites; D013424:Sulfanilamides; D009569:Nitric Oxide