Nitrite and Nitrate Analyses

7/27/2019 Nitrite and Nitrate Analyses

1/26

Nitrite and Nitrate Analyses: A ClinicalBiochemistry Perspective

GRAHAM ELLIS,1 IAN ADATIA,2 MEHRDAD YAZDANPANAH,1,3 and SINIKKA K. MAKELA3

1Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College Street,Toronto, ON M5G 1L5, Canada, 2Divisions of Cardiology, Critical Care Medicine and Pediatrics,University of Toronto, and 3Division of Clinical Biochemistry, The Hospital for Sick Children, 555

University Avenue, Toronto, ON M5G 1X8, Canada

Objective: To review the assays available for measurement of nitrite

and nitrate ions in body fluids and their clinical applications.Design and Methods: Literature searches were done of Medlineand Current Contents to November 1997.Results: The influence of dietary nitrite and nitrate on the concen-trations of these ions in various body fluids is reviewed. An overviewis presented of the metabolism of nitric oxide (which is converted tonitrite and nitrate). Methods for measurement of the ions arereviewed. Reference values are summarized and the changesreported in various clinical conditions. These include: infection,gastroenterological conditions, hypertension, renal and cardiacdisease, inflammatory diseases, transplant rejection, diseases ofthe central nervous system, and others. Possible effects of environ-mental nitrite and nitrate on disease incidence are reviewed.Conclusions: Most studies of changes in human disease have beendescriptive. Diagnostic utility is limited because the concentrationsin a significant proportion of affected individuals overlap with thosein controls. Changes in concentration may also be caused by diet,

outside the clinical investigational setting. The role of nitrite andnitrate assays (alongside direct measurements of nitric oxide inbreath) may be restricted to the monitoring of disease progression,or response to therapy in individual patients or subgroups. Associ-ations between disease incidence and drinking water nitrate contentare controversial (except for methemoglobinemia in infants).Copyright 1998 The Canadian Society of Clinical Chemists

KEY WORDS: nitrates, nitrites, nitric oxide, nitric-oxide synthase, arginine, human disease

Introduction

I

nterest in the measurement of nitrite and nitrate

in various body fluids has increased in recentyears (15). These ions are ingested as part of thediet and are also produced endogenously from nitricoxide (NO). In this article, we shall review theeffects of dietary intake on nitrite and nitrate con-centrations in plasma and urine. We shall discuss

the metabolism of NO to nitrite and nitrate and

summarize methods of measuring these ions in bodyfluids. We shall comment on the clinical relevance ofthe measurements. Finally, we shall discuss possi-ble associations between dietary intake and diseaseincidence.

Dietary intake of nitrite and nitrate

Nitrite and nitrate ions are both highly soluble inwater. With few exceptions, they form water-solublesalts with almost all cations. When taken orally,they are readily absorbed from the proximal smallintestine as reviewed by Walker (6). About 25% of

orally ingested available nitrate is actively secretedinto the saliva. This nitrate is then partially con-verted to nitrite by oral bacteria (7) and to NO bystomach acids, helping to reduce gastrointestinaltract infection (6,8). The transport systems of ni-trates and nitrites have only recently become betterunderstood in bacteria (9), in plants (10), and inmammalian cells (11), where a nitrate and H

cotransporter seems important. Nitrites and ni-trates are added as preservatives and texture en-hancers to various foods, such as meat products andcheeses (12), but they are also a natural part of thediet. The concentrations in drinking water are usu-

ally 10 mg/L in the absence of bacterial contami-nation (13). In areas where drinking water concen-trations are high, steps are taken to lower them (14)in order to avoid nitrate-induced methemoglobin-emia in infants. Vegetables, especially beets, celery,and leafy vegetables like lettuce and spinach arerich in nitrates (6,15,16). Other vegetables containnitrate at lower concentrations, but because they areconsumed in greater quantity, they may contributemore nitrate to the diet. Santillana et al. (17) sum-marized findings from 710 commercial food samplesavailable in Spain. Cured meats had the highestmean nitrite content (36 g/g) and canned vegeta-bles the highest mean nitrate content (88 g/g).

Correspondence: Dr. Graham Ellis, Department of Clin-ical Biochemistry, St. Johns Hospital at Howden, HowdenRoad West, Livingston, West Lothian, EH54 6PP, UnitedKingdom.

Manuscript received January 6, 1998; accepted March24, 1998.

Clinical Biochemistry, Vol. 31, No. 4, 195220, 1998Copyright 1998 The Canadian Society of Clinical Chemists

Printed in the USA. All rights reserved0009-9120/98 $19.00 .00

PII S0009-9120(98)00015-0

CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998 195


2/26

Improper storage and bacterial contamination mayincrease nitrite (18). In Western diets, assessed bydietary survey, the mean adult daily dietary intakeof nitrite (0.1 mg) is much lower than that of nitrate(80 mg) (19). Others have found corresponding val-ues of 1.4 mg and 54 mg in a 9 to 24-year-old Finnishpopulation (20). In some European countries, meandaily intake may be as high as 8.7 mg of nitrite and

(in vegetarians) as high as 194 mg of nitrate (6).Excessive nitrite or nitrate intake could potentiallygenerate N-nitroso compounds, that are carcino-genic (21,22), but this is still controversial (6).

Nitrite and nitrate are excreted in the kidneys.The nitrate is excreted in the urine as such or afterconversion to urea (23). Clearance of nitrate fromblood to urine approximates 20 mL/min in adults(24), indicating considerable renal tubular reabsorp-tion of this ion. Tubular reabsorption is also sup-ported by studies of the urinary excretion of totalnitrate plus nitrite (NOx) concentrations in responseto diuretics (25). There is little detectable nitrite or

nitrate in feces (26). There is some loss of nitrate (40mol/L) or nitrite (3 mol/L) in sweat, but this is nota major route of excretion (27). (These values repre-sent means for 6 subjects after a 40 min sauna.)Patients on antibiotics had much lower sweat ni-trite, implying a role for skin bacteria in nitritesynthesis.

Variability in dietary intake (28) may contributegreatly to the variability of plasma nitrate and tourinary excretion. As would be expected, in con-trolled studies, the daily urine output increasedwith higher intake (26). Plasma nitrate increasedfrom about 30 mol/L to about 200 mol/L in re-sponse to a high-nitrate diet and to about 300mol/L after 500 mg of oral potassium nitrate (29).The ions disperse widely into body fluid spaces,giving a large volume of distribution: 0.28 L/kg (30),similar to that of the dog at 0.21 L/kg, equivalent tothe extracellular volume (31). A diet high in nitratemay still elevate urinary excretion during the nextday (29,32). There was a wide variability in plasmaNOx over the course of 1 day in five individualstaking their normal Japanese diet, as measured by 4hourly samples (33). Between-day variation was alsoobserved by Wang et al. (34). Some authors haverecommended that random urines should be takenimmediately after getting up in the morning (35), as

the second fasting sample of the morning (36), orjust prior to dinner (32) to reduce this variability.Excretion is then related to urine creatinine. Onegroup has recommended that there should be di-etary restriction of nitrates and nitrites for at least48 h prior to collection, if excretion is to be regardedas a measure of NO metabolism (29). Others haveused a 15 h fast, with distilled water as the onlyfluid permitted (37,38).

If a diet low in nitrite and nitrate is needed,Westfelt et al. (39) recommends the exclusion ofalcoholic beverages, caviar, charcuteries (cooked orcured delicatessen-style meats), cheese, herbs, pick-led fish, roots and vegetables during the study and

for 2 days before the studies are undertaken. Viini-kka (2) noted that many foodstuffs contain consid-erable amounts of nitrate and listed meat, vegeta-bles, fish, melon, herb and black teas, maltbeverages and wine as examples. Others have usedlow-nitrate diets containing approximately 210mol/d (40) or180 mol/d (41); the latter referencelists both permitted and avoidance food groups. A

low nitrate diet for at least 3 to 4 days was found byWang et al. (34) to minimize the influences of theJapanese diet on plasma NOx concentrations and24 h urinary excretion, expressed in relation tocreatinine excretion. However, these authors col-lected samples only between the third to the sixthday of the low nitrite and nitrate diet. Plasma levelstaken at 0600 h were found to be consistent after 4days of a well-balanced Japanese hospital diet (33).In contrast, morning urines (the second fastingsample of the morning in 12 healthy volunteers,expressed in relation to creatinine) seemed littleaffected by two days of low nitrate (180 mol/d)

diet, compared to the normal Scottish diet or anitrate-supplemented diet (36).Nitrate-containing drugs have been used for many

years for the treatment of angina. They produce NO(42,43) and give rise to urine nitrate. Because thetherapeutic doses are relatively small comparedwith normal dietary intake, their contribution toexcretion is generally quite small.

Nitric oxide (NO)

In humans, NO is produced endogenously fromvarious sources, the most important of which isL-arginine (4,5,29,39,4448). Arginine is oxidized

by NO synthase(s) (NOS, EC 1.14.13.39) to producecitrulline and NO. There are at least three isoen-zymes of NOS (4953); one is located almost exclu-sively in vascular endothelium. The NO that itproduces acts as a relaxing factor on smooth musclecells of the blood vessel wall, causing vasodilatation(44,54). By so doing, it is an important determinantof blood pressure (55). It is constitutively expressed.

A second (inducible) NOS is produced in macro-phages and many other cell types in response toinflammation or infection (52,5663). The NO pro-duced by the macrophage NOS not only damages theagent under attack but also causes apoptosis of the

macrophage (64). NO may also induce apoptosis inother cell types (6567). A third isoenzyme (neuro-nal NOS) is found constitutively in brain, neuronaltissue, neuroblastomas, skeletal muscle, -cells ofthe pancreatic islets, and also epithelial cells ofbronchioli uterus, and stomach (52). All threeisozymes are found in kidney (68). The activities ofthe two constitutively expressed NOS isozymes arenot constant. They are calcium and calmodulin-dependent enzymes. Transient increases in intracel-lular calcium concentrations result in short-livedbursts of activity. This enables NO to act as aneurotransmitter/neuromodulator by diffusion (62,6971) or by S-nitrosylation, nitrosothiol exchange,

ELLIS ET AL.

