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Letters |
Divisions of
1
Gastroenterology and Hepatology, and
2
Nephrology, Department of Medicine, University Hospital Groningen, 9700 RB Groningen, The Netherlands
a Address correspondence to this author at: Division of Gastroenterology and Hepatology, Department of Medicine, University Hospital Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Fax 31-50-3614756; e-mail h.moshage{at}med.rug.nl.
To the Editor:
Nitric oxide (NO·) plays an important role both in maintaining normal homeostasis and in the pathogenesis of various disorders (1). NO· has a short biological half-life and is rapidly converted into its stable metabolites, nitrite and nitrate (1)(2). In plasma, nitrite is rapidly oxidized to nitrate (2). Determination of nitrite and nitrate (NOx) in body fluids like plasma and urine is widely used as a marker of NO· production (3). However, bacteria in urine are known to produce nitrite, and leukocytes in urine sediments contain NO synthase activity (4). Therefore, it has been suggested that NOx determinations in urine are reliable only when precautions have been taken to prevent bacterial growth in the urine specimens. Indeed, Smith et al. (4) have shown that incubation of urine samples that contain bacteria leads to increased nitrite concentrations in these samples. Therefore, urine is often collected on ice and/or in the presence of antibiotics or organic solvents (5)(6)(7)(8). The objective of this study was to establish the effect of different storage times and temperatures on NOx concentrations in urine and to define optimal collection and storage protocols for NOx determination in urine.
NOx was determined in urine of 7 healthy volunteers and in urine of 10 individuals after kidney transplantation, as described previously (2)(9), except that the final NADPH concentration was increased to 250 µmol/L to improve recovery at higher NOx concentrations. Recovery of exogenously added nitrate from five randomly selected urine samples ranged from 91% to 110% (mean, 102%) for 100 µmol/L added nitrate and from 80% to 103% (mean, 94%) for 200 µmol/L added nitrate. The mean NOx concentration in the seven healthy volunteers was 895 µmol/L (range, 533-1354 µmol/L), in accordance with previously reported values (2)(5)(10). The mean NOx concentration in the 10 individuals after kidney transplantation was substantially lower (mean, 303 µmol/L; range, 55–836 µmol/L).
Within 1 h after voiding, urine samples were placed in glass tubes, capped, and incubated for 4, 8, and 24 h at 4, 20, and 37 °C. Aliquots were taken from the tubes and snap-frozen in liquid nitrogen. The NOx concentration was expressed as a percentage of the concentration measured in an aliquot of the urine sample that was immediately snap-frozen in liquid nitrogen after voiding (0-value).
Fig. 1
demonstrates that urinary NOx concentrations are stable for at
least 24 h when stored at 4 °C. At 20 °C, a sudden and
dramatic decrease in NOx concentration was noted in 2 of the 17 samples
between 8 and 24 h of incubation. At 37 °C, markedly decreased
NOx concentrations were observed in 6 of the 17 samples within 24
h. The decrease in NOx concentration was observed in normal urine
samples and in urine samples from individuals after kidney
transplantation.
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The explanation for the decrease in NOx concentration is not clear. One
possibility is the presence of bacteria that are able to reduce nitrate
and nitrite. Another possibility is the release of a factor that
interferes with the NOx assay, e.g., an inhibitor of the enzyme,
nitrate reductase. This enzyme is necessary to convert nitrate into
nitrite, which is subsequently measured in the Griess assay. To
investigate this possibility, known amounts of nitrate were added to
urine samples in which the sudden decrease in NOx concentration had
occurred. Recovery of nitrate in these samples was near-quantitative
(84–88%), suggesting that no factor is released that interferes with
the NOx assay. NOx concentrations did not increase during incubation at
37 °C, compared with the 0-value in any of the urine samples tested,
even in those samples with bacteriuria 
10 CFU/mL
(10 colony-forming units/L). This contrasts with the
finding of Smith et al. (4). The reason for this discrepancy
is not clear, but may be related to the fact that Smith et al. measured
nitrite specifically, whereas in our study, the sum of nitrite and
nitrate (NOx) was determined. Moreover, in our study only two urine
samples contained >10 CFU/L, and none of our samples had
urinary sediments containing leukocytes. In conclusion, our results
demonstrate that NOx concentrations can be reliably determined in urine
samples stored at 4 °C for at least 24 h, without additional
precautions. Serious artifacts can occur after storage >4 h at room
temperature and at increased temperature, causing gross underestimation
of urinary NOx concentration.
References
The following articles in journals at HighWire Press have cited this article:
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  H. Moshage Simple and Reliable Measurement of Nitric Oxide Metabolites in Plasma Clin. Chem., October 1, 2009; 55(10): 1881 - 1882. [Full Text] [PDF]
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