on the dynamics of nitrite, nitrate and other biomarkers of nitric oxide production in inflammatory...
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Nitric Oxide 22 (2010) 155–167
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Nitric Oxide
journal homepage: www.elsevier .com/locate /yniox
On the dynamics of nitrite, nitrate and other biomarkers of nitric oxideproduction in inflammatory bowel disease
Fumito Saijo a, Alexandra B. Milsom a, Nathan S. Bryan a, Selena M. Bauer a, Thorsten Vowinkel c,Marina Ivanovic b, Chris Andry b, D. Neil Granger c, Juan Rodriguez d, Martin Feelisch a,*
a Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USAb Department of Pathology, Boston University School of Medicine, Boston, MA 02118, USAc Department of Physiology, LSU Health Sciences Center, Shreveport, LA, USAd Department of Physics, Centenary College of Louisiana, Shreveport, LA, USA
a r t i c l e i n f o
Article history:Received 29 July 2009Revised 20 November 2009Available online 11 December 2009
Keywords:InflammationUlcerative colitisInflammatory bowel diseaseNitric oxideNitriteNitrosationAscorbateRedox signaling5-AminosalicylateHypoxia
1089-8603/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.niox.2009.11.009
* Corresponding author. Address: Experimental MeClinical Sciences Research Institute, Warwick MedicWarwick, Gibbet Hill Road, Coventry CV4 7AL, Unit7652 8372; fax: +44 (0)24 7615 0589.
E-mail address: [email protected] (M. Feelisch).
a b s t r a c t
Nitrite and nitrate are frequently used surrogate markers of nitric oxide (NO) production. Using rat mod-els of acute and chronic DSS-induced colitis we examined the applicability of these and other NO-relatedmetabolites, in tissues and blood, for the characterization of inflammatory bowel disease. Global NOdynamics were assessed by simultaneous quantification of nitrite, nitrate, nitroso and nitrosyl speciesover time in multiple compartments. NO metabolite levels were compared to a composite disease activityindex (DAI) and contrasted with measurements of platelet aggregability, ascorbate redox status and theeffects of 5-aminosalicylic acid (5-ASA). Nitroso products in the colon and in other organs responded in amanner consistent with the DAI. In contrast, nitrite and nitrate, in both intra- and extravascular compart-ments, exhibited variations that were not always in step with the DAI. Extravascular nitrite, in particular,demonstrated significant temporal instabilities, ranging from systemic drops to marked increases. Thelatter was particularly evident after cessation of the inflammatory stimulus and accompanied by pro-found ascorbate oxidation. Treatment with 5-ASA effectively reversed these fluctuations and the associ-ated oxidative and nitrosative stress. Platelet activation was enhanced in both the acute and chronicmodel. Our results offer a first glimpse into the systemic nature of DSS-induced inflammation and reveala greater complexity of NO metabolism than previously envisioned, with a clear dissociation of nitritefrom other markers of NO production. The remarkable effectiveness of 5-ASA to abrogate the observedpattern of nitrite instability suggests a hitherto unrecognized role of this molecule in either developmentor resolution of inflammation. Its possible link to tissue oxygen consumption and the hypoxia that tendsto accompany the inflammatory process warrants further investigation.
� 2009 Elsevier Inc. All rights reserved.
Introduction
Nitric oxide (NO) is a free radical capable of reacting with a vari-ety of molecules in biological fluids and tissues. These interactionsproduce not only the oxidation products, nitrite and nitrate, butalso lead to formation of nitrosyl (NO-heme) species and modifica-tion of thiols and amines to produce S- and N-nitroso products [1].The level of these NO-related substances, in fluids and in tissues, iswidely assumed to reflect the activity of NO-synthases, includingthe inducible form of NO-synthase (iNOS) which is known to beexpressed at high levels during inflammation [2], such as in inflam-
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dicine & Integrative Biology,al School, The University ofed Kingdom. Tel.: +44 (0)24
matory bowel disease (IBD) [3]. The link between iNOS expressionand inflammation serves as a rationale for considering increasedlevels of NO-related products as evidence of inflammatory condi-tions. Nitrite and nitrate have been used extensively as markersof inflammation, largely because of their ease of determinationusing a variety of assays, [4] including the widely available Griessreaction that can measure the combined concentrations of both an-ions (NOx) in body fluids [5].
The use of NO-related products as markers of inflammationpresupposes that only iNOS activity affects their concentrationsin the compartment of interest. However, it is well known that ni-trite and nitrate from the diet can also affect NOx levels assessed influids [6] and in tissues [7]. There is mounting evidence that nitriteis not as biologically inert as previously assumed, i.e. that it is abiochemically and pharmacologically active molecule [7–12] andthat its levels are maintained within a tight concentration range(200–500 nM) across many mammalian species [13], suggesting
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some level of regulation. Further evidence for some form of nitritehomeostasis in plasma is gleaned from experiments recently con-ducted in rats sustained on a low NOx diet. These experiments re-vealed an unusual compartmentalization of this anion, showingcomplete depletion in the extravascular compartment with essen-tially unaltered levels in plasma [7]. If the level of nitrite in plasmawas indeed regulated, then the question arises as to whether thesystemic concentration of this NO product is able to increase atall during a local inflammatory event. In addition to assessing themerit of NO products as markers of inflammation, we were inter-ested in understanding how increased NO production localized toan inflammatory site, such as the colon in colitis, can affect NO sta-tus in distal tissues. This interest is grounded on evidence fromclinical studies of colon inflammation showing that this diseasecan trigger several extra-intestinal problems that appear to beassociated with a decreased bio-availability of NO, including en-hanced platelet aggregation [14–17] and thromboembolic events[18–21].
This study sought to investigate the dynamics of a wide spec-trum of NO markers, including nitrite, nitrate, nitroso and nitrosylproducts, in the circulation and in tissues throughout the body ofan exemplary rodent species, following the induction of acuteand chronic colitis. Both forms of colitis are characterized by muco-sal inflammation of the large bowel, leading to an increased NOproduction secondary to iNOS upregulation [22–30]. A state ofacute and chronic colitis that is clinically and histologically remi-niscent of human ulcerative colitis (UC) can be induced in rodentsby administration of dextran sulfate sodium (DSS) [31]. In thisstudy, the in vivo assessment of NO-products was complementedby measurements of ascorbate redox status and platelet aggrega-bility; in addition, the effects of the therapeutic drug 5-aminosali-cyclic acid (5-ASA) were investigated. Our results demonstrate thatcolitis elicits significant NO-related reactions that are complex andat times counter-intuitive, suggesting they may not be suited asmarkers of disease activity until their behavior is better under-stood. Additionally, our results raise the prospect that nitrite playsa hitherto unrecognized role in either development or resolution ofIBD and that fluctuations in nitrite concentration are intimatelylinked to tissue oxygen consumption and the hypoxia that tendsto accompany the inflammatory process.
