Nitrite Adulteration of Workplace Urine Drug-Testing Specimens I ...

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Experimental The commercial product Klear was purchased from a local merchant. The package insert directs the donor to "Add the contents of one tube of Klear to each urine sample...For large samples of more than 4 oz. use two tubes". The urine test strip Chemstrip TM was used to screen for nitrite and was obtained from Boehringer Mannheim (Indianapolis, IN). Specimens for determination of nitrite concentrations In order to determine the concentration of nitrite in urine, the following specimen types were employed: clean-catch specimens for medical urinalysis from patients who had tested positive for nitrite with the Chemstrip and other specimens that were positive by culture for nitrate-reducing pathogens; specimens from patients taking medications that contained nitrate, nitro, or azo groups, nitroglycerin, isosorbide dinitrate, a combination of nitroglycerin and isosorbide dinitrate, isosorbide-5-mononitrate, nitro- furantoin, nifedipine, phenazopyridine, nitroprusside, ranitidine, and metronidazole; specimens submitted for drug testing that demonstrated that they may have been adulterated with nitrite by a GC-MS assay and the Chemstrip nitrite test; and specimens submitted for employment drug testing that were certified as negative. In order to determine if nitrite concentration increased with time from bacterial metabolism at room temperature, some medical urinalysis and negative drug-test specimens were analyzed after receipt, then held at room temperature 6-8 days and retested. Identification and quantitation of nitrite in urine Nitrite analysis was performed on a Lachat QuikChem AT TM automated continuous flow analyzer (Lachat Instruments, Milwaukee, WI) using U.S. Environmental Protection Agency (EPA) method 353.2, Determination of Nitrate-Nitrite Nitrogen by Automated Colorimetry, wavelength, 543 nm (3). The method is authorized for nitrite and nitrate testing in drinking water. When operated without the cadmium sulfate reduction step, the method is specific for nitrite; any nitrate present does not cross-react. The detection limit of nitrite in urine is 0.612g/mL, which is expressed in nitrogen from nitrite ion. Because nitrite measurements in biological matrices and in the medical literature are reported primarily in micrograms-per-milliliter nitrite ion, the concentrations obtained by the EPA method were mul- tiplied by the factor 3.284 to convert to micrograms-per-milliliter nitrite ion. Negative interference occurred from residual chlo- rine, iron, copper, and other metals. According to the EPA pro- cedure, there are no known substances in drinking water that give a positive interference. The testing was performed by the Utah State Public Health Laboratory (Salt Lake City, UT), which is certified by the EPA. The statistical significance of nitrite concentration changes over time was evaluated by the method of randomized, paired comparison. Results and Discussion Nitrite in urine may arise from internal sources (i.e., for- mation in tile body) or from external sources. 90 Journal of Analytical Toxicology, Vol. 22, March/April 1998 Nitrite in urine from internal sources Mammalian biochemistry. In 1981, it was determined that rodents and humans excrete larger amounts of nitrate than could be accounted for from the diet, suggesting that mammalian cells have the biochemical processes necessary to synthesize oxides of nitrogen (4,5). Studies from many disciplines converged to identify nitric oxide radical (NO.) as the precursor of endogenously formed nitrite and nitrate, l-Arginine is metab- olized to citrulline and NO., which requires molecular oxygen and reduced NADP, and is catalyzed by the enzyme nitric oxide synthase (NOS). NO. is oxidized further to nitrite and nitrate, which are eventually excreted primarily in the urine. NO. is an important secretory chemical involved in the regulation of the cardiovascular, nervous, and immune systems and among the most vigorously researched entities in biochemistry and the medical sciences. NOS occurs in three isoforms (6). In neu- ronal tissues (Type I, nNOS) and in endothelium (Type III, eNOS) the enzyme is constitutive; they are normal tissue components. Type II (iNOS) is nonconstitutive and is discussed under Patho- logical Conditions. The NO. produced from nNOS activity functions in neurotransmission. NO. from eNOS functions as the primary regulator of blood vessel dilation, and also has throm- bolytic activity, as part of the body's physiological processes. The amount of nitrite/nitrate formed from NO. by nNOS and eNOS activities is very low (picomoles). The nitrite formed by actions of all three NOS enzymes is taken up by red blood cells and interacts with oxygenated hemoglobin to produce nitrate ion and methemoglobin (7). Therefore, nitrate concentration in urine from NOS activity is much greater than the nitrite concentration. With regard to the ratio of nitrate to nitrite in urine, Moshage et al. (8) reported a study of 13 healthy volunteers who consumed diets not regulated for nitrate content. The data indicated average concentrations of 61 IJg/mL nitrate and 0.2 !ag/mL nitrite. The nitrite