Nitrate reductase

THU (purity>99% w/w by HPLC chromatogram) was synthesized and provided by the Research and Development Institute, The Government Pharmaceutical Organization, Bangkok, Thailand. Phenylephrine (Phe), thiobarbituric acid (TBA), 5,5 dithio-bis-2-nitrobenzoic acid (DTNB), ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), glutathione (GSH), butylated hydroxytoluene (BHT), sulfanilamide, dinitrophenylephrinenylhydrazine (DNPH), N-1-nepthylethylenediamine dihydrochloride (NED), 1,1,3,3-tetraethoxypropane, bromophenol blue, 2-mercaptoetanol, lucigenin and guanidine were purchased from Sigma-Aldrich Pte. Ltd. (Singapore). Nitrate reductase, nicotinamide adenine dinucleotide phosphate (NADPH) and glucose-6-phosphate dehydrogenase were obtained from Roche Applied Sciences (Mannheim, Germany). Acetylcholine chloride (ACh), sodium nitroprusside (SNP), trichloroacetic acid (TCA), metaphosphoric acid (MPA), 1-methyl-2 vinyl-pyridinum trifate (M2VP), lucigenin, Tween and skimmed milk were obtained from Fluka Chemika Co. Ltd (Buch, Switzerland). Mouse monoclonal anti-eNOS was obtained from BD Biosciences (CA, USA). Anti-mouse IgG antibody, a rabbit polyclonal anti-inducible nitric oxide symthase (iNOS) antibody and anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals used were of analytical grade quality.

Nitrite levels in the media were measured in duplicate by Griess reaction with sodium nitrite as the standard as described [54 (link),57 (link)]. Briefly, nitrate in 0.1 mL media was first reduced at 37 °C for 20 min with 10 µL nitrate reductase (0.4 U/mL, Roche Diagnostics GmbH, Mannheim, Germany). Media samples were then mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 25% (v/v) H₃PO₄) and incubated for 5 min at room temperature, and the optical density was measured at 523 nm on an ELISA photometer [54 (link),57 (link)]. The data were normalized to the cartilage wet weight.

Quantification of nitrite, the stable metabolite of nitric oxide, was measured colorimetrically via Griess reaction. Aliquots of plasma or pancreas homogenates were preincubated for 30 min at room temperature with 50 μM nicotinamide adenine dinucleotide phosphate (Sigma-Aldrich, St. Louis, MO, USA) and 24 mU nitrate reductase (Roche Diagnostics Gmbh, Mannheim, Germany), and then the samples were treated with 0.2 U lactate dehydrogenase (Roche) and 0.5 mol sodium pyruvate for 10 min. The coloration was developed by adding Griess reagent (Merck KGaA, Darmstadt, Germany; 1:1, vol/vol). Finally, after 10 min at room temperature, absorbance was recorded by 96 well plate microtiter at λ 540 nm. Nitrite levels were determined using a standard curve and expressed as nanomoles of NO₂⁻/NO₃⁻ per ml of plasma or NO₂⁻/NO₃⁻ per milligram of protein. Protein concentration was measured using TAKE 3 nanodrop.

For the determination of nitrite and nitrate levels in GCF samples, 300 ml of phosphate buffer (50 mM, pH: 7.4) was added onto each sample and the samples were mixed vigorously for the extraction of nitrite and nitrate into buffer. Nitrite and nitrate content of the samples extracts was determined using Griess reagent (Grisham et al., 1996 (link)). The sum of nitrate and nitrite was measured by the same assay after reduction of nitrate to nitrite with nitrate reductase from Aspergillus niger (Roche Diagnostics, Mannheim, Germany). Nitrite and nitrate levels in both saliva and plasma were determined by the same method without any pretreatment. The nitrate concentrations were calculated by subtracting the nitrite level from the total nitrite level (nitrite + nitrate). This assay is in the routine research analyze protocol of the laboratories of Hacettepe University Faculty of Medicine Department of Biochemistry for more than a decade and its sensitivity and specificity is being tested routinely by the university staff, together with other routine tests. However, commercial standards were implemented to confirm its sensitivity and specificity in the present study.

Nitrate concentrations were evaluated according to the Griess method, as described previously [23 (link)]. Fifteen microliters of serum or homogenised spleen tissue from each mouse was incubated for 3 hours at room temperature with 5 μL of nitrate reductase (5 U/mL; Boehringer Mannheim, Laval, Quebec, Canada) and 15 μL of NADPH (1.25 mg/mL; Boehringer). Following incubation, 100 μL of Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride, and 1% orthophosphoric acid; Sigma Chemical Co. St. Louis, MO, USA) and 100 μL of trichloroacetic acid (10% aqueous solution) were added, and this mixture was incubated for 10 min at room temperature. Subsequently, protein precipitates were removed by centrifugation at 14,000 rpm for 5 min. Next, 100 μL of each supernatant was transferred to a 96-well flat-bottom plate. Concentrations were evaluated in an ELISA reader (Stat Fax Plate Translator, USA, to 540 nm) using a standard curve with sodium nitrate (Sigma), which was diluted in similarly prepared pooled sera from uninfected control CBA/Ca mice.

Serum nitrate concentrations were evaluated according to the Griess method as described previously [29 (link)]. Briefly, 15 μL of serum from each mouse was incubated with 5 μL of nitrate reductase (5 U/mL; Boehringer Mannheim, Laval, Quebec, Canada) and 15 μL of NADPH (1.25 mg/mL; Sigma-Aldrich) for 15 min at room temperature; 100 μL of Griess reagent (Sigma-Aldrich) and 100 μL of trichloroacetic acid (10% aqueous solution) were added. The protein precipitate was then removed by centrifugation at 14,000 rpm for 5 min, and 100 μL of each supernatant was transferred to a 96-well flat-bottom plate. Concentrations were evaluated by an ELISA reader (Stat Fax Plate Translator, NY, USA) at 540 nm using a standard curve comprising sodium nitrate (Sigma-Aldrich) diluted in pooled serum from uninfected control mice treated in the same manner as the experimental samples.