Methods
Materials
All chemicals were from Sigma Chemicals Co. (St. Louis, MO)unless otherwise stated.
Induction of colitis
Male Wistar rats (Harlan, Indianapolis; 200–400 g; housed3/cage) were kept on a normal 12/12 light/dark cycle, with accessto food (2018 rodent diet, Harlan Teklad) and water ad libitum. Aminimum of 7 days was allowed for acclimatization to the institu-tional housing environment before experimental use. Rats wereadministered 5% DSS (MP Biomedicals) dissolved in tap water for7 days (day 0–6, ad libitum) to induce acute colitis, followed by4 days of tap water without DSS to assess recovery. Chronic colitiswas induced by three cycles of DSS treatment, where one cycle in-cluded 7 days 5% DSS followed by 7 days water, and measurementswere performed on the final day of three complete cycles (i.e. oneweek after cessation of the last DSS administration). A group of ani-mals that received tap water only served as time-matched controls.A minimum of 3 animals/group was used for each time point in theacute study, whereas 5 (control) and 6 (DSS) animals/group were
used in the chronic study. All protocols described were carried outin strict accordance with the National Institutes of Health Guidelinesfor the Care and Use of Laboratory Animals (NRC 1996) and were ap-proved by the Institutional Animal Care and Use Committee of theBoston University School of Medicine.
Clinical assessment of colitis
The extent of colitis was assessed using a composite diseaseactivity index (DAI) [32,33] determined by scoring changes inweight, hemoccult positivity (as determined by a guaiac paper test(ColoScreen; Helena Laboratories Inc., Beaumont, TX, USA) andstool consistency. The scores (0–4) for weight change, the presenceof blood in the stools and stool consistency were combined and thefinal score divided by 3 where 0 represents no symptoms and 4represents maximum symptoms. Following sacrifice colon sampleswere assessed for myeloperoxidase (MPO) activity and histopa-thological signs of disease progression.
Myeloperoxidase assay
Colon samples were rinsed with cold phosphate buffered saline,blotted dry and immediately frozen in liquid nitrogen. Followingstorage at �80�C, tissues were thawed, weighed and assessed forMPO activity using the o-dianisidine method as described [34,35].
Histology
Colon cross sections were fixed with 10% neutral-buffered for-malin and embedded in paraffin. Full-thickness sections werestained with hematoxylin/eosin and examined by a blindedpathologist for evidence of pathological changes and neutrophilinfiltration.
NO-related biochemistry
To provide a comprehensive assessment of NO biochemistryduring development of colon inflammation the NO-related metab-olites, nitrite and nitrate as well as S- and N-nitroso species andnitrosylated heme species were measured in blood (plasma andred blood cells), brain, heart, liver, kidney, colon, aorta and inferiorvena cava (IVC). In addition, cellular redox status was assessed inthe same compartments by measurement of the redox coupleascorbate/dehydroascorbate, and platelet activity was analyzed inwhole blood as detailed underneath. Analyses were performed onday 0 (control); days 1, 3, 5 and 7 of DSS administration, and ondays 9 and 11 (i.e. 2 and 4 days after cessation of DSS administra-tion) to assess recovery in the acute model. In the chronic model,measurements were taken following three cycles of DSS treatmentas described above.
Experimental protocol
Heparinized (0.07 U/g i.p.) rats were anesthetized with diethyl-ether (2 min) and killed by cervical dislocation. After thoracotomy,a catheter was inserted into the infrarenal part of the abdominalaorta, and organs were flushed free of blood by retrograde in situperfusion with air-equilibrated PBS (50 mM, pH 7.4) supplementedwith N-ethylmaleimide (NEM)/ethylenediamine tetraacetic acid(EDTA) (10/2.5 mM) at a rate of 10 ml/min. The superior vena cavawas cut directly after the aortic cannulation to provide an exit portfor blood and buffer. After 2 min of perfusion, the brain was re-moved, followed by the heart. Liver, kidneys, and colon were indi-vidually cannulated and perfused through the portal vein, renalartery, and mesenteric artery, respectively, and excised 2–3 minapart. The colon was removed from the cecum to the pelvic floor
F. Saijo et al. / Nitric Oxide 22 (2010) 155–167 157
and prepared for histology and assessment of MPO activity. Tho-racic aorta and IVC were harvested last. This order of extractionwas based on the relative metabolic rates of the organs and wasstrictly adhered to throughout the study to minimize tissue hypox-ia. Excised tissues were blotted dry on filter paper, weighed, cutinto small pieces and homogenized immediately in ice-coldNEM/EDTA-containing perfusion buffer (5:1 v/w) by using eithera Teflon-glass Potter-Elvehjem or a Wheaton glass–glass homoge-nizer immersed in an ice bath. Tissue homogenates were kept onice in the dark and analyzed within 1–4 min of preparation.
Quantification of NO metabolites
Nitroso species (i.e. the sum of S- and N-nitroso compounds)were quantified by a technique that uses reductive denitrosationfollowed by chemiluminescence detection of NO in the gas phaseas described in detail elsewhere [36]. NO-heme species were quan-tified by direct injection of cell or tissue homogenates into an oxi-dizing solution comprised of 0.05 M ferricyanide in PBS [1]. Totalamounts of NO adducts were calculated by comparison of peakareas of the samples to those of freshly prepared standards. Theoxidative decomposition products of NO, nitrite and nitrate, weremeasured in aliquots of tissue homogenates and blood after meth-anol precipitation of proteins (1:1 vol/vol) using a dedicated HPLCsystem (ENO-20; EiCom, Kyoto) as described [1].
NO-related responses are reported either as concentrations orfold-changes relative to the average basal levels. Most of the basallevels measured in our current study (see Table 1) were similar tothose reported in earlier studies, although some differences werenoted which might be attributed to opposed light cycle conditions(animals used in this study were kept on a normal rather than a re-versed 12 h/12 h light/dark cycle), strain (different local supplier)or weight differences (final animal weights differed between theacute and chronic study). With the concentration of NO-hemeproducts being noticeably lower under these conditions, no at-tempt was made to analyze the time course of these species. Inview of our current lack of knowledge about the identity and sig-nificance of individual proteins undergoing NO-dependent post-translational modification during inflammation, in some cases weopted to collapse the data obtained for S- and N-nitroso com-pounds into a single value.
