Quantifying Nanomolar Levels of Nitrite in Biological Samples by HPLC-Griess Method: Special Reference to Arterio-Venous Difference in vivo

Abstract

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. NO 2

Different Disappearance Rates of Plasma Nitrite (NO2-) Contribute to Apparent Steady-State Arterio-Venous Differences in Anesthetized Animals

“…It is also unclear whether plasma NO 2 Ϫ levels reflect in vivo changes in NO 2 Ϫ levels. 10,12,18) Exogenously administered NO 2 Ϫ or NO resulted in a large A-V difference in plasma NO 2 Ϫ , with arterial blood preferentially reflecting the changes, 10,14,17) presumably due to greater contributions by RBCs or tissue as a buffering compartment under a state of lower oxygenation. A possible difference in the uptake rate of RBCs could not be large enough to explain such a large A-V difference, and contributions of tissue factors would be a great concern.…”

“…8 min from blood sampling observed by us previously. 10,14) Therefore, rapid sample processing is critical for more accurate estimation of plasma NO 2 Ϫ levels. We applied an identical procedure to rats and guinea pigs to examine whether species differences impact the evaluation of plasma NO 2 Ϫ concentrations under steady-state conditions.…”

“…[10][11][12][13][14][15][16] The A-V difference may reflect consumption of NO 2 Ϫ to form NO during peripheral circulation for preservation of blood flow at lower oxygen tension. 12) Although we have previously reported a small but significant A-V difference under steadystate conditions, 10,14,17) these observations are complicated by a report that, in isolated blood, uptake of NO 2 Ϫ by RBCs is faster in venous blood than in arterial blood, 15) a scenario that may cause artificial apparent A-V differences when arterial and venous blood samples are treated after some delay. If this situation is indeed the case, our previous work reporting kinetic parameters 10) should also be re-evaluated.…”

“…When we removed the reduction column and increased the loading volume to 100 ml, the sensitivity and detection limit for NO 2 Ϫ improved to 1 nM and 2 nM, respectively. 10,14) Special attention was paid to exclude possible sources of NO 2 Ϫ and NO 3 Ϫ contamination during the entire procedure. Unless otherwise specified, all laboratory wares were washed five times with pure water (resistance above 18.3 MW and almost completely free of NO x contamination; MILLI-Q SP, Millipore, Bedford, MA, U.S.A.) to minimize NO 2 Ϫ and NO 3 Ϫ contamination.…”

“…Unless otherwise specified, all laboratory wares were washed five times with pure water (resistance above 18.3 MW and almost completely free of NO x contamination; MILLI-Q SP, Millipore, Bedford, MA, U.S.A.) to minimize NO 2 Ϫ and NO 3 Ϫ contamination. 14,23) Hb and metHb Measurements Arterial and venous blood (0.6 ml each) were sampled with syringes equipped with heparin sheets, and 10 ml was used to determine total Hb levels. Photometric quantification of Hb content was performed using the Hemoglobin B-test Wako (SLS-hemoglobin method; Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions.…”