Diode Laser Spectrometer for Diagnostic Assessment of Exhaled Air Components.

The main requirements for a screening test are simplicity, non-invasiveness, safety of testing procedures, high processing speed, and ability to detect diseases at an early stage. A multichannel gas analyzer for assessment of exhaled air composition (diode laser spectrometer), non-invasive screening, and biomedical testing was developed on the basis of near-infrared diode lasers with fiber output. The device measures the following exhaled air components: 12CO2, 13CO2, CH4, NH3, H2O, and H2S. The concentration of molecules was measured in a multi-pass Herriot cell with a reference length of 40 cm, 1.8 L volume, and a total optical path length of 26 m. Three diode lasers manufactured by NTT Electronics (Japan) were used in the work. Detection of CH4 was carried out in the 1.65 µm wavelength range, 12CO2, 13CO2, and H2S levels were measured in the 1.60 µm range, NH3 and H2O in the 1.51 µm range. All measurements were taken in real time. Clinical testing of the spectrometer was carried out at V.M. Buyanov City Clinical Hospital of Moscow Department of Health. More than 150 patients were examined. The tests included analysis and measurement of these molecular components in the exhaled air of patients with various diseases. The content of these components was studied in conditions of various changes in the human physiological state (dosed physical activity, relaxation, psychoemotional stress, etc.). The studies have demonstrated efficacy of using the developed hardware system for assessment of exhaled air components in order to reveal functional disorders in various diseases of the digestive system, cardiorespiratory system, diseases caused by impaired nitrogen-excreting function of the kidneys, etc.

Mechanism of inhibition of catalase by nitro and nitroso compounds.

Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested--nitroarginine, nitroglycerol, nitrophenol, and furazolidone--gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.

Proposed mechanism of nitrite-induced methemoglobinemia.

A scheme of development of nitrite-induced oxyhemoglobin oxidation in erythrocytes based on the analysis of experimental data is proposed. It was found that, contrary to widespread opinion, direct oxidative-reductive interaction between hemoglobin and nitrite is absent or negligible under physiological conditions. The driving stage of this process is methemoglobin-catalyzed peroxidase oxidation of nitrite. The product of the oxidation (presumably NO2*) directly oxidizes oxyhemoglobin to methemoglobin-peroxide complex without hydrogen peroxide release into the environment. The oxidant itself is reduced to nitrite or oxidized to nitrate as a result of interaction with another NO2* molecule. Thus, the stoichiometry of the process depends on the ratio of rates of these two reactions. Substances that are able to compete with nitrite for peroxidase and therefore to prevent the nitrite oxidation effectively protect hemoglobin from oxidation. Catalase is not able to destroy methemoglobin-peroxide complexes, but it can prevent their production in the course of interaction of methemoglobin and free peroxide by destroying the latter.

Nitrite-catalase interaction as an important element of nitrite toxicity.

It was established that nitrite in the presence of chloride, bromide, and thiocyanate decreases the rate of hydrogen peroxide decomposition by catalase. The decrease was recorded by the permanganatometric method and by a method of dynamic calorimetry. Nitrite was not destroyed in the course of the reaction and the total value of heat produced in the process was not changed by its presence. These facts suggest that nitrite induces inhibition of catalase with no change in the essence of the enzymatic process. Even micromolar nitrite concentrations induced a considerable decrease in catalase activity. However, in the absence of chloride, bromide, and thiocyanate inhibition was not observed. In contrast, fluoride protected catalase from nitrite inhibition in the presence of the above-mentioned halides and pseudohalide. As hydrogen peroxide is a necessary factor for triggering a number of important toxic effects of nitrite, the latter increases its toxicity by inhibiting catalase. This was shown by the example of nitrite-induced hemoglobin oxidation. The naturally existing gradient of chloride and other anion concentrations between intra- and extracellular media appears to be the most important mechanism of cell protection from inhibition of intracellular catalase by nitrite. Possible mechanisms of this inhibition are discussed.