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Effect of Hemolyzed Plasma on the Batch Measurement of Nitrate by Nitric Oxide Chemiluminescence

¹ Vascular Biology Program, Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, N6B 1B8 Canada
Departments of
² Medical Biophysics,
³ Anaesthesia,
⁴ Respirology, and
⁵ Pharmacology-Toxicology, University of Western Ontario, London, Ontario, N6A 5C1 Canada

^aaddress correspondence to this author at: Department of Medicine and Pharmacology-Toxicology, University of Western Ontario, London, Ontario, N6A 5C1 Canada; fax 519-663-3789, e-mail dfreeman@uwo.ca

Nitric oxide (NO) is a potent vasodilator and regulator of vascular tone, a neurotransmitter, and a cytotoxic agent (1)(2). In aqueous aerobic environments, the primary decomposition product of NO is nitrite (NO₂^-) (3), with further oxidation to nitrate (NO₃^-) being dependent on the presence of additional oxidizing species such as oxyhemoproteins (3). Collectively, these NO oxidation products are referred to as NO_x^-. Several analytical techniques have been used to quantify NO_x^- species, including spectrophotometric assays based on the Griess reaction (4)(5) and enzymatic reduction of NO₃^- (6), gas chromatography–mass spectrometry (7), chromatographic flow systems (8), and chemiluminescence (9)(10)(11).

NO_x^- may be important in sepsis (12)(13)(14)(15)(16). Of possible analytical importance is intravascular hemolysis during sepsis (17)(18)(19). The effect of hemolysis on NO_x^- analysis is unknown. Other potential causes of intravascular hemolysis include drug-induced hemolytic anemia, hemolytic transfusion reactions, and artificial heart valves (20), and hemolysis can also occur during blood collection (21) and inappropriate blood storage. The objectives of our study were (a) to determine whether hemolysis interferes with the determination of plasma NO₃^- by NO chemiluminescence batch methodology and (b) to determine whether the interference could be eliminated by sample pretreatment.

We purchased helium and oxygen from Praxair. Other chemicals were from Sigma-Aldrich. All chemicals were reagent-grade quality. Deionized water was used to prepare all solutions. Blood samples were obtained from healthy human volunteers by venipuncture with heparin as anticoagulant. Aqueous NO₃^- calibrators (25 µmol/L) were prepared in deionized water. Digitonin in phosphate-buffered saline (PBS), at final concentrations of 0, 20, 45, and . . . [Full Text of this Article]

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References

The following articles in journals at HighWire Press have cited this article:

				R. M. Bateman, C. G. Ellis, and D. J. Freeman Optimization of Nitric Oxide Chemiluminescence Operating Conditions for Measurement of Plasma Nitrite and Nitrate Clin. Chem., March 1, 2002; 48(3): 570 - 573. [Full Text] [PDF]