J Forensic Sci, January 2016, Vol. 61, No.

1
doi: 10.1111/1556-4029.12918
TECHNICAL NOTE Available online at: onlinelibrary.wiley.com

TOXICOLOGY

Hui Yan,1 M.Sc.; Xiangyi Zhuo,1 B.Sc; Baohua Shen,1 M.Sc.; Ping Xiang,1 Ph.D.; and Min Shen,1 M.Sc.

Determination of Nitrite in Whole Blood by

High-Performance Liquid Chromatography with
Electrochemical Detection and a Case of
Nitrite Poisoning*

ABSTRACT: Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue-
baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed
conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini-cartridges to remove hydrophobic
compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite
was 13.8 min and the detection limit of nitrite in whole blood was 0.4 lmol/L. The calibration curve was linear (r2 = 0.9999) over the concen-
tration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined
using the present method. The basal levels for nitrite in human blood was determined with 7.1  0.9 lmol/L (n = 12).

KEYWORDS: forensic science, forensic toxicology, nitrite, blood, ion chromatography, electrochemical detection, intoxication

Nitrite is widely used for various purposes, such as coronary
artery dilation, coloring meat, preventing rust, fertilizing crops,
and producing explosives (1). However, nitrite is a major toxic-
ity hazard to children and is responsible for the blue-baby syn-
drome (2). Infants are particularly sensitive because a large
proportion of hemoglobin in infants is in the fetal hemoglobin
form, which is more readily oxidized to methemoglobin than
adult hemoglobin (3). Diet is the primary method of exposure to
nitrite. Vegetables, meat, cured-meat products, and drinking
water are the nitrite sources for humans. After reacting with
amides and secondary amines, various N-nitroso compounds are
generated, which are toxic, mutagenic, teratogenic, and carcino-
genic (4).
Nitrite in biological specimens is analyzed using the Griess
assay (5), ion chromatography (6), gas chromatography-mass
spectrometry (GC-MS) (7), and capillary electrophoresis (CE)
(8). Among these methods, ion chromatography is the most com-
mon for a simple analysis of nitrite. Most of previous reports
used plasma (6), serum (9), urine (10), and saliva (4) as the
specimens, but there are few reports for whole-blood samples. In
blood, the half-life for nitrite is notably short because nitrite in
the bloodstream immediately reacts with oxyhemoglobin to form
1
Department of Forensic Toxicology, Institute of Forensic Science, Min-
istry of Justice, Shanghai Key Laboratory of Forensic Medicine, 1347 Road
Guangfu Xi, Shanghai 200063, China.
*Supported by grants from the National Natural Science Foundation of
China (81302614), the Science and Technology Commission of Shanghai
Municipality (13ZR1443000, 14DZ2270800), Ministry of Finance, PR China
(GY2013G-7).
Received 18 June 2014; and in revised form 18 Jan. 2015; accepted 23
Jan. 2015.
methemoglobin and nitrate. However, whole-blood analysis is
particularly essential for hemolysed blood samples in forensic
practices because plasma or serum is unobtainable. Nitrite was
determined in blood samples from four different individuals
using a flow injection analysis (FIA) and an IC method, where
the concentrations range was 0.1–0.4 lmol/L (11,12). The blood
sample volumes in both methods are quite large (1.5 mL).
Another IC method with a detection limit of 4.3 lmol/L could
not detect the endogenous nitrite levels in whole blood of seven
healthy subjects, which was possibly because of the lack of
sensitivity (1).
Therefore, we aimed to develop a sensitive and selective
method to quantify nitrite in human blood, which can help to
diagnose nitrite poisoning because excess nitrite can enter the
blood circulation from diet, oral medicines, or the environment.
To prevent the rapid metabolism of nitrite to nitrate in the ery-
throcytes, a particular method to prepare blood samples must be
identified.
Materials and Methods
Material
HPLC-grade acetonitrile and methanol were from Sigma-
Aldrich (St Louis, MO). The nitrite IC standard solution
(1000 mg/L) was purchased from NSI Solutions (Raleigh, NC).
All other chemicals were of analytical grade or higher purity,
and the aqueous solutions were prepared with distilled water
(resistivity 18.2 MΩ/cm), which was purified using a Milli-Q
system (Millipore, Molsheim, France). One cc cartridges of On
Guard II RP, On Guard II Ag and On Guard II Na were

