Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method.
Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.
Ricart-Jané
D
D
Departament de Bioquímica i Biologia Molecular, Unitat B, Facultat de Biologia, Universitat de Barcelona, Spain.
Llobera
M
M
López-Tejero
M D
MD
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D019343:Citric Acid / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D005029:Ethylenediamines; D016166:Free Radical Scavengers / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D008297:Male; D009566:Nitrates / Q000097:blood*; D009573:Nitrites / Q000097:blood*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013424:Sulfanilamides
D000925:Anticoagulants; D005029:Ethylenediamines; D016166:Free Radical Scavengers; C095000:Griess reagent; D009566:Nitrates; D009573:Nitrites; D013424:Sulfanilamides; D019343:Citric Acid; D006493:Heparin; D004492:Edetic Acid