serum neopterin, nitric oxide, inducible nitric oxide synthase and tumor necrosis factor-α levels...

Clin Chem Lab Med 2008;46(8):1149–1155 � 2008 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2008.213 2007/542Article in press - uncorrected proof

Serum neopterin, nitric oxide, inducible nitric oxide

synthase and tumor necrosis factor-a levels in patients

with ischemic heart disease

Vidosava B. Djordjevic1,*, Ivana Stojanovic2,

Vladan Cosic1, Lilika Zvezdanovic1, Marina

Deljanin-Ilic3, Senada Dimic2, Braca Kundalic2,

Tatjana Cvetkovic2 and Tatjana Jevtovic-

Stoimenov2

1 Center for Medical Biochemistry, Clinical Center,Nis, Serbia2 Institute of Biochemistry, Faculty of Medicine, Nis,Serbia3 Institute for Cardiovascular and RheumaticDiseases, Niska Banja, Serbia

Abstract

Background: Atherosclerosis, a chronic inflammatorydisease, underlies the pathogenesis of coronaryartery disease. The present study assessed the diag-nostic possibilities of inflammatory biomarkers,serum neopterin, nitrite/nitrate (NO2

–/NO3–), inducible

nitric oxide synthase (iNOS) and tumor necrosis fac-tor-a (TNF-a), and their correlation with risk factors inpatients with acute coronary syndromes and stableangina pectoris.Methods: We studied 44 patients with chronic stableangina pectoris, 46 with unstable angina, 55 withacute ST-elevation myocardial infarction and 39 age-matched healthy volunteers (control group). Serumneopterin, iNOS and TNF-a were determined withcommercially available enzyme linked immunosor-bent assay methods and NO2

–/NO3– by the modified

cadmium-reduction method.Results: Mean serum neopterin levels were signifi-cantly higher in patients with unstable and stableangina pectoris in comparison to control subjects(p-0.01 and p-0.05, respectively). Serum NO2

–/NO3–

values were significantly elevated (p-0.01) only inpatients with unstable angina. ST-elevation myocar-dial infarction patients with cardiac death during fol-low-up showed significantly lower baseline neopterinvalues (p-0.001), and higher NO2

–/NO3– levels

(p-0.05) in comparison to those without adverseevents. Significantly higher NO2

–/NO3– values

(p-0.05) were also found in patients who had myo-cardial reinfarction. Serum iNOS and TNF-a in allpatient groups were within control ranges. A strongcorrelation was found between neopterin and bothsmoking (p-0.01) and triglycerides (p-0.05) in un-

*Corresponding author: Prof. Vidosava B. Djordjevic, PhD,Institute of Biochemistry, Faculty of Medicine, Bul. drZorana Djindjica 81, 18000 Nis, SerbiaPhone: q381-18-535-666, Fax: q381-18-238-770,E-mail: [email protected] December 10, 2007; accepted March 31, 2008;previously published online June 20, 2008

stable angina patients. In stable angina patients,neopterin, iNOS and TNF-a significantly correlatedwith hypertension (p-0.01) and triglycerides(p-0.05). A significant difference in neopterin concen-tration was found between smokers and non-smokers(p-0.05).Conclusions: The results of this study suggest that instable angina patients, if studied over time, serumneopterin or NO2

–/NO3– levels may indicate future

plaque instability. In ST-elevation myocardial infarc-tion patients, neopterin and/or NO2

–/NO3– levels may

identify patients at long-term risk of death or recur-rent acute coronary events after myocardialinfarction.Clin Chem Lab Med 2008;46:1149–55.

Keywords: inducible nitric oxide synthase (iNOS);ischemic heart diseases; neopterin; nitric oxide;tumor necrosis factor-a (TNF-a).

Introduction

Neopterin, nitric oxide (NO), inducible nitric oxidesynthase (iNOS) and tumor necrosis factor-a (TNF-a)are known markers of inflammation. Inflammationplays a key role in the initiation and the evolution ofatherosclerosis (1). The inflammatory mechanism canbe maintained and enhanced by multiple factors, suchas activated macrophages and lymphocytes, as wellas native vascular wall cells, which secrete cytokinesand other substances implicated in the atheroscleroticinflammatory process. Activated lymphocytes in ath-erosclerotic plaques produce interferons, which acti-vate macrophages. These then produce and releaseneopterin (2), TNF-a, interleukin-8 (IL-8) and NO (3).By modification of the redox state, neopterin activatesthe common proinflammatory transcriptional factor,nuclear factor kB (NF-kB) (4), which in turn upregu-lates proinflammatory genes. Activated NF-kB hasbeen indentified in situ in the smooth muscle cells,macrophages and endothelial cells of human athero-sclerotic plaques (5).

