Glycated hemoglobin A1c as a modern biochemical marker of glucose regulation/Glikoziliranihemoglobin A1c kao savremenibiohemijski marker glikoregulacije.
Human hemoglobin (Hb) is not chemically homogenous. In erythrocytes of a healthy adult person, there are three different hemoglobins: fetal Hb (HbF) and two hemoglobins that belong to adults (HbA and HbA2), HbA being dominant. One pair of [alpha]-chains is same for all three fractions of Hb, while the other globin chain pair is different for every Hb: [gamma], [beta] and [delta]-chain. In 1950s, HbA1 was isolated from HbA using chromatographic analysis and other three fractions HbA1a, HbA1b and HbA1c . Glycation is the nonenzymatic addition of a sugar residue to amino groups of proteins. Glycated hemoglobins distinguish one from the other by the type of added carbohydrate and place of glycation. The place of glycation is usually N-terminal part of [beta]-globin chain, but glycation can happen on other parts of [beta]-chain or on [alpha]-chain [2, 3] (Table l).
HbA1c is the most important fraction of glycated HbA1, which is 75-80% of its total amount. It is formed irreversibly by nonenzymatic glycation, bonding two molecules of glucose for every N-terminal part of [beta]-globin chain . Chemically, N[H.sub.2]-terminal valine residue is condensed with aldehyde glucose group. First unstable Schiff base is made (aldimine or pre-HbA1c). In normoglycemia, Schiff base (aldimine or pre-HbA1c) can dissociate again on glucose and HbA or in hyperglycemia, it can undergo the Amadori rearrangement to form stable ketoamine HbA1c, which cannot dissociate because human erythrocytes do not have enzymes which are necessary for its degradation. A similar process of glycation occurs on other plasma proteins [2, 3].
Clinical Significance of HbA1c
Over the last few years, HbA1c became a necessary parameter in following the regulation of diabetes mellitus, as well as a criterion for diagnosing this disease. In 1976, Koening et al. showed its strong correlation  to glucose levels. The studies were done on patients with diabetes type 1 and 2 [5-7] and they showed a good correlation with the degree of glycemic control. During the 1970s and 1980s [1, 3], kinetic studies showed that synthesis intensity and concentration of HbA1c depended exclusively on the previous glucose blood level and the lifespan of erythrocytes. A HbA1c concentration represents the integrated value for glucose over the preceding 6 to 8 weeks, compared to glycemia which represents the current glucose blood level . That is why HbA1c has become firmly established as a biochemical marker of long-term blood glucose concentrations and as a measure of the success rate of diabetes treatment .
Besides that, big randomized studies such as the Diabetes Control and Complications Trials (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have shown clearly that HbA1c is a predictor of diabetic microvascular complications. The DCCT study, which was carried out on 1400 patients with diabetes mellitus type 1 from 1983 to 1993 [5, 6], documented the direct relationship between HbA1c and the absolute risk for retinopathy, neuropathy and nephropathy. The risk for retinopathy continuously increased with increasing the HbA1c level. The HbA1c concentrations lower by 10% were associated with 45% lower risk for retinopathy development. In the UKPDS study conducted on more than 5000 subjects of both sexes with type 2 diabetes between 1977 and 1997 , 1% reduction in HbA1c was associated with the risk reduction of 37% for microvascular disease and 21% for deaths related to diabetes.
Individual studies [5-7, 10] showed that HbA1c could also be a risk marker for the development of macrovascular complications, primarily for cardiovascular disease although the obtained results were not significant.
A strong linear correlation between the levels of HbA1c and glycemia in the diabetic patients was found using the results from a big retrospective study, primarily the DCCT and the UKPDS [5-7], as well as from the studies of other authors [11-13], and it was the cause to perform the A1C-Derived Average Glucose (ADAG) study. This study included 507 healthy controls and patients with diabetes type 1 and 2 and stable glycemic control between 2006 and 2008 . It confirmed a strong correlation between HbA1c and glycemia regardless of the sex, presence of diabetes or its type, which resulted in a mathematical equation of a new parameter showing glycemia in a three-month period: the estimated glyceamia value (eAG-estimated Average Glucose): 1.5944 x % HbA1c--2.5944 (mmol/l). It is useful to calculate eAG because it makes it easier to understand the meaning of HbA1c as a marker of the long term glycemic control for patients [15-17] and expresses HbA1c in the same manner as when the patients perform their self-control. However, India and China, as the countries with lot of diabetic patients, were not the part of the ADAG study and neither were children or pregnant women, so the results cannot be applied generally and in all diabetic patient groups [14, 18].
