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Optimization of nitric oxide chemiluminescence operating conditions for measurement of plasma nitrite and nitrate.

The oxidized nitric oxide (NO) metabolites nitrite (N[O.sub.2.sup.-]) and nitrate (N[O.sub.3.sup.-]) have been used as markers of NO synthase activity in several clinical and experimental studies including those investigating sepsis (1-4), septic shock (5), endotoxemia (6, 7), cardiac transplant (8), hypertension (9), and cancer (10). Although the NO chemiluminescence methodology has been used to measure N[O.sub.2.sup.-] and N[O.sub.3.sup.-] (11-13), there are few reports evaluating the operating conditions of the assay.

The operating efficiency of the NO chemiluminescent system is governed by several variables: driving pressure and flow rate of the carrier gas (14,15), which determine the degree of mixing in reducing solution and dispersion of NO in the carrier gas stream; chemiluminescent reaction chamber pressure ([P.sub.RC]); and the selectivity (16), pH (11), temperature (13), and concentration of the reducing agents used to convert N[O.sub.2.sup.-] and N[O.sub.3.sup.-] to NO. Yang et al. (13) studied the efficiency of conversion of both N[O.sub.2.sup.-] and N[O.sub.3.sup.-] to NO using various reducing agents over a range of operating temperatures; however, no studies on the relationship between carrier gas flow rate and [P.sub.RC] have been reported.

The present study had three primary objectives: (a) to determine the optimal operating conditions for carrier gas flow rate and [P.sub.RC] to achieve maximum efficiency of the chemiluminescent response for both N[O.sub.2.sup.-] and N[O.sub.2.sup.-] determinations; (b) to determine the detection limit and linearity of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] responses on the basis of these optimal operating conditions; and (c) to evaluate the recovery of N[O.sub.2.sup.-] and N[O.sub.3.sup.-]from plasma and deproteinized plasma under optimized operating conditions.

We purchased potassium iodide, sodium nitrite, sodium nitrate, and glacial acetic acid from Sigma, vanadium (III) trichloride, and hydrochloric acid from Aldrich Chemical Co., and helium and oxygen from Praxair.

The NO chemiluminescence system used in these experiments consisted of four main components: helium carrier gas (set at 35 psi), two in-parallel purge vessels with condensers connected by T-valves, a nitric oxide analyzer (NOA) with inlet valve and chemiluminescence reaction chamber (Sievers 270B, NO Chemiluminescence Analyzer, Sievers Instruments), and a vacuum pump (Edwards Pump) to draw the carrier gas into the reaction chamber. Helium carrier gas passed through a chemical trap (2 mol/L NaOH) before entry into the NOA to remove volatile acids. Connecting tubing was 3.2 mm i.d. (890 FEP; Nalge Nunc International). One purge vessel (10-mL capacity) was dedicated to KI reduction at room temperature, whereas the other (12 mL-capacity and fitted with a cold-water condenser) was dedicated to VIII) reduction at 90[degrees]C. The helium flow rate into either purge vessel was controlled by a flow meter (Matheson Gas Products) placed upstream of the purge vessels. Operating pressure of the NO chemiluminescence reaction chamber was controlled by a needle valve at the entrance to the NOA. Inlet oxygen pressure, used to generate ozone, was 6 psi. In brief, N[O.sub.2.sup.-] or N[O.sub.3.sup.-] calibrators were injected (10 [micro]L) into a purge vessel containing either KI or VIII) and converted to NO. NO was then stripped from the reducing medium by a helium carrier gas and transported, via the chemical trap, to the chemiluminescence reaction chamber where it reacted with ozone to generate the chemiluminescent signal as described previously (17).

Operating conditions were adjusted to evaluate efficiency of the NO chemiluminescent response at different gas flow rate and [P.sub.RC] combinations. Plots of NO chemiluminescence responses for various flow rates against [P.sub.RC] are called isoflow curves. The strength, concentration, and temperature of reducing agents used were established according to recommendations from the NOA manufacturer (18) and by Yang et al. (13). N[O.sub.2.sup.-] was reduced to NO in KI solution (50 mg x 1 [mL.sup.-1] of water x 4 [mL.sup.-1] of glacial acetic acid) at room temperature (22[degrees]C) to minimize foaming of the reducing solution. N[O.sub.3.sup.-] was reduced to NO in VIII) (3.5 mL; 0.05 mol/L in 0.8 mol/L HCl) at 90[degrees]C.