Neopterin is involved in both the activation of con-stitutive NOS and iNOS (6) and TNF-a synthesis andactivity regulation (7). The secretion of NO is partiallyregulated by the autocrine or paracrine action ofTNF-a (3). iNOS is not normally present in restingcells, but its expression may be induced by immuno-logical stimuli in myocytes, macrophages and endo-thelial cells (8, 9). Single stimulation of smooth mus-cle cells with either neopterin or TNF-a caused iNOSgene expression and consequent NO production (7).

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NO exerts many antiatherogenic properties, includinginhibition of platelet aggregation and oxidative mod-ification of low-density lipoprotein cholesterol, mod-ulation of both platelet- and monocyte-endothelialinteractions and inhibition of smooth muscle prolif-eration. As endothelial dysfunction is one of the ear-liest signs of atherosclerosis, reduced NO productionis considered as one of the earliest and more impor-tant markers of endothelial dysfunction. However,both defective NO synthesis and the stimulation ofiNOS and NO overproduction may play a role in ath-erogenesis (10).

As atherogenesis is a chronic inflammatory diseaseassociated with the production of a number of inflam-matory molecules showing interdependence, thisstudy was designed to determine the relationshipbetween serum levels of neopterin, nitrites/nitrates(NO2

–/NO3–), iNOS and TNF-a in patients with clini-

cally distinct manifestations of ischemic heartdisease.

Patients and methods

We studied 145 individuals admitted to the Institute for Car-diovascular and Rheumatic Diseases ‘‘Niska Banja’’ for theassessment of angina chest pain. A total of 44 (14 femalesand 30 males, aged 62"6.5 years) had chronic stable angina(SAP group), 46 (15 females and 31 males, aged61.5"10.5 years) unstable angina (USAP group), and 55 (20females and 35 males, aged 59"13 years) acute ST-elevationmyocardial infarction (STEMI group). SAP was defined astypical exertional chest pain relived by rest, glyceryl trinitrateadministration, or both, with positive responses to exerciseelectrocardiogram and/or positive stress echocardiographytesting. In all patients, symptoms were stable for at least3 months before study entry. None of the patients in thisgroup had previous myocardial infarction or myocardialrevascularization, cardiac valve disease, cardiomyopathy,malignant arrhythmias, acute or chronic liver disease, renalfailure or inflammatory diseases. Unstable angina wasdefined according to Hamm and Braunwald (11), and onlythose patients with class III unstable angina were includedin the study. All USAP patients had angina at rest during thelast 48 h before admission and all had diagnostic ST-seg-ment changes, T-wave changes, or both, and negative car-diac enzymes and troponin I. Acute myocardial infarction(AMI) was diagnosed in 55 patients by chest pain persistinglonger than 30 min, concomitant changes on the electrocar-diogram at the admission to hospital and elevated troponinI levels. All 55 patients had STEMI, which was defined assignificant ST-elevation (in two adjacent leads and )0.1 mVin leads I–III, aVF, aVL, V4–V6, and )0.2 mV in leads V1–V3),according to the Guidelines of the European Society of Car-diology (12).

All patients underwent a detailed clinical analysis. Dataabout age, sex, vascular risk factors, including hypertension,diabetes mellitus, smoking habit, obesity, family history,physical inactivity, cholesterol and triacylglycerol levels werecollected just after admission for all the patients. Also, allpatients were asked for current medications. Venous bloodsamples from patients were taken within 24 h after admis-sion, in the morning after 12 h fasting, and standard analyseswere performed the same days; at the same time, neopterin,NO2

–/NO3–, iNOS and TNF-a sera were collected and stored

in aliquots at –808C until analyses.

Simultaneously, we tested 39 age- and sex-matchedhealthy volunteers (control group). Controls were recruitedfrom the Department for Blood Transfusion of the ClinicalCentre Nis from blood bank donors. All volunteers were freeof any acute infectious disease and any history of hyperten-sion, diabetes or ischemic heart disease.

All patients and healthy individuals gave written informedconsent before study entry and the study was approved bythe local Ethics Committee.