Redox status
The redox status of plasma and tissues was assessed by quanti-fication of the concentrations of the reduced and oxidized forms ofascorbate using the dinitrophenylhydrazine assay and expressed asascorbate/dehydroascorbate ratio. Samples were treated as de-scribed by Carr et al. [37] and read at 524 nm using a 96-well plate
Table 1Basal concentrations of nitrite, nitrate, nitroso, and nitrosyl species in plasma and tissues of(means ± SEM; n = 3 for acute study, time-matched to day 7 of DSS administration; n = 5–
reader. An additional blank was run in which 2,4-dinitrophenylhy-drazine was not added until after the addition of sulfuric acid to ac-count for tissue-specific background coloration of samples [38].Total ascorbate concentrations in tissues were determined by thedifference in readings of the 2,6-dichlorophenol-indophenol(DCIP)-treated and control samples by comparison with a standardcurve of ascorbic acid.
5-ASA administration
To investigate the effects of 5-ASA on the development of acutecolitis a further set of animals received 5-ASA at a dose level of50 mg/kg/day with the drinking water, starting from day 5 ofDSS treatment throughout the duration of the study period to mi-mic a therapeutically relevant scenario. NO-related metabolites,ascorbate/dehydroascorbate and platelet activity in blood and tis-sue were analyzed as described above on days 7, 9 and 11 (i.e. after2, 4 and 6 days of 5-ASA administration).
Platelet aggregation
Platelet aggregability was assessed in freshly obtained wholeblood, sampled from the inferior vena cava of DSS-treated and con-trol rats and diluted 1:1 with PBS, using a Whole Blood Aggregom-eter (Chronolog Model 590D). Amplitude and slope of the resultingchanges in impedance following addition of either collagen (2 lM)or adenosine diphosphate (5 lM) were evaluated using the Aggro-link software (Chronolog).
Statistical analysis
Data from the acute study were analyzed with a one-way ANO-VA followed by Dunnett’s post hoc test. Comparisons between twogroups, as in the chronic study were analyzed using an unpaired ttest. All analyses were performed using GraphPad Prism (version 4,GraphPad Software, San Diego). All values are reported as mean-s ± SEM. Statistical significance was set at p < 0.05. Values reportedas fold-increases were calculated from the averages at a giventime-point to corresponding time-matched controls, and theirrespective SEM through propagation of errors.
Results
Disease assessment
The development of colitis was accompanied by reduced bodyweight gain until day 3 of DSS administration and an averageweight loss thereafter, leading to body weights varying from �1%
Wistar rats enrolled in the acute (grey shaded) and chronic (unshaded) model of colitis6 for chronic study, corresponding to end of the treatment cycle).
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(day 5) and �9% (day 7) on DSS to �6% and �10% on days 2 and 4off DSS compared to the weight at day 0. In agreement with pub-lished data [39–44], the DAI increased gradually over the time ofDSS administration, reaching a maximum of 3.11 ± 0.05 on the lastday of DSS administration (day 7), and subsequently decreased to1.55 ± 0.17 at the 4th day after DSS cessation. The full DAI timecourse is shown in Fig. 1. The induction and extent of colitis wasfurther confirmed by blinded histological assessment, whichshowed inflammatory injury in the colon of DSS-treated animalsincluding crypt damage, mucosal ulceration, loss of goblet cells,and submucosal edema. Significant leukocyte recruitment to thecolon was observed at day 7 of DSS administration, with MPOactivity remaining increased on day 11 (p < 0.05).
Colonic nitrosative stress and systemic NO metabolite changes
Consistent with previous findings [1], the concentrations of ni-troso and nitrosyl species present in blood and tissues at baseline(day 0) were found to be in the range of 10–100 pmol/g wet tissueweight, with distinctly different ratios in different compartments.The major nitroso compounds present in plasma and in the colonwere mercury-stable species (presumably RNNOs), whereas thosein venous tissue were almost exclusively of the RSNO type (datanot shown). In all other compartments a mixture of S- and N-nitro-so products, along with nitrosyl heme species prevailed. A complexpicture of changes in NO-related metabolites was apparent, bothduring administration and cessation of DSS (Fig. 2). In the colon,DSS-induced inflammation was accompanied by a transient de-crease in total nitros(yl)ation products (i.e. the sum of all S/N-ni-troso and heme nitrosyl species), followed by an increase withpeak concentrations on day 7 (p < 0.01), coinciding with the peakin DAI, and a return to control levels by day 11 (Fig. 1). These alter-ations were accompanied by dynamic changes in the ratio of indi-vidual colon nitroso and nitrosyl products with a shift from amineto thiol nitrosation products and the appearance of nitrosyl hemespecies on day 7 of DSS administration, indicative of a significantchange in local NO production and metabolism. Overall, shifts inmetabolite composition and concentration were organ-specificand most dramatic on days 1, 5 and 7 of DSS application, suggest-ing the involvement of multiple competing pathways affecting lo-cal NO metabolite profiles. Importantly, these changes wereevident also in tissues other than the colon, revealing the systemicnature of DSS-induced inflammation.
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Fig. 1. Disease activity index (DAI) and concentration of total nitroso compounds inthe colon of DSS-treated rats. Data displayed as mean ± SEM, n = 3/day. DAI wassignificantly increased from day 3 of DSS application on and remained significantly(p < 0.01) elevated 4 days after cessation of DSS administration (asterisks omittedfor sake of clarity). Colon nitrosation was significantly (**p < 0.01) increased on day7 of DSS administration.
Plasma and colon nitrite and nitrate versus DAI
Plasma nitrite and nitrate exhibited a time course that paralleledthe DAI up to and including day 2 following cessation of DSS (seeFig. 3A). By day 4 after DSS cessation, however, both anions experi-enced a resurgence in concentration, in sharp contrast to the DAIwhich continued to drop. The time course for changes in nitriteand nitrate concentrations in the colon is shown in Fig. 3B and con-trasted in the same figure with the kinetics of the DAI. While nitrateexhibits a relatively small reduction during the course of the dis-ease, nitrite experiences two surges, including a 2.5-fold-increase3 days after DSS administration and a 2.2-fold-increase after its ces-sation. A considerable decline in colon nitrite is observed in theintervening time, particularly at day 2 after DSS cessation when lev-els of the anion dropped from a basal level of 1.4 lM to below ourdetection limit of about 0.01 lM. Thus, NOx species in the colonexperience changes very distinct from those seen in plasma.
