A method for rapid fluorometric assay of intracellular nitric oxide (NO) formation was developed for use in cultured neuronal cells. In a cell-free system 2,7-dichlorofluorescin (DCF), a non-fluorescent species, is oxidized by NO to dichlorofluorescein, a fluorescent compound. Addition of NO to a solution containing DCF increased the fluorescent signal within 10 s and continued to increase slowly over a 10-min period. The intensity of the fluorescence was dependent upon the concentration of NO. In DCF-loaded PC12 cells, addition of NO markedly increased fluorescence (limit of detection = 16 microM NO) and pretreatment with reduced hemoglobin (Hb) inhibited the NO-mediated increase of fluorescence in both the cell-free system and PC12 cells. In PC12 cells loaded with DCF, the NO generator sodium nitroprusside (SNP) produced a rapid increase of fluorescence. To rule out the possibility that reactive oxygen species (ROS) mediated the increased of fluorescence, superoxide dismutase (SOD) and catalase were added to the cuvette. The enzymes did not alter the fluorescence generated after addition of NO to PC12 cells. This assay was used to determine the ability of glutamate to stimulate NO production in cerebellar granule cells. When 10 microM glutamate was added to DCF-loaded cerebellar granule cells, a rapid increase in fluorescence was noted. The fluorescence was blocked approximately 50% after addition of either Hb or SOD, or by pretreatment with NG-nitro-L-arginine methyl ester (300 microM), a nitric oxide synthase (NOS) inhibitor. It was concluded that glutamate stimulated intracellular generation of both NO and ROS, and at least 50% of the oxidation of DCF was attributed to intracellular generation of NO. These results demonstrate that oxidation of DCF by NO can be used to measure intracellular generation of NO and by adding either Hb or SOD to the cell system, the extent of oxidation of DCF attributed to NO and ROS can be determined.

Analysis of Variance↗

PubMedDate not supplied

Quantification of expired metabolites following potassium cyanide administration: a new method.

A sensitive and specific radiolabel method for measuring expired hydrogen cyanide (HCN) or carbon dioxide (CO2) derived from cyanide was developed. An ethanol collecting solution containing a 10(-2) M of cobalt chloride trapped 88% of the H14CN passed through the solution following acid volatilization of a known amount of K14CN. The range of linearity, r = 0.998, exceeded the 0.01 to 0.1 mumoles tested for measuring the pulmonary metabolites. The cobalt chloride collecting solution trapped less than 0.02% of the 10 to 100 mumoles of 14CO2 generated by acid hydrolysis of 14C-sodium bicarbonate (NaH14CO3). Introduction of a second collecting solution specific for CO2, composed of ethanol: ethanolamine (2:1, v/v), was used to collect the CO2 derived from cyanide. Following the subcutaneous (sc) administration of 4.6 mg/kg of potassium cyanide (KCN) and 4.5 microcuries (microCi) K14CN, 1 to 2% and 2 to 3% of the dose was expired as H14CN and 14CO2, respectively.

Animals↗

PubMedDate not supplied

Pharmacokinetic analysis of potassium cyanate in mice using whole blood and expired air.

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Animals↗