196 CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998


3/26

and related mechanisms (72). Inducible NOS con-tains tightly bound calcium and calmodulin and it isgenerally insensitive to ambient calcium concentra-tion (49,73) Hormones, such as estradiol, may up-regulate NO synthesis (74). As much as 90% ofcirculating nitrite is derived from the L-arginine:NOpathway (38). On a low-nitrate controlled diet (210mol/d) about one-half the urine nitrate originated

from plasma arginine (40).NO can also be produced from recycled nitrite ornitrate in various ways. Acidification of nitrite mayrelease NO in the stomach (8). NO may be derivedfrom urine nitrate in infected urine (75). NO produc-tion has been described in infarcted heart tissue(76). The process is independent of NOS because it isunaffected by specific NOS inhibitors. NO is alsoproduced in the mouth of both the rat (77) andhumans (78), or in human sweat (27). In theseinstances, commensal bacteria may play a role insynthesis and the NO that is produced may serve asa defense mechanism against pathogens.

NO is used therapeutically in patients with pul-monary hypertension and other conditions (79,80).Serum nitrates and nitrites (measured as a com-bined total) or plasma nitrate by high-performanceliquid chromatography (HPLC) increase with NOinhalation (24,81). Most (90%) of the 15NO that isinhaled is retained and at least 70% is recovered inurine as 15N nitrate (39).

NO has an unpaired electron and this makes itschemistry and biochemistry (82) and its pharmacol-ogy (83) complex. Its biological half-life is 5 s or less(84,85). Some of the NO produced by various tissuesrapidly binds to heme groups in enzymes such asguanylyl cyclase, in effector cells such as the smooth

muscle cells of the blood vessel wall (86) or platelets(87). This increases cGMP and in this way NO actsas an intermediate or second messenger of hor-mone action (88). In some cells, NO action is effectedthrough intracellular calcium cycling (89). Residualamounts of NO react with water to form nitrite,which in the presence of heme groups in proteinssuch as myoglobin, hemoglobin, or enzymes, rapidlyoxidizes to nitrate and the corresponding met-hemeprotein (85). For example, when NO diffuses into thered cell, it reacts rapidly with oxyhemoglobin to formmethemoglobin and nitrite (90). In the presence ofoxygen and methemoglobin reductase, these are

rapidly converted back to hemoglobin and to nitrate.NO also reacts with thiol groups to form nitrosothi-ols, such as nitrosocysteine or nitrosoalbumin (91,92) or nitrosohemoglobin (93,94). The associationbetween thiols and NO is relatively weak and nitro-sothiols can exert long-acting endothelium-derived-releasing-factor-like effects by slowly releasing NO.

As nitrosohemoglobin becomes deoxygenated, con-formational changes take place and NO is released;this may act on the endothelium to cause vasodila-tation where it may be most needed (94). Becausealbumin is so abundant and it has one free cysteineresidue, much of the protein-bound releasable NOcirculates as the albumin-adduct. Advanced glyca-

tion adducts may quench NO release from nitroso-thiols, reducing their effectiveness as NO donors(91). The exact role has not been determined for agroup of proteins described in insect salivary glandsthat bind and readily release NOnitrophorins(95), though they may act to increase blood supply tothe bite area. Their mammalian counterparts (ded-icated high capacity NO carriers) have not been

described.NO has important effects as a bactericidal agentwhen it is released by macrophages during infection(58,59,96). This defense mechanism seems to havebeen conserved through evolution (97). Peroxyni-trite (ONOO) is formed from NO and superoxide(O2

) (98) and may be responsible for some of thecytotoxic effects of these molecules (57,99). Whenprotonated, ONOO gives rise to hydroxyl (OH)and nitrogen dioxide (NO2

) radicals, which caninduce tissue damage (100105). Nitrite may alsoproduce the nitrogen dioxide radical when oxidizedby hydrogen peroxide, catalyzed by heme peroxi-

dases (106). Tissue damage may result from tyrosinenitration (107,108). In mammalian smooth musclecells, NO may inhibit proliferation by influencingcyclin-dependent kinases that are critical to the cellcycle (109).

Methods of measuring nitrite and nitrate inplasma and urine

Methods for these analyses have been reviewed(2,110,111). Nitrite is difficult to measure reliably inblood because it is unstable, being rapidly oxidizedto nitrate. When added to blood, 16% of added nitriteremained after 30 min at room temperature; at 60

min, the corresponding fraction was 5%. At 4C,the percentages were 69% and 55%. Once plasma isseparated from blood (avoiding hemolysis), both ni-trite and nitrate are stable at 20 C for at least 1year (3). Urine nitrate and nitrite are stable in theabsence of microbial contamination. Acid pH de-stroys nitrite, so acid preservatives should beavoided. Samples should be collected on ice, possiblywith the addition of antibiotics or isopropanol to theurine during the collection (4). After collection,freezing is recommended for long-term storage.

Nitrite concentrations in plasma and urine areusually only a small fraction (5%) of the nitrate

concentrations, reflecting in part the dietary intakesof these ions and also their bioreactivity. A combinedmeasurement of nitrate plus nitrite (termed NOx) isoften used with colorimetric assays because theprocedures can be simplified and the stringent sam-ple preparation requirements for nitrite analysiscan be avoided. If investigators have no interest innitrite, serum can be used for NOx determinations.Because nitrite is usually present at much lowerconcentration than nitrate in body fluids, NOx isalmost synonymous with nitrate in most instances.There is some confusion in the literature over no-menclature. Some authors have used nitrite tomean NOx because nitrite is the component mea-

NITRITE AND NITRATE ANALYSES



4/26

sured after reduction of nitrate (112116). This errorhas been noted by others (117). In this review, whenauthors have used the abbreviation NOx to denotesomething other than a combined total concentra-tion of nitrite plus nitrate, e.g., nitrite plus nitrosocompounds, then we have explained their usageand we have used quotes (NOx) to denote this.Proposals to standardize nomenclature in NO re-

search have recently been published (118).

COLORIMETRIC METHODS

Nitrite is reactive and used extensively in dyemanufacturing. Over 50 colorimetric methods wereproposed for its determination; many based on theformation of azo dyes (119). Currently, there areoccasional references to the use of the Brucinemethod, in which nitrite is oxidized to nitrate bypermanganate and boiled with strychnine to pro-duce a yellow color (35). However, most recent clin-ical papers use colorimetric methods based on the

Griess reaction. Nitrite is reacted with sulfanil-amide and N-(1-naphthyl)ethylenediamine (3) toproduce an azo dye. Nitrate may also be measured ifit is first converted to nitrite with the enzymenitrate reductase from bacteria (Escherichia coli,

Pseudomonas oleovorans) (111) or fungi (Aspergil-lus) (120). Alternatively, metallic reduction overcopper-plated cadmium (121,122) or granulated cad-mium (123) can be used. There are a number of

variants of the Griess reaction, designed to elimi-nate interference from both reagents (120,124) andthe material assayed. Plasma assays require proteinremoval, e.g., by precipitation with zinc hydroxide(125), zinc sulfate (3), or by ultrafiltration (61).

When the methods are compared against gas chro-matography-mass spectrometry, other sources of in-terference become evident, such as free reducedthiols and other poorly characterized plasma con-stituents (126). Drugs, such as L-arginine analogs,may interfere also (127,128). Recoveries have beenreported to be 91.5%, compared with copper-cad-mium reduction (120) or 95% for nitrate and 100%for nitrite in serum (124). However, when comparedwith mass spectrometry, the variant of the Griessreaction that was used for urine gave recoveries

varying between 3080% (126). There are severalautomated methods based on the Griess reaction

and flow injection analysis (121) or HPLC. Methodsbased on HPLC are discussed subsequently. Vari-ants of the method have used alternate couplingreagents for nitrite determination (129).

ULTRAVIOLET SPECTROPHOTOMETRIC METHOD

A direct continuous kinetic spectrophotometricmethod has been described for urine and serumnitrate (130). The nitrate is reduced by NADPHusing nitrate reductase from Aspergillus. A similarone-step assay used the method of standard addi-tions to adjust for possible reaction interferents inserum (131).

FLUORIMETRIC ASSAYS

Nitrite (and nitrate after reduction) have alsobeen measured fluorimetrically. In one method, ni-trite enables the acid-catalyzed ring closure of 2,3-diaminonaphthalene to form highly fluorescent 2,3-diaminonaphthotriazole (132,133). The method is50100 times more sensitive than the Griess reac-

tion and was used on serum or plasma and tissueculture media. With minor adaptation, these meth-ods may be used for determination of S-nitrosothiols(134). A simple and highly selective fluorimetricmethod for determining nitrite with Rhodamine 6Ghas been described in which Rhodamine 6G is oxi-dized in sulfuric acid medium. The assay has beenapplied to the analysis of milk (135).

METHODS USING ELECTRODES OR BIOANALYTICALELEMENTS

Several nitrate or nitrite-specific electrodes and

bioanalytical elements have been designed. Theyhave usually been shown to work with aqueoussamples (136143). One has been used with saliva(144) and another with milk (145). Electrochemicaldetection of nitrite has been used with HPLC (de-scribed subsequently).

METHODS USING NO ANALYZERS

NO analyzers have been designed to measure NOin air samples by chemiluminescence in the pres-ence of ozone. The ozone is produced by an electricaldischarge acting on oxygen supplied from a cylinder.The first product from NO is excited nitrogen diox-

ide, which emits chemiluminescence as it returns toits ground state (146). The analyzers have beenrefined and are used in clinical investigation tomonitor both NO and NO2 when NO is used in thetherapy of pulmonary vascular disease (79,80), andNO production in exhaled breath in asthma andother conditions (147149). With this instrumenta-tion, exhaled NO is easier to measure than nitriteand nitrate. It has also been applied to the study ofNO metabolism in various conditions. However, theapparent production rate of exhaled NO varies withexhalation technique. Moreover, the site of origin ofthe exhaled NO is unclear. Much of the measured

exhaled NO may be produced in the nasopharynx,unless stringent precautions are used (147), or inthe airways, rather than at the level of the alveolus(150).

In order to measure nitrite or nitrate, the NOanalyzers are coupled to an enclosed vessel throughwhich an inert gas, such as nitrogen or helium, ispurged (151). Then selective chemical reduction ofeither nitrite or nitrate to nitric oxide can be moni-tored directly as sequential samples are added toexcess of the appropriate reagent. Acetic acid/iodideare often used for nitrite or vanadium III/NaOH fornitrate. Various oxidants have been tested and com-pared (152). It is subject to interference by a number

ELLIS ET AL.



5/26

of arginine analogs currently investigated as poten-tial pharmacological agents (127). The method hasalso been applied to saliva (153). In a modification ofthe method to include a photolysis step (154), orthermolysis, followed by nitrate reductase (155),S-nitrosothiols can be quantified.

CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) methods have beenapplied to a wide range of analyses and they showpromise for the future (156). CE procedures havebeen described for assay of nitrite and nitrate inserum or plasma (37,157160) and urine (158161).Nitrite and nitrate show good resolution with ashort run-time of 422 min. Both anions may bequantified with a single injection after minimalsample preparation, such as ultrafiltration. Capil-lary isotachyphoresis methods permit assay of S-nitroso compounds in addition to nitrite and nitrate(162). Micellar electrokinetic capillary chromatogra-

phy has also been used for determination of nitriteand nitrate in extracts prepared from milk and blood(163). In this technique, migration of the ions ismodified by inclusion of a positively charged surfac-tant, such as dodecyltrimethylammonium bromide.CE is a microtechnique, suitable for small volumesof fluid. It has been used experimentally for mea-surement of airway surface fluid from rats (164) andto assess nitrite and nitrate content of single neu-rons (165). Despite the availability of alternativeapproaches for nitrate and nitrite analysis usingcapillary electrophoresis, CE equipment and exper-tise are not yet widely available in clinical laborato-ries.

GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASSSPECTROMETRY

Gas chromatography has been used for the anal-ysis of nitrite in water (166) and saliva (167) and fornitrate analysis in rat urine (168). GC-MS methodsuse 15N labeled internal standards or isotope dilu-tion techniques and derivatization with pentafluoro-benzyl bromide (169), benzene (168), or toluene (24).They form the basis of reference methods for nitriteand nitrate in plasma and urine (126) and water(170). The specificity of a commonly used GC-MS

method for nitrite in plasma (171) has been ques-tioned on the basis of 15N enrichment studies (38).Nitrate recovery from rat serum was as low as 67%,with one method (168).

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

High-performance liquid chromatography (HPLC)is used for nitrite/nitrate measurement in environ-mental studies and a direct UV spectrophotometricmethod (at 215 nm) is approved by the US Environ-mental Protection Agency for this purpose (172). Anumber of similar methods are available for mea-surements in foods (17,28), urine and serum (24,

173176). The methods are generally based on sep-aration by ion-exchange HPLC, although some havebeen described for nonbiological matrices that usereversed phase chromatography on C18 with ion-pairing reagents such as cetyltrimethylammonium

(177). The binding of ion-pairing reagents to C18columns is often difficult to predict (178) and HPLCcolumns with intrinsic ion-exchange functionality

are probably more reliable. Plasma assays requireultrafiltration or protein precipitation and addi-tional sample preparation by some form of chroma-tography. Similar preparation (without protein pre-cipitation) is used for urine (24,173). Not all methodshave been fully described and validated. Dedicatedion chromatographs (with conductimetric or spectro-photometric detection) can also be used for theseions (179181). Chloride ion has to be removed toprevent interference when conductimetric detectionis used because of its high concentration in biologicalsamples. Sensitive HPLC assays for nitrite in foods(182) and tissues (183,184) have been developed

using electrochemical detection. A method has alsobeen described for whole blood, in which nitrite isdetected electrochemically and nitrate by spectro-photometry (185). It is unclear whether there wouldbe significant interference from nitrosohemoglobinwhen whole blood is analyzed. Ion-exchange HPLChas also been used followed by copper-cadmiumreduction and the colorimetric Griess reaction tomeasure plasma, urine and cell culture nitrite andnitrate (173,186).

CONTAMINATION AVOIDANCE

It is important that clinicians and laboratory

workers be ever vigilant against possible contami-nation of nitrite and nitrate assays (37). Glasswareis often contaminated with nitrate and sometimesnitrite, unless it is sold for use in trace analysis (e.g.,HPLC vials). Ultrafiltration filters are contami-nated with nitrate (possibly from the azide, presum-ably added to preserve the cellulose or other filter-matrix) (174). We have generally found plasticdisposable wares clean. Disposable glass transferpipettes, gloves, and glove powder are potentialsources of contamination (187). We have found thatblood tubes containing anticoagulants such asEDTA from some manufacturers contain nitrate.Some heparinized syringes are contaminated andrubber stoppers in vacutainer tubes may leach ni-trate. Others have noted nitrite contamination ofblood tubes containing citrate or EDTA anticoagu-lants (126). Type A water (18 M) should be used forreagent preparation and final rinsing of glassware.

Clinical correlates

REFERENCE VALUES

Sex differences

In one study (188), 22 men had mean plasmanitrate (27 mol/L) approximately twice that of 18




6/26

women (14 mol/L) and they exhaled more NO (34vs. 20 ppb), even after correction for body weight.Diet was uncontrolled in this study and no estimatesof intake were given, but samples were taken after a12-h fast. Others have observed higher values forplasma nitrate in men and have noted increasing

values with age and other factors associated withatherogenesis, such as lipids, glucose, and blood

pressure (189). Giovannoni et al. (124) did not ob-serve gender differences of this magnitude. Meanserum NOx concentrations were 39 mol/L in 12men and 33 mol/L in 12 women. These authors alsonoted a two- to three-fold increase in values inplasma, compared with serum. However, they didnot seem to exclude contamination from the antico-agulant as a possible reason for the observed differ-ences. No difference was found between serum andplasma in another study (3). Men and postmeno-pausal women had similar serum NOx concentra-tions (190). Reference values have been summarizedin two reports (2,126); each contains information on

12 studies. Dietary restriction in 12 German sub-jects reduced plasma concentrations and urinaryexcretion by approximately one-third to one-half(126,169).

Reports on the effects of the menstrual cycle onvalues are conflicting. Plasma NOx values increasein adult women in association with follicular devel-opment (74). In this latter study, there was a posi-tive correlation of plasma NOx with plasma estra-diol in control women and in those undergoinghormone therapy for in vitro fertilization. Diet wasuncontrolled, but most samples were fasting. Subse-quent studies by these authors showed increasedplasma NOx in postmenopausal women when they

were treated with estrogens (191). In a similarstudies, serum NOx concentrations increased inwomen (but not in men) following 3 days of trans-dermal estradiol (190) or in women following 12months of therapy (192). In contrast, Jilma et al.(188) found no changes in breath NO or plasmanitrate over the course of the menstrual cycle in 18women and in a small group of 5 ovulating women,Morris et al. (193) found no changes in exhaled NOand early-morning urinary nitrite/creatinine ratiosover 30 days. However, amenorrheic women withhyperprolactinemia had lower serum NOx than con-trols and values increased after successful therapy

(194).Values in children

In a group of 90 normal Japanese children, those3 years old had higher NOx/creatinine ratios thanolder children, values showed no gender differences,and were independent of age after about 12 years(35,195). Plasma and urine NOx were also measuredin a small group of 16 healthy children (196). Valuesfor urine NOx (not nitrite) in 17 children have alsobeen presented (113). In groups of East Indianchildren and adults, mean salivary nitrate concen-trations (2136 ppm) were approximately twice or

three times those of nitrite (1217 ppm) (197). Ce-rebrospinal fluid (CSF) NOx is age related in chil-dren. In 35 British children with various neurologi-cal conditions in whom no disturbances ofmonoamine neurotransmitter metabolism or infec-tion were suspected, values are highest in the first 2years after birth and appear to decrease after thistime to 17 years (198).

GENERAL CONSIDERATIONS

Because NO is involved in so many diverse activ-ities in the body, one might expect that its metabo-lism would change in a wide variety of conditions,leading to alterations in the concentrations of itsmetabolites in blood and urine. While this is true tosome extent, many of the physiological activitiesinvolve one of the two constitutive NOS isoenzymesthat change little in disease. However, other pro-cesses are associated with the many tissues andorgans that have inducible NOS isozymes. These

enzymes can be massively induced by infection orinflammation. The enzyme activity of a fully-in-duced macrophage can be approximately 1000-foldthat of an endothelial cell (45).

URINE NITRITE AS A MARKER OF URINARY TRACTINFECTION

Nitrite is produced from urine nitrate by bacterialnitrate reductase (75) and small quantities mayperhaps arise by alternate pathways, involving NOS(199). Some nitrite is converted to NO in acidifiedurine (75), but much remains. Elevated urine nitritemay assist in the diagnosis of urinary tract infec-

tions and test strips for this purpose have beenavailable for many years. Some have found thatstrips for bacterial leukocyte esterase are more ef-fective than those for urine nitrite (200). Nitrofuran-toin may reduce the effectiveness of nitrite teststrips (201). The merits of using these test strips hasbeen the subject of much debate. Results should notbe used to direct antibiotic therapy (202), they hadlow positive predictive value (203205), but theymay be better than urinalysis (204,206) and theyhave merit in certain specific situations (204,206208). Others have reported that they perform well(209).

NITRITE AND NITRATE IN OTHER INFECTIONS

In sick hospitalized patients, dietary intake, ifany, is less likely to contribute to plasma or urinenitrate and nitrite. For this reason, increases inthese ions may become more reliable markers ofdiseases, because of alterations in NO synthesis,degradation or excretion. This may apply in septicshock (210217), in patients with endotoxemia dueto severe cirrhosis (218,219), or in patients withsystemic inflammatory response syndrome (220,221), and multiple organ failure (222). There weresignificant differences between plasma NOx (by the

ELLIS ET AL.



7/26

Griess reaction) in controls and infants with sepsisand septic shock; mean values were: 35, 126, and582 mol/L for the respective groups (212). It isunclear whether the assessment of efficacy of thepossible use of NOS inhibitors in septic shock (223)would be assisted by measurements of plasma/urinenitrite or nitrate. Plasma nitrite was increased in

AIDS with pulmonary involvement (224) and wasincreased in other AIDS-related infection (225). Oth-ers saw no change in NO production in AIDS pa-tients without severe infection (41). Serum NOx wasincreased in trypanosomiasis (226).

NITRITE AND NITRATE IN GASTROENTEROLOGY

Possible low vegetable consumption could en-hance the utility and selectivity of plasma and urinenitrate in patients with gastrointestinal diseases,unmasking higher values in gastroenteritis (36,227229) (Figure 1). The precise role of NO and other freeradicals in secretory diarrhea is still to be deter-mined (230). In inflammatory bowel disease, urinenitrate/creatinine ratios were about four-fold thoseof patients with non-inflammatory disorders (231).However, others found no significant differencesbetween median serum nitrate in ulcerative colitis,

Crohn disease, and healthy controls; but patientswith active disease had higher values than thosewith inactive disease (232). Greatly increased serumNOx may help predict those patients with ulcerativecolitis who require a change in medical therapy orsurgery (233). Urine nitrate in 14 patients declinedin parallel with their recovery in response to therapy(234). Plasma NOx was significantly increased insmall groups of patients with collagenous colitis andalso those with lymphocytic colitis; at colonoscopy,NO sampled from the lumen of the colon, close to themucosal surface, was also increased in 4 of 5 pa-tients with collagenous colitis compared with that in10 controls (235).