254 © 2015 American Academy of Forensic Sciences


purchased from Dionex (Sunnyvale, CA). The syringe filters
(PES, 0.22 lm, 13 mm) were from ANPEL (Shanghai, PRC).
Instrumentation
A Thermo Scientific Dionex ICS 5000 ion chromatograph
(Sunnyvale, CA) was used. It is composed of four modules: AS-
DV autosampler, Single Pump (SP) module, Eluent Generator
(EG) module, and Detector/Chromatography (DC) module. Instru-
ment control, acquisition, and data processing were performed
using the Chromeleon software. Chromatographic separation was
achieved using an Ion Pac AG11-HC (50 mm 9 4 mm i.d.)
guard column and an Ion Pac AS11-HC (250 mm 9 4 mm i.d.)
analytical column that was eluted with potassium hydroxide
(KOH), which was produced using an online automatic eluent
generation system (EGC KOH). The KOH gradient program was
as follows: isocratic 5 mmol/L KOH for 25 min, gradient from 5
to 50 mmol/L KOH for 0.2 min, isocratic 50 mmol/L KOH for
9.8 min, and re-equilibration over 10 min with isocratic 5 mmol/
L KOH. The flow-rate was 1.0 mL/min throughout the procedure
and there was no buffer. The total run time was 45 min. An
Anion Self Regenerating Suppressor (ASRS) 300 operating at
112 mA was used for the suppressed conductivity detection. The
column temperature and detector temperature were set at 30°C
and 35°C, respectively. Direct conductivity detection was used.
Sample Preparation
In a 1.5 mL polypropylene centrifuge tube, 200 lL of blood
sample and 800 lL of acetonitrile were added for deproteiniza-
tion. The mixture was briefly vortex-mixed for 10 sec and cen-
trifuged at 9588 g for 3 min in a Minispin Plus
microcentrifuge (Eppendorf, Hamburg, Germany). The super-
natant was transferred to another tube and evaporated to
FIG. 1––Representative chromatograms for measurement of a standard solution containing nitrite (2.2 lmol/L) (a) and a blood sample (50-fold dilution)
taken from a patient suffering nitrite poisoning (b).
YAN ET AL. . ANALYSIS OF NITRITE IN WHOLE BLOOD 255
dryness under a N2 flow at 75°C. The residue was reconsti-
tuted in 20 mL of deionized water, vortexed for 10 sec, filtered
through a 0.22 lm filter, and successively purified with On
Guard II RP, On Guard II Ag, and On Guard II Na cartridges.
The On Guard II RP cartridges were conditioned with metha-
nol (10 mL) and subsequently deionized water (15 mL); then,
it was left for 30 min. The On Guard II Ag and Na cartridges
were conditioned with deionized water (10 mL) and left for
30 min. A 250 lL aliquot of the final filtrate was analyzed
using ion chromatography.
Venous blood samples were collected from twelve healthy
volunteers (no dietary restriction) and a patient who suffered
severe acute nitrite poisoning.
Method Validation
The following elements were used to validate the method: lin-
earity, detection limit, quantification limit, precision, and accu-
racy. The quantitative analysis was performed using the external
calibration method with the peak heights. The linearity of this
method was assessed using the aqueous standards at various con-
centrations (2–2200 lmol/L, 8 plots). The calibration curve
(y = ax + b) was constructed from the plots of the peak heights
(y, lS) of the analyte concentrations (x, lmol/L) of the calibra-
tion standards. The analyte concentrations of the unknown sam-
ples were determined by interpolating the calibration curves. The
method sensitivity was assessed by determining the low limit of
detection (LOD) and low limit of quantification (LOQ). The
LOD calculation was based on a signal-to-noise ratio of 3, and
LOQ was defined as the concentration of the lowest calibration
standard that was used. The method precision was determined
by injecting the calibration samples and two QC samples
(n = 4). The precision, which was determined based on 22 and
217 lmol/L nitrite-spiked blood samples was expressed as the
256 JOURNAL OF FORENSIC SCIENCES