Aqueous 30 [micro]mol/L calibrator samples of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] were prepared by diluting N[O.sub.2.sup.-] and N[O.sub.3.sup.-] into deionized water or fresh plasma obtained from heparinized rat whole blood. Plasma samples were also deproteinized by acetonitrile (1:1 by volume) to limit potential interference from hemoglobin (17). Supernatants were collected by centrifugation (30008 for 3 min at 4[degrees]C).

Kruskal-Wallis one-way ANOVA on ranks was used to assess differences in the time of NO chemiluminescence signal onset over various operating conditions (SigmaStat 2.0; SP55 Inc.). Dunnett's method was used for pairwise multiple comparison with the highest flow group (flow rate,126 mL/min) acting as the control group. Regression analysis was performed with SigmaPlot 5.00 (SP55 Inc. Chicago, IL). P <0.05 was considered statistically significant.

[FIGURE 1 OMITTED]

Isoflow curves of NO chemiluminescence responses from 300 pmol of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] in both aqueous solution and plasma are depicted in Fig. 1. In all cases, for a given flow rate, the maximum NO signal was dependent on [P.sub.RC]. As helium carrier gas flow rates increased, from 42 to 126 mL/min, maximum NO signal response was obtained at increasingly higher [P.sub.RC]. These responses demonstrated that the maximum NO signal was dependent on both flow rate and [P.sub.RC]. The detection limit and linearity of the NO chemiluminescent response for aqueous N[O.sub.2.sup.-] and N[O.sub.3.sup.-] conversion to NO at optimal low (flow rate, 42 ml/min; [P.sub.RC],1133 Pa), medium (flow rate, 69 ml/min; [P.sub.RC], 1100 Pa), and high (flow rate, 126 ml/min; [P.sub.RC], 1100 Pa) flow rate states are shown in Table 1. Both the detection limit and linear range were dependent on operating conditions with the lowest detection limit and greatest linear range being achieved at a high flow rate. Although greater NO signals were obtained at a low flow rate, detection limit was improved at a high flow rate because the signal-to-noise ratio was increased. Additionally, the onset of NO signal at the high flow rate (12.8 [+ or -] 0.2 s) decreased (P < 0.05) compared to low flow rate (29.4 [+ or -] 0.2 s). Under high flow rate conditions, recovery of nitrite (10-100 000 pmol) in plasma and deproteinized plasma ranged from 92.2% [+ or -] 6.8% to 100.2% [+ or -] 2.5% and 87.3% [+ or -] 5.5% to 111.7% [+ or -] 8.9%, respectively. Nitrate recovery (10-100 000 pmol) was similar (i.e., in plasma, 101% [+ or -] 2.3% to 96.1% [+ or -] 1.2%; in deproteinized plasma, 89% [+ or -] 1.6% to 101.7% [+ or -] 2.2%, respectively). Recovery at 1 pmol, although detectable, was not readily quantifiable electronically because of increased background signal. The decrease in sensitivity in plasma, relative to aqueous samples, was attributable to increased background signal in both plasma and deproteinized plasma.

Isoflow curves show that NO chemiluminescent signal is dependent on both flow velocity of helium carrier gas and [P.sub.RC]. This is consistent with earlier reports that chemiluminescent signal is related to the rate of carrier gas flow (14,15). Increasing the rate of the carrier gas into the purge vessel should increase the rate of mass transfer of NO from the liquid phase into the gas phase, thereby increasing the efficiency of the separation process and also decreasing the axial dispersion of the NO in the helium flow stream. However, the probability of a successful collision between NO and ozone within the reaction chamber may decrease because of decreased NO residence time, thereby reducing signal intensity; although, this is offset somewhat by higher [P.sub.RC], despite the likelihood of enhanced nonradiative collisional quenching of the excited state of N[O.sub.2] (N[O.sub.2]*) (18).

We determined the maximum NO chemiluminescent signal for all flow rates tested and observed that as flow rates increased, the maximum signal not only decreased, but occurred at higher [P.sub.RC] (Fig. 1). This indicated a complex relationship among flow, [P.sub.RC], and NO signal. Interestingly, this complex relationship is similar in some respects to the efficiency profile of gas and liquid chromatographic separations, where the maximum efficiency is related to the carrier gas velocity at which the theoretical plate height is minimized (19). At high flow rates, the detection limit was enhanced (Table 1) because of increased peak stability at low concentrations (i.e., electronic area integration was improved.)

In conclusion, a range of carrier gas flow rates have been reported (100 mL/min of [N.sub.2] to 200 mL/min of helium) for chemiluminescence-based analysis of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] (11-13). On the basis of our results, we recommend the use of a high carrier gas/mobile phase flow rate at optimized [P.sub.RC]. Despite a reduction in NO chemiluminescent signal response, this increases sensitivity and linear response of the assay and has the additional benefit of decreasing analysis time.

