Reference Interval for Human Plasma Nitric OxideEnd Products

JULIAN DIAZ,1,2 ENRIQUE SERRANO,1 FRANCISCO ACOSTA,3 and LUIS F. CARBONELL2

1Department of Biochemistry and the 3Department of Anesthesiology, University Hospital“Virgen de la Arrixaca” of Murcia, Spain, 2Department of Physiology, University of Murcia

School of Medicine, Spain

Introduction

In recent years, it has become apparent that nitricoxide (NO) has a number of biologically important

functions. This molecule acts as an intercellular andintracellular messenger of many physiological pro-cesses (1,2). In mammalian cells, NO is synthesizedvia the enzyme NO synthase with the basic aminoacid L-arginine acting as substrate and molecularoxygen as cosubstrate. In biological samples, NO israpidly deactivated by oxidation to nitrite and ni-trate by physically dissolved oxygen and water. NOresearch has expanded explosively in the past 10years. The results have demonstrated that NO is apluripotential molecule that acts as both an auto-crine and paracrine mediator of homeostasis, anddisturbance of its metabolism can be linked withmany pathophysiological events (1,2).

NO is a stable colorless gas, which is moderatelysoluble in water. In solution, NO is oxidated tonitrate and nitrite (NO has a half-life of under 30 s)(2). Once produced, NO can be inactivated by super-oxide anions and protected by the presence of super-oxide dismutase. In this respect, NO may be consid-ered a free radical (FR) scavenger and thus, acytoprotective factor. However, under appropriateconditions, NO can form a potent oxidant, peroxyni-trite, with half-life of 1 s (2).

Peroxynitrite and its degradation products havebeen linked to oxidative stress, nitrozation of severaltyrosine molecules that regulate enzyme functionand signal transduction, sodium channel inactiva-tion and interaction with transitional metals. Underphysiological conditions, it will combine with proteinbound thiol groups to form stable, biologically activeS-nitrosyl compounds and may also circulate as anS-nitrosoadduct of albumin. Finally, each of theseinteractions can contribute to cell injury and the

effect of NO during oxidative stress injury in humandiseases still remains controversial (1-3).

However, because NO is extremely reactive andshort-lived making its direct detection difficult (2),determination of NO end products is, therefore, ofpractical importance in determining the effects ofFR in biological systems. In recent years, severalmethods have been proposed for measuring theseparameters levels in biological samples (3–5). Manyliterature reports are for individual analytes, andare based on small numbers, unspecified statisticalevaluation and reported reference intervals forplasma vary widely (3–6). This may be due tomethological variations, problems related to thesampling, time required for analysis and limitationscorresponding to each method. These differencesmake it difficult to choose the best procedure. There-fore, we undertook the current study to providereliable reference intervals for human plasma, in-cluding the establishment of possible sex-relateddifferences.

Materials and methods

SUBJECTS

With the consent of our hospital’s Clinical Re-search Committee, we measured plasma NO endproducts in plasma of 200 nonhospitalized adultsubjects, selected for absence of known organic dis-ease and were carefully screened for infectious,hypertension, alcoholism, malignant, and other se-rious disorders. The mean age was 42 years (range18–65) and all persons enrolled in the study hadsimilar lifestyles and dietary habits (6). To addition-ally check their state of health they were subjectedto a conventional biochemical screening and hema-tological analysis (7). Subjects were classified in twogroups according to sex, group A (men) and group B(women).

Correspondence: Julian Diaz, M.D., C/ Jose MariaMortes Lerma, 32 Dpl, Pta 14. 46014-Valencia, Spain.

Received December 11, 1997; accepted April 22, 1998.

Clinical Biochemistry, Vol. 31, No. 6, 513–515, 1998Copyright © 1998 The Canadian Society of Clinical Chemists

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CLINICAL BIOCHEMISTRY, VOLUME 31, AUGUST 1998 513

SAMPLE COLLECTION AND SPECIMEN PREPARATION

Blood was collected from overnight fasted subjectsby venipuncture into 5 mL evacuated tubes contain-ing EDTA/K3 solution as anticoagulant (6). Aftercentrifugation (2500 3 g) for 10 min in a centrifugecooled to 4° C, the supernatant plasma was removedcarefully, to avoid contamination with platelets,within 30 min after sample collection. Plasma sam-ples were stored at -80° C until analyzed (usuallywithin 30 d) with no freeze-thaw cycles in trace-element-free tubes to maintain the stability of theplasma samples (4,5).