Systemic changes in nitrite, nitrate, redox and tissue nitrosation status
Nitrite concentrations in all organs studied exhibited changesremarkably similar to one another and to those observed in the co-lon (see Fig. 4 for absolute changes at select time points and Fig. 5 forfractional changes over the entire time course). These included (1) ageneral decline in nitrite, bottoming at levels below our detectionlimit at day 2 following DSS cessation, superimposed with (2) aspike at day 3 into the DSS treatment, and (3) a profound surge byday 4 after DSS cessation. In the course of these changes, the redoxcouple ascorbate/dehydroascorbate (Asc/DHA) remained steady ex-cept at day 4 after cessation of DSS administration, when a statisti-cally significant redox change occurred, characterized by a decreasein ascorbate (p < 0.05) and an increase in the corresponding oxida-tion product, DHA (p < 0.05) (Fig. 5A). Increases in nitrite concentra-tions after DSS cessation correlated well (R2 = 0.89, data not shown)with decreases in the logarithm of the AA/DHA ratio, strongly sug-gesting the existence of a redox-related link between the changesin ascorbate and nitrite. In contrast to the many-fold changes exhib-ited by nitrite and AA/DHA (Fig. 5A), and total ascorbate (notshown), nitrate remained largely unchanged throughout the acutephases of the disease (Fig. 5B). In all organs studied, tissue nitrosa-tion levels (i.e., the concentrations of S- and N-nitroso productscombined) exhibited remarkably similar changes in response toDSS administration (see Fig. 5C). Those trends included a moderatedecline during the first 5 days of DSS administration, followed by asudden rise on the last day of the treatment. The sudden rises in tis-sue nitrosation levels at day 7 are in stark contrast to the nitritedownward trends observed during that period in the same organs.
Platelet aggregation during acute colitis
Irrespective of the test stimulus applied, platelet aggregabilitywas found to be elevated both during acute DSS administration(Fig. 6) and following cessation (not shown), although the differ-ences to untreated controls did not reach statistical significance.Given that a certain percentage of UC cases is associated with ahypercoagulable state we reasoned that the changes observed inthe acute model may be indicative of a stage that precedes thisstate and may become more pronounced only in the chronic set-ting. To investigate this possibility a more chronic form of colitiswas induced in a separate set of animals and the above measure-ments repeated.
Chronic colitis model
The chronic model of colitis yielded a DAI of 1.3 ± 0.1 comparedto time-matched controls (p < 0.01). Changes in absolute concen-
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Fig. 2. Systemic changes in concentration of NO-related metabolites in blood and exemplary tissues of the rat during development of acute colon inflammation during DSSadministration (days 0–7) and following cessation of DSS treatment (recovery phase; days 9–11). Nitros(yl)lation products are exemplified by S-nitrosothiols (white),N-nitrosamines (black) and nitrosyl heme species (grey), whereas nitrate (black) and nitrite (grey) represent products of oxidative NO metabolism (NOx). Data aremeans ± SEM; n = 3/day/tissue.
F. Saijo et al. / Nitric Oxide 22 (2010) 155–167 159
tration of NO metabolites in blood and tissues are shown in Fig. 7,and fractional changes from control levels are summarized for NO-metabolites and AA/DHA ratios in Table 2. As observed during DSSadministration in the acute model, plasma nitrite levels remainedsignificantly elevated in the chronic model (mean fractionalchange of 2.26 over 11 days for the acute model versus 2.25 forthe chronic model). In contrast to the changes in tissue nitrite con-centrations observed during acute DSS administration, whichdropped to levels below our detection limit, those levels remainedeither unchanged or increased in the chronic model. Tissue nitrosa-tion also exhibited a general tendency toward systemic increasesthat was particularly evident in targets of the cardiovascular sys-tem (exemplified by effect on heart and aorta; see Fig. 7 and Table.2), whereas changes in cellular redox status and nitrate showed noclear systemic trends. While plasma nitrate in particular remainedalmost unchanged in the chronic model, platelet aggregation(Fig. 6) was elevated to levels comparable to those observed inthe acute model, with differences reaching statistical significancewhen compared to age and time-matched controls.
5-ASA treatment
Histological assessment of the colon in those animals that re-ceived 5-ASA between day 5 of DSS administration through today 4 after removal from DSS revealed no measurable reductionin colon damage. However, 5-ASA treatment was accompaniedby a reduction in DAI compared to animals receiving DSS alone(2.70 ± 0.07, day 7; 1.22 ± 0.40, day 11). The difference in DAIwas not due to reduced weight loss, which was comparable be-tween the 5-ASA treatment group and the DSS-treated controlgroup that received only water in the recovery phase (�7% and
�11% at days 9 and 11, respectively). Similarly, no changes inMPO activity were observed compared to animals treated withDSS alone. Given the short time the animals received 5-ASA treat-ment on top of DSS these results are perhaps not surprising. Unex-pectedly, however, 5-ASA had a dramatic effect on NO-relatedproducts and redox status. Co-administration of 5-ASA togetherwith DSS completely abolished the increased formation of nitrosat-ed species to levels virtually indistinguishable from those ofcontrol animals (data not shown). Moreover, 5-ASA treatment lar-gely prevented the drop in tissue nitrite concentrations at theheight of DSS-induced inflammation and fully prevented the sys-temic increase in nitrite and oxidative stress observed in allcompartments on the fourth day of recovery following DSS admin-istration (Fig. 8). In addition to the abrogation of oxidative stressfollowing DSS cessation a trend towards a more reduced statusbecame apparent in several compartments with 5-ASA administra-tion, in particular in the brain. The significance of this finding re-mains unclear at present. In contrast, blood nitrate levelsremained unchanged with 5-ASA treatment compared to DSSadministration alone (data not shown).
Discussion
The present study provides a first comprehensive assessment ofthe global dynamics of a series of NO-related metabolites duringdevelopment, manifestation and remittance of DSS-induced coloninflammation in a rodent model in vivo. Our results reveal an unex-pected level of complexity concerning the fate of NO and are atodds with the contemporary (and often indiscriminate) use ofNO-related metabolites as universal markers of enhanced NOSactivity which typically accompanies inflammatory events. These
Fig. 3. Inflammatory response to continuous administration of DSS over a period of7 days (area shaded in grey). The blue and green curves contrast the time course ofnitrite and nitrate, respectively, with that of the DAI, in plasma (A) and in the colon(B). Each time point represents a fold-change from basal levels, calculated from theratio of the mean concentration (n = 3) at the day indicated on the X axis comparedto the basal level (n = 3) assessed at day 0 (Controls). Error bars were omitted forclarity.
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Fig. 4. Blood and tissue concentration of nitrite in controls (white bars, day 0),following 7 days DSS treatment (grey bars, day 7) and 4 days after treatmentcessation (black bars, day 11). Data displayed as means ± SEM, n = 3/day/tissue.Where * represents p < 0.05 and ** represents p < 0.01 versus. matched control (day0). IVC, inferior vena cava; RBC, red blood cells.
