Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species - PubMed (original) (raw)

Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species

Enika Nagababu et al. Free Radic Biol Med. 2007.

Abstract

Recent studies have demonstrated that plasma nitrite (NO2-) reflects endothelial nitric oxide synthase activity and it has been proposed as a prognostic marker for cardiovascular disease. In addition, NO2- itself has been shown to have biological activities thought to be triggered by reduction back to NO in blood and tissues. The development of sensitive and reproducible methods for the quantitative determination of plasma NO2- is, therefore, of great importance. Ozone-based chemiluminescence assays have been shown to be highly sensitive for the determination of nanomolar quantities of NO and NO-related species in biological fluids. We report here an improved direct chemiluminescence method for the determination of plasma NO2- without interference of other nitric oxide-related species such as nitrate, S-nitrosothiols, N-nitrosamines, nitrated proteins, and nitrated lipids. The method involves a reaction system consisting of glacial acetic acid and ascorbic acid in the purge vessel of the NO analyzer. Under these acidic conditions NO2- is stoichiometrically reduced to NO by ascorbic acid. Fasting human plasma NO2- values were found in the range of 56-210 nM (mean=110+/-36 nM). This method has high sensitivity with an accuracy of 97% and high precision (CV<10%) for determination of plasma nitrite. The present method is simple and highly specific for plasma NO2-. It is particularly suited for evaluating vasculature endothelial NO production that predicts the risks for cardiovascular disease.

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Figures

Fig.1

Fig.1

A) Chemiluminescence signals obtained under acidic conditions. The purge vessel contained 87.5% glacial acetic acid in absence and presence of 50 mM ascorbic acid and/or 200mM sulfanilamide in a total volume of 8 ml. Samples injected into the purge vessel were: 20 pmoles nitrite, 100 pmoles of each RSNO (GSNO or albumin-SNO), RNNO (_N_-nitrosodimethylamine or _N_-nitroso-N-methylurea), nitrooleate, nitrotyr (nitrotyrosine) or Hb(II)NO (iron nitrosylhemoglobin). ASC indicates the addition of ascorbic acid; Sulf indicates the addition of sulfanilamide. B) Release of NO from S- nitrosoalbumin by KI or I3- or ascorbate in glacial acetic acid. The reaction mixture for I3- chemiluminescence assay consisted of 7 ml glacial acetic acid, 0.5 ml (1M) KI solution and 0.5 ml (0.35 M) I2 solution. The reaction mixture for KI assay consisted of 7 ml glacial acetic acid and 1 ml (0.5 M) KI solution. The reaction mixture for ascorbate consisted of 7 ml glacial acetic acid and 1 ml (0.5 M) ascorbate solution. 50 pmoles S-nitrosoalbumin was injected into the purge vessel.

Fig.1

Fig.1

A) Chemiluminescence signals obtained under acidic conditions. The purge vessel contained 87.5% glacial acetic acid in absence and presence of 50 mM ascorbic acid and/or 200mM sulfanilamide in a total volume of 8 ml. Samples injected into the purge vessel were: 20 pmoles nitrite, 100 pmoles of each RSNO (GSNO or albumin-SNO), RNNO (_N_-nitrosodimethylamine or _N_-nitroso-N-methylurea), nitrooleate, nitrotyr (nitrotyrosine) or Hb(II)NO (iron nitrosylhemoglobin). ASC indicates the addition of ascorbic acid; Sulf indicates the addition of sulfanilamide. B) Release of NO from S- nitrosoalbumin by KI or I3- or ascorbate in glacial acetic acid. The reaction mixture for I3- chemiluminescence assay consisted of 7 ml glacial acetic acid, 0.5 ml (1M) KI solution and 0.5 ml (0.35 M) I2 solution. The reaction mixture for KI assay consisted of 7 ml glacial acetic acid and 1 ml (0.5 M) KI solution. The reaction mixture for ascorbate consisted of 7 ml glacial acetic acid and 1 ml (0.5 M) ascorbate solution. 50 pmoles S-nitrosoalbumin was injected into the purge vessel.

Fig. 2

Fig. 2

Ascorbate concentration dependent nitrite reduction to NO under acidic conditions. The reaction mixture contained 7 ml of glacial acetic acid and varying concentration of ascorbic acid in a total volume of 8 ml. 25 pmoles nitrite was injected into the purge vessel. A.U, Arbitrary Units.

Fig.3

Fig.3

Chemiluminescence signals of nitrite and standard curve. (A) 100 μl of 10 nM to 5120 nM nitrite was injected into reaction mixture contained 7 ml glacial acetic acid and 1 ml (0.5 M) ascorbic acid solution (inset shows standard curve obtained by integrating the area under the curve using the Origin program) (B) Standard curve for nitrite from 10nM to 640 nM. The values are the mean of 3 experiments with duplicate determinations for each experiment.

Fig.3

Fig.3

Chemiluminescence signals of nitrite and standard curve. (A) 100 μl of 10 nM to 5120 nM nitrite was injected into reaction mixture contained 7 ml glacial acetic acid and 1 ml (0.5 M) ascorbic acid solution (inset shows standard curve obtained by integrating the area under the curve using the Origin program) (B) Standard curve for nitrite from 10nM to 640 nM. The values are the mean of 3 experiments with duplicate determinations for each experiment.

Fig.4

Fig.4

The reduction of nitrite to NO by the ASC reductive chemiluminescence assay is comparable with I3- reductive chemiluminescence assay. The reaction mixture for I3- (square) and ASC chemiluminescence assay (circle) was as mentioned in figure 1B. 10 μl to 250 μl of 0.2 μM nitrite that corresponds to 10 pmoles, 20 pmoles, 30 pmoles, 40 pmoles and 50 pmoles were injected into the purge vessel. Values are the mean of 3 experiments.

Fig.5

Fig.5

Chemiluminescence signals of plasma nitrite: Peak 1, 100 μl plasma was injected into 8 ml of 87.5% glacial acetic acid reagent. This peak was completely suppressed in the presence of 200mM sulfanilamide (sulfa) in 87.5% glacial acetic acid. Peak 2, 100 μl plasma was injected into7 ml glacial acetic acid and 1 ml (0.5M) ascorbic acid. This peak was almost completely suppressed in the presence of 200 mM sulfa in 87.5% glacial acetic acid and 1 ml (0.5M) ascorbic acid.

Fig.6

Fig.6

Flow diagram for determination of plasma nitrite using (A) I3- reductive chemiluminescence method and (B) ascorbate reductive chemiluminescence method.

Fig.7

Fig.7

Nitrite consumption in blood: Nitrite (1μM) was added to venous blood and kept on ice. The first aliquot was centrifuged within 10 sec to separate plasma. This plasma nitrite value was considered as 100%. Additional aliquots were centrifuged at different times followed by a determination of the nitrite in the supernatant.

Fig.8

Fig.8

Nitrite levels in human plasma: 100μl plasma was injected into the purge vessel that contained 7 ml of glacial acetic acid and 1 ml of 0.5 M ascorbic acid at 37 °C. Values are the mean of quadruplicates for each sample.

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