Increased NO production might also be expectedin these conditions from other evidence. Colon mu-cosa cultured from biopsy specimens showed 4 to10-fold higher NOS activity and 4 to 8-fold highernitrite production in areas of active Crohn disease orulcerative colitis, compared to those from normalcontrols, and values were lower when biopsies werecultured in the presence of methylprednisolone orketotifen (236). Similar increases in mucosal NOS inCrohn disease were seen by others (237). Also mu-cosal L-citrulline (an indirect measure of NO syn-thesis) is higher in biopsies from patients withactive colitis (238). Topical application of L-NAME

(N-omega-nitro-L-arginine methyl ester, a competi-tive inhibitor of NOS) reduced colonic inflammationin a rat model of ulcerative colitis, suggesting thatNO synthesis was associated with the inflammationin this condition (239).

In mice, oral arginine supplementation protectedagainst bacterial translocation from the gut to otherorgans after lipopolysaccharide-insult (240), sug-gesting an important role for NO synthesis in hostdefense from pathogen entry via the gut. The defen-sive roles of NO production by the gut in gastroin-testinal disease probably outweigh its potentiallyharmful effects (241). Increased nitrite concentra-

tions were found in the gastric juice of patients withchronic atrophic gastritis (242). The affected groupalso had slightly higher gastric pH, which might beexpected to increase the stability of nitrite. Nitriteproduction from nitrate by commensal bacteria inthe mouth may serve to protect against Candida andother pathogens (78). The NO (nitrergic) nervoussystem plays an important role in gut motility andsecretion and in other functions (243). Pyloric steno-sis in infants appears linked to a neuronal NOS genepolymorphism (244), and Hirshprung disease is as-sociated with low neuronal NOS in intestinal nervefibers (245), but we are unaware of any studies onNOx production in these conditions.

Figure 1 (A) Urinary nitrate:creatinine molar ratios and (B) serum nitrate concentrations (mol/L) in healthy volunteercontrols and in patients with rheumatoid arthritis or infectious gastroenteritis. Bars show the mean SD. Broken lineshows mean 2SD for controls. Group means significantly different from controls are indicated by *p 0.005 and **p 0.001. (Reproduced with permission from Lippincott-Raven Publishing from Grabowski PS, England AJ, Dykhuizen R, etal. Elevated nitric oxide production in rheumatoid arthritis. Detection using the fasting urinary nitrate:creatinine ratio.

Arthritis Rheum 1996; 39: 6437 [36].)




8/26


9/26

were also increased in skin biopsy specimens (112).Yet in another study, plasma nitrate concentrationswere similar in controls, and patients with systemiclupus erythematosis, thrombotic throbocytopenicpurpura and primary antiphospholipid syndrome(269). In Kawasaki disease, a pediatric systemicpanvasculitis associated with immunological abnor-malities, more urinary NOx and neopterin were

excreted at certain stages of the disease than incontrols (270). Mean early morning urine NOx/cre-atinine was increased in eight patients with Ka-wasaki disease; values rose with initial therapy anddeclined to normal as the disease resolved (271).

NITRITE AND NITRATE IN HYPERTENSION AND IN RENALAND CARDIOVASCULAR DISEASE

The role of NO in the regulation of blood flow andarterial pressure has been reviewed (272,273). Micelacking the gene for endothelial NOS are hyperten-sive (274). The blood pressure of spontaneously

hypertensive rats can be lowered for several weeks(relative to controls) by (non-genomic) delivery of thegene for human NOS (275). This procedure in-creased urine and serum NOx, along with urine andaortic cGMP. The endothelium itself has a multi-factorial role in blood pressure modulation (86), andits dysfunction is important in atherogenesis (47,276). The interaction of the NO system with agingand atherosclerosis in general (277), and with homo-cysteine metabolism in relation to atherothrombosisare not fully understood (278,279).

NO has multiple effects within the kidney (5,280283) and, as previously mentioned, renal tissuecontains all three isozymes of NOS and their precise

role in normal physiology, hypertension and diabe-tes has not been fully elucidated (68). The use ofselective and nonselective NOS inhibitors and NOdonors may help elucidate the role of NO productionwithin the complex physiology of the renal tubule(284,285) and its diuretic and natriuretic responses(286). In rats, chronic specific inhibition of neuronalNOS (found also in the macula densa cells of thekidney) caused an increase in blood pressure, possi-bly related to tubuloglomerular feedback and tran-siently decreased GFR (287). Because nitrate andnitrite are excreted by the kidney, renal functionand clearance have to be considered if they are

intended to be used as markers of NO production invarious conditions. In 71 patients in an intensivetherapy unit, there was a strong positive correlationbetween NOx and creatinine concentrations in se-rum (288). Mean plasma nitrate was approximatelythree- to four-fold higher than normal in patientswith renal disease prior to dialysis and fell to belownormal following the procedure (289).

NO may have a role in the initiation of hypertension(290). Dubeyet al. (290) showed that in the one kidney,one clip hypertensive rat model, increasing serumNOx correlated with systolic blood pressure. Corre-sponding human studies in renovascular hypertensiondo not show significant changes. In 38 children with

hypertension, plasma NOx was increased comparedwith 16 normal children. Values in all but one patientwith renovascular hypertension were normal, as wereabout one-half the remaining patients with renal pa-renchymal disease. Elevated plasma NOx was associ-ated in part with the reduced GFR in the patients withparenchymal disease (196). In nephrotic syndrome,

urine NOx (referred to as nitrite) was increased inchildren with minimal change nephrotic syndromeand normal in patients with focal segmental glomeru-losclerosis or IgA nephropathy (113,114) (Figure 3). Anumber of endogenous inhibitors of NOS have beendescribed: N(G)-monomethyl-L-arginine (L-NMMA),asymmetrical dimethyl arginine (ADMA) and sym-metric dimethyl arginine (SDMA). They are increasedin patients with hypertension, especially those withlow GFR and they may have physiological effects invivo, but it is uncertain whether they are a contribu-tory cause of the hypertension or a consequence of itand its related disease processes (291). In Bartters

syndrome, a normotensive, renal potassium-wastingdisease, mean NOx/creatinine ratios, in mol/molcreatinine, (0.45) were approximately twice those ofcontrols (0.25) or patients with pseudo-Bartter syn-drome (0.28), and urinary c-GMP excretion was alsoincreased (292).

The protective role of estrogens on the coronaryvasculature may in part be related to their complexeffects on NOS (293). Short-term oral high-doseantioxidant therapy lowers blood pressure in normo-tensives and hypertensives and increases nitriteexcretion, implying a role for NO in this effect (294).Some of the deleterious effects of oxidized LDL alsorelate to interactions with the NO system (295,296).

Figure 3 Urinary NOx (not nitrite) excretion(nmol/mg creatinine) in healthy controls (CONT), childrenwith minimal change nephrotic syndrome (MCNS) inremission (REM) or relapse (REL), with focal segmentalglomerulosclerosis (FSGS), or IgA nephropathy (IgAN).Bars indicate group means; the asterisks indicate a pvalue of0.025 versus other patient groups. (Reproduced

with permission from Mosby, Inc. from Trachtman H,Gauthier B, Frank R, Futterweit S, Goldstein A, TomczakJ. Increased urinary nitrite excretion in children withminimal change nephrotic syndrome. J Pediatr 1996; 128:1736 [113].)




10/26

Plasma cholesterol and LDL were inversely corre-lated with plasma NOx, taken at 0600 h on awell-balanced hospital diet (33). The role of NO inheart failure and cardiomyopathy is not known(297), but cardiomyopathy is frequently associatedwith disorders in which there is higher than normalNO production. A high-capacity inducible NOS iso-form is present in the myocardium of patients withidiopathic dilated cardiomyopathy and plasma ni-

trate was significantly increased in 39 patients withheart failure compared with 62 normal controls.Mean concentrations were 51 and 25 mol/L, but

values overlapped considerably between the groups(298). Increases are also reported in chronic heartdisease (299) and in infants with heart failure due toseptal defects prior to cardiopulmonary bypass(300). In ischemic heart tissue, NO can be produceddirectly from nitrite and the significance of thispathway has not been evaluated (76). Urine andplasma nitrate measurements were unhelpful indetermining the cause of low systemic vascularresistance syndrome after cardiopulmonary bypass

(301) or of primary Raynauds phenomenon (302).After transesophageal variceal ligation in patientswith portal hypertension, there was a prompt fall inserum nitrate (from 39 to 29 mol/L) associated withthe changes in hemodynamics (303). However, therewas no indication that serum nitrate would serve topredict a good long-term clinical outcome.

The literature on nitrate and nitrite in preeclamp-sia is confusing. Mean serum nitrate concentrationswere higher in 20 women with preeclampsia (47mol/L) compared with 20 healthy pregnant (31mol/L) and 12 nonpregnant (32 mol/L) controls.There was no dietary restriction and nitrate wouldbe unhelpful in diagnosis because of extensive over-

lap between the groups. It was not clear whether theincreases were due to increased production or de-creased renal clearance (304) (Figure 4). Increaseswere found in pregnant women throughout preg-nancy (mean NOx 30 mol/L), compared to nonpreg-

nant controls and values were even higher (meanNOx 45 mol/L) in preeclampsia (305) (Figure 5). Inthis study, blood was taken after a 1215 h fast.Plasma NOx correlated positively with systolic bloodpressure in the preeclamptic patients. Previous au-thors saw no change in NOx (not nitrite, as statedby the authors) in maternal serum at 3335 weeksgestation; at delivery, fetal side umbilical venousserum showed a slight increase over maternal (116).

Also both nitrate and nitrite were lower in pre-eclampsia in the (non-fasting) group studied bySeligman et al. (115) and serum NOx (not nitrite)was inversely correlated with blood pressure in

preeclampsia. Davidge et al. (306) found that urineNOx (not nitrite)/creatinine ratios were lower in 14women with preeclampsia than in 20 normotensivepregnant women (0.37 vs. 0.69 mol NOx/mg creat-inine), but mean plasma NOx was not significantlydifferent (33 vs. 26 mol/L). Mean amniotic fluidnitrite decreases in late pregnancy at 3741 weeks;exhaled NO is unchanged (307).