relative standard deviation (%R.S.D.) of the relative peak twelve healthy volunteers were analyzed for nitrite. The mean
heights. The interday precision was also evaluated by performing basal nitrite concentration that was measured using this method
four replicates each day for 3 days to determine of two spiked was 7.1  0.9 lmol/L. The reported nitrite concentrations in
QC samples (22, 217 lmol/L). The recovery of added nitrite in human biological fluids are summarized in Table 1. The nitrite
blood (with endogenous nitrite) on three spiked QC samples was concentration range in the circulation is 0.1–20 lmol/L. The
investigated using the standard addition method. nitrite concentrations significantly vary in biological fluids even
when the same methodology was used. Variability in dietary
intake may significantly contribute to the variability in circulat-
Results and Discussion ing nitrite and to urinary excretion. Classically, nitrite in biologi-
cal specimens was determined using the Griess assays,
Method Development
where nitrite is diazotized with the amino group of sulfanil-
A series of analytical procedures has been devised to eliminate amide and then reacted with N-1-naphthyl- ethylenediamine to
interfering constituents in whole blood. Proteins in blood sam- form a colored product (14). These methods are subject to vari-
ples, particularly hemoglobin, may considerably interfere with ous interferences and lack of specificity. Other methods such
the quantitative determination of nitrite using ion chromatogra-
phy. Preik-Steinhoff et al. (11) eliminated proteins by the precip-
itation with sodium hydroxide followed by ultrafiltration using
TABLE 1––Basal nitrite concentrations in biological matrices.
commercially available cartridges. However, most ultrafiltration
cartridges are contaminated with nitrite and nitrate, which results Nitrite
in higher nitrite and nitrate concentrations in ultrafiltered cases Concentration
compared to nonultrafiltered plasma samples (13). Acetonitrile Biological (mean  SD)
was selected for the protein precipitation in this study; thus, it Matrix (lmol/L) Detection References
immediately disrupted all blood cells and irreversibly denatured Plasma 0.16  0.059 CE Wang et al. (8)
all proteins. The following use of On Guard II RP cartridges Plasma 6.1  2.3 CE Zuni
c et al. (21)
was notably useful to remove hydrophobic compounds from the Plasma 3.5  1.6 HPLC-UV Smith et al. (22)
Plasma 3.1  0.4 HPLC-UV Radisavljevic et al.
blood extracts. (23)
Nitrite detection is susceptible to the interference of chloride Plasma 0.55  0.16 HPLC-UV Tsikas et al. (24)
anion, which is present at notably high levels in blood (approxi- Plasma 0.71  0.46 HPLC-UV/ECD Stratford et al. (25)
mately 100 mmol/L). Silver acetate or silver fluoride can remove Plasma 1.3  0.8 HPLC-ECD Jedlickova et al. (6)
the chloride ions but give intense acetate or fluoride peaks that Plasma 1.3–13 (range) Griess Moshage et al. (5)
Plasma 0.45 CE Leone et al. (15)
overlap with the nitrite peak and other peaks (1). In this study, Plasma 0.6  0.27 GC-MS Rhodes et al. (7)