After recruitment, baseline characterization and bloodcollection, the patients were followed up for 1 year. Majorclinical events during follow-up were readmission to thecoronary care unit with class III unstable angina, non-fatalmyocardial infarction and cardiac death.

Neopterin serum concentrations were determined usinga commercially available immunoassay (ELISA kit, IBL,Hamburg, Germany). Limit of detection was 0.7 nmol/L.

NO2–/NO3

– serum concentrations were measured by themodified cadmium-reduction method of Navaro-Gonzalvezet al. (13) based on the produced nitrite determination bydiazotization of sulfanilamide and coupling to naphthylenediamine. Detection limits in serum were 2–250 mmol/L.

iNOS serum activity was determined by a commerciallyavailable Quantikine human iNOS immunoassay (ELISA kit,R&D Systems Europe, Ltd., Abingdon, UK). Limit of detectionwas 0.15 U/mL.

Serum TNF-a levels were estimated by a Quantikinehuman TNF-a immunoassay (ELISA kit, R&D SystemsEurope, Ltd.). Limit of detection was 1.6 pg/mL.

All other biochemical parameters were carried out by anautoanalyzer Olympus AU400 (Olympus, Tokyo, Japan) andby an immunoanalyzer AxSYM (Abbott Laboratories, AbbottPark, IL, USA).

Statistical analysis was performed to identify differencesin serum concentrations of inflammatory markers, as well asto correlate their levels to standard biochemical markers andrisk factors. Comparisons between controls and patientgroups were carried out using analysis of variance followedby two-sided Dunnett’s test (for multiple comparisons) orStudent’s non-paired t-test as appropriate. Linear regressionanalysis was used to assess the relationships between stud-ied inflammation markers and risk factors. The results areexpressed as mean"SD. All statistical calculations were per-formed using the Statistical Package for the Social Sciences(SPSS) computer program (SPSS Inc., Chicago, IL, USA).

Results

Baseline characteristics and risk factors of patientswith ischemic heart diseases are shown in Table 1.All patient groups were matched for age. SAP patientshad a higher prevalence of smoking and physicalinactivity. USAP patients showed a higher prevalenceof hypertension and hypertriglyceridemia, and morethan 50% of STEMI patients had hypertension, highcholesterol and smoking habits. Therapeutic charac-teristics of patients are presented in Table 2. Almostall patients were on multiple drug therapy. SAPpatients were mostly treated with aspirin, nitrates, b-blockers and statins, USAP patients with aspirin, clo-pidogrel, nitrates, b-blockers (all studied patients) andstatins, while almost all STEMI patients received aspi-rin, clopidogrel, nitrates, b-blockers, statins, angio-tensin converting enzyme (ACE) inhibitors and lowmolecular weight (LMW) heparin.



Djordjevic et al.: Inflammatory markers in patients with ischemic heart disease 1151


Table 1 Baseline characteristics and risk factors in patients with ischemic heart diseases.

STEMI patients USAP patients SAP patients Controls(ns55) (ns46) (ns44) (ns39)

Age, years 59"13 61.5"10.5 62"6.5 58"9.1Sex (male/female), n 35/20 30/16 30/14 23/16Blood biochemical data

Glucose, mmol/L 6.4"2.2*** 6.3"2.9** 5.6"1.2* 5.1"0.8Total cholesterol, mmol/L 5.9"1.5 5.7"1.7 5.6"1.2 5.4"1.2Triglycerides, mmol/L 2.1"1.0* 2.9"1.6*** 2.1"1.0* 1.8"1.0Urea nitrogen, mmol/L 7.9"4.2*** 8.8"4.0*** 7.4"4.9*** 4.8"1.4Creatinine, mmol/L 100.8"35.9** 113.5"49.4*** 96.2"27.4* 85.4"13.4Troponin I, ng/mL 22.8"38.4*** 3.1"7.7* 0 0

Coronary risk factorsHypertension, n (%) 43 (78%) 40 (87%) 35 (80%) 0 (0%)Diabetes mellitus, n (%) 17 (31%) 16 (35%) 3 (7%) 0 (0%)Smoking, n (%) 31 (56%) 21 (46%) 30 (68%) 10 (26%)Obesity, n (%) 16 (29%) 5 (11%) 7 (16%) 0 (0%)Family history, n (%) 24 (44%) 17 (37%) 21 (48%) 9 (23%)Physical inactivity, n (%) 14 (25%) 2 (4%) 44 (100%) 19 (49%)

*p-0.05, **p-0.01, ***p-0.001.