Nitrate excretion and 15N arginine studies wereused to study newborns with persistent pulmonaryhypertension (308). The authors concluded thatthere was decreased arginine utilization during theacute vasoconstrictive stage of the disease, possiblyleading to insufficient endogenous NO production in

Figure 4 Serum nitrate concentration (mol/L) forwomen with preeclampsia, matched healthy pregnant andnonpregnant women. Bars indicate group means. (Repro-duced with permission from Blackwell Science Ltd., fromSmarason AK, Allman KG, Young D, Redman CW. Ele-

vated levels of serum nitrate, a stable end product of nitricoxide, in women with pre-eclampsia. Br J Obstet Gynaecol1997; 104: 53843 [304].)

Figure 5 Serum NOx concentration (means and SEM)for women with preeclampsia (PE), healthy normotensivepregnant (NTP) and nonpregnant (NP) women, andwomen with essential hypertension (EHT). Values repre-

sent means SEM and n the number of samples (*p 0.0001; p 0.005; ANOVA with Fisers tests). (Repro-duced with permission from S. Karger AG, Basel fromNobunaga T, Tokugawa Y, Hashimoto K, et al. Plasmanitric oxide levels in pregnant patients with preeclampsiaand essential hypertension. Gynecol Obstet Invest 1996;41: 18993 [305].)

ELLIS ET AL.



11/26

this condition. Others found that urinary NOx con-centrations by the Griess reaction were lower innewborns with persistent pulmonary hyperten-sion than in controls (309). They also attributedthis to reduced pulmonary NO production in thesepatients because of their lung disease. Valuesincreased transiently after the initiation of extra-corporeal membrane oxygenation (ECMO). This was

believed to be due to enhanced lung production ofNO with the improved tissue oxygenation caused bythe ECMO. There was considerable overlap betweencontrols and patients. In newborns receiving bloodor indomethacin, NO metabolism (assessed by urinenitrite excretion) was affected by these treatments(310).

ARGININE SUPPLEMENTATION AND THE REGULATION OFBLOOD PRESSURE AND BLOOD FLOW

Endothelial NO, synthesized from arginine, is animportant second messenger for blood pressure

control (53,311). Feeding arginine-deficient diets torats after small-intestine resection causes hyperten-sion (312). An infant with arginosuccinate lyasedeficiency, who was unable to synthesize arginine,was hypertensive prior to arginine replacement(313).

Arginine infusion induces hypotension and diure-sis/natriuresis in man (314). The blood-pressure-lowering effects of arginine infusion are short termin patients with essential hypertension, producingsignificant change on only the first of four infusiondays (315). Both hypertension and angiotensin-con-

verting enzyme therapy affect the renal response toL-arginine infusion (316). Arginine infusion in neo-

nates with possible endothelial dysfunction and de-fective NO synthesis improved oxygenation (317). Inshort-term experiments, arginine infusion improvedthe circulation in affected ischemic limbs of patientswith atherosclerosis (318). In dogs undergoing car-dioplegic arrest and coronary artery ligation andrelease, arginine supplementation of the cardiople-gia solution reduced postischemic injury (319). Itinhibits platelet aggregation in healthy subjects(320).

In partially nephrectomized rats, oral argininegave some protection against renal failure (321).Its chronic use in drinking water has also been

associated with attenuated cardiac hypertrophyin spontaneously hypertensive rats (322). Inhumans, oral L-arginine supplementation (0.10.2 g/kg) may increase NO excretion and plasmanitrate, without affecting blood pressure (323).Oral supplementation with lower doses mayslightly increase insulin without effect on nitriteconcentrations in plasma or nitrate excretion(324). In men with coronary artery disease, oralL-arginine, taken over a 3-day period, improvedendothelium-dependent dilatation of the brachialartery and reduced monocyte/endothelial cell ad-hesion (325).

The proposed beneficial effects of L-arginine may

result in part from its ability to act as a scavenger ofsuperoxides as well as a substrate for NO synthesis(326,327). If L-arginine therapy becomes establishedpractice for specific indications, plasma or urinenitrate or NOx measurements along with exhaledNO may be useful in the assessment of the treat-ment regimes. However, there may be contraindica-tions to arginine therapy. It enhances lipid peroxi-dation in poorly perfused rat kidney (328). In mousemalnutrition, supplementation with arginine alonemay not be beneficial (329). Also some of the bene-ficial effects of the low protein diet in renal diseaseare believed to be a direct consequence of argininerestriction (330).

Figure 6 Plasma NOx concentration (nitrite plusnitroso compounds, mol/L) for patients after liver trans-plant: (A) by clinical status, (B) by rejection grade on theday of biopsy confirmation. Bars show group means.(Reproduced with permission from Williams and Wilkenfrom Devlin J, Palmer RMJ, Gonde CE, et al. Nitric oxidegeneration a predictive parameter of acute allograft rejec-tion. Transplantation 1994; 58: 5925 [333].)




12/26

NITRITE AND NITRATE IN TRANSPLANTATION ANDREJECTION

NO is produced by various cells in the liver andmay serve as a second messenger and as a defenseagainst invading microorganisms, parasites and tu-mor cells (331). Exhaled NO was increased in aseries of patients with hepatopulmonary syndrome

and values returned to normal in one patient aftertransplantation (332). Plasma acid-labile NOx (ni-trite and nitroso compounds, measured by chemilu-minescence) was increased in liver transplant rejec-tion in fifty consecutive patients. There was overlapin values between the groups, but the highest valueswere seen in Grade 2 and 3 rejection (333) (Figure6). In this study, plasma nitrate did not correlatewith rejection. Samples, with no reported anticoag-ulant, were separated within 20 min, to avoid thereported rapid conversion of nitrite to nitrate inwhole blood (3). Others have criticized the use ofplasma nitrite as a marker of NO production, be-

cause of its instability (334). Plasma nitrate wasincreased in patients with moderate to severe livertransplant rejection, but not in those with mildrejection. Mean values (mol/L) were 50 in thesevere rejection group, compared with approxi-mately 20 in groups with no or mild rejection at thetime of liver biopsy. Values fell to normal aftersuccessful antirejection therapy with glucocorticoids(335). Mild increases in plasma nitrate were foundin many patients in the postoperative period, per-haps linked to temporarily ineffective anti-rejectiontherapy (336). But rats show increased NO synthe-sis following partial hepatectomy (337), so the rise innitrate following transplantation might be a nonspe-

cific response to injury.In rats, heart transplant rejection was associatedwith an eight-fold increase in urine nitrate excre-tion, compared with controls. Unfortunately, rejec-tion in transplanted animals that had been treatedwith dexamethasone and cyclosporine was muchless dramatic, leading to only two- to three-foldincreases (338). These increases were probably dueto effects on the inducible NOS isoenzyme of themyocardium as well as increased macrophage NOSactivity. Urine nitrate is increased in human hearttransplant patients during rejection. In a thoroughstudy of 86 patients, mean values (mol/mmol cre-

atinine) were 100 in patients with no evidence ofrejection and 128 in those showing rejection (339).The magnitude of these differences compared withintra- and interindividual differences was insuffi-cient to make the test clinically useful for the diag-nosis of rejection. However, Benvenuti et al. (340)found that serum NOx correlated with the severityof heart transplant rejection, as assessed by histo-logical grade and a cutoff value of 20mol/L could beused to help detect Grade 2 rejection.

Serum NOx has been shown to increase in exper-imental small bowel rejection in the rat. Concentra-tions fell rapidly with the onset of anti-rejectiontherapy (341). In renal transplantation, urine nitrite

dipstick methods may not have sufficient sensitivityto detect bacteriuria (342).

NITRITE AND NITRATE IN DISEASES OF THE BLOOD SYSTEM

Plasma NOx concentrations were increased in

acute painful crises in sickle cell disease whencompared with healthy control subjects (90). Thereasons for this increase were not clear, but it isunlikely that routine NOx measurements will find arole in monitoring such patients because the in-crease was quite modest (mean NOx 50 vs. 30mol/L) and sickle patients had elevated valueswhen not in crisis; their mean NOx was 42 mol/L.

Cell-free hemoglobin can remove NO rapidly (343)and by doing so, its presence may lead to abnormalthromboregulation as summarized by Marcus et al.(344). This may be relevant in the development andpossible introduction of hemoglobin-based bloodsubstitutes (343).

Figure 7 NOx concentrations (mol/L) in inducedsputum in patients with asthma and normal controls.

Bars represent mean values. (Reproduced with permis-sion from Mosby, Inc. from Kanazawa H, Shoji S, YamadaM, et al. Increased levels of nitric oxide derivatives ininduced sputum in patients with asthma. J Allergy Clin

Immunol 1997; 99: 6249 [347].)

ELLIS ET AL.



13/26

NITRITE AND NITRATE IN RESPIRATORY DISEASES AND INSMOKERS

The bronchiolar lavage fluid mean NOx concen-tration was 20-fold increased in 20 lung cancerpatients compared with 8 smoker controls, presum-ably due to cytokine activation (345). NOx in bron-chio-alveolar lavage fluid was increased in 5 of 13

immunosuppressed children with pneumonia, com-pared with 31 controls. The mean value (approxi-mately 21 mol/L) was about twice that of thecontrols; serum NOx was similar in the two groups(346). NOx by the Griess reaction is increased ininduced sputum from patients with asthma (347).Mean concentrations (mol/L) were 1086 in 18 asth-matics and 577 in 10 controls. There was littleoverlap in values (Figure 7). However, exhaled NO(148,149,348,349) may give a more direct measure-ment of NO production in lung than its metabolites,nitrite or nitrate, and simple, sensitive NO analyz-ers are increasingly used in clinical medicine.

The effects of smoking on NO metabolism arecomplex. Cigarette smoke is an excellent source ofNO (up to 500 ppm), that can nitrate tyrosine (107).Its NO concentration varies widely, depending uponhow the tobacco was grown and manufactured (350).Smoking impairs endothelium-dependent vessel re-laxation (350), but other effects are somewhat unex-pected. Smoking, particularly heavy smoking, isparadoxically associated with lower serum NOx(351). Moreover, exhaled NO increases after quittingsmoking (352), perhaps implying better lung func-tion.

CSF AND PLASMA NITRITE AND NITRATE IN NERVOUSSYSTEM DISORDERS

In addition to acting as a neurotransmitter (353,354), NO may cause damage to nerve cells, but itsrole in various diseases is not well understood (51,62,63,355,356). Whether it causes benefit or harmmay depend on the stage of the disease process(357). Male mice deficient in neuronal NOS exhibitbad behavior (358,359), but we know of no clinicalstudies where nitrite or nitrate have been measuredin behavioral disorders.