the On Guard II Ag cartridges with On Guard II Na are applied Plasma 3.3  1.5 CE Ueda et al. (26)
to remove the chloride ions and avoid contamination of the sepa- Plasma 0.23–0.43 FIA-Griess Lauer et al. (27)
ration column and autosuppressor by Ag ions. (range)
Plasma 0.35  0.013 FIA-Griess Kleinbongard et al.
With the described sample preparation, nitrite in human blood (28)
was detected at 13.8 min using anion-exchange chromatography. Plasma 9 Griess Dembny et al. (29)
The addition of nitrite standard confirmed its identity and clearly Plasma 0.2  0.1 HPLC-Griess Ishibashi et al. (30)
separated from other types of disturbing peak (Fig. 1). Plasma 0.2  0.02 FIA Kleinbongard et al.
The method linearity was assessed using aqueous standards. (31)
Plasma 0.22  0.07 Griess Giustarini et al. (32)
The nitrite calibration curves appeared linear from 2 to Plasma 3.6  0.8 GC-MS Tsikas et al. (33)
2200 lmol/L. The linearity regression equation was Serum 4.2  3.9 HPLC-UV Monaghan et al. (34)
y = 0.0031x + 0.0019 (R2 = 0.9999), where x represents the Serum 0.14 Chemiluminescence Farell et al. (35)
concentration of nitrite and y represents the peak height. The Serum 5–20 (range) SIA-Griess Pinto et al. (9)
Serum 3.3  2.1 Griess Phizackerley and
limit of detection of this assay for nitrite in blood under this Al-Dabbagh (36)
experimental condition was 0.4 lmol/L at a signal-to-noise ratio Serum 4.9  1.2 Griess Miranda et al. (37)
of 3:1. The limit of quantification was 2 lmol/L for the lowest Serum 3.7  0.6 Griess Sastry et al. (38)
calibration standard. The recovery of added nitrite in the physio- Serum 0.5  0.07 GC-MS Keimer et al.(39)
logically relevant concentration range (4.3–435 lmol/L) was Blood 0.58  0.12 HPLC-ECD Preik-Steinhoff et
al. (11)
investigated using the standard addition method and found to be Blood 0.1–0.4 (range) FIA-Griess Schulz et al. (12)
64  8% (n = 6). The method accuracy is inevitably affected, Cerebrospinal 0.23  0.01 HPLC-UV/ECD Zecca et al. (40)
which may be associated the nitrite decrease in blood because of fluid
the action of hemoglobin in vitro during the sample treatment. Saliva 80–148 (range) HPLC-ECD Helalehet al. (4)
Urine 4 (mean) Griess Moshage et al. (5)
The method precision, which was determined based on 22 and Urine 0.2–1.6 (range) GC–MS Tsikas et al. (10)
217 lmol/L nitrite spiked blood samples, was expressed as the Urine 0.49  0.25 GC–MS Tsikas et al. (33)
relative standard deviation (%R.S.D.) of the relative peak lmol/mmol
heights. The intraday precision were 10.5% and 3.4%, and the creatinine
interday precision during 3 days (n = 12) were 14.9% and 4.9% Urine 380  61 HPLC-UV Radisavljevic et al.
(23)
for 22 and 217 lmol/L nitrite in the blood samples, respectively. Urine 0.6  0.03 GC-MS Keimer et al. (39)
Urine 14  5 HPLC-Griess Kurioka et al. (41)
(1.9–8.1
Nitrite Levels in Human Biological Fluids lmol/mmol
creatinine)
The proposed method was applied to determine the nitrite
concentrations in human blood samples. The blood samples of FIA, Flow injection analysis; SIA, Sequential injection analysis.

