CHANGES IN SERUM HOMOCYSTEINE LEVEL FOLLOW TWO DIFFERENT TRENDS IN PATIENTS DURING EARLY POST MYOCARDIAL INFARCTION PERIOD Introduction Homocysteine

Th e evolution of homocysteine (Hcy) changes after acute myocardial infarction is still not elucidated. Serum Hcy concentration has been shown to increase between acute and convalescent period after myocardial infarction and stroke. Also a decrease in serum Hcy during acute phase was observed. It is still not clear whether the Hcy is a culprit or an innocent bystander in cardiovascular diseases. Addressing the discrepancies in Hcy changes in patients with acute myocardial infarction might give insight in Hcy role in cardiovascular diseases and off er implications both for the clinical interpretation and patients risk stratifi cation. Th e aim of the study was to evaluate serum Hcy concentration changes during early post myocardial infarction. Th e study included  patients with AMI from the Clinics for Heart Diseases and Rheumatism at University of Sarajevo Clinics Centre. For Hcy analysis blood was collected on day  and  after the AMI onset. Serum Hcy concentration was determined quantitatively with fl uorescent polarisation immunoassay on AxSYM system. Cluster analysis revealed two groups of AMI patients with diff erent trends of serum Hcy changes. Increase in serum Hcy concentration was observed in  (,) patients (AMI  group), while in  (,) patients a decrease was observed (AMI  group). On day , patients in AMI  group had signifi cantly higher mean Hcy concentration compared to AMI  group of patients (,±, and ,±, μmol/L p<,). On day , no signifi cant diff erence in mean Hcy level between AMI  and AMI  group of patients was observed (,±, vs. ,±, μmol/L respectively). Signifi cant diff erences between AMI  and AMI  patients were observed in VLDLC levels and CK-MB activity on day . CHANGES IN SERUM HOMOCYSTEINE LEVEL FOLLOW TWO DIFFERENT TRENDS IN PATIENTS DURING EARLY POST MYOCARDIAL INFARCTION PERIOD Amina Valjevac1*, Alen Džubur 2, Emina Nakaš-Ićindić1, Almira Hadžović-Džuvo1, Asija Zaćiragić1, Orhan Lepara1, Amila Arslanagić 2 1 Institute of Physiology and Biochemistry, Faculty of Medicine, University of Sarajevo, Čekaluša ,   Sarajevo, Bosnia and Herzegovina 2 Clinic for Heart Diseases and Rheumatism, University of Sarajevo Clinics Centre, Bolnička ,   Sarajevo, Bosnia and Herzegovina * Corresponding author


Introduction
Homocysteine is a sulfur-containing amino acid that functions as a key intermediate in methionine metabolism.It is produced as a by-product of methyl-transfer reactions, which are important for the DNA synthesis, methylated proteins, neurotransmitters and phospholipids ().Hcy might be involved in initiation and progression of atherosclerosis through several mechanisms including increased production of reactive oxygen species, endothelial dysfunction, infl ammation and smooth muscle cell proliferation ().Elevations in serum Hcy levels have been associated with cardiovascular diseases ().This standpoint mostly relies on results obtained from case-control studies which have consistently related serum Hcy elevation with myocardial infarction, stroke and atherothrombosis.Prospective observational studies, on the other hand, have been less convincing ().In patients with acute myocardial infarction (AMI) and stroke, subsequent tissue damage might be responsible for elevation of serum Hcy levels and thus explain observed association between hyperhomocysteinemia and atherosclerosis in patients with cardiovascular diseases ().Several studies have evaluated changes in serum Hcy concentration in patients presenting with acute coronary syndrome and stroke (, , , ).It has been observed that an increase in serum Hcy levels between acute and convalescent period in AMI patients might have influence on the evolution and the degree of myocardial injury (), but the results on Hcy levels changes during post myocardial infarction are conflicting.Recent study reported a decrease in serum Hcy concentration in a group of patients with AMI but the underlying cause is still not understood ().Addressing the discrepancies in Hcy behaviour during the early post myocardial infarction period has implications both for the clinical interpretation and patients risk stratification.During myocardial infarction acute phase reactions could alter biochemical and laboratory parameters and also Hcy levels.Th erefore, the aim of the study was to evaluate serial serum homocysteine concentration during early post myocardial infarction and to identify possible determinants of different homocysteine concentration changes.

