Reference interval for human plasma nitric oxide end products

Clinical Biochemistry, Vol. 31, No. 6, 513–515, 1998 Copyright © 1998 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/98 $19.00 1 .00

PII S0009-9120(98)00051-4

Reference Interval for Human Plasma Nitric Oxide End Products JULIAN DIAZ,1,2 ENRIQUE SERRANO,1 FRANCISCO ACOSTA,3 and LUIS F. CARBONELL2 Department 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 1

Introduction n recent years, it has become apparent that nitric oxide (NO) has a number of biologically important functions. This molecule acts as an intercellular and intracellular messenger of many physiological processes (1,2). In mammalian cells, NO is synthesized via the enzyme NO synthase with the basic amino acid L-arginine acting as substrate and molecular oxygen as cosubstrate. In biological samples, NO is rapidly deactivated by oxidation to nitrite and nitrate by physically dissolved oxygen and water. NO research has expanded explosively in the past 10 years. The results have demonstrated that NO is a pluripotential molecule that acts as both an autocrine and paracrine mediator of homeostasis, and disturbance of its metabolism can be linked with many pathophysiological events (1,2). NO is a stable colorless gas, which is moderately soluble in water. In solution, NO is oxidated to nitrate and nitrite (NO has a half-life of under 30 s) (2). Once produced, NO can be inactivated by superoxide anions and protected by the presence of superoxide dismutase. In this respect, NO may be considered a free radical (FR) scavenger and thus, a cytoprotective factor. However, under appropriate conditions, NO can form a potent oxidant, peroxynitrite, with half-life of 1 s (2). Peroxynitrite and its degradation products have been linked to oxidative stress, nitrozation of several tyrosine molecules that regulate enzyme function and signal transduction, sodium channel inactivation and interaction with transitional metals. Under physiological conditions, it will combine with protein bound thiol groups to form stable, biologically active S-nitrosyl compounds and may also circulate as an S-nitrosoadduct of albumin. Finally, each of these interactions can contribute to cell injury and the

I

Correspondence: Julian Diaz, M.D., C/ Jose Maria Mortes Lerma, 32 Dpl, Pta 14. 46014-Valencia, Spain. Received December 11, 1997; accepted April 22, 1998. CLINICAL BIOCHEMISTRY, VOLUME 31, AUGUST 1998

effect of NO during oxidative stress injury in human diseases still remains controversial (1-3). However, because NO is extremely reactive and short-lived making its direct detection difficult (2), determination of NO end products is, therefore, of practical importance in determining the effects of FR in biological systems. In recent years, several methods have been proposed for measuring these parameters levels in biological samples (3–5). Many literature reports are for individual analytes, and are based on small numbers, unspecified statistical evaluation and reported reference intervals for plasma vary widely (3– 6). This may be due to methological variations, problems related to the sampling, time required for analysis and limitations corresponding to each method. These differences make it difficult to choose the best procedure. Therefore, we undertook the current study to provide reliable reference intervals for human plasma, including the establishment of possible sex-related differences. Materials and methods SUBJECTS With the consent of our hospital’s Clinical Research Committee, we measured plasma NO end products in plasma of 200 nonhospitalized adult subjects, selected for absence of known organic disease and were carefully screened for infectious, hypertension, alcoholism, malignant, and other serious disorders. The mean age was 42 years (range 18 – 65) and all persons enrolled in the study had similar lifestyles and dietary habits (6). To additionally check their state of health they were subjected to a conventional biochemical screening and hematological analysis (7). Subjects were classified in two groups according to sex, group A (men) and group B (women). 513

DIAZ

SAMPLE

COLLECTION AND SPECIMEN PREPARATION

Blood was collected from overnight fasted subjects by venipuncture into 5 mL evacuated tubes containing EDTA/K3 solution as anticoagulant (6). After centrifugation (2500 3 g) for 10 min in a centrifuge cooled to 4° C, the supernatant plasma was removed carefully, to avoid contamination with platelets, within 30 min after sample collection. Plasma samples were stored at -80° C until analyzed (usually within 30 d) with no freeze-thaw cycles in traceelement-free tubes to maintain the stability of the plasma samples (4,5). SPECIMEN