as high-performance liquid chromatography (HPLC) (4), gas
chromatography–mass spectrometry (GC–MS) (10), and capillary
electrophoresis (CE) (15) are also applied to measure nitrite in
biological fluids. Despite of a specific identification for nitrite,
the GC–MS methods require a derivatization reaction prior to
analysis, which is a complicated process. Among various types
of high-performance liquid chromatography, the conductimetry
detector is universal for ionic substances.
When the diet is poor in nitrite or nitrate, the blood concentra-
tion of nitrite may indicate nitric oxide (NO) synthesis using
the endothelial NO synthase (NOS), which is important in
regulating the vascular tone, neurotransmission, host immunity,
nutrient metabolism, and entire-body homeostasis (16). Kelm
et al. (17) reported that an increase in serum concentrations of
nitrite from 0.402  0.059 to 0.977  0.082 lmol/L (n = 12)
was significantly correlated with an increase in forearm
blood flow in response to the infusion of acetylcholine (an
endothelium-dependent vasodilator). The concentration of serum
nitrite sensitively reflects the changes in endothelial NO forma-
tion in human forearm circulation. Kohli (18) observed that
plasma levels of nitrite in streptozotocin-induced diabetic rats
(0.53  0.05 lmol/L) had lower nitrite plasma levels than non-
diabetic rats (0.71  0.08 lmol/L).
Case Report
A 30-year-old woman was sent to hospital by her husband.
After quarreling with the family, she ingested a small quantity of
sodium nitrite powder to attempt suicide several minutes before
she reached the hospital, which resulted in hypoxemia and disor-
der of consciousness. Her lips and extremities were markedly
cyanotic. The blood sample was collected approximately a quar-
ter after admission, and promptly sent to our laboratory for toxi-
cological analysis. The blood nitrite ion was 44 lmol/L (i.e.,
2 lg/mL), which is much higher than concentrations in Table 1
and consistent with the clinical symptoms of nitrite poisoning.
After proper therapy with specific antidote, the patient eventually
recovered a week later. An accidental ingestion of substance that
contains nitroso-substances may release a large amount of nitrite
in body. Nitrite (50 lmol/L) was measured for a human blood
specimen of a male subject immediately after he sniffed isobutyl
nitrite gas (1).
Nitrite can induce systemic toxicity only after it is absorbed
into the gastrointestinal tract. The acute toxic effects of nitrite
are well documented, such as methemoglobinemia, vasodilata-
tion, hypotension, tachycardia, collapse, nausea, vomiting,
abdominal cramps, and diarrhea. Methemoglobin levels above
70% are likely fatal because methemoglobin cannot transport
oxygen (19). The present ion chromatographic method for nitrite
may help to find the cause of poisoning, for other compounds
such as chlorate, it can also cause methemoglobinemia. How-
ever, in the postmortem case, most nitrite in whole blood is
converted into nitrate during the postmortem interval between
the death and sample collection, which may be a limitation of
this study. Multiple specimen types such as blood, bile, and vit-
reous humor were tested for both nitrite and nitrate in a case of
suicidal sodium nitrite poisoning (20). A 34-year-old male
ingested sodium nitrite and caused death within 1 h. The time
interval between death and autopsy was 5 h. The concentrations
of nitrite and nitrate in blood were 0.55 and 30.0 lg/mL, respec-
tively. Analysis of the blood for methemoglobin was also per-
formed, and 90% of the hemoglobin had been converted to
methemoglobin.
YAN ET AL. . ANALYSIS OF NITRITE IN WHOLE BLOOD 257
Conclusions
A sensitive method to determine nitrite in human blood using
ion chromatography was successfully developed and validated.
The combination of the specifically developed sample prepara-
tion with electrical conductivity detection yields a low detection
limit of 0.4 lmol/L in human blood and requires a small amount