Methods for Determining HbA1c and their Standardization
So far, there have been more than 30 different methods for determining glycated Hb. They differ one from another by the way of Hb separation from its glycated form. These methods are based on the charge differences (ion-exchange chromatography, high-performance liquid chromatography (HpLC), electrophoresis and isoelectric focusing), structural differences (immunoassay and affinity chromatography) or chemical analysis (photometry and spectrophotometry) . The first methods determined the values of total glycated Hb and nowadays methods for measuring the largest fraction of HbA1c, whose concentration is a reliable marker for assessing the degree of glycation, are used.
In the beginning, there were numerous obstacles in HbA1c determination in routine practice because there were great differences between different methods and laboratory results due to the lack of method standardization. Therefore, a study work group of the DCCT and AACC (American Association for Clinical Chemistry) for HbA1c standardization was formed in 1993 and established the National Glycohemoglobin Standardization Program (NGSP). This group suggested measuring HbA1c using only reference method . However, the NGSP was not accepted in all countries of the world especially in those which had their own program of standardization, such as Japan or Sweden. Besides that, the suggested reference method was not specific because it was used for measuring different forms of glycated Hb. Because of that, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) work group presented a new reference method for HbA1c determination in 1995, which was completely developed and accepted by all the IFCC members in 2001. This method is very specific and precise, but because of its complexity and high price, it cannot be used in routine clinical work. Therefore, the IFCC method of standardization, which has been supported by the development of the global reference laboratory network, makes it obligatory to standardize all laboratory instruments and tests for HbA1c determination according to the IFCC reference method, regardless of the applied methodology .
According to the European Union directive from 1998, all products distributed in Europe should be accompanied by the document stating that they are traceable with the IFCC reference method . The correlation between the NGSP and IFCC method is strong, but the IFCC method is more specific and precise because it measures only HbA1c and not other fractions of glycated Hb. The values obtained by this method are about 1.5-2% lower than the ones obtained using NGSP method.
An immunoassay is the most frequently used method for determining HbA1c in routine laboratory practice. HbA1c is usually measured in venous blood using latex agglutination . The test is based on the use of monoclonal antibody against HbA1c. These antibodies do not recognize unstable intermediary forms of glycation or other forms of glycated Hb (HbA1a or HbA1b) or other forms of Hb (HbF, HbS etc.), so there is no need for the sample pre-treatment in order to eliminate them and the possibility of false positive result appearance is reduced to minimum . In routine practice, besides the immunoassay, ion-exchange chromatography is frequently used. It requires the sample pre-treatment in order to remove labile intermediary forms of HbA1c. Despite that, there is a great possibility of getting false high results in case of the presence of great quantities of labile intermediary forms of HbA1c and in case of bonding non-carbohydrate compounds in uremia, alcoholism, different poisonings and chronic treatment with high doses of acetylsalicylic acid . This has to be taken into consideration when interpreting the results. It is important to emphasize that fasting is not necessary for HbA1c determination.
The sample is taken from the venous blood, and ethylendiamintetraacetic acid (EDTA), oxalate or fluoride is used as an anticoagulant. The use of heparin is limited. If the analysis is not done immediately, it is recommended to store venous samples, not erythrocyte hemolysate .
Reporting and Interpretation of Results of HbAle Determination
According to the NGSP standardization program, HbA1c values were expressed as the percentage proportion of HbA1c of the total Hb (%HbA1c).