SPECIMEN PREPARATION

Blank, standards, and plasma samples weredeproteinized before analysis as follows. Pipette aportion of 100 mL of the blank, standards andsamples into 1.5 mL Eppendorf tubes containing 400mL of ethanol. Treated specimens were mixed byvortexing. They were centrifuged at 13000 g for 10minutes. Four hundred microliters of supernatantwere transferred to 1.5 mL Eppendorf tubes, andput in a draughty heater at 60° C until total evapo-ration. Once that ethanol was evaporated, blank,standards, and plasma samples were dissolved with100 mL of phosphate buffer (50 mmol/L, pH 7.5).

REAGENTS AND KIT

Ethanol and sodium nitrite were obtained fromMerck Co. (Germany) and Sigma Co. (United King-dom), respectively. Nitric oxide end products (ni-trates plus nitrites) were analyzed by Nitric OxideColorimetric AssayR (Boehringer Mannheim, Ger-many). The principle of this analysis is as follows.The nitrates present in the sample were stoichio-metrically reduced to nitrites by incubation of sam-ple for 60 min at 37° C, in the presence of the enzymenitrate reductase (Aspergillus species, BoehringerMannheim, Germany), reduced NADPH and FAD(final concentrations: phosphate buffer [50 mmol/L,pH 7.5], nitrate reductase [20 mU], FAD [5 mmol/L],NADPH [0.6 mmol/L], and 100 mL of deproteinizedplasma). The nitrite formed reacts with sulfanil-amide and N-(1-naphthyl)-ethylenediamine dihy-drochloride to give a red-violet diazo chromophore,which is measured on the basis of its absorbance inthe visible range at 550 nm on a 96-well microtiterread plate (5,6). Absorbance was measured on amicroplate reader. Concentrations were determinedfrom a linear standard curve that was obtained byusing sodium nitrate under the experimental condi-tions described previously (between 15.3 and 122.6mmol/L). Specimens were analyzed in duplicate.

ANALYTICAL PERFORMANCE

Detection limit

The detection limit was determined as describedby Gatautis and Pearson (8). A sample containing

nitrite at a concentration three- to five-fold that ofreagent blank was measured 10 times, and thedetection limit was calculated as 2 SD/mean.

Linearity

The levels of standard calibration solutions (15.3,30.6, 61.3, and 122.6 mmol/L) were determined intriplicate. Linear regressions and the correlationcoefficient were then calculated.

Precision

To determine between-run and within-run preci-sions, we froze aliquots of plasma from a controlsubject at 280° C, thawing these only before analy-sis. Within-run precision was calculated from 10assays done on the same day. Between-run preci-sions was calculated from 20 assays done over30 d.

Analytical recovery

Analytical recovery was measured by evaluatingthe analytical recovery of standard additions.Known quantities of the 200 mmol/L standard solu-tion were added to plasma from healthy subjectsbefore adding the reagents. After homogenizing thesample, NO end products were measured as de-scribed.

Statistical analysis

The statistical parameters (mean, standard devi-ation, fractiles, coefficient of variation, regression,unpaired Student’s t-test and Kolmogorov-Smirnovtest) were determined with the SPSS statisticalpackage (SPSS Inc., Chicago, IL, USA).

Results and discussion

Assays of nitrite and nitrate have become increas-ingly important in recent years for health care andeconomic reasons. The final products of NO in vivoare nitrite and nitrate. The relative proportion ofnitrate and nitrite is variable and cannot be pre-dicted with certainly. Therefore, the best index oftotal NO production is the sum of both nitrate andnitrite. Nitrates and nitrites are always producedwhen oxidative stress process occur in biologicalsystems, and it is of interest to identify and measurethese compounds as an index of the extend of FR andas an aid to elucidate the role of FR as causativeagents in certain pathological conditions. In addi-tion, the detection of an oxidative stress process invivo and assay of secondary molecules released(whether or not they are responsible for tissuelesions) require the development of quantitativemethods that satisfy the analytical criteria of withinand between-run precisions, sensitivity and accu-racy.

The linearity displayed by measuring standard

DIAZ ET AL.

514 CLINICAL BIOCHEMISTRY, VOLUME 31, AUGUST 1998

solutions was excellent in the assay range thatcorrespond to plasma concentrations. The correla-tion coefficient of the regression line (r 5 0.978, p ,0.001) in the standard range from 15.33 to 122.60mmol/L was suitable, indicating that this methodcould be used in plasma samples. Detection limitwas 2.00 mmol/L. This satisfactory sensitivity issufficient for the method to applied to humanplasma. Within-run and between-run precisionshown in Table 1. Analytical recovery was 90 to100% when the final concentration of NO end prod-ucts was ,100 mmol/L and one of the advantages ofusing this method for assaying plasma samples thenmonitoring patients, is primarily, the satisfactoryanalytical recovery.