160 F. Saijo et al. / Nitric Oxide 22 (2010) 155–167
findings are of relevance not only for the monitoring of diseaseprogression and for biomarker research, but may have importantimplications for the role of NO and nitrite in inflammation in gen-eral. The appreciation that iNOS induction during inflammationcan be sometimes beneficial and on other occasions detrimental[3,45], depending in part on whether the enzyme is induced inblood cells or tissues [46], does little to answer the question asto whether NO is protective or deleterious [47,48]. This has createda situation, characterized as ‘‘NO paradox”, that not only continuesto confuse clinical and basic investigators but represents a signifi-cant impediment to research progress in this area. By addressingthis situation from an analytical angle, our study contributes to abetter understanding of the chemical biology of NO and its reactionproducts in the setting of gastrointestinal inflammation, an impor-tant prerequisite for defining novel treatment strategies aimed atmodulating NO bio-availability. The demonstration that 5-ASAleads to almost complete normalization of the NO metabolite pro-file and oxidative stress-induced perturbations in tissues, evenwhen administered well after colon inflammation is established,further suggests that the systems-based experimental paradigm
used in the present investigation may hold promise for futuremechanistic studies and drug discovery efforts.
NO metabolites as markers of inflammation – prospects andlimitations
While elevated NOS activity has been found in experimental ro-dent models of colon inflammation as well as in patients withUlcerative Colitis and Crohn’s Disease, the relationship betweenthis activity and the severity of bowel inflammation remains ill de-fined [49,50]. This may be due, in part, to the fact that most indi-viduals from whom colon biopsy samples are obtained typicallyreceive either corticosteroids or other anti-inflammatory medica-tions all of which have the potential to interfere with NOS expres-sion and activity. Information in the literature about iNOSexpression in DSS colitis (in particular changes over time) is sparse,sometimes conflicting, and differs between species and strains. Weopted not to determine message or protein expression for endothe-lial or inducible NOS in our present study. We reasoned that suchmeasurements may be of little help in interpreting the changes inNO metabolite patterns observed without concomitant informa-tion about true NOS activity (as opposed to a potential activity asmeasured by citrulline or oxyHb assay, which is typically deter-mined in the presence of optimal cofactor and substrate supply),reactive oxygen species production and NO/nitrite consumptionprocesses. While we could speculate about oxidative stress levelstaking into account the ascorbate redox status measured, NO/ni-trite consumption is likely to change dynamically as inflammationprogresses or animals recover. Thus, all these processes have thepotential to affect NO availability and metabolic profile even ifNO production progressively increased or remained constant.Other complications include the multitude of reactions NO andits metabolites can undergo in a biological environment, the chem-ical instability of some of the reaction products and analytical chal-lenges related to their quantification at trace level in biologicalsamples. Sampling frequency and logistical challenges related tosample processing further limit the mechanistic insight one cangain from an analysis of clinical specimen in this particular areaof research, emphasizing the need to address issues of greater com-plexity in animal models of disease such as the one employed inthis study.
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C
Fig. 5. Comparisons of the changes in redox status (expressed as AA/DHA ratio; A)as well as in the concentrations of nitrate (B) and nitroso products (C) with those innitrite (solid curves) across various organs and in the circulation, in response to DSSadministration over a course of 7 days followed by 4 days of recovery. Each timepoint represents a fold-change from basal levels, calculated from the ratio of themean concentration (n = 3) at the day indicated on the X axis compared to the basallevel (n = 3) assessed at day 0 (Controls). Error bars were omitted for clarity. AorticAA/DHA levels were not determined due to limited sample size.
Collagen ADP
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30 ** *
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Agg
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tion
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s]
Fig. 6. Platelet aggregation as assessed by addition of collagen and adenosinediphosphate (ADP) to whole blood under control conditions (white bars, n = 6) andfollowing 7 days of DSS administration (grey bars, n = 4) and in the chronic model ofcolon inflammation (white hatched bars, n = 9) with time-matched controls (greylined bars, n = 4). Data displayed as means ± SEM (*p < 0.05, **p < 0.01 versus.control).
F. Saijo et al. / Nitric Oxide 22 (2010) 155–167 161
Another formidable confounding factor is dietary intake. Rodentdiets are known to contain varying amounts of NOx which, if notcontrolled for, may contribute to poor comparability betweenstudies. Since stress and/or sickness are typically accompanied byreduced food intake progression of disease may translate intochanges in bodily NOx supply and their concentration in tissues.Since accurate quantification of food consumption is only possiblewith individually housed animals (which itself is a stressor thatmay impact on the inflammatory process) weight gain is oftenused as a proxy for food intake. In agreement with other investiga-tions a variable degree of weight loss was observed in DSS-treatedanimals in the present study. However, with regard to its relevanceto NO metabolite concentration this data has to be interpretedwith caution as loss in body weight may be due to changes inmetabolism, extracellular fluid volume depletion and other factorsunrelated to food intake.
Irrespective of these limitations, both nitrite and nitrate seem totrack the DAI in our acute colitis model, with the exception of thesudden surge in nitrite/nitrate observed four days after DSS cessa-tion (discussed below). In the chronic model, however, nitratelevels remained essentially unaltered (fractional change of1.27 ± 0.19) while nitrite levels more than doubled (2.25 ± 0.31).The finding that nitrate increases significantly only in the acutemodel of inflammation suggests that plasma nitrite may be abetter overall marker of inflammatory events. Consequently, anyplasma assay that combines nitrite with nitrate into one singlemarker of NO synthesis (i.e. NOx assays) will not provide a moresensitive disease index since only nitrite appears to be changingconsistently during inflammation. In fact, since plasma nitrite con-centrations are usually an order of magnitude lower than those ofnitrate, any changes in nitrite may be overwhelmed by samplingerrors in nitrate. Thus, our results suggest that plasma nitrite by it-self may be better suited as a marker of inflammatory activity thanassays that combine nitrite and nitrate values. However, caution isrecommended in associating the time-course of this product tooclosely to the DAI, given our observation that its levels reboundedeven as the DAI subsided (Fig 3A).
A surprising finding from our acute model is the extent of NOresponses (Figs. 2–5) that occurs in the extravascular compart-ment. Our results clearly indicate that the levels of nitrite and
Colon
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Nitroso/Nitrosyl Species Nitrite/Nitrate
Fig. 7. Systemic changes in concentration of NO-related metabolites in blood and representative organs during chronic colon inflammation. Nitros(yl)lation products areexemplified by S-nitrosothiols (white), N-nitrosamines (black) and nitrosyl heme species (grey), whereas nitrate (black) and nitrite (grey) represent products of oxidative NOmetabolism (NOx). Data are means ± SEM, n = 5–6/day/tissue.
Table 2Global changes of NO-related metabolites in plasma and representative organs duringchronic colon inflammation. Fractional changes in nitrite, nitrate and total nitrosoproducts (RXNO) and redox status (expressed as the ratio of ascorbic acid/dehydro-ascorbate, AA/DHA) in rats subjected to three cycles of DSS treatment compared toage- and weight-matched controls. Data displayed as means ± SEM from n = 5–6animals. ND, not determined.
