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

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 nitro-
glycerin 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 litera-
ture 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 con-
centration 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 mam-
malian cells have the biochemical processes necessary to syn-
thesize 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 func-
tions 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 con-
centration. 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 patholog-
ical conditions and produces NO. under these listed condi-
tions: 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 concentra-
tions from iNOS are greater than that from normal physiolog-
ical 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 pico-
moles, 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