Since 2007, the IFCC has been recommending to show the HbA1c values in mmol of HbA1c on mol of total Hb (mmol/mol). It is possible to convert the results from the IFCC units to the NGSP ones by using a simple mathematical equation--the so called "master equation" . Other conversion equations for IFCC transformation values into the values of HbA1c of other programs of standardization have been developed. However, it is still disputable which units should be used for the result presentation, whether mmol/mol or % HbA1c. According to the globally accepted consensus from 2007 , HbA1c should be presented in both IFCC and NGSP units, i.e. in mmol/mol and in % HbA1c, respectively; however, the final decision is up to each country individually. In the United States, the results are shown as %HbA1c together with calculated eAG , and in some other countries in % HbA1c and in mmol/mol with the tendency to adopt only the IFCC units. As recommended, the IFCC units (mmol/mol) have been used in our country since September 1st, 2011 .
HbA1c concentration in blood depends exclusively on the lifespan of erythrocytes and blood glucose level. Still, when interpreting the results, it is important to have on mind that glucose levels in the previous month determine about 50% of HbA1c level, while glucose levels in the first month determine only 25% of its value . The levels of HbA1c are not affected by daily glucose fluctuations, recent physical activity, food intake or an acute illness [3, 19]. Falsely high results can be obtained when the labile intermediary forms of HbA1c are present in the blood, especially when the methods of electrophoresis or ion-exchange are used . In case of acute glucose level changes, the concentrations of labile intermediary forms change suddenly, while in cases of prolonged hyperglycemia, they bond irreversibly into HbA1c ketoamines. Therefore, labile intermediary forms reflect current glycemia. In healthy persons, they make 5-8% of total HbA1 and in diabetic patients up to 30% depending on the degree of glycemic control.
When interpreting the results, it is important to know whether the erythrocytes of tested person have the normal lifespan. The HbA1c values could be falsely low in the patients with hemolytic disease due to a large share of young erythrocytes or in other conditions where the lifespan of erythrocytes is shortened, as well as in recent significant blood loss. Falsely high values could be found in sideropenic anemia probably due to the enlarged share of old erythrocytes in blood. Falsely high or low values of HbA1c could be found in different hemoglobinopathies (HbF, HbS, HbC etc.) or in the presence of noncarbohydrate compounds bonded to Hb, such as in uremia, alcoholism, liver disease, different poisonings or applications of some medications . In these cases, it is necessary to compare the HbA1c concentrations with the previous HbA1c results of the same person but not with the target values. Instead of HbA1c in these cases, some other glycated proteins, e.g. fructosamine, can be measured as an alternative indicator of glycemic control .
Recommendations for Clinical Practice
The current recommendations of the American Diabetes Association (ADA) for clinical practice  and the National Guidelines of Good Clinical Practice for Diabetes Mellitus of the Ministry of Health of the Republic of Serbia  recommend the HbA1c determination at least twice a year in patients with the achieved therapeutic goal and stabile glycemic control, and every three months in patients with a significant treatment change or when therapeutic goals have not been achieved (Table 2).
For prevention of microvascular diabetic complications, the therapeutic target values of HbA1c in adults is below 7% HbA1c with the tendency to be as close to the reference values as possible.
In case of previously diagnosed diabetes in pregnancy, HbA1c should be measured every 4-8 weeks, and the therapeutic goal is less than 6% HbA1c, while in gestational diabetes, HbA1c determination is not recommended.
In order to diagnose diabetes mellitus, it is recommended to determine HbA1c, and the "cut-off value is [greater than or equal to] 6.5% HbA1c. It is necessary to confirm the diagnosis of diabetes mellitus by multiple determinations of HbA1c. Point-of-care tests should not be used for setting up the diagnosis of diabetes.
Abbreviations Hb --hemoglobin HbA1c --glycated hemoglobin A1c DCCT --Diabetes Control and Complications Trials UKPDS --United Kingdom Prospective Diabetes Study EPIC-Norflok --European Prospective Investigation of Cancer and Nutrition ADAG --A1c-Derived Average Glucose Study eAG --estimated Average Glucose NGSP --National Glycohemoglobin Standardization Program IFCC --International Federation of Clinical Chemistry and Laboratory Medicine
Rad je primljen 20. I 2014.
Recenziran 10. IV 2014.
Prihvacen za stampu 9. V 2014.
[1.] Kahn R, Fonseca V. Translating the A1c Assay. Diabetes Care. 2008; 31(8):1704-7.