Table 2 Therapeutic characteristics of patients with ische-mic heart diseases.

STEMI USAP SAPpatients, % patients, % patients, %

Aspirin 100 98 92Clopidogrel 100 80 13LMW heparin 89 70 0Nitrates 94 98 92b-Blockers 94 100 96Ca-antagonists 9 50 8Diuretics 19 37 33Statins 78 72 92Fibrinolytics 39 0 0ACE inhibitors 87 50 71Amiodarone 13 0 8Farin 6 0 8Adrenaline 4 0 0Xylocaine 2 0 0

LMW, low molecular weight; ACE, angiotensin convertingenzyme.

Table 3 Serum neopterin, NO2–/NO3

–, iNOS and TNF-a in patients with ischemic heart diseases.

Group n Neopterin, NO2–/NO3

–, iNOS, TNF-a,nmol/L mmol/L U/mL pg/mL

Controls 39 10.9"6.5 101.8"41.4 2.7"2.3 13.6"2.2SAP patients 44 17.9"11.0* 99.7"36.4 2.2"1.2 12.7"1.3USAP patients 46 19.8"17.3**,a 125.2"31.4**,b 2.8"1.5 13.2 "3.5STEMI patients 55 13.5 "9.9 111.8 "37.5 2.8"1.4 13.8"3.7

*p-0.05 vs. controls, **p-0.01 vs. controls, ap-0.05 vs. STEMI patients, bp-0.01 vs. SAP patients.

Serum concentrations of inflammatory markers aregiven in Table 3. Neopterin levels in SAP patients(17.9"11.0 nmol/L) and USAP patients(19.8"17.3 nmol/L) were significantly higher than incontrols (10.9"6.5 nmol/L) (p-0.05 and p-0.01,respectively). Also, neopterin values in USAP patientswere higher than neopterin values in STEMI patients(13.5"9.9 nmol/L, p-0.05). STEMI patients whodied due to cardiac death during follow-up showedsignificantly lower baseline neopterin values(9.46"2.4 nmol/L, p-0.001) in comparison to STEMI

patients without adverse events (14.5"2.2 nmol/L).Further, in smokers with SAP a significant elevationin neopterin (25.1"23.0 nmol/L, p-0.05) compared tonon-smokers with SAP (14.8"6.8 nmol/L) wasobserved. With the exception of this difference, otherstudied markers did not show any significant differ-ence between risk factors positive and risk factorsnegative in any patient group.

A significant increase in NO2–/NO3

– was observedin USAP patients (125.2"31.4 mmol/L, p-0.01) incomparison to the control group (101.8"41.4 mmol/L)and the SAP group (99.7"36.4 mmol/L, p-0.01). Also,a significant increase in NO2

–/NO3– was noted in

both STEMI patients who died during follow-up andSTEMI patients who had myocardial reinfarction(163.7"55.9 mmol/L and 157.0"64.0 mmol/L,respectively).

Both serum iNOS and TNF-a values were similarwithin groups. Average iNOS values were 2.2"1.2U/mL in SAP patients, 2.8"1.5 U/mL in USAP patientsand 2.8"1.4 U/mL in STEMI patients, and there wasno significant difference in comparison to controls(2.7"2.3 U/mL) or within the groups. TNF-a concen-trations were 12.7"1.3 pg/mL in SAP patients,13.2"3.5 in USAP patients and 13.8"3.7 pg/mL inSTEMI patients.

Different correlation patterns between neopterinand biomarkers (Table 4) were observed for eachstudied group. In healthy individuals, serum neop-terin levels correlated with inflammation biomarkers,lipid status, as well as cardiac markers. In SAP and





Table 4 Correlation between neopterin and other biomarkers.

STEMI USAP SAP Controls

Inflammation biomarkersF rs0.39 rs0.36ESR rs0.54TNF-a rs0.51 rs–0.53 rs–0.26NO rs0.22*iNOS rs0.27 rs0.39 rs0.35

Lipid profileChol rs–0.55LDL-C rs–0.41HDL-C rs–0.58 rs0.42TG rs0.53ApoA rs–0.57ApoB rs–0.42Lp(a) rs0.39OxLDL rs–0.39

Cardiac markersLDH rs0.35AST rs0.44ALT rs0.32BNP rs0.38Cat rs–0.32 rs0.62

For all parameters (except NO): p-0.01. *p-0.05. F, fibrinogen; ESR, erythrocyte sedimentation rate; Chol, cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; apo, apolipoprotein; Lp(a),lipoprotein (a); OxLDL, oxidized LDL; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotrans-ferase; BNP, brain natriuretic peptide; Cat, catalase.