Much of the clinical neurology literature relatingto changes in CSF and plasma nitrate and nitrite is

conflicting. In two infants with Hemophilus influen-zae meningitis, CSF nitrite concentration was in-creased. This was regarded as evidence for enhancednitric oxide production in this condition (360). CSFnitrite was also increased in all six patients withbacterial meningitis and declined with successfultherapy (361). In patients with recent stroke, argi-nine and nitrite were both increased in CSF andtheir concentrations correlated (362). The extent ofpossible overlap in values between affected patientsand unaffected controls was not given, so it isdifficult to assess whether CSF nitrite measure-ments could have a diagnostic role in this condition.Minimal changes in CSF nitrite and nitrate oc-

curred during the treatment of adult head injurypatients with hypothermia, but values were slightlyhigher in non-survivors than in those who survived(363). CSF NOx was unchanged in Parkinsons dis-ease (364) and in aseptic meningitis, multiple scle-rosis and Guillain-Barre syndrome (365), or in Par-kinsons disease, spinocerebellar ataxia andamyotrophic lateral sclerosis (366). It was un-

changed in Huntingtons and Alzheimers disease,amyotrophic lateral sclerosis, and HIV infection, butvalues were significantly increased in a small groupof patients with bacterial and viral meningitis (367).Others (224) described similar negative findings forCSF, but noted some increases in serum nitrite ingeneralized infections that may affect the brain,such as cytomegalovirus in AIDS.

Lower than normal mean CSF nitrate was foundin Alzheimers and Parkinsons diseases and in mul-tiple system atrophy (368). But there were nochanges in fasting plasma NOx (mean concentration33 mmol/L) or CSF NOx (mean 5 mol/L) in the

group of patients with Alzheimers studied by Na-varro et al. (369) and CSF nitrite was increased inanother Parkinsons disease group (370). Plasmanitrate was unchanged in Parkinsons disease ac-cording to another report (371). CSF nitrite andnitrate concentrations were statistically signifi-cantly higher in patients with lumbar spondylosiscompared with those of controls, and the authors feltthat NO may play a role in lumbar pain or nervedamage in sciatica and that CSF nitrite/nitrate maybe used as a diagnostic parameter of spinal diseases(372). Unfortunately, individual values for the 18affected patients and 18 controls with other CNSdiseases are not shown, but there was significant

overlap between the two groups with mean (SEM)values (mol/L) of 4.48 (0.34) and 3.48 (0.22), respec-tively. Serum NOx was increased in a group ofdemyelinating diseases including multiple sclerosis,inflammatory neurological diseases and AIDS pa-tients studied by Giovannoni et al. (373). Mean NOx(mol/L) for the groups were 66, 56 and 58, com-pared with noninflammatory neurological disease,41, and normal controls, 33. There was significantoverlap in values between the groups.

On the basis of higher CSF NOx seen in youngerchildren (who were shorter in height and spinallength than older children), Surtees et al. (374)

proposed that a rostrocaudal NOx gradient existedin CSF and that most CSF NOx originated from thebrain.

NITRITE AND NITRATE MEASUREMENTS IN MISCELLANEOUSCONDITIONS

Plasma nitrate concentrations were elevated inpatients after burn injury. The highest values werein patients with 1040% of the body involved (176).Minor burns are not associated with significantlyincreased plasma NOx; increased values were foundonly when there was therapeutic cerium nitratecontamination of the burn area, or generalized




14/26

stress due to smoke inhalation injury (375). Thenitrate concentration in human tear fluid (like thatof the black vulture, Coragyps atratus) is lower thanthat of serum (181), but there are no reports of itsuse in the diagnosis of eye conditions (in eitherspecies); although the NO system is very importantin many intraocular processes and in eye irritation(376).

NO plays an important role in the initiation ofapoptosis in various cell types, such as neuronalcells, pancreatic cells, chondrocytes and macro-phages (6467,377). NO interactions with the p53tumor suppressor system are under intensive studyand NO may actually support tumor growth (378).Plasma and urine NOx assays have not been usedextensively to monitor apoptosis or tumor progres-sion clinically, except where they are associatedwith inflammation. Plasma and CSF NOx weregenerally unhelpful in malaria (379), but higherplasma NOx was associated with deeper coma incerebral malaria (380). NO plays an important role

in intercellular communication in bone (381,382),but its metabolites have not been studied in boneand joint diseases, except in arthritis and the spon-dyloarthropathies (382).

ENVIRONMENTAL NITRITE AND NITRATE: POSSIBLE EFFECTSON THE INCIDENCE OF DIABETES AND OTHER DISEASES

There are tenuous connections between the NOsystem and several processes in diabetes (383). NOand cytokines are associated with islet cell death indiabetes (384386) and high glucose concentrationsinduce NOS expression and superoxide anion inhuman aortic endothelial cell culture, implying a

possible role for NO in the vascular complications ofdiabetes (387). NO mediates increased blood flow topancreatic islets during hyperglycemia (388) andexperimental hyperinsulinemia increases urinaryNOx excretion (258). Type I diabetics have evidenceof arterial endothelial dysfunction (389) and specificNOS inhibitors may influence urinary albumin lossand the formation of advanced glycation end prod-ucts in the diabetic rat kidney (390). Alternativeinhibitors may increase blood pressure and causeproteinuria in other rat models (391). There areearly indications that tissue advanced glycation endproducts may be important modulators of NO-medi-

ated responses (392).However, because of the wide variability of di-etary nitrate intake (393), it is difficult to under-stand how disease incidence in childhood diabetescould be causally associated with higher nitrateconcentrations in drinking water (394). The relativerisk was 1.27. Others have found no associationsbetween Type I diabetes and child or maternalnitrate intake from food and water, but they found apositive association with dietary nitrite (relativerisk 2.3 for the fourth quartile of intake) (395).Drinking water (particularly well water) may be-come contaminated by nitrate from fertilizer run-offor from waste water contamination and this may

present an increasing problem (396). Associationswith the concentration of nitrate in drinking waterand disease incidence have also been described forbladder cancer (397), non-Hodgkins lymphoma(398,399), gastric and prostate cancer (400), sponta-neous abortion in four women (401), chromatic/chromosome breaks in children (402), and thyroidenlargement (403).

In other studies, associations between environ-mental contamination and cancer incidence weredeemed hypothetical and unproven (404). Bladdercancer incidence showed no association with drink-ing water nitrate, but it was linked to water chlori-nation (405). No association was found with braintumors, but drinking water nitrate concentrations(16 mg/L) were not exceptionally high (406). How-ever, pediatric brain tumor incidence appeared as-sociated with maternal cured meat consumptionassessed retrospectively (407). There was no associ-ation between nitrate intake (median 99 mg fromfoods and 4 mg from drinking water) and gastric

cancer in a recent Netherlands study of over 120,000men and women with ages 5569 (408). In someareas where drinking water was seriously contami-nated (up to mean nitrate 135 mg/L), leukocyteenzyme abnormalities were found and nitrosamineswere detected in urine; but some water sampleswere mutagen-positive by the Ames test, so nitratesmay not have been the only contaminants (409). Inthis study, salivary nitrite, salivary nitrate, andurine nitrate showed rough correlations with drink-ing water nitrate concentrations. If these associa-tions with disease incidence are confirmed, it will beimportant to show what components in vegetariandiets protect against their high nitrate/nitrite con-

tent (6). Several have been suggested (407,410413).Methemoglobin reductase is less well developed in

infants and this may account for their well-recog-nized greater susceptibility to methemoglobinemiafrom drinking high concentrations of nitrate indrinking water or other environmental sources(414417) or from therapeutic NO (418). Accidentalsubstitution of nitrite for nitrate as a food preserva-tive attests to its potential for toxicity (419).

Conclusions

Assays for nitrite and nitrate in plasma and urine,along with measurements of exhaled NO, have beenused to study various aspects of NO metabolism. Inmost instances, because of its greater stability invivo, nitrate is the more predominant ion. Occasion-ally, nitrite or labile NOx (nitrite and nitrosocompounds) may have higher predictive value thannitrate. The involvement of NO in a wide range ofbiochemical and physiological functions tends toreduce the clinical utility of nitrite and nitratemeasurements for individual disease conditions orpathological processes. Changes in metabolism thatmay involve constitutive NOS isozymes, or slowlydeveloping, possibly chronic, conditions may not

ELLIS ET AL.



15/26

register against the background noise of NO syn-thesis or nitrate intake. Variable intake of nitriteand nitrate from dietary sources, or less impor-tantly, medications and NO intake from smokingand air pollution additionally confound interpreta-tion, outside the experimentally-controlled environ-ment. Plasma and urine nitrite and nitrate mea-surements may have a diagnostic or monitoring role

for individual patients with conditions in whichinducible NOS is massively up-regulated, such asinfection, rejection, and inflammation.

Acknowledgement

This work was supported by grants from The PhysicianServices Incorporated Foundation.

References

1. Kostka P. Free radicals (nitric oxide). Anal Chem1995; 67: 411R6R.

2. Viinikka L. Nitric oxide as a challenge for the clinical

chemistry laboratory. Scand J Clin Lab Invest 1996;56: 57781.3. Moshage H, Kok B, Huizenga JR, Jansen PL. Nitrite

and nitrate determinations in plasma: a criticalevaluation. Clin Chem 1995; 41: 8926.

4. Moshage H. Nitric oxide determinations: much adoabout NO-thing. Clin Chem 1997; 43: 5536.

5. Kone BC. Nitric oxide in renal health and disease.Am J Kidney Dis 1997; 30: 31133.

6. Walker R. The metabolism of dietary nitrites andnitrates. Biochem Soc Trans 1996; 24: 7805.

7. van Maanen JM, van Geel AA, Kleinjans JC. Modu-lation of nitrate-nitrite conversion in the oral cavity.Cancer Detect Prev 1996; 20: 5906.

8. McKnight GM, Smith LM, Drummond RS, Duncan

CW, Golden M, Benjamin N. Chemical synthesis ofnitric oxide in the stomach from dietary nitrate inhumans. Gut 1997; 40: 2114.

9. Omata T. Structure, function and regulation of thenitrate transport system of the cyanobacterium Syn-echococcus sp. PCC7942.Plant Cell Physiol 1995; 36:20713.

10. Meharg AA, Blatt MR. NO3-transport across theplasma membrane of Arabidopsis thaliana roothairs: kinetic control by pH and membrane voltage. J

Membr Biol 1995; 145: 4966.11. Chow C-W, Kapas A, Romanek R, Grinstein S.

NO3-induced pH changes in mammalian cells. Ev-

idence for an NO3-H cotransporter. J Gen Physiol

1997; 110: 185200.