[2.] Bunn HF, Haney DN, Kamin S, Gabbay KH, Gallop PM. The biosynsthesis of human hemoglobin A1c. J Clin Invest. 1976; 57:1652-9.
[3.] Mortensen HB, Christophersen C. Glucosylation of human haemoglobin A in red blood cells studied in vivo: kinetics of the formation and dissociation of haemoglobin A1c. Clin Chem Acta. 1983; 134:317-26.
[4.] Koenig RJ, Peterson CM, Kilo C, Cerami A, Williamson JR. Hemoglobin A1c as an indicator of the degree of glucose intolerance in diabetes. Diabetes. 1976; 25:230-2.
[5.] Diabetes control and complications trial research group. The effect of intensive diabetes treatment on the development and progression of long-term complications in insulin-dependent diabetes mellitus: diabetes control and complications trial. N Engl J Med. 1993; 329:978-86.
[6.] Diabetes control and complications trial research group. The association between glycaemic exposure and long-term diabetic complications in the diabetes control and complications trial. Diabetes. 1995; 44:968-83.
[7.] UK prospective diabetes study group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998; 352:837-53.
[8.] Bunn HF. Evaluation of glycosylated hemoglobin in diabetic patients. Diabetes. 1981; 30:613-7.
[9.] Mitrovic M, Pantelinac P, Radosavljevic J, Bajkin I, Todorovic Cilas Lj. Mesto i uloga insulinskih analoga u savremenoj terapiji secerne bolesti. Med Pregl. 2006; 59(11-12):539-44.
[10.] Khaw KT, Wareham N, Luben R, Bingham S, Oakes S, Welch A, et al. Glycated haemoglobin, diabetes, and mortality in men in Norfolk cohort of European Prospective investigation of cancer and nutrition (EPIC-Norfolk). BMJ. 2001; 322:15-8.
[11.] Azim W, Omair M, Khan MOA, Shaheen N, Azim S. Correlation between glycated haemoglobin and random plasma glucose levels for the screening of diabetes mellitus. Int J Pathol. 2010; 8(2):59-62.
[12.] Nathan DM, Turgeon H, Regan S. Relationship between glycated haemoglobin levels and mean glucose levels over time. Diabetologia. 2007; 50:2239-44.
[13.] Riet E, Alssema M, Rijkelijkhuizen JM, Kostense PJ, Nijpels G, Dekker JM. Relationship between A1c and glucose levels in the general dutch population (The New Hoorn Study). Diabetes Care. 2010; 33:61-6.
[14.] Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, Heine RJ, the A1c-Derived Average Glucose (ADAG) Study Group. Translating the A1C assay into estimated average glucose values. Diabetes Care. 2008; 31(8):1473-8.
[15.] American Diabetes Association. Standards of medical care in diabetes-2013. Diabetes Care. 2013; 36 Suppl 1:S11-66.
[16.] The American Diabetes Association, European Association for the Study of Diabetes, International Federation of Clinical Chemistry and Laboratory Medicine, and the Inter national Diabetes Federation--Consensus Committee. Consensus statement on the worldwide standardization of the hemoglobin A1C measurement. Diabetes Care. 2007; 30:2394-9.
[17.] Bozkaya G, Ozgu E, Karaca B. The association between estimated average glucose levels and fasting plasma glucose levels. Clinics. 2010; 65(11):1077-80.
[18.] Sacks DB. Correlation between Hemoglobin A1c (HbA1c) and average blood glucose: can HbA1c be reported as estimated blood glucose concentration? J Diabetes Sci Technol. 2007; 1(6):801-3.
[19.] Sacks DB, Bruns DE, Maclaren NK, McDonald JM, Parrott M. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem 2002; 48:436-72.
[20.] Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Official Journal of the European Communities 1998; 331:1-3.
[21.] Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem. 2001; 47:153-63.
[22.] Geistanger A, Arends S, Berding C, Hoshino T, Jeppsson JO, Little R, et al. IFCC Working Group on Standardization of HbA1c. Statistical methods for monitoring the relationship between the IFCC reference measurement procedure for hemoglobin A1c and the designated comparison methods in the United States, Japan and Sweden. Clin Chem. 2008; 54(8):1379-8.