of sample. Using this method, the basal levels for nitrite in
human blood were determined with 7.1  0.9 lmol/L (n = 12).
It is also useful in the detection and quantification of nitrite in
other biological systems and investigation of nitrite-poisoning
cases.
References
1. Suzuki O, Watanabe K, Okamoto N, Nozawa H, Ishii A. Simultaneous
analysis of nitrite and nitrate in whole blood by ion chromatography.
J Liq Chromatogr Relat Technol 2005;28:3077–85.
2. Guti
errez AJ, Rubio C, Caballero JM, Hardisson A. Nitrites. In: Wexler
P, editor. Encyclopedia of toxicology. 3rd rev. edn. London,U.K.: Aceda-
mic, 2014;532–5.
3. Abdollahi M, Khaksar MR. Sodium nitrite. In: Wexler P, editor. Ency-
clopedia of toxicology. 3rd rev. edn. London, U.K.: Acedamic,
2014;334–7.
4. Helaleh MIH, Korenaga T. Ion chromatographic method for simultaneous
determination of nitrate and nitrite in human saliva. J Chromatogr B
2000;744:433–7.
5. Moshage H, Kok B, Huizenga JR, Jansen PLM. Nitrite and nitrate deter-
minations in plasma: a critical evaluation. Clin Chem 1995;41:892–6.
 Determination of nitrate and nitrite by
6. Jedlickov
a V, Paluch Z, Alus
ık S.
high-performance liquid chromatography in human plasma. J Chromatogr
B 2002;78:193–7.
7. Rhodes PM, Leone AM, Francis PL, Struthers AD, Moncada S. The
L-arginine:nitric oxide pathway is the major source of plasma nitrite in
fasted humans. Biochem Biophys Res Commun 1995;209:590–6.
8. Wang X, Adams E, Van Schepdael A. A fast and sensitive method for
the determination of nitrite in human plasma by capillary electrophoresis
with fluorescence detection. Talanta 2012;97:142–4.
9. Pinto PCAG, Lima JLFC, de Sousa Saraiva MLMF. Sequential injection
analysis of nitrites and nitrates in human serum using nitrate reductase.
Clin Chim Acta 2003;337:69–76.
10. Tsikas D. Methods of quantitative analysis of the nitric oxide metabolites
nitrite and nitrate in human biological fluids. Free Radical Res
2005;39:797–815.
11. Preik-Steinhoff H, Kelm M. Determination of nitrite in human blood by
combination of a specific sample preparationwith highperformance
anion-exchange chromatography and electrochemical detection.
J Chromatogr B 1996;685:348–52.
12. Schulz K, Kerber S, Kelm M. Reevaluation of the Griess method for
determining NO/NO 2 in aqueous and protein-containing samples.
Nitric Oxide 1999;3:225–34.
13. Ricart-Jan
e D, Llobera M, Lopez-Tejero MD. Anticoagulants and other
preanalytical factors interfere in plasma nitrate/nitrite quantification by
the Griess method. Nitric Oxide 2002;6:178–234.
14. Tsikas D. Analysis of nitrite and nitrate in biological fluids by assays
based on the Griess reaction: appraisal of the Griess reaction in the
L-arginine/nitric oxide area of research. J Chromatogr B 2007;851:51–70.
15. Leone AM, Francis PL, Rhodes P, Moncada S. A rapid and simple
method for the measurement of nitrite and nitrate in plasma by high-per-
formance capillary electrophoresis. Biochem Biophys Res Commun
1994;200:951–7.
16. Wu G, Morris SM Jr. Arginine metabolism: nitric oxide and beyond.
Biochem J 1998;336:1–17.
17. Kelm M, Preik-Steinhoff H, Preik M, Strauer BE. Serum nitrite sensi-
tively reflects endothelial NO formation in human forearm vasculature:
evidence for biochemical assessment of the endothelial L-arginine-NO
pathway. Cardiovasc Res 1999;41:765–72.
18. Kohli R, Meininger CJ, Haynes TE, Yan W, Self JT, Wu G. Dietary
L-arginine supplementation enhances endothelial nitric oxide synthesis in
streptozotocin-induced diabetic rats. J Nutr 2004;134:600–8.
19. Hunault CC, van Velzena AG, Adrienne JAMS, Ronald CS, Jan M.
Bioavailability of sodium nitrite from an aqueous solution in healthy
adults. Toxicol Lett 2009;190:48–53.
258 JOURNAL OF FORENSIC SCIENCES
20. Standefer JC, Jones AM, Street E, Inserra R. Death associated with
nitrite ingestion: report of a case. J Forensic Sci 1979;24(4):768–71.
 c G, Spasi
c S, Jeli
c-Ivanovi
c Z. Simple and rapid method for
21. Zuni