Subjects have fasted for at least 12 h and, conse-quently, the confounding variable of serum nitratefrom dietary intake is minimized because the half-life of ingested nitrate in serum is about 5 h (6). Foreach group, there was a representative sample asreference population, according to the IFCC guide-lines (9,10). From the initial 100 individuals in eachgroup, we discarded the results of those who, in thediagnosis, had some disease and more than onebiochemical or hematological measurement altered.For both groups, the distribution of plasma NO endproducts concentration values followed a Gaussianfrequency distribution, as verified by the Kolmog-orov-Smirnov test. Aberrant values were excludedaccording to the IFCC guidelines (9). Betweengroups, the composite distributions were not signif-icantly different from the distribution for either sexseparately. Accordingly, we calculated both para-metric (mean 6 2SD) and nonparametric (0.05–0.95fractiles) reference intervals (Table 2). When allmen were compared with all women, no significantdifferences were found, when use this assay, whichis based on the commercially available colorimetricdetermination by the Griess reaction after enzy-matic reduction of nitrate to nitrite.

In conclusion, this simple, reproducible, and sen-sitive assay can be adapted by clinical laboratoriesfor the routine monitoring NO plasma end productsin human disorders.

Acknowledgements

This work was supported in part by Fondo de Investi-gaciones Sanitarias (Madrid, Spain, Grants: FIS 96/1631and FIS 97/5249), plus a grant from Fundacion para elDesarrollo del Trasplante Hepatico and Novartis Farma-ceutica S.A. (Madrid, Spain).

References

1. Davies MG, Fulton GJ, Hagen PO. Clinical biology ofnitric oxide. Br J Surg 1995; 82: 1598–1610.

2. Beckman JS, Crow JP. Pathological implications ofnitric oxide, superoxide and peroxynitrite formation.Biochem Soc Trans 1993; 21: 330–4.

3. Body SC, Hartigan PM, Shernan SK, Formanek V,Hurford WE. Nitric oxide: delivery, measurement andclinical application. J Cardiothorac Vac Anesth 1995;9: 748–63.

4. Green LC, Wagner DA, Glogowski J, Skipper PL,Wishnok JS, Tannenbaum SR. Analysis of nitrate,nitrite and 15Nitrate in biological fluids. Anal Bio-chem 1982; 126: 131–8.

5. Roselli M, Imthurn B, Macas E, Keller PJ, Dubey RK.Circulating nitrite/nitrate levels increase with follic-ular development: indirect evidence for estradiol me-diated NO-release. Biochem Biophys Res Commum1994; 202: 1543–52.

6. Wagner DA, Schultz DS, Deen WM, Young VR, Tan-nenbaum SR. Metabolic fate of an oral dose of 15N-labeled nitrate in humans: effect of diet supplementa-tion with ascorbic acid. Cancer 1983; 43: 1921–5.

7. Diaz J, Tornel PL, Martinez P. Reference intervals forblood ammonia in healthy subjects determined bymicrodiffusion. Clin Chem 1995; 7: 1048.

8. Gatautis V, Pearson KH. Separation of plasma caro-tenoids and quantitation of beta carotene usingHPLC. Clin Chim Acta 1987; 166: 195–206.

9. IFCC. The theory of reference values. Part 5. Statis-tical treatment of collected reference values. Determi-nation of reference limits. J Clin Chem Clin Biochem1983; 21: 749–60.

10. Solberg HE, Establishment and use of reference val-ues. In: Burtis CA, Ashwood ER, Eds. Tietz texbook ofclinical chemistry. 2nd ed. Pp. 454–84. Philadelphia,PA: WB Saunders, 1994.

TABLE 1Between-Run and Within-Run Precision for Nitric Oxide

End Products Assay

Low Pool High Pool

Within-Run Precision (n 5 10)Mean (mmol/L) 25.3 52.7Standard deviation (mmol/L) 1.0 2.1

Coefficient of variation (%) 4.1 4.0Between-Run Precision (n 5 20)

Mean (mmol/L) 25.7 52.8Standard deviation (mmol/L) 1.2 2.4Coefficient of variation (%) 4.8 4.6

TABLE 2Reference Intervals for Human Plasma Nitric Oxide End

Products Concentration (mmol/L)

Reference Intervals

Groups Mean (Mean 6 2SD)(0.05–0.95Fractiles)

Group A(men; n587)

18.8NS 12.2-25.5 12.9-26.4

Group B(women; n 5 83)

20.0 13.1-26.8 12.1-26.0

NSNo significantly compared with women mean.

HUMAN PLASMA NITRIC OXIDE ENDPRODUCTS

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