concentration was only 0.4% of the total nitrate plus nitrite. Hovenga et al. (9) reported a normal range of nitrate plus nitrite in urine of 0-124 1Jg/mL, which is consistent with the data of Moshage et al. (8) cited here previ- ously. Pathological conditions. During inflammatory and infec- tious processes, cytokines and endotoxins stimulate several cell types to synthesize a third form of NOS, inducible NOS (Type II, iNOS), iNOS activity is implicated in a wide range of pathological conditions and produces NO. under these listed conditions: sepsis, asthma, rheumatoid arthritis, atherosclerotic lesions, tuberculosis, inflammatory bowel disease, surgical diversions of the urinary tract, allograft rejection, Alzheimer's disease, and multiple sclerosis (6). Although nitrite concentrations from iNOS are greater than that from normal physiological processes involving NO., they are relatively modest. Moncada et al. (10) in their literature review found that for nNOS and eNOS, concentrations of NO. released are in picomoles, whereas concentrations of NO, released from iNOS are 1000-fold greater, in nanomoles. Sasajima et al. (11) reported an increase in urinary nitrite excretion of up to 48 IJg/mL in the healing phase of ulcerative colitis. Wagner and Tannenbaum (12), while doing nitrate balance studies in humans on low nitrate diets (120 pmoles per day), observed that one person in the study became ill with nonspecific intestinal diarrhea. The
Journal of Analytical Toxicology, Vol. 22, March/April 1998 subject's average daily output of nitrate was approximately 750 lJmoles, but the nitrate output went to 4500 pmoles, a sixfold increase, on the third day of illness. By the fifth day of illness, nitrate output began to approach pre-illness levels. They repli- cated this pattern in the rat by injecting a pyrogenic factor from Escherichia coli, and obtained a ninefold increase in nitrate output over baseline. The highest level obtained, 4500 pmoles per day, assuming average daily urinary output of 1500 mL, would produce a nitrate concentration of about 186 IJg/mL. Nitrite was not independently measured, but assuming that nitrite is less than 1% of the total as indicated in the Moshage et al. (8) study, it would be less than 2 pg/mL. Urinary tract infection. Such enterobacteriaceae as Escherichia coli, Klebsiella, Proteus, Staphylococcus, and Pseu- domona are frequent causes of urinary tract infections. When numbers of these organisms are present in bladder urine in the range of ix 10S/mL, nitrate is reduced to nitrite by the microbial enzyme nitrate reductase. A semiquantitative test for nitrite was recommended as early as 1922 as an indicator of a significant urinary tract infection (13) and is still used extensively, employing commercially available urine test strips. These strips are based upon the Griess test for nitrite (14). The Chemstrip indicates a limit of detection of 0.5 pg/mL. Ten clean-catch specimens were obtained from a clinical urinalysis laboratory and the nitrite quantitated by the EPA method (Table I) to assess the concentration of nitrite associated with medical specimens which are test-strip nitrite positive. In order to determine if an increase in nitrite may occur upon incubation, the specimens were stored at room temperature for 8 days and retested. There was no significant change (p = 0.40) due to the extended holding; six of the specimens decreased and four increased in nitrite concentration. The data from 18 clean-catch specimens for which culturing indicated an enterobacteriaceae infection of approximately 100,000 colony-forming units per milliliter of urine are also listed in Table I. Averages and ranges for specimens 1-8 (initial and 7-day incubations) and all 18 specimens indicated no appre- ciable difference in the average or the ranges (p = 0.93). With this group, two specimens decreased in nitrite concentration while six specimens increased. The highest nitrite level in any of the 28 specimens in these two categories was 35.5 IJg/mL. These data are consistent with the Chemstrip product literature, which indicated that urine specimens infected with nitrate reducing organisms do not usually produce more than 100 IJg/mL nitrite (15). As far as is presently known, nitrate reductase enzymes capable of reducing nitrate to nitrite have not been identified in humans or other mammals (16). In addition to the nitrite formed from microbial nitro reductase activity, some portion of the nitrite measured in urinary tract infection is contributed by stimulation of iNOS activity by endotoxins from the enterobacteriaceae organisms (17). Nitrite in urine from external sources In the environment, nitrogen in the combined state occurs mostly as potassium and sodium nitrates. From the environment, Table I. Nitrite Concentrations in Urine Specimens that Were Nitrite Positive by Test Strip and in Specimens Positive for Enterobacteriaceae by Culture Nitrite (pg/mL) Nitrite (pg/mL) Test strip nitrite positive Day 0 Day 8 Enterobacteriaceae positive Day 0 Day 7 Average Range 1 18.4 1 5.9 4.6 2 33.5 2 7.2
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Nitrite Adulteration of Workplace Urine Drug-Testing Specimens I ...

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