PREPARATION

Blank, standards, and plasma samples were deproteinized before analysis as follows. Pipette a portion of 100 mL of the blank, standards and samples into 1.5 mL Eppendorf tubes containing 400 mL of ethanol. Treated specimens were mixed by vortexing. They were centrifuged at 13000 g for 10 minutes. Four hundred microliters of supernatant were transferred to 1.5 mL Eppendorf tubes, and put in a draughty heater at 60° C until total evaporation. Once that ethanol was evaporated, blank, standards, and plasma samples were dissolved with 100 mL of phosphate buffer (50 mmol/L, pH 7.5). REAGENTS

AND KIT

Ethanol and sodium nitrite were obtained from Merck Co. (Germany) and Sigma Co. (United Kingdom), respectively. Nitric oxide end products (nitrates plus nitrites) were analyzed by Nitric Oxide Colorimetric AssayR (Boehringer Mannheim, Germany). The principle of this analysis is as follows. The nitrates present in the sample were stoichiometrically reduced to nitrites by incubation of sample for 60 min at 37° C, in the presence of the enzyme nitrate reductase (Aspergillus species, Boehringer Mannheim, 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 deproteinized plasma). The nitrite formed reacts with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo chromophore, which is measured on the basis of its absorbance in the visible range at 550 nm on a 96-well microtiter read plate (5,6). Absorbance was measured on a microplate reader. Concentrations were determined from a linear standard curve that was obtained by using sodium nitrate under the experimental conditions described previously (between 15.3 and 122.6 mmol/L). Specimens were analyzed in duplicate. ANALYTICAL

PERFORMANCE

Detection limit The detection limit was determined as described by Gatautis and Pearson (8). A sample containing 514

ET AL.

nitrite at a concentration three- to five-fold that of reagent blank was measured 10 times, and the detection 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 in triplicate. Linear regressions and the correlation coefficient were then calculated. Precision To determine between-run and within-run precisions, we froze aliquots of plasma from a control subject at 280° C, thawing these only before analysis. Within-run precision was calculated from 10 assays done on the same day. Between-run precisions was calculated from 20 assays done over 30 d. Analytical recovery Analytical recovery was measured by evaluating the analytical recovery of standard additions. Known quantities of the 200 mmol/L standard solution were added to plasma from healthy subjects before adding the reagents. After homogenizing the sample, NO end products were measured as described. Statistical analysis The statistical parameters (mean, standard deviation, fractiles, coefficient of variation, regression, unpaired Student’s t-test and Kolmogorov-Smirnov test) were determined with the SPSS statistical package (SPSS Inc., Chicago, IL, USA). Results and discussion Assays of nitrite and nitrate have become increasingly important in recent years for health care and economic reasons. The final products of NO in vivo are nitrite and nitrate. The relative proportion of nitrate and nitrite is variable and cannot be predicted with certainly. Therefore, the best index of total NO production is the sum of both nitrate and nitrite. Nitrates and nitrites are always produced when oxidative stress process occur in biological systems, and it is of interest to identify and measure these compounds as an index of the extend of FR and as an aid to elucidate the role of FR as causative agents in certain pathological conditions. In addition, the detection of an oxidative stress process in vivo and assay of secondary molecules released (whether or not they are responsible for tissue lesions) require the development of quantitative methods that satisfy the analytical criteria of within and between-run precisions, sensitivity and accuracy. The linearity displayed by measuring standard CLINICAL BIOCHEMISTRY, VOLUME 31, AUGUST 1998

HUMAN PLASMA NITRIC OXIDE ENDPRODUCTS

TABLE 1 Between-Run and Within-Run Precision for Nitric Oxide End Products Assay

Within-Run Precision (n 5 10) Mean (mmol/L) Standard deviation (mmol/L) Coefficient of variation (%) Between-Run Precision (n 5 20) Mean (mmol/L) Standard deviation (mmol/L) Coefficient of variation (%)