12. Gloria MBA, Vale SR, Vargas OL, Barbour JF,Scanlan RA. Influence of nitrate levels added tocheesemilk on nitrate, nitrite, and volatile nitro-samine contents in gruyere cheese. J Agricult FoodChem 1997; 45: 35779.

13. Kross BC, Hallberg GR, Bruner DR, CherryholmesK, Johnson JK. The nitrate contamination of privatewell water in Iowa. Am J Public Health 1993; 83:2702.

14. Kapoor A, Viraraghavan T. Nitrate removal fromdrinking water: review. J Environ Eng Assoc 1997;123: 37180.

15. Meah MN, Harrison N, Davies A. Nitrate and nitritein foods and the diet. Food Addit Contam 1994; 11:51932.

16. Vallance P. Dietary nitrate: poison or panacea. Gut1997; 40: 288.

17. Santillana MI, Ruiz E, Nieto MT, de Alba M. Highperformance ion chromatography determination ofnitrite and nitrate in foodstuffs. J Liqu Chromatogr1993; 16: 156171.

18. Hunt J, Turner MK. A survey of nitrite concentra-tions in retail fresh vegetables. Food Addit Contam1994; 11: 32732.

19. van Vliet JJ, Vaessen HA, van den Burg G, Scho-thorst RC. Twenty-four-hour duplicate diet study1994; nitrate and nitrite: method development andintake per person per day. Cancer Lett 1997; 114:3057.

20. Laitinen S, Virtanen SM, Rasanen L, Penttila PL.Calculated dietary intakes of nitrate and nitrite byyoung Finns. Food Addit Contam 1993; 10: 46977.

21. Hecht SS. Approaches to cancer prevention based onan understanding of N nitrosamine carcinogenesis.

Proc Soc Exp Biol Med 1997; 216: 18191.22. Hill MJ. Endogenous N-nitrosation. Eur J Cancer

Prev 1996; 5: 4750.23. Green LC, Ruiz de Luzuriaga K, Wagner D, et al.

Nitrate biosynthesis in man. Proc Natl Acad SciUSA 1981; 78: 7764 8.

24. Wennmalm , Benthin G, Edlund A, et al. Metabo-lism and excretion of nitric oxide in humans. Anexperimental and clinical study. Circ Res 1993; 73:11217.

25. Suto T, Losonczy G, Qiu C, et al. Acute changes inurinary excretion of nitrite nitrate do not neces-sarily predict renal vascular NO production. Kidney

Int 1995; 48: 12727.26. Bednar C, Kies C. Nitrate and vitamin C from fruits

and vegetables: impact of intake variations on ni-trate and nitrite excretions of humans. Plant Foods

Hum Nutr 1994; 45: 7180.27. Weller R, Pattullo S, Smith L, Golden M, Ormerod A,

Benjamin N. Nitric oxide is generated on the skinsurface by reduction of sweat nitrate. J Invest Der-matol 1996; 107: 32731.

28. van Vliet JJH, Vaessen H, Vandenburg G, Scho-thorst RC. Twenty four hour duplicate diet study1994: nitrate and nitrite: method development andintake per person per day. Cancer Lett 1997; 114:3057.

29. Jungersten L, Edlund A, Petersson AS, WennmalmA. Plasma nitrate as an index of nitric oxide forma-tion in man: analyses of kinetics and confoundingfactors. Clin Physiol 1996; 16: 36979.

30. Jungersten L, Edlund A, Petersson AS, WennmalmA. Plasma nitrate as an index of endogenous nitricoxide formation in man. Analysis of kinetics, con-

founding factors, and response to immunologicalchallenge. Circulation 1993; 88: I369.31. Zeballos GA, Bernstein RD, Thompson CI, et al.

Pharmacodynamics of plasma nitrate/nitrite as anindication of nitric oxide formation in conscious dogs.Circulation 1995; 91: 29828.

32. Egberts J, Soederhuizen W. Urine samples beforedinner are preferable when studying changes inendogenous nitrate production under uncontrolleddietary conditions. Clin Chim Acta 1996; 254: 1418.

33. Tanaka S, Yashiro A, Nakashima Y, Nanri H, IkedaM, Kuroiwa A. Plasma nitrite/nitrate level is in-versely correlated with plasma low-density lipopro-tein cholesterol level. Clin Cardiol 1997; 20: 3615.

34. Wang J, Brown MA, Tam SH, Chan MC, Whitworth




16/26

JA. Effects of diet on measurement of nitric oxidemetabolites. Clin Exp Pharmacol Physiol 1997; 24:41820.

35. Tsukahara H, Hiraoka M, Hori C, Miyanomae T,Kikuchi K, Sudo M. Age-related changes of urinarynitrite/nitrate excretion in normal children. Nephron1997; 76: 3079.

36. Grabowski PS, England AJ, Dykhuizen R, et al.Elevated nitric oxide production in rheumatoid ar-thritis. Detection using the fasting urinary nitrate:creatinine ratio. Arthritis Rheum 1996; 39: 6437.

37. Leone AM, Francis PL, Rhodes P, Moncada S. Arapid and simple method for the measurement ofnitrite and nitrate in plasma by high performancecapillary electrophoresis. Biochem Biophys Res Com-mun 1994; 200: 9517.

38. Rhodes P, Leone AM, Francis PL, Struthers AD,Moncada S, Rhodes PM. The L-arginine:nitric oxidepathway is the major source of plasma nitrite infasted humans.Biochem Biophys Res Commun 1995;209: 5906.

39. Westfelt UN, Benthin G, Lundin S, Stenqvist O,Wennmalm A. Conversion of inhaled nitric oxide to

nitrate in man. Br J Pharmacol 1995; 114: 16214.40. Castillo L, Beaumier L, Ajami AM, Young VR. Wholebody nitric oxide synthesis in healthy men deter-mined from [15N]-arginine-to-[15N]citrulline label-ing. Proc Natl Acad Sci USA 1996; 93: 114605.

41. Evans T, Rasmussen K, Wiebke G, Hibbs JB. Nitricoxide synthesis in patients with advanced HIV infec-tion. Clin Exp Immunol 1994; 97: 836.

42. Cederqvist B, Persson MG, Gustafsson LE. Directdemonstration of NO formation in vivo from organicnitrites and nitrates, and correlation to effects onblood pressure and to in vitro effects. Biochem Phar-macol 1994; 47: 104753.

43. Benedini F, Bertolini G, Gromo G, Mizrahi J, Sala A.The discovery of a new organic nitrate: an overview.

J Cardiovasc Pharmacol 1995; 26(Suppl 4): S15.44. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric

oxidea physiologic messenger. Ann Intern Med1994; 120: 22737.

45. Anggard E. Nitric oxidemediator, murderer, andmedicine. Lancet 1994; 343: 1199206.

46. Davies MG, Fulton GJ, Hagan P-O. Clinical biologyof nitric oxide. Br J Surg 1995; 82: 1598610.

47. Dattilo JB, Makhoul RG. The role of nitric oxide invascular biology and pathobiology. Ann Vasc Surg1997; 11: 30714.

48. Farrell AJ, Blake DR. Nitric oxide. Ann Rheum Dis1996; 55: 720.

49. Knowles RG, Moncada S. Nitric oxide syntheses in

mammals. Biochem J 1994; 298: 24958.50. Schinikerth VB, Vanhoutte PM. Nitric oxide synthe-ses in vascular cells. Exp Physiol 1995; 80: 885905.

51. Sparrow JR. Inducible nitric oxide synthase in thecentral nervous system. J Mol Neurosci 1994; 5:21929.

52. Nathan C, Xie QW. Regulation of biosynthesis ofnitric oxide. J Biol Chem 1994; 269: 137258.

53. Moncada S. Nitric oxide. J Hypertens 1994; 12:S35S9.

54. Magazine HI. Detection of endothelial cell-derivednitric oxide: current trends and future directions.

Adv Neuroimmunol 1995; 5: 47990.55. Cooper CJ, Landzberg MJ, Anderson TJ, et al. Role

of nitric oxide in the local regulation of pulmonary

vascular resistance in humans. Circulation 1996; 93:26671.

56. MacMicking J, Xie QW, Nathan C. Nitric oxide andmacrophage function. Annu Rev Immunol 1997; 15:32350.

57. Miller RA, Britigan BE. The formation and biologicsignificance of phagocyte-derived oxidants. J Inves-tig Med 1995; 43: 3949.

58. Miller MJS, Grisham MB. Nitric oxide as a mediator

of inflammation? You had better believe it. Media-tors Inflamm 1995; 4: 38796.

59. Jungersten L, Edlund A, Hafstrom LO, Karlsson L,Petersson AS, Wennmalm A. Plasma nitrate as anindex of immune system activation in animals andman. J Clin Lab Immunol 1993; 40: 14.

60. Saleh D, Barnes PJ, Giaid A. Increased production ofthe potent oxidant peroxynitrite in the lungs ofpatients with idiopathic pulmonary fibrosis. Am J

Respir Crit Care Med 1997; 155: 17639.61. Tracey WR, Tse J, Carter G. Lipopolysaccharide-

induced changes in plasma nitrite and nitrate concen-trations in rats and mice: pharmacological evaluationof nitric oxide synthase inhibitors. J Pharmacol ExpTher 1995; 272: 10115.

62. Dawson TM, Snyder SH. Feature article: gases asbiological messengers: nitric oxide and carbon mon-oxide in the brain. J Neurosci 1994; 14: 514759.

63. Dawson VL, Dawson TM. Nitric oxide in neuronaldegeneration. Proc Soc Exp Biol Med 1996; 211:3340.

64. Shimaoka M, Iida T, Ohara A, et al. NOC, a nitric-oxide-releasing compound, induces dose dependentapoptosis in macrophages. Biochem Biophys ResCommun 1995; 209: 51926.

65. Blanco FJ, Ochs RL, Schwarz H, Lotz M. Chondro-cyte apoptosis induced by nitric oxide. Am J Pathol1995; 146: 7585.

66. Troy CM, Derossi D, Prochiantz A, Greene LA,

Shelanski ML. Downregulation of Cu/Zn superoxidedismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J Neurosci 1996; 16: 25361.

67. Nicotera P, Bonfoco E, Brune B. Mechanisms fornitric oxide-induced cell death: involvement of apo-ptosis. Adv Neuroimmunol 1995; 5: 41120.

68. Galle J, Wanner C. Impact of nitric oxide on renalhemodynamics and glomerular function: modulationby atherogenic lipoproteins? Kidney Blood Press Res1996; 19: 215.

