Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

doi:10.1016/S0891-5849(03)00406-4

Free Radical Biology and Medicine

Volume 35, Issue 7, 1 October 2003, Pages 790-796

https://doi.org/10.1016/S0891-5849(03)00406-4 Get rights and content

Abstract

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 ± 23 nmol/l), monkeys (367 ± 62 nmol/l), minipigs (319 ± 24 nmol/l), dogs (305 ± 50 nmol/l), rabbits (502 ± 21 nmol/l), guinea pigs (412 ± 44 nmol/l), rats (191 ± 43 nmol/l), and mice (457 ± 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(−/−) and NOS-inhibited wild-type mice revealed that 70 ± 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.

Introduction

Among various mediators released by the endothelium, nitric oxide (NO) plays an important role in regulating vascular tone and inhibiting smooth muscle cell proliferation, blood cell adhesion, and lipid peroxidation 1, 2, 3. NO is released abluminally and into the vascular lumen. Because of the rapid distribution and metabolism of NO, a reliable quantification of its basal production in vivo has been rather challenging. The majority of intravascular NO is inactivated by its reaction with hemoglobin to form nitrate [4]. Nitrate concentrations are influenced by a variety of NO synthase (NOS) independent factors, including dietary nitrate intake, formation of saliva, bacterial nitrate synthesis within the bowel, denitrifying liver enzymes, inhalation of atmospheric gaseous nitrogen oxides, and renal function 5, 6. Due to these factors and the high background level, small changes in plasma nitrate concentrations may not sensitively reflect acute changes in NOS activity [7]. Apart from the predominant metabolism of NO to nitrate, a minor portion of NO undergoes conversion to nitrosated and nitrosylated species and nitrite, respectively 8, 9. Stimulation of endothelial NOS (eNOS) has been shown to result in an acute change in plasma nitrite concentration in the human forearm vasculature [7]. The significance of plasma nitrite in reflecting constitutive NOS-activity is not known. The reported basal nitrite concentrations in plasma of mammals range from “nondetectable” [10] over nanomolar (450 nmol/l) [11] to μmolar (26 μmol/l) [12] levels. These enormous differences in nitrite levels can be rationalized, considering the emerging confounding factors and variations in blood sampling and sample processing as well as the methodological problems inherent to the analytical procedures used. Some methods simply do not possess the sensitivity to allow a precise measurement of nitrite in the proposed physiological concentration range. In addition, the analysis might be affected by proteins, varying redox conditions, and trace contamination with nitrite during sample processing [13].

Using three distinct analytical methods we demonstrate here the uniform occurrence of nanomolar levels of plasma nitrite in different mammalian species, indicative of comparable constitutive NOS activity.

Section snippets

Chemicals and solutions

N^G-monomethyl-l-arginine (l-NMMA) was acquired from Clinalfa (Schwalbach, Germany). Sodium nitrite and nitrate were purchased from Merck (Darmstadt, Germany). All other chemicals were from Sigma-Aldrich (Taufkirchen, Germany or St. Louis, MO, USA).

Characteristics of the study population

To determine the basal plasma nitrite concentration the following species were investigated: 24 healthy human volunteers, 3 monkeys (Maccaca mulatta), 4 Göttinger minipigs, 6 dogs (foxhounds), 7 rabbits (New Zeeland White), 11 rats (Wistar), 5 guinea

Plasma nitrite in mammals

In human plasma, nitrite values were determined using three distinct analytical methods (see Table 1). Measurement of plasma nitrite correlated significantly: (FIA/CLD: r = 0.96; p > .001; n = 18; CLD/HPLC: r = 0.97; p > .001; n = 14). Concentration of plasma nitrite in healthy human volunteers amounted to 305 ± 23 nmol/l (n = 24). Plasma nitrite concentrations of all other investigated species were also in the nanomolar range: monkeys (367 ± 62 nmol/l, n = 3), minipigs (319 ± 24 nmol/l, n =

Discussion

Our data demonstrate the uniform presence of nanomolar concentrations of plasma nitrite, indicative of comparable levels of NOS activity, in various mammals under basal conditions, and suggest that the majority of plasma nitrite is derived from constitutive NOS-activity. In contrast, acute NOS inhibition had no immediate effect on plasma nitrate concentrations due to higher baseline levels and different pharmacokinetic properties.

Acknowledgements

This work was supported by the Biomedizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, by grants from the Deutsche Forschungsgemeinschaft (DFG) (Sche 479/1-1, and Sonderforschungsbereich 612), and National Institutes of Health grant HL69029 (to M.F.). T.R. is a research fellow funded by the DFG Ra 969/1-1. The technical assistance of S. Matern, G. Doemer, C. Ferfers, and B. Berke is gratefully acknowledged.

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Original contributionPlasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Abstract

Introduction

Section snippets

Chemicals and solutions

Characteristics of the study population

Plasma nitrite in mammals

Discussion

Acknowledgements

J. Inorg. Biochem.

Biochim. Biophys. Acta

J. Chromatogr. B Biomed. Appl.

Biochem. Biophys. Res. Commun.

Methods Enzymol.

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

Life Sci.

Biosynthesis and metabolism of endothelium-derived nitric oxide

Annu. Rev. Pharmacol. Toxicol.

Regulation and functional consequences of endothelial nitric oxide formation

Ann. Med.