[23.] Majkic Sing N, Lalic N. Zajednicki zakljucci o standardizaciji i novim preporukama izvestavanja rezultata odrecivanja HbA1c. Informator republicke strucne komisije za medicinsku i klinicku biohemiju. 2009; 1-3.
[24.] Tahara Y, Shima K. Kinetics of HbA1c, glycated albumin, and fructosamine and analysis of their weight functions against preceding plasma glucose level. Diabetes Care. 1995; 18:440-7.
[25.] Republicka strucna komisija za izradu i implementaciju vodica dobre klinicke prakse. Nacionalni vodic dobre klinicke prakse DIABETES MELLITUS. Drugo izmenjeno i dopunjeno izdanje. Beograd: Agencija za akreditaciju zdravstvenih ustanova Srbije; 2012.
Suncica KOJIC DAMJANOV, Mirjana DERIC and Nevena EREMIC KOJIC
University of Novi Sad, Faculty of Medicine
Department of Pathological Physiology Clinical Centre of Vojvodina Centre for Laboratory Medicine
Corresponding Author: Mr sc. med. Suncica Kojic Damjanov, Klinicki centar Vojvodine, Centar za laboratorijsku medicinu, 21000 Novi Sad, Hajduk Veljkova 1-7, E-mail: firstname.lastname@example.org
Table 1. Hemoglobin species in adult erytrocites Tabela 1. Vrste hemoglobina u eritrocitima odraslih osoba Hemoglobin species Globin Vrsta hemoglobina Globin Adult hemoglobin A/Adultni [[alpha].sub.2] hemoglobin A (HbA) [[beta].sub.2] Adult hemoglobin A2/Adultni [[alpha].sub.2] hemoglobin A2 (HbA2) [[delta].sub.2] Fetal hemoglobin/Fetalni [[alpha].sub.2] hemoglobin (HbF) [[gamma].sub.2] Total glycated hemoglobin Ukupni glikozilirani [[alpha].sub.2] hemoglobin (HbA1+A0) [[beta].sub.2] + sugar/secer Glycated hemoglobin species/Vrsta glikoziliranog hemoglobina Glycated hemoglobin A0 [[alpha].sub.2] [[beta].sub.2]-lysine or [alpha]-chain + sugar Glikozilirani hemoglobin [[alpha].sub.2] [[beta].sub.2]-lizin A0 (HbA0) ili [alpha]-lanac + secer Glycated hemoglobin A1/Glikozilirani [[alpha].sub.2] [[beta].sub.2]-valine hemoglobin + sugar A1 (HbA1 = HbA1a + HbA1b + HbA1c) [[alpha].sub.2] [[beta].sub.2]-valin + secer Glycated hemoglobin A1a [[alpha].sub.2] [[beta].sub.2]-valine + fructose-1,6-diphosphate Glikozilirani hemoglobin A1a [[alpha].sub.2] [[beta].sub.2]-valin + (HbA1a) fruktoza-1,6-difosfat or [[alpha].sub.2] [[beta].sub.2]-valine + glucose-6-phosphate ili [[alpha].sub.2] [[beta].sub.2]-valin + glukoza-6-fosfat Glycated hemoglobin A1b [[alpha].sub.2] [[beta].sub.2]-valine + pyruvic acid Glikozilirani hemoglobin [[alpha].sub.2] [[beta].sub.2]-valin + A1b (HbA1b) piruvicna kiselina Glycated hemoglobin A1c [[alpha].sub.2] [[beta].sub.2]-valine + glucose Glikozilirani hemoglobin [[alpha].sub.2] [[beta].sub.2]-valin + A1c (HbA1c) glukoza Pre-HbA1c (unstable Schiff labile intermediar of HbA1c base, aldimine) Pre-HbA1c (nestabilna Sifova labilni intermedijer HbA1c baza, aldimin) Tabela 1. Vrste hemoglobina u eritrocitima odraslih osoba Hemoglobin species Part of total Hb Vrsta hemoglobina Udeo u ukupnom Hb Adult hemoglobin A/Adultni 90-97% hemoglobin A (HbA) Adult hemoglobin A2/Adultni 2-5% hemoglobin A2 (HbA2) Fetal hemoglobin/Fetalni 2% hemoglobin (HbF) Total glycated hemoglobin Ukupni glikozilirani 3-9% hemoglobin (HbA1+A0) Glycated hemoglobin species/Vrsta glikoziliranog hemoglobina Glycated hemoglobin A0 Glikozilirani hemoglobin 1% A0 (HbA0) Glycated hemoglobin A1/Glikozilirani hemoglobin A1 (HbA1 = HbA1a + HbA1b + HbA1c) 5-8% Glycated hemoglobin A1a Glikozilirani hemoglobin A1a (HbA1a) -- Glycated hemoglobin A1b -- Glikozilirani hemoglobin A1b (HbA1b) Glycated hemoglobin A1c 4-6% Glikozilirani hemoglobin (80% HbA1) A1c (HbA1c) Pre-HbA1c (unstable Schiff base, aldimine) Pre-HbA1c (nestabilna Sifova 5-8% baza, aldimin) Hb--hemoglobin Table 2. Clinical practice recommendations for HbA1c testing Tabela 2. Preporuke za odredivanje glikoliziranog hemoglobina A1c u klinickoj praksi HbA1c testing in adult patients with diabetes/Odredivanje HbA1c kod odraslih pacijenata sa dijabetesom * at least twice a year in patients who have achieved treatment goals and have stable glycemic control/najmanje 2 puta godisnje kod pacijenata sa postignutim terapijskim ciljem i stabilnom glikemijskom kontrolom * every 3 months in patients whose therapy has changed significantly or who have not achieved glycemic goals/svaka 3 meseca kod pacijenata kod kojih je znacajnije menjana terapija ili nisu postignute ciljne vrednosti glikemije Prevention of microvascular complications in diabetes/Prevencija mikrovaskularnih komplikacija u dijabetesu * target value of HbA1c < 7% (53 mmol/mol), as close to the reference values as possible/ciljna vrednost HbA1c < 7% (53 mmol/mol), sto blize referentnim vrednostima * more or less stringent HbA1c target values may be appropriate in some patients/kod odrecenih pacijenata mogu se primeniti vise ili manje stroge ciljne vrednosti HbA1c HbA1c testing in pregnant women with diabetes/Odrecivanje HbA1c kod trudnica sa dijabetesom * pre-existing diabetes://prethodno postojeci dijabetes: every 4-8 weeks/svakih 4-8 nedelja target value of HbA1c < 6% (42 mmol/mol)/cijna vrednost HbA1c < 6% (42 mmol/mol) * gestational diabetes/gestacijski dijabetes: not recommended/ne preporucuje se HbA1c testing in children and adolescents with diabetes/Odredivanje HbA1c kod dece i adolescenata sa dijabetesom * at least 3-4 times a year, and in younger children up to 6 times a year/najmanje 3-4 puta godisnje, a kod manje dece i do 6 puta godisnje * HbA1c as a diagnostic criterion for diabetes: [greater than or equal to] 6.5% (48 mmol/mol) * (as in adults)/HbA1c kao dijagnosticki kriterijum za dijabetes: > 6,5% (48 mmol/mol)* (kao kod odraslih) HbA1c as a criterion for making diabetes diagnosis/HbA1c kao kriterijum za postavljanje dijagnoze dijabetesa * HbA1c [greater than or equal to] 6,5% (48 mmol/mol)* [right arrow] diabetes mellitus * HbA1c 5,7-6,4% (39-46 mmol/mol)* [right arrow] increased risk for diabetes (prediabetes)/povecan rizik za dijabetes (predijabetes) * results should be confirmed by repeating HbA1c testing/rezultate je potrebno potvrditi ponavljanjem odredivanja HbA1c
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Seminar for phisicians/Seminar za lekare u praski|
|Author:||Damjanov, Sunciea Kojic; Deric, Mirjana; Kojic, Nevena Eremic|
|Date:||Sep 1, 2014|
|Previous Article:||Drug induced lung disease--amiodarone in focus/Lekovima izazvana plucna bolest sa posebnim osvrtom na amiodaron.|
|Next Article:||Modulatory role of nitric oxide in cardiac performance/Modulatorna uloga azot-oksida na srcane performance.|