Nitrite Nitrate Total RXNO AA/DHA
Plasma 2.25 ± 0.31 1.27 ± 0.19 1.05 ± 0.67 NDBrain 1.51 ± 0.42 0.77 ± 0.20 1.19 ± 0.29 0.98 ± 0.08Heart 1.73 ± 0.57 2.03 ± 0.46 3.96 ± 1.69 0.46 ± 0.19Liver 4.77 ± 2.99 1.14 ± 0.46 2.28 ± 0.79 0.45 ± 0.16Kidney 1.46 ± 1.00 0.88 ± 0.24 1.44 ± 0.30 0.62 ± 0.13Colon 1.24 ± 0.28 0.85 ± 0.20 1.44 ± 0.43 1.41 ± 0.27Aorta 1.26 ± 0.35 1.14 ± 0.45 7.45 ± 5.34 ND
162 F. Saijo et al. / Nitric Oxide 22 (2010) 155–167
nitroso products, but not nitrate, are significantly altered synchro-nously in all organs including the colon. Additionally, the data ob-tained in the chronic model (Figs. 7 and 8, Table 2) indicate that thecolon exhibits no particular pattern of NO-metabolites that woulddifferentiate it significantly from other organs. Thus, given the sim-ilarity in changes in NO-metabolite profiles between the colon(where supposedly inflammation is confined to) and other organs,it appears that these species may not serve as markers suitable forconfirmation or localization of inflammation in tissues, neither inthe acute nor in the chronic stage.
Compartmentalization of nitrite between intra- and extravascularspace
Another surprising finding from our study is the degree of com-partmentalization of nitrite between intra- and extravascularspace. For instance, a salient feature in the nitrite signals from
the colon and from other organs (Fig. 5A) is the presence of twosurges, at days 3 after initiation of DSS treatment and at day 4 fol-lowing its withdrawal. The first nitrite surge which is prominent inthe colon but also present in other tissues, may originate from theinduction of iNOS in macrophages and other cells in those tissues.Interestingly, there is no evidence that this added nitrite diffusesinto the intravascular compartment, which is surprising consider-ing the relatively large volume of extravascular spaces that experi-ence increases in nitrite. Further evidence of compartmentalizationis seen in the dramatic loss in extravascular nitrite observed in theintervening time between the two surges. This loss occurs whilelevels of nitrite in the intravascular compartment continue toincrease above basal values. Further evidence for nitrite compart-mentalization can be gleaned from our 5-ASA data, which demon-strates that therapeutic administration of this drug selectivelyaffects nitrite in the extravascular compartment.
The degree of compartmentalization between the extra- andintravascular compartments is surprising, given the establishedability of this anion to cross cell membranes through ion channelsand possibly also via passive transport in the form of nitrous acid(HNO2) [51]. This level of compartmentalization is reminiscent ofthose from another recent study [7]. In that particular study, ani-mals sustained on a low NOx diet exhibited a systemic loss of ni-trite from all tissues except the circulation where levelsremained unchanged compared to those of animals on a normaldiet. Thus, the low NOx diet also did not lead to a loss of tissue ni-troso products when nitrite was lost. The similarities between thefindings of that study with those reported here might be part of acommon reaction channel yet to be characterized that is activatedunder certain physiological stresses. In the following section weelaborate further on the nature of this response and the difficultyin reconciling it with our current understanding of NO biochemis-try in inflammation.
Brain
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Fig. 8. Comparison of changes in nitrite concentration and ascorbate redox status in blood and tissues of DSS-treated animals in the absence (A and B) and presence (C and D)of 5-ASA. Upper panel: (A) Blood and tissue concentrations of nitrite and (B) tissue concentrations of ascorbate and dehydroascorbate (DHA; displayed as a ratio of ascorbate/DHA) in controls on day 0 (white bars), day 7 (grey bars) and day 11 (black bars). Lower panel: (C) Blood and tissue concentrations of nitrite and (D) tissue concentrations ofascorbate and dehydroascorbate (DHA; displayed as a ratio of ascorbate/DHA) on day 0 (white bars), day 7 (grey bars) and day 11 (black bars) of DSS administration withconcomitant 5-ASA treatment starting on day 5. Data displayed as means ± SEM from 3 animals/day (*p < 0.05). IVC, inferior vena cava; RBC, red blood cells. No ascorbate/DHA measurements were performed in vascular tissue and blood due to limited sample availability.
F. Saijo et al. / Nitric Oxide 22 (2010) 155–167 163
A need to re-examine the chemical biology of NO and nitrite ininflammation
Most NO-metabolites found in vivo, including nitrite, nitrate,and nitrosation products are thought to arise primarily from theNO autoxidation pathway, in which NO and O2 react to formNO2, from which either N2O4 or N2O3 are formed:
2NOþ O2 ! 2NO2 ð1Þ
2NO2�N2O4 ð2Þ
NOþ NO2�N2O3 ð3Þ
These higher N-oxides can subsequently lead to the formation ofNOx and nitroso products according to the following reactions:
N2O4 þ RXH! RXNOþ NO�3 þHþ ð4Þ
N2O3 þH2O! 2NO�2 þ 2Hþ ð5Þ
N2O4 þH2O! NO�2 þ NO�3 þ 2Hþ ð6Þ
N2O3 þ RXH! RXNOþ NO�2 þHþ ð7Þ
Thus, if inflammatory responses were associated with an in-crease in NO synthesis while the availability of other reactants(O2, H2O, RXH) was kept essentially constant, one would expectthat levels of nitrite, nitrate, and nitroso species all would rise
simultaneously in inflamed tissues to eventually end up in the cir-culation. However, this is not what we observe. Instead, nitrite lev-els in tissues drop even as nitrosation in that compartment rises,while no appreciable changes in nitrate concentration are appar-ent. Furthermore, our data suggest that these products behave dif-ferently in the circulation and are not in equilibrium with theircounterparts in tissues.
The departure from the chemistry outlined in reactions (1)–(7)might be attributed to a shift in NO-biochemistry driven by in-creased levels of a third reactant, namely superoxide (O2
�). Thisoxygen-derived radical anion, which is not merely a by-productof aerobic respiration but fulfills important signaling functions[52], is produced at much higher levels during inflammatory re-sponses. Superoxide reacts very rapidly with NO to formperoxynitrite:
O�2 þ NO! ONOO� ð8Þ
Scavenging of NO by this reaction would reduce its bio-avail-ability, leading to lower levels of nitrite from reactions (5)–(7).While a decrease in NO bio-availability might account for the low-er levels of nitrite in tissues and the increased platelet aggregabil-ity in the circulation, much of the product peroxynitrite decaysreadily to form nitrate,
ONOO� ! NO�3 ð9Þ
leading to what we should have registered as increased levels of ni-trate in tissues. As clearly seen from Fig. 3B for the colon, and ech-oed in the data for other organs (Fig. 5B), nitrate either remains
164 F. Saijo et al. / Nitric Oxide 22 (2010) 155–167
unchanged or even decreases somewhat during this period. Thisscenario also fails to account for the seemingly contradictory risein tissue nitrosation (Fig. 5C) when NO bio-availability is presum-ably diminished. The significant rise in nitrite in the circulation ob-served at days 5 and 7 (Fig. 3A), at a time when all tissuessupposedly produce much less of it (Figs. 4 and 5), also cannot berationalized as a consequence of superoxide production.