Table 5 Correlation between inflammatory markers and risk factors.

Hypertension Cholesterol TG DM Smoking Family history Physical inactivity

STEMINeopterin rs–0.22 rs0.19

p-0.01 p-0.05iNOS rs0.25 rs0.25 rs0.17

p-0.01 p-0.01 p-0.05TNF-a rs–0.27 rs0.20

p-0.01 p-0.05USAP

Neopterin rs–0.21 rs0.30p-0.01 p-0.01

iNOS rs0.26 rs0.22 rs0.33p-0.05 p-0.05 p-0.01

TNF-a rs0.40p-0.01

SAPNeopterin rs0.26 rs0.22

p-0.01 p-0.05iNOS rs0.22 rs0.30

p-0.05 p-0.01TNF-a rs–0.39 rs0.27

p-0.01 p-0.05

TG, triglyceride; DM, diabetes mellitus.

STEMI patients, neopterin correlated with inflamma-tion biomarkers, whilst in USAP patients, neopterincorrelated with inflammation biomarkers and lipidstatus. Correlations between studied inflammatorymarkers and risk factors (Table 5) showed specificpatterns for each patient group.

Discussion

To the best of our knowledge, this is the first studyaimed at evaluating serum neopterin, NO, iNOS and

TNF-a in ischemic heart disease clinically expressedas SAP, USAP or AMI. This study clearly demon-strates that serum neopterin and NO2

–/NO3– are sig-

nificantly increased in USAP patients, whilst onlyneopterin levels are significantly elevated comparedto controls in SAP patients. The results related topatients with ischemic heart diseases are dissimilar.Some studies have shown that plasma neopterin waselevated in USAP or AMI patients in comparison tostable angina (14, 15). It is also elevated in USAPpatients associated with the presence of complex vul-nerable coronary lesions (16, 17). Others have found





a significant neopterin increase in women with USAPand in those with SAP with cardiac events in com-parison to chronic SAP patients (17). Similar resultswere published by Smith et al. (18). There is also evi-dence that neopterin and some other inflammatorymarkers are higher in patients with periphery arterydisease than in those with coronary artery disease.Our results are consistent with studies concerningunstable angina pectoris. In USAP patients, signifi-cantly higher serum interferon-g (IFN-g) levels weredocumented (19). In addition, we observed a strongcorrelation between neopterin and both smoking(p-0.01) and triglycerides (p-0.01) in USAP patients.However, we found a significant serum neopterin lev-el increase in patients with SAP. This finding suggeststhat disease activity may be present despite clinicalstability.

Ongoing inflammation in stable plaques may pre-dispose the patient to rapid disease progression andthe development of unstable syndromes (20). In rela-tion to our patients, elevated neopterin levels may bea consequence of the presence of multiple risk fac-tors, such as hypertension, smoking, total physicalinactivity (100% patients) and high concentration oftriglycerides. Serum neopterin significantly correlatedwith both hypertension (p-0.01) and hypertriglyceri-demia (p-0.05). There is evidence suggesting thatvery low-density lipoprotein can activate inflamma-tory functions of vascular endothelial cells via acti-vation of the common proinflammatory transcriptionfactor NF-kB (21). Remnant lipoproteins have beenshown to stimulate the expression of monocytechemo-attractant protein-1 (MCP-1) by endothelialcells and to increase monocyte adherence to theendothelium. The patients with hypertriglyceridemiashowed increased platelet activation and alterationsin the hemostatic and fibrinolytic system (22).

There is evidence that obese and overweight sub-jects have elevated neopterin levels, such as adultscompared to healthy adolescents (23). Only 16% ofour SAP patients were obese and we did not find anycorrelation between neopterin and obesity, neither inthis nor in two other patient groups. However, smok-ing may be a serious factor, as a significant differencein neopterin levels was found between smokers (whohad two-fold higher neopterin levels) and non-smok-ers (p-0.05). SAP patients taking statins (92%) havebeen shown to downregulate neopterin production bymonocyte in vitro. A few clinical studies that investi-gated the relationship between neopterin levels andthe use of statins have obtained controversial results(24, 25). In this study, no patient group showed anycorrelation between neopterin and statins. Further,serum neopterin concentrations are associated withthe development of adverse cardiovascular events inpatients with chronic SAP, but this association wasindependent of the severity of coronary artery disease(16). We also showed that serum neopterin correlatedsignificantly with some other inflammatory markers,such as fibrinogen, TNF-a and iNOS, and that thispattern is different between various patient groups.