This research was supported by Medical Research Council Grant MA-13941 (to C.G.E.). R.M.B. was supported by the Spoerel Research Fellowship, Department of Anesthesia, University of Western Ontario.

References

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(2.) Satoi S, Kamiyama Y, Kitade H, Kwon AH, Takahashi K, et al. Nitric oxide production hepatic dysfunction in patients with postoperative sepsis. Clin Exp Pharmacol Physiol 2000;27:197-201.

(3.) Wong HR, Carcillo JA, Burckart G, Shah N, Janosky JE. Increased serum nitrite and nitrate concentrations in children with the sepsis syndrome. Crit Care Med 1995;23:835-42.

(4.) Bateman RM, Jagger JE, Sharpe MD, Ellsworth ML, Mehta S, Ellis CG. Erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density during sepsis. Am J Physiol Heart Circ Physiol 2001;280: H2848-56.

(5.) Strand OA, Leone A, Giercksky KE, Kirkeboen KA. Nitric oxide indices in human septic shock. Crit Care Med 2000;28:2779-85.

(6.) Brovkovych V, Dobrucki LW, Brovkovych S, Dobrucki I, Kalinowski L, et al. Nitric oxide measurements during endotoxemia. Clin Chem 2001;47:1068-74.

(7.) Deshmukh DR, Ghole VS, Marescau B, De Deyn PP. Effect of endotoxemia on plasma and tissue levels of nitric oxide metabolites and guanidino compounds. Arch Physiol Biochem 1997;105:32-7.

(8.) Benvenuti C, Bories PN, Loisance D. Increased serum nitrate concentration in cardiac transplant patients. A marker for acute allograft cellular rejection. Transplantation 1996;61:745-9.

(9.) Wu CC, Yen MH. Higher level of plasma nitric oxide in spontaneously hypertensive rats. Am J Hypertens 1999;12:476-82.

(10.) Szaleczky E, Pronai L, Nakazawa H, Tulassay Z. Evidence of in vivo peroxynitrite formation in patients with colorectal carcinoma, higher plasma nitrate/nitrite levels, and lower protection against oxygen free radicals. J Clin Gastroenterol 2000;30:47-51.

(11.) Braman RS, Hendrix SA. Nanogram nitrite and nitrate determination in environmental and materials by vanadium (III) reduction with chemiluminescence detection. Anal Chem 1989;61:2715-8.

(12.) Cox RD, Frank CW. Determination of nitrate and nitrite in blood and urine by chemiluminescence. J Anal Toxicol 1982;6:148-52.

(13.) Yang F, Troncy E, Francoeur M, Vinet B, Vinay P, et al. Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence. Clin Chem 1997;43:657-62.

(14.) Aoki T. Continuous flow determination of nitrite with membrane separation/ chemiluminescence detection. Biomed Chromatogr 1990;4:128-30.

(15.) Pai TG, Payne WJ, LeGall J. Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes. Anal Biochem 1987;166:150-7.

(16.) Greenberg SS, Xie J, Spitzer JJ, Wang JF, Lancaster J, et al. Nitro containing L-arginine analogs interfere with assays for nitrate and nitrite. Life Sci 1995;57:1949-61.

(17.) Bateman RM, Ellis CG, Sharpe MD, Mehta S, Freeman DJ. Effect of hemolyzed plasma on the batch measurement of nitrate by nitric oxide chemiluminescence. Clin Chem 2001;47:1847-51.

(18.) Operation and Service Manual. Boulder, CO: Sievers Instruments, 1992.

(19.) Berezkin VG, Malyukova IV, Avoce DS. Use of equations for the description of experimental dependence of the height equivalent to a theoretical plate on carrier gas. J Chromatogr A 2000;872:111-8.

Ryon M. Bateman, [1,2] Christopher G. Ellis, [1,2] and David J. Freeman [3] * (1 Vascular Biology Program, Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, N6B 1B8 Canada; Departments of [2] Medical Biophysics and [3] Medicine, University of Western Ontario, London, Ontario, N6A 5C1 Canada; * address correspondence to this author at: Department of Medicine, University of Western Ontario, University Hospital 339 Windermere Rd., London, Ontario, N6A 5A5 Canada; fax 519-663-3789, e-mail dfreeman@uwo.ca)
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Title Annotation:Technical Briefs
Author:Bateman, Ryon M.; Ellis, Christopher G.; Freeman, David J.
Publication:Clinical Chemistry
Date:Mar 1, 2002
Words:2227
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