Determination of Nitrate in Biological Fluids Using Nitrate Reductase in a Flow System.

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The concentration of nitrate in biological fluids was determined using nitrate reductase (NR) in a flow system. A merging zone method was applied, in which a zone of NR and that of nitrate in separate streams were merged, and allowed to react. The decrease in NADPH caused by the reaction between NR and nitrate was measured at 340 nm. The conditions were as follows: length of the reaction coil used for the enzymatic reaction was 250 cm; 0.1 M PIPES buffer (pH 7.5) was used as the first carrier (C1); 0.1 M PIPES buffer (pH 7.5) containing 0.1 mM NADPH was used as the second carrier (C2); the reaction coil was immersed in a water bath maintained at 32°C. Under these conditions, a linear calibration curve (r = 0.994) was plotted for the concentration of nitrate between 1 and 100 μM with a detection limit (S/N = 3) of 0.2 μM. The present method was applied to determine the amount of nitrate in serum, plasma and urine using samples that had not been deproteinized. The concentration of nitrate within each sample was calculated from differences observed in the peak areas obtained in the absence or presence of nitrate reductase. The recovery test of the nitrate added to biological fluids indicated the applicability of the present method to the determination of nitrate in them. The nitrate concentrations determined using this technique correspond well with those of other methods.

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