69. Snyder SH. Nitric oxide: first in a new class ofneurotransmitters? Science 1992; 257: 4946.

70. Brann DW, Bhat GK, Lamar CA, Mahesh VB. Gas-eous transmitters and neuroendocrine regulation.

Neuroendocrinology 1997; 65: 38595.

71. Brenman JE, Bredt DS. Synaptic signaling by nitricoxide. Curr Opin Neurobiol 1997; 7: 3748.72. Stamler JS, Toone EJ, Lipton SA, Sucher NJ. (S)NO

signals: translocation, regulation, and a consensusmotif. Neuron 1997; 18: 6916.

73. Griffith OW, Stuehr DJ. Nitric oxide synthases:properties and catalytic mechanism. Annu Rev

Physiol 1995; 57: 70736.74. Rosselli M, Imthurm B, Macas E, Keller PJ, Dubey

RK. Circulating nitrite/nitrate levels increase withfollicular development: indirect evidence for estra-diol mediated NO release. Biochem Biophys ResCommun 1994; 202: 154352.

75. Lundberg JON, Carlsson S, Engstrand L, Morcos E,Wiklund NP, Weitzberg E. Urinary nitrite: more

ELLIS ET AL.



17/26

than a marker of infection. Urology 1997; 50: 18991.

76. Zweier JL, Wang P, Samouilov A, Kuppusamy P.Enzyme-independent formation of nitric oxide inbiological tissues. Nat Med 1995; 1: 8049.

77. Duncan C, Dougall H, Johnston P, et al. Chemicalgeneration of nitric oxide in the mouth from theenterosalivary circulation of dietary nitrate. Nat

Med 1995; 1: 54651.

78. Dougall HT, Smith L, Duncan C, Benjamin N. Theeffect of amoxycillin on salivary nitrite concentra-tions: an important mechanism of adverse reactions?

Br J Clin Pharmacol 1995; 39: 4602.79. Zapol WM, Bloch KD. Nitric oxide and the lung. In:

Lenfant C, Ed. Lung biology in health and disease;vol. 98. Pp. 1469. New York: Marcel Dekker, Inc.,1997.

80. Adatia I, Wessel DL. The use of inhaled nitric oxidein congenital heart disease. In: Weir EK, Archer SL,Reeves JT, Eds. Nitric oxide and radicals in the

pulmonary vasculature. Pp. 46391. Armonk, NY:Futura Publishing Company, Inc., 1996.

81. Valvini EM, Young JD. Serum nitrogen oxides dur-ing nitric oxide inhalation. Br J Anaesth 1995; 74:3389.

82. Stamler JS, Singel DJ, Loscalzo J. Biochemistry ofnitric oxide and is redox-activated forms. Science1992; 258: 1898902.

83. Edwards AD. The pharmacology of inhaled nitricoxide. Arch Dis Child 1995; 72: F127F30.

84. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide:physiology, pathophysiology, and pharmacology.

Pharmacol Rev 1991; 43: 109 42.85. Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE,

Byrns RE. Oxidation of nitric oxide in aqueous solu-tion to nitrite but not nitrate: comparison with enzy-matically formed nitric oxide from L-arginine. Proc

Natl Acad Sci USA 1993; 90: 81037.

86. Luscher TF, Noll G. The pathogenesis of cardiovas-cular disease: role of the endothelium as a target andmediator. Atherosclerosis 1995; 118: S81S90.

87. Rao GHR, Raij L, Lester BB, White JG. Inhibition ofagonist-induced human platelet activation by nitricoxide. In: Moncada S, Higgs EH, Eds. Nitric oxide

from L-arginine: a bioregulatory system. Pp. 35563.Amsterdam: Exerpta Medica, 1990.

88. Busse R, Fleming I. Regulation and functional con-sequences of endothelial nitric oxide formation. Ann

Med 1995; 27: 33140.89. Milbourne EA, Bygrave FL. Do nitric oxide and

cGMP play a role in calcium cycling? Short review.Cell Calcium 1995; 18: 20713.

90. Rees DC, Cervi P, Grimwade D, et al. The metabo-

lites of nitric oxide in sickle-cell disease. Br JHaematol 1995; 91: 8347.91. Farkas J, Menzel EJ. Proteins lose their nitric oxide

stabilizing function after advanced glycosylation.Biochem Biophys Acta-Gen Subj 1995; 1245: 30510.

92. Butler AR, Rhodes P. Chemistry, analysis, and bio-logical roles of S-nitrosothiols. Anal Biochem 1997;249: 19.

93. Jia L, Bonaventura J, Stamler JS. S-nitrosohaemo-globin: a dynamic activity of blood involved in vas-cular control. Nature 1996; 380: 2216.

94. Stamler JS, Jia L, Eu JP, et al. Blood flow regulationby S-nitrosohemoglobin in the physiological oxygengradient. Science 1997; 276: 20347.

95. Champagne DE, Nussenzveig RH, Ribeiro JM. Puri-

fication, partial characterization, and cloning of ni-tric oxide-carrying heme proteins (nitrophorins)from salivary glands of the blood-sucking insect

Rhodnius prolixus. J Biol Chem 1995; 270: 86915.96. Lewis RS, Tamir S, Tannenbaum SR, Deen WM.

Kinetic analysis of the fate of nitric oxide synthe-sized by macrophages in vitro. J Biol Chem 1995;270: 293505.

97. Franchini A, Conte A, Ottaviani E. Nitric oxide: an

ancestral immunocyte effector molecule. Adv Neuro-immunol 1995; 5: 46378.

98. Pfeiffer S, Gorren ACF, Schmidt K, et al. Metabolicfate of peroxynitrite in aqueous solution: reactionwith nitric oxide and pH dependent decomposition tonitrite and oxygen in a 2/1 stoichiometry. J BiolChem 1997; 272: 346570.

99. Zingarelli B, Day BJ, Crapo JD, Salzman AL, SzaboC. The potential role of peroxynitrite in the vascularcontractile and cellular energetic failure in endotoxicshock. Br J Pharmacol 1997; 120: 25967.

100. Pryor WA, Squadrito GL. The chemistry of peroxyni-trite: a product from the reaction of nitric oxide withsuperoxide. Am J Physiol 1995; 268: L699722.

101. Moro MA, Darley-Usmar VM, Goodwin DA, et al.Paradoxical fate and biological action of peroxyni-trite on human platelets. Proc Natl Acad Sci USA1994; 91: 67026.

102. Muijsers RB, Folkerts G, Henricks PA, Sadeghi-Hashjin G, Nijkamp FP. Peroxynitrite: a two-facedmetabolite of nitric oxide. Life Sci 1997; 60: 183345.

103. Beckman JS, Koppenol WH. Nitric oxide, superox-ide, and peroxynitrite: the good, the bad, and ugly.

Am J Physiol 1996; 271: C142437.104. Szabo C. The pathophysiological role of peroxynitrite

in shock, inflammation, and ischemia-reperfusioninjury. Shock 1996; 6: 7988.

105. Gross SS, Wolins M. Nitric oxide: pathophysiologicalmechanisms. Annu Rev Physiol 1995; 57: 73769.

106. van der Vliet A, Eiserich JP, Halliwell B, Cross CE.Formation of reactive nitrogen species during perox-idase-catalyzed oxidation of nitrite. A potential ad-ditional mechanism of nitric oxide-dependent toxic-ity. J Biol Chem 1997; 272: 761725.

107. Eiserich JP, Vossen V, ONeill CA, Halliwell B, CrossCE, van der Vliet A. Molecular mechanisms of dam-age by excess nitrogen oxides: nitration of tyrosineby gas-phase cigarette smoke. FEBS Lett 1994; 353:536.

108. Yi D, Smythe GA, Blount BC, Duncan MW. Per-oxynitrite-mediated nitration of peptides: character-ization of the products by electrospray and combinedgas chromatography-mass spectrometry. Arch Bio-chem Biophys 1997; 344: 2539.

109. Ishida A, Sasaguri T, Kosaka C, Nojima H, OgataJ. Induction of the cyclin-dependent kinase inhibitorp21(Sdi1/Cip1/Waf1) by nitric oxide-generating vaso-dilator in vascular smooth muscle cells. J Biol Chem1997; 272: 100507.

110. Grisham MB, Johnson GG, Lancaster JR Jr. Quan-titation of nitrate and nitrite in extracellular fluids.

Methods Enzymol 1996; 268: 23746.111. Granger DL, Taintor RR, Boockvar KS, Hibbs JB Jr.

Measurement of nitrate and nitrite in biologicalsamples using nitrate reductase and Griess reaction.

Methods Enzymol 1996; 268: 14251.112. Belmont HM, Levartovsky D, Goel A,et al. Increased

nitric oxide production accompanied by the up regu-lation of inducible nitric oxide synthase in vascular




18/26

endothelium from patients with systemic lupus ery-thematosus. Arthritis Rheum 1997; 40: 1810 6.

113. Trachtman H, Gauthier B, Frank R, Futterweit S,Goldstein A, Tomczak J. Increased urinary nitriteexcretion in children with minimal change nephroticsyndrome. J Pediatr 1996; 128: 1736.

114. Trachtman H. Urinary nitrite versus nitrate excre-tion in children: reply. J Pediatr 1997; 130: 161.

115. Seligman SP, Buyon JP, Clancy RM, Young BK,Abramson SB. The role of nitric oxide in the patho-genesis of pre-eclampsia. Am J Obstet Gynecol 1994;171: 9446.

116. Lyall F, Young A, Greer IA. Nitric oxide concentra-tions are increased in the fetoplacental circulation inpre-eclampsia. Am J Obstet Gynecol 1995; 173:7148.

117. Tsukahara H, Hata I, Sudo M. Urinary nitrite versusnitrate excretion in children. J Pediatr 1997; 130:161.

118. Moncada S, Higgs A, Furchgott R. InternationalUnion of Pharmacology Nomenclature in Nitric Ox-ide Research. Pharmacol Rev 1997; 49: 13742.

119. Sawicki E, Stanley TW, Pfaff J, DAmico A. Compar-

ison of fifty-two spectrophotometric methods for thedetermination of nitrite. Tantala 1963; 10: 64155.120. Verdon CP, Burton BA, Prior RL. Sample pretreat-

ment with nitrate reductase and glucose-6-phos-phate dehydrogenase quantitatively reduces nitratewhile avoiding int

Date post:	02-Apr-2018
Category:	Documents
Upload:	azimah-hamidon
View:	225 times
Download:	0 times

Nitrite and Nitrate Analyses

Documents