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Novel methods of measuring nitric oxide and nitrite concentrations using cobinamide and cobalamin

  • Author(s): Duan, Kailin Catherine
  • et al.
Abstract

Nitric oxide (NO) is an important signaling molecule produced by isoforms of nitric oxide synthase in mammals. Methods of measuring NO must take into consideration the low concentrations (nanomolar to micromolar) at which it is found in the body. We developed a novel method of direct nitric oxide measurement by measuring the absorbance change of the binding of nitric oxide to cobinamide(II) (Cbi), a vitamin B12 analogue. The absorbance values of NO-Cbi change linearly at 366 nm and 469 nm as increasing amounts of NO are added. The Ka of nitric oxide binding to Cbi was found to be 5.10 × 10⁸ 1/ M. The LOD and LOQ of the Cbi method performed in water were measured at 1.98 [mu]M and 4.39 [mu]µM, respectively. Because this method was only viable in anaerobic systems, we used cobalamin(III) (Cbl) to measure nitrite concentrations. At 375 nm, the absorbance values of NO₂- Cbl increase linearly as nitrite concentration increases. The Ka of this method was measured at 7.23 × 10⁷ 1/M, and the LOD and LOQ in water were measured at 0.672 [mu]M and 3.91 [mu]M, respectively. Compared to the Griess reagent assay, the Cbl(III) method was less sensitive. Improvements upon both the Cbi(II) method and the Cbl(III) method are required for their potential use as cost- effective, sensitive, and easy methods of determining nitric oxide or nitrite concentrations in cell culture. However, the Cbl(III) method may be a viable method of taking nitrite and nitrate measurements in biological samples

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