Low Pool

High Pool

25.3 1.0 4.1

52.7 2.1 4.0

25.7 1.2 4.8

52.8 2.4 4.6

solutions was excellent in the assay range that correspond to plasma concentrations. The correlation coefficient of the regression line (r 5 0.978, p , 0.001) in the standard range from 15.33 to 122.60 mmol/L was suitable, indicating that this method could be used in plasma samples. Detection limit was 2.00 mmol/L. This satisfactory sensitivity is sufficient for the method to applied to human plasma. Within-run and between-run precision shown in Table 1. Analytical recovery was 90 to 100% when the final concentration of NO end products was ,100 mmol/L and one of the advantages of using this method for assaying plasma samples then monitoring patients, is primarily, the satisfactory analytical recovery. Subjects have fasted for at least 12 h and, consequently, the confounding variable of serum nitrate from dietary intake is minimized because the halflife of ingested nitrate in serum is about 5 h (6). For each group, there was a representative sample as reference population, according to the IFCC guidelines (9,10). From the initial 100 individuals in each group, we discarded the results of those who, in the diagnosis, had some disease and more than one biochemical or hematological measurement altered. For both groups, the distribution of plasma NO end products concentration values followed a Gaussian frequency distribution, as verified by the Kolmogorov-Smirnov test. Aberrant values were excluded according to the IFCC guidelines (9). Between groups, the composite distributions were not significantly different from the distribution for either sex separately. Accordingly, we calculated both parametric (mean 6 2SD) and nonparametric (0.05– 0.95 fractiles) reference intervals (Table 2). When all men were compared with all women, no significant differences were found, when use this assay, which is based on the commercially available colorimetric determination by the Griess reaction after enzymatic reduction of nitrate to nitrite. In conclusion, this simple, reproducible, and sensitive assay can be adapted by clinical laboratories for the routine monitoring NO plasma end products in human disorders.

CLINICAL BIOCHEMISTRY, VOLUME 31, AUGUST 1998

TABLE 2 Reference Intervals for Human Plasma Nitric Oxide End Products Concentration (mmol/L) Reference Intervals Groups

Mean

(Mean 6 2SD)

(0.05–0.95 Fractiles)

Group A (men; n587) Group B (women; n 5 83)

18.8NS

12.2-25.5

12.9-26.4

20.0

13.1-26.8

12.1-26.0

NS

No significantly compared with women mean.

Acknowledgements This work was supported in part by Fondo de Investigaciones Sanitarias (Madrid, Spain, Grants: FIS 96/1631 and FIS 97/5249), plus a grant from Fundacion para el Desarrollo del Trasplante Hepatico and Novartis Farmaceutica S.A. (Madrid, Spain).

References 1. Davies MG, Fulton GJ, Hagen PO. Clinical biology of nitric oxide. Br J Surg 1995; 82: 1598 –1610. 2. Beckman JS, Crow JP. Pathological implications of nitric 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 and clinical 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 Biochem 1982; 126: 131– 8. 5. Roselli M, Imthurn B, Macas E, Keller PJ, Dubey RK. Circulating nitrite/nitrate levels increase with follicular development: indirect evidence for estradiol mediated NO-release. Biochem Biophys Res Commum 1994; 202: 1543–52. 6. Wagner DA, Schultz DS, Deen WM, Young VR, Tannenbaum SR. Metabolic fate of an oral dose of 15Nlabeled nitrate in humans: effect of diet supplementation with ascorbic acid. Cancer 1983; 43: 1921–5. 7. Diaz J, Tornel PL, Martinez P. Reference intervals for blood ammonia in healthy subjects determined by microdiffusion. Clin Chem 1995; 7: 1048. 8. Gatautis V, Pearson KH. Separation of plasma carotenoids and quantitation of beta carotene using HPLC. Clin Chim Acta 1987; 166: 195–206. 9. IFCC. The theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. J Clin Chem Clin Biochem 1983; 21: 749 – 60. 10. Solberg HE, Establishment and use of reference values. In: Burtis CA, Ashwood ER, Eds. Tietz texbook of clinical chemistry. 2nd ed. Pp. 454 – 84. Philadelphia, PA: WB Saunders, 1994.

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