the measurement of nitrite and nitrate in human plasma and cere-
brospinal fluid by capillary electrophoresis. J Chromatogr B 1999;
727:73–9.
22. Smith CCT, Stanyer L, Betteridge DJ. Evaluation of methods for the
extraction of nitrite and nitrate in biological fluids employing high-per-
formance anion-exchange liquid chromatography for their determination.
J Chromatogr B 2002;779:201–9.
23. Radisavljevic Z, George M, Dries DJ, Gamelli RL. Determination of
intracellular and Extracellular Nitrite and Nitrate by Anion Chromatogra-
phy. J Liq Chromatogr Relat Technol 1996;19:1061–79.
24. Tsikas D, Rossa S, Sandmann J, Frolich JC. High-performance liquid
chromatographic analysis of nitrite and nitrate in human plasma as S-ni-
troso-N-acetylcysteine with ultraviolet absorbance detection. J Chro-
matogr B 1999;724:199–201.
25. Stratford MRL, Dennis MF, Cochrane R, Parkins CS, Everett SA. The
role of nitric oxide in cancer improved methods for measurement of
nitrite and nitrate by high-performance ion chromatography. J Chro-
matogr A 1997;770:151–5.
26. Ueda T, Maekawa T, Sadamitsu D, Oshita S, Ogino K, Nakamura K.
The determination of nitrite and nitrate in human blood plasma by capil-
lary zone electrophoresis. Electrophoresis 1995;16:1002–4.
27. Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, et al.
Plasma nitrite rather than nitrate reflects region al endothelial nitric oxide
synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad
Sci USA 2001;98:12814–9.
28. Kleinbongard P, Dejam A, Lauer T, Jax T, Kerber S, Gharini P, et al.
Plasma nitrite concentrations reflect the degree of endothelial dysfunction
in humans. Free Radic Biol Med 2006;40:295–302.
29. Dembny KD, Roza AM, Johnson C, Adams MB, Pieper GM. Heparin
interferes with the determination of plasma nitric oxide by inhibition of
enzymatic conversion of nitrate to nitrite by nitrate reductase. Clin Chim
Acta 1998;275:107–14.
30. Ishibashi T, Matsubara T, Ida T, Hori T, Yamazoe M, Aizawa Y, et al.
Negative NO3 difference in human coronary circulation with severe
atherosclerotic stenosis. Life Sci 1999;66:173–84.
31. Kleinbongard P, Rassaf T, Dejam A, Kerber S, Kelm M. Griess method
for nitrite measurement of aqueous and protein-containing samples.
Methods Enzymol 2002;359:158–68.
32. Giustarini D, Dalle-Donne I, Colombo R, Milzani A, Rossi R. Adapta-
tion of the Griess reaction for detection of nitrite in human plasma. Free
Radical Res 2004;38:1235–40.
33. Tsikas D, B€oger RH, Bode-B€oger SM, Gutzki FM, Fr€olich JC. Quantifi-
cation of nitrite and nitrate in human urine and plasma as pentafluo-
robenzyl derivatives by gas chromatography–mass spectrometry using
their 15N-labelled analogs. J Chromatogr B 1994;661:185–91.
34. Monaghan JM, Cook K, Gara D, Crowther D. Determination of nitrite
and nitrate in human serum. J Chromatogr A 1997;770:143.
35. Farell AJ, Blake DR, Palmer RM, Moncada S. Increased concentrations of
nitrite in synovial fluid and serum samples suggest increased nitric oxide
synthesis in rheumatic diseases. Ann Rheum Dis 1992;51:1219–22.
36. Phizackerley PJ, Al-Dabbagh SA. The estimation of nitrate and nitrite in
saliva and urine. Anal Biochem 1983;131:242–5.
37. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric
method for simultaneous detection of nitrate and nitrite. Nitric Oxide
2001;5:62–71.
38. Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotomet-
ric determination of serum nitrite and nitrate by copper-cadmium alloy.
Anal Biochem 2002;306:79–82.
39. Keimer R, Stutzer FK, Tsikas D, Troost R, Gutzki FM, Fr€olich JC. Lack
of oxidative stress during sustained therapy with isosorbide dinitrate and
pentaerythrityl tetranitrate in healthy humans: a randomized, double-blind
crossover study. J Cardiovasc Pharmacol 2003;41:284–92.
40. Zecca L, Rosati M, Renella R, Galimberti M, Ambrosini A, Fariello RG.
Nitrite and nitrate levels in cerebrospinal fluid of normal subjects. J Neu-
ral Transm 1998;105:627–33.
41. Kurioka S, Koshimura K, Sugitani M, Murakami Y, Nishiki M, Kato Y.
Analysis of urinary nitric oxide metabolites in healthy subjects. Endocr J
1999;46:421–8.
Additional information and reprint requests:
Hui Yan, M.Sc.
Department of Forensic Toxicology
Institute of Forensic Science
Ministry of Justice
Guangfu Xi Road 1347
Shanghai 200063
China
E-mail: yanh501@163.com