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Technical Briefs |
1
Vascular Biology Program, Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, N6B 1B8 Canada
Departments of
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Medical Biophysics,
3
Anaesthesia,
4
Respirology, and
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Pharmacology-Toxicology, University of Western Ontario, London, Ontario, N6A 5C1 Canada
aaddress correspondence to this author at: Department of Medicine and Pharmacology-Toxicology, University of Western Ontario, London, Ontario, N6A 5C1 Canada; fax 519-663-3789, e-mail dfreeman{at}uwo.ca
Nitric oxide (NO) is a potent vasodilator and regulator of vascular tone, a neurotransmitter, and a cytotoxic agent (1)(2). In aqueous aerobic environments, the primary decomposition product of NO is nitrite (NO2-) (3), with further oxidation to nitrate (NO3-) being dependent on the presence of additional oxidizing species such as oxyhemoproteins (3). Collectively, these NO oxidation products are referred to as NOx-. Several analytical techniques have been used to quantify NOx- species, including spectrophotometric assays based on the Griess reaction (4)(5) and enzymatic reduction of NO3- (6), gas chromatography–mass spectrometry (7), chromatographic flow systems (8), and chemiluminescence (9)(10)(11).
NOx- may be important in sepsis (12)(13)(14)(15)(16). Of possible analytical importance is intravascular hemolysis during sepsis (17)(18)(19). The effect of hemolysis on NOx- analysis is unknown. Other potential causes of intravascular hemolysis include drug-induced hemolytic anemia, hemolytic transfusion reactions, and artificial heart valves (20), and hemolysis can also occur during blood collection (21) and inappropriate blood storage. The objectives of our study were (a) to determine whether hemolysis interferes with the determination of plasma NO3- by NO chemiluminescence batch methodology and (b) to determine whether the interference could be eliminated by sample pretreatment.
We purchased helium and oxygen from Praxair. Other chemicals were from Sigma-Aldrich. All chemicals were reagent-grade quality. Deionized water was used to prepare all solutions. Blood samples were obtained from healthy human volunteers by venipuncture with heparin as anticoagulant. Aqueous NO3- calibrators (25 µmol/L) were prepared in deionized water. Digitonin in phosphate-buffered saline (PBS), at final concentrations of 0, 20, 45, and 90 µmol/L, was used to control the degree of whole-blood hemolysis (21). Whole blood was also treated by dilution (1:2 by volume) with lysis buffer (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.14 mmol/L EDTA, pH 7.2) to obtain complete hemolysis. Whole blood was diluted 1:2 by volume with digitonin solutions (35 °C) and lysis buffer and mixed gently for 5 min. Supernatants were collected by centrifugation (10 min at 1500g and 18 °C) and refrigerated. Hemoglobin (Hb) content was estimated by hemoximetry (OSM2 Hemoximeter; Radiometer) and determined quantitatively (plasma hemoglobin test reagent set; Sigma Diagnostics). The Hb plasma solutions were supplemented with NO3- (NO3-/Hb) to produce a final concentration of 25 µmol/L. Solutions were left at room temperature for 10 min to allow sample equilibration.
In a separate series of tests, Hb was removed from the mildly hemolyzed sample (20 µmol/L digitonin) and the completely hemolyzed sample (lysis buffer) by precipitation with ethanol (1:2 by volume). Samples were mixed vigorously for 1 min and left standing at room temperature for 15 min before supernatants were collected by centrifugation (2 min at 5000g and 4 °C). Background NOx- (endogenous NO2-/NO3-) was subtracted from NO chemiluminescence signals. Digitonin at 1000 µmol/L had no effect on the NO3- calibrator signal.
A NO chemiluminescence analyzer (NOA) system (Sievers 270B, NO Chemiluminescence Analyzer; Sievers Instruments) was used to detect NO3-. NO3- was chemically reduced under reflux conditions to NOgas in the Radical PurgerTM by hot (92 °C, circulating hot water bath) vanadium(III) (3.5 mL of 0.05 mol/L VCl3 in 0.8 mol/L HCl, pH 0.48) and subsequently stripped and carried to the NOA reaction chamber (under reduced pressure; Edwards Pump) by helium. The internal reaction chamber pressure was adjusted to 800 Pa. Within the reaction vessel, NOgas reacted with ozone to generate oxygen and the excited state NO2 species, which decayed to give a weak infrared (>600 nm) chemiluminescence signal that was detected and amplified by a photon multiplier tube (22)(23). Output signals in mV were recorded on a strip recorder (Chromatopak C-R1A; Shimadzu) with the areas of the NO peaks (mV · s) being electronically integrated from baseline to baseline.
To test the effects of hemolysis on NO3- detection, a batch protocol was used: a 25 µmol/L aqueous NO3- calibrator was injected five times to establish a baseline NO signal (Pre-Hb) and was immediately followed by 10 repeated injections of a NO3--supplemented hemolyzed plasma sample (NO3-/Hb) and then by 5 injections of the aqueous NO3- calibrator (Post-Hb). Ten-microliter injections (gas-tight syringe; Hamilton Company) were used for all calibrators and samples. The influence of Hb accumulation in the purge vessel on NO3- determination was calculated by the difference in NO signal between the Pre-Hb and Post-Hb aqueous NO3- calibrators. Duplicate batch runs of each treatment were performed.
All values are reported as mean ± SE unless otherwise stated. For all tests of significance, P <0.05 was considered statistically significant. A t-test (Sigma Stat 2.0; Jandel Scientific) was used to assess the influence of the Hb on the difference between Pre-Hb and Post-Hb NO3- calibrators. CVs were based on the combined results of aqueous NO3- calibrators from duplicate batch experiments (n = 10).