The increased production of nitroso products at the peak of thedisease activity invites consideration of a third scenario involving arecently proposed mechanism for thiol nitrosation via a radicalmechanism [53]. In this mechanism, NO2 produced via reaction 1reacts directly with thiols to produce a thiyl radical, RS� via the fol-lowing reaction:
NO2 þ RSH! RS� þ NO�2 þHþ ð10Þ
Once formed, the RS� radical can react very rapidly with NO toproduce the nitroso product RSNO via a radical–radical reaction:
NO� þ RS� ! RSNO ð11Þ
If inflammation somehow produces conditions that favor reac-tions (10) and (11) over reactions (2)–(7), the net result wouldbe a chemical shift that produces less nitrate in favor of nitroso(and nitration) products and nitrite. The increased nitrite, however,is not consistent with the observed disappearance of this anionfrom tissues, including the colon.
The difficulty in reconciling current models of NO biochemistrywith the observed dynamics of NO products in our model of acutecolitis incites the question of whether additional mechanisms areintervening to modulate nitrite, nitrate, or nitroso products. For in-stance, several mechanisms have been proposed in the literaturethat could reduce nitrite to NO in vivo and generate nitroso prod-ucts through the nitrosative chemistry described above. Mecha-nisms include interaction of nitrite with the mitochondrialelectron transport chain, haemoglobin and myoglobin, xanthineoxidase, cytochrome P450, and simple protonation to form nitrousacid [10]. In addition, nitrite may be consumed in reactions withperoxidases/H2O2, which are known to be present at high levelsin inflamed tissues (and documented by MPO measurements in co-lon tissue in this study), leading to the formation of nitrotyrosine,nitrated fatty acids and other nitration products [54,55] whichwould go largely undetected in our assay systems. While thesemay readily account for the diminishing levels of nitrite we ob-serve, they could only account for the increased nitrosation if weassumed the increased NO fluxes from nitrite reduction enhancedthe nitrosative chemistry described above. However, that samenitrosative chemistry would inevitably yield increased levels of ni-trate, a trend we do not observe. To complicate matter further, theaction of peroxidase/H2O2 on nitrite gives rise to NO2 to eitherform S-nitrosothiols via reactions (10) and (11) or nitrite via reac-tions (3) and (5). Furthermore, the increased platelet aggregabilitywe measured at the peak of the disease activity (i.e., at day 7 of DSSadministration) would also argue against increased systemic NObio-availability during the course of the disease, as NO is wellknown to inhibit platelet activation.
A possible way to resolve the apparent conflict between the for-mation of nitroso products without increased NO or other NOproducts, such as nitrate, is to consider an alternative mechanismthat produces nitrosation from nitrite without the intermediacyof NO or nitrosative species such as N2O3. The existence of such amechanism was indeed demonstrated in a recent study of the bio-chemical response of rat tissues to nitrite, both in vivo and in vitro[7]. This study found that tissue nitrosation was directly correlatedwith nitrite levels. A unique feature of this process, which involvesa cooperative action between thiols and hemes, is its use of nitriteas a substrate to produce nitrosation products without the inter-mediacy of NO. If the same mechanism was responsible for the
nitrosation observed in the present study, our results would sug-gest that this may be part of an enzymatic process that is signifi-cantly enhanced in inflammation.
Links between nitrite, ascorbate status and the pathology of gutinflammation
Many of the findings discussed above point to an unusualbehavior of nitrite during the course of the acute inflammatoryprocess, i.e. one that extends beyond a passive role as an inertend-product of NO formation. One of those findings relates to theextraordinary surge in the levels of this anion that take place onthe fourth day after withdrawal of DSS, i.e. at a time when iNOSexpression is supposedly no longer massively up-regulated. Impor-tantly, this surge is observed in every organ examined, and appearsto be correlated with a significant oxidative event in those organsas indicated by the drop in their AA/DHA ratio (Fig. 5A). The con-nection between nitrite and the redox status in those tissues is fur-ther strengthened by the relation between the drop in the log ofthe AA/DHA ratio and the magnitude of the nitrite surge(R2 = 0.89). The response is particularly large in extra-colonic tis-sues such as the liver and kidney (Fig. 5A), but its mechanism re-mains unclear at present. Dehydroascorbate formation is unlikelya consequence of direct chemical interaction between ascorbateand nitrite given that intracellular concentrations of the former ex-ceed those of the latter by at least an order of magnitude in mosttissues. Rather, it may originate from an impaired capacity of themucosa to reduce DHA back to ascorbate, which has been observedin colonic biopsies from IBD patients [56]. Regardless of the under-lying mechanism, an impairment of ascorbate-mediated antioxida-tive defense may render inflamed tissue more susceptible tooxidative damage. Moreover, these changes lead to an impairedavailability of reduced ascorbate in tissues and may therefore af-fect hydroxylation processes and other reactions in which ascor-bate is acting as a specific cofactor rather than an antioxidant.
Another curious connection between nitrite and acute colitis isgleaned from the results obtained during co-administration of 5-ASA with DSS. This drug is highly effective in the setting of acuteinflammatory bowel disease in the clinic and has become the goldstandard for the therapy of ulcerative colitis [57]. Its intestinalanti-inflammatory effects are believed to be mediated via interac-tion with the peroxisome proliferator-activated receptor-c [58]. Inthis study, no significant change in either weight gain or MPOactivity was observed following 5-ASA administration, while intes-tinal bleeding and effects on stool consistency were clearly re-duced. Yet, both circulating and tissue nitrite levels (Fig. 8C)showed substantial reversal toward basal levels, both at the peakof the DAI (day 7) and at day 4 after DSS withdrawal when nitritewould otherwise surge (Fig. 8A). While we cannot exclude the pos-sibility that reduced food intake may contribute to the progressivedecrease in tissue NOx levels once colitis is established, we do notbelieve that dietary changes can account for the dramatic surge innitrite following cessation of DSS treatment for two reasons: (1) re-duced body weight gain persisted well into the recovery phase; (2)there was no difference in weight loss after DSS cessation betweenthe group that received only water and the one that received5-ASA, although the surge in nitrite was completely abrogated inthe latter. The prevention or reversal of the nitrite surge toward ba-sal levels was accompanied by a notable attenuation of oxidativestress (compare Fig. 8B and D). It has been demonstrated thatthe p-amino phenol moiety of 5-ASA confers both potent antioxi-dant effects [59] and a reactive nitrogen oxide species scavengingability [60] to the molecule. Both of these actions might work inconcert to reduce nitrite accumulation and prevent the occurrenceof oxidative and nitrosative stress. These results appear to be ofparticular relevance in the context of concerns that the nitrite
F. Saijo et al. / Nitric Oxide 22 (2010) 155–167 165
produced during inflammatory conditions in the gut may enhance,via formation of carcinogenic N-nitrosamines, the risk for coloncancer [61,62]. Of note, nitrite has recently been suggested to mod-ulate T-cell function and cytokine release [63]. Given the signifi-cance of T-lymphocytes in the disease process [64] the avoidanceof abrupt fluctuations in nitrite concentration may prove benefi-cial. Our biochemical findings with 5-ASA would seem to lendsupport to the contemporary interest in its broader use for chemo-prevention of colorectal cancer, one of the more fearsome compli-cations of IBD [65,66].