Schumacher et al. (26) observed more pronouncedchanges in neopterin levels in patients with AMI than

in those with chronic coronary artery disease. Simi-larly, patients with a non-Q-wave AMI had higherneopterin levels than patients with USAP (27). Also,in patients with AMI before thrombolytic therapy,neopterin levels were found to be significantly higherthan in patients with stable coronary artery diseaseand control subjects. Furthermore, 4 h after AMI a sig-nificant decrease was observed followed by anincrease, reaching values significantly different fromall values other than the initial one, after 72 h (26).During follow-up for 1 year, we observed that STEMIpatients who died due to cardiac death showed sig-nificantly lower baseline neopterin values in compar-ison with STEMI patients without adverse events. The1-year clinical follow-up of patients with USAP or non-Q-wave AMI showed no changes in neopterin andprocalcitonin levels, whereas some other inflamma-tory markers, such as C-reactive protein, serum amy-loid A, fibrinogen and IL-6 were increased (28).

With regard to NO, it is documented that in myo-cardium it is produced in both endothelial cells andcardiomyocytes (29). NO effects may be either pro-tective or deleterious. Protective effects are based onmultifunctional properties of NO. The deleteriouseffects lay in the possibility to be reduced or to reactwith oxygen free radicals, mostly superoxide, produc-ing dangerous radicals, such as peroxynitrite (30).Myocardial preconditioning also requires both NOand superoxide synthesis, but preconditioning in turnattenuates the overproduction of NO, superoxide andperoxynitrite during a subsequent episode of ische-mia and reperfusion, thereby protecting the heart(30). A number of studies suggest that NO plays acentral role in adaptation on intermittent hypoxia.Protective mechanisms related to NO include stimu-lated NO synthesis, restriction of NO overproduction,non-enzymatic sources of NO and NO autoregulation.Ischemia of myocardium leads to the activation ofmyocardial iNOS and the consequent NO increase(31). Akiyama et al. (32) found a significant increasein plasma NO2

–/NO3– values after experimental myo-

cardial infarction which may be a result of NO over-production in neutrophils (33). Contrary to thisfinding, we did not find any significant difference inNO production neither in STEMI nor in SAP patients.We could not ignore the fact that AMI is associatedwith oxidant stress and an excessive production ofsuperoxide which could rapidly react with and inac-tivate NO. This NO trapping may be a reason for low-er NO values in AMI patients in comparison to USAPpatients. But, we also noted that STEMI patients whodied and who had reinfarction during follow-upshowed significantly higher baseline NO2

–/NO3– lev-

els in comparison to the values of those withoutadverse events. We observed a significant increase inUSAP patients. This result may be explained by NOrelease from aggregated platelets, as platelet acti-vation, despite aspirin treatment (34), as well as plate-let resistance to the antiaggregatory effects of NOdonors (35), may exist in ischemic heart diseasepatients.

In healthy arterial wall, iNOS mRNA is expressed atlow levels. In atherosclerotic vessels, an increased





expression of iNOS mRNA and protein was confirmedin macrophages, foam cells and the vascular smoothmuscle cells (36). These phenomena were observedeither in the majority of early lesions or in alladvanced atherosclerotic lesions. An increase insuperoxide production, peroxynitrite formation, aswell as nitrotyrosine was detected (36). In spite ofthese in situ findings, although expected, we did notobserve any significant difference in serum iNOSactivity in any patient groups. In our previous article(37), we found a significant increase in plasma NOSactivity in children with vasculitis. However, it shouldbe taken into account that there is a stronger acti-vation of endothelium in vasculitis syndrome in com-parison to chronic evolution of atherogenesis.

In conclusion, the results of this study suggest thatin SAP patients, if studied over time, serum neopterinor NO2

–/NO3– levels may indicate future plaque insta-

bility. In STEMI patients, neopterin and/or NO2–/NO3

–

levels may identify patients at long-term risk of deathor recurrent acute coronary events after myocardialinfarction.

Acknowledgements

This work was supported by the Ministry of Science andEnvironmental Protection of Serbia.

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serum neopterin, nitric oxide, inducible nitric oxide synthase and tumor necrosis factor-α levels...

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