The effect of different degrees of hemolysis on
NO3- determination is shown in
Fig. 1
, A–E and G. As Hb accumulated in the purge vessel to
concentrations >1000 µg (Fig. 1
, C, D, and G), the NO
chemiluminescence response of the hemolyzed plasma samples decreased
sequentially with repeated injections. The effect of injecting
hemolyzed plasma samples on the subsequent determination of aqueous
NO3- calibrators was assessed
by comparing the NO responses of the calibrators before (Pre-Hb) and
after (Post-Hb) injections of hemolyzed plasma. The results are
summarized in Table 1
. As the degree of hemolysis increased from 2.9% to
30%, the difference between the values obtained for the Pre-Hb
and Post-Hb aqueous NO3-
calibrators increased from 5.2% (P = 0.047) to 19%
(P <0.001), respectively, showing that sample hemolysis
inhibited not only the plasma
NO3- signal, but also that of
the calibrator by a carryover effect.
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When mildly and completely hemolyzed samples were precipitated with
ethanol (Fig. 1, F and H
), there was no evidence of sequential
decreases in NO3-/Hb signal,
nor was there a significant difference in the NO signal of the aqueous
calibrator before and after exposure to precipitated samples (Table 1
).
Recovery of the NO signal was nearly quantitative from the hemolyzed
plasma samples, 112% and 115% with 20 µmol/L digitonin and lysis
buffer, respectively.
The NO chemiluminescence technique, based on the chemical reduction of NOx- species to NO and subsequent reaction with ozone, has several advantages over other methods. These advantages include increased accuracy and precision, and reduced susceptibility to sample interference from colored species, suspended materials (24), and nitro-organic compounds (10). Braman and Hendrix (10) showed that reduction of NO2- and NO3- by vanadium(III) to NO was both temperature and pH dependent. Yang et al. (25) confirmed the suitability of vanadium(III) as the reductant of choice and indicated that a temperature of 80–90 °C should be used to reduce both NO2- and NO3- to NO. Although NO3- was the focus of this study, the conditions used would also detect NO2-.
It has been reported that plasma protein causes extensive foaming in
the purge vessel and that this interferes with the chemical reduction
of NO2- and
NO3- (25). We found
that simply diluting the plasma fraction of whole blood threefold in
PBS (Fig. 1B
) eliminated the requirement for deproteinization before
batch processing recommended by Yang et al. (25). Although
plasma protein may interfere with
NOx- determinations, we found
that the NO chemiluminescent response was compromised by the presence
of Hb in the absence of extensive foaming. In fact, foaming was
encountered only with the highest accumulations of Hb, i.e., >1000
µg, corresponding to samples with 19% and 30% hemolysis, whereas
measurement of aqueous NO3-
calibrators was affected by much lower degrees of hemolysis
(2.9–7.0%) in the absence of foaming (Table 1
). This suggests that
the Hb itself is capable of interfering with the
NOx- reduction; additional
tests showed that neither free iron nor glutathione produced the
effect.
When Hb was removed from hemolyzed samples by ethanol precipitation, no
deterioration of plasma NO3-
signal or decrease between Pre-Hb and Post-Hb aqueous
NO3- calibrators was detected.
We wondered whether sample dilution accounted for the improvement in
Post-Hb aqueous NO3- signal.
With respect to the completely hemolyzed sample, a threefold dilution
would have been equivalent to a sample with 
10% hemolysis. This
degree of hemolysis, according to Table 1
, would still have produced a
significant decrease in the signal produced by the Post-Hb aqueous
NO3- calibrators.
Because this was not the case, we believe that simple dilution of Hb
alone cannot account for the improvement in the NO chemiluminescent
signal.
According to Braman and Hendrix (10), the chemiluminescent method of detecting NO using vanadium(III) is superior to other methods because many samples can be analyzed sequentially without the necessity of sample prereduction or other preparation. Our results suggest that sample hemolysis ranging from 2.9% to 30% (note that 1–2% hemolysis gives a pinkish color to plasma) may compromise the accurate detection of NOx- species because Hb accumulates in the purge vessel. Ethanol precipitation can be used to clean up the sample with near-quantitative recovery of NOx- species. Other precipitants were not evaluated in the chemiluminescent system. As NOx- measurements gain increasing clinical acceptance in patient monitoring, it will be important to recognize which patient samples are hemolyzed so that corrective measures can be taken to ensure accurate analysis.
Acknowledgments
This research was supported by Medical Research Council Grant MA-13941 (to C.G. Ellis). R.M. Bateman was supported by the Spoerel Research Fellowship, Department of Anesthesia, University of Western Ontario.
References
The following articles in journals at HighWire Press have cited this article:
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  R. M. Bateman, C. G. Ellis, and D. J. Freeman Optimization of Nitric Oxide Chemiluminescence Operating Conditions for Measurement of Plasma Nitrite and Nitrate Clin. Chem., March 1, 2002; 48(3): 570 - 573. [Full Text] [PDF]
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) were followed
by repeated injections of the
NO3--supplemented hemolyzed plasma samples
(NO3-/Hb; 
and 
) and a second series of
aqueous NO3- calibrators. Whole-blood
hemolysis was induced by digitonin (0, 20, 45, and 90 µmol/L) and
lysis buffer. Panels A–D show the effect of Hb
accumulation on NO3- determination.
Panels E–H compare the effect of mildly hemolyzed
untreated (20 µmol/L digitonin) and completely hemolyzed (lysis
buffer) samples with ethanol-treated samples. 
, accumulated Hb.
Values on x axis indicate number of injections.
Vertical dashed lines demarcate
NO3- calibrators from hemolyzed samples.