Thus, parallels between the behavior of nitrite described aboveand known disease progression/complications of colitis seem ines-capable. First, ulcerative colitis is a disease characterized by recur-ring cycles of acute illness and remission. Our study shows that anunderlying product of the inflammatory process, nitrite, alsoundergoes considerable cycling throughout the disease, dippingto vanishingly low levels and suddenly rising to values many-foldabove basal ones. Second, IBD is often accompanied by complica-tions in distal organs, including the liver and the kidney, for rea-sons that are yet unclear [67]. Our findings show that those twoorgans are the main targets of nitrite overload and significant oxi-dative stress. Third, our findings show that the drug 5-ASA, whichis effective in the treatment of ulcerative colitis, also normalizesthe levels of nitrite in the circulation and in the extravascular com-partment. Together, these parallels raise the prospect that an inti-mate connection exists between nitrite and IBD. Whether thisrelationship is causal or consequential and whether nitrite playsa beneficial or detrimental role in gut inflammation remains tobe determined.
Chronic gastrointestinal inflammation is accompanied by spe-cific alterations in oxygen supply and utilization as well as increasedtissue metabolism, rendering inflamed mucosal tissue hypoxic [68].Tissue hypoxia leads to activation of the hypoxia-responsivetranscription factor HIF-1a, and this response is subject to redoxmodulation by the combined production of NO and reactive oxygenspecies [69]. Whilst mitochondrial reactive oxygen species produc-tion is enhanced in hypoxia, [70] mitochondrial NO consumption isreduced [71], with important consequences for local NO availabilityand redox signaling. Hypoxia is linked to inflammation via thetranscription factor, NF-jB [72]. Ascorbate is a cofactor of prolylhyroxylases, and inhibition of HIF hydroxylases has been shownto be protective in rodent models of colitis [68]. Thus, changes in cel-lular redox status secondary to alterations in NO and ascorbateavailability may affect hypoxic signaling and key inflammatorymediators in a complex fashion via several different angles. Nitriteis known to be consumed by mammalian tissues under hypoxic con-ditions [1], with NO being produced along the process [10] The for-mation of hydrogen sulphide (H2S), produced in the gut bycommensal sulfate-reducing bacteria or in tissues by mammalianenzymatic processes, has been suspected to be involved in the etiol-ogy of IBD, possibly as a result of impaired detoxification [73]. Ofnote, H2S formation is markedly upregulated in inflammation andknown to affect NO availability [74]. It is possible, therefore, thatthis cross-talk between mediators of inflammatory and hypoxicprocesses is sufficient to explain the fluctuations in nitrite observedin the present study, and tissue NO metabolite profiling an informa-tive tool to study this cross-talk in vivo. Given the effectiveness of5-ASA to prevent and/or abolish the alterations in NO-relatedmetabolite levels and redox status, this systems cross-talk wouldseem to deserve further investigation.
Conclusions
This study investigated the formation and fate of nitrite, nitrate,and nitroso products, following the induction of colitis in a rodent
model of colitis in vivo. Our main findings are summarized asfollows:
(1) Plasma nitrite and nitrate correlate well with disease activ-ity, but only in the acute case and during the onset of the inflam-matory response. In the recovery phase, both plasma anions riseagain to very high levels 4 days after withdrawal of the inflamma-tory stimulus, even when disease activity continues to fall. In thechronic stage, nitrite shows a significant increase in plasmawhereas nitrate remains essentially unchanged. Our findingssuggest that plasma nitrite by itself provides a more consistentindication of colitis, although rises of this indicator may not be syn-chronized with either progression or resolution of inflammation.
(2) In the extravascular space during acute colitis, only nitrosoproducts correlate well with disease activity. This response, how-ever, seems to be systemic in nature inasmuch as qualitativelyidentical transient rises in nitroso products are also seen in othertissues. At the same time nitrite levels in all organs, including thecolon, actually drop as the disease intensifies, while nitrateremains essentially unchanged. Thus, none of these NO-relatedproducts appear to be suitable as biomarkers of inflammation intissues.
(3) Our assessment of NO metabolite profiles during inflamma-tion reveals dynamical features of NO biochemistry that cannot befully rationalized in terms of classical oxidative and nitrosativetextbook chemistry. In particular, the dynamics of nitrite and itscompartmentalization between intravascular and extravascularspaces seems to imply that this anion is under the control of yetunidentified mechanisms that are activated as part of the inflam-matory response. A potential major factor in this context may bethe degree of tissue hypoxia accompanying the inflammatory pro-cess, and this relationship deserves further investigation. In anycase, our systems analysis of individual NO-related metabolitesin vivo has unmasked a richer complexity of NO biochemistry aspreviously appreciated from analyses of NOx levels in blood alone.
(4) Finally, our study brings to light the destabilizing effect thatcolon inflammation has on the redox state and on nitrite levels inother tissues. The level of oxidative stress appears to be particu-larly significant in the liver and kidneys, organs well known to suf-fer from complications of IBD. This destabilizing effect waseffectively abolished by the therapeutic drug 5-ASA, suggestingthat nitrite may play a key role in the modulation of inflammation.
Acknowledgments
The authors thank Dr. A. Stucchi for his invaluable advice andcomments on the manuscript, and Drs. B.O. Fernandez and M.F.Garcia-Saura for insightful discussions. This work was supportedin part by grants from the National Institute of Health (HL 69029to M.F., P20 RR16456 from the BRIN/INBRE Program of the NCRRto J.R.), a Ruth Kirschstein Cardiovascular Training Grant (toN.S.B.), and the Strategic Appointment Scheme of the Medical Re-search Council, UK (to M.F.).
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