Materials and Methods
Patients Th e study included  patients with AMI ( males and  females), admitted within  hours of symptom onset to the Intensive Care Unite at the Clinics for Heart Disease and Rheumatism at University of Sarajevo Clinics Centre.Th e diagnosis of acute myocardial infarction was based according to the World Health Organization criteria () including one of the main criteria: typical rise and gradual fall in troponine levels or cratine kinase (CK) elevation of at least twice the upper normal limit, with CK-MB isoenzyme concentration of at least  of the peak CK value with the at least one addition minor criteria: presence of ischemic chest pain of at least  minutes duration, dynamic ST-segment elevation or depression of  mm or more in at least  adjacent leads or prior coronary intervention.Th e exclusion criteria were diabetes mellitus, renal failure (creatinine levels ≥ μmol/L), hypothyroidism, malignant disease, treatment with anticonvulsants, theophylline, niacin or hormonal therapy which might influence Hcy levels and established defi ciency of vitamin B and folate.Approval for the study was obtained by the local Ethics Committee.All procedures on human subjects were performed in the accord with the Helsinki Declaration of .All subjects included in the study singed informed consent upon careful explanation of the study procedure.Subjects underwent history and clinical examination.Clinical details included risk factor assessment for coronary artery disease.Smoking status, history of hypertension or diabetes mellitus and details of treatment received before the admission was recorded.Routine blood chemistry including serum lipid profi le, uric acid and fi brinogen level as well as international normalized ratio (INR) and partial thromboplastin time (aPTT) were carried out on admission in all patients, while serum glucose, creatinine, urea, blood cell count, hematocrit, sedimentation rate, liver enzymes, C-reactive protein and troponine I, CK-MB and CK were determined on  nd and  th day upon the AMI onset using standard techniques.Serum blood samples for Hcy measurements were obtained on  nd and  th day after AMI onset.Fasting blood samples Patients in AMI  group had signifi cant increase in platelets count from day  to day  (,±, vs. ,±,; p<,).Our study of serial Hcy changes in patients with AMI revealed two diff erent patterns of Hcy changes in early post infarction period which might refl ect two distinct populations of AMI patients.Although further research is necessary, possible explanation for the observed fi ndings could be a diff erent genetic background, vitamin and oxidative status of patients with AMI.

Hcy assay
Serum Hcy was measured by fl uorescence polarization immunoassay (Homocysteine; Abbott) on an automated analyzer (IMx system; Abbott).Optimal procedures in blood sample collection and handling were followed to prevent the passage of Hcy from red cells to plasma and thus ensure reliable measurements.Th e reference ranges for Hcy concentration in our laboratory is ,-, μmol/L for males and ,-, μmol/L for females.Hyperhomocysteiemia were defi ned as serum Hcy concentration ≥ , percentiles of our reference values which is , μmol/L for males and , μmol/L for females.

Statistical analysis
For normal distributed variables, values are expressed as mean±SEM.Signifi cant diff erences in serial Hcy values were tested using a t-test for paired samples since Hcy values followed normal distribution.Diff erences in mean between groups were tested using a t-test and diff erences in median by use of Mann-Whitney's test.Associations between continuous variables were tested with Spearman's rank or Pearson correlation analysis where appropriate.Two-tailed p values <, were considered statistically significant.Statistical analysis was performed using SPSS statistical software system (version ., SPSS Inc, Chicago, Illinois, SAD).

Results
Mean Th ese repair processes require the methylation of DNA, RNA and proteins -reactions that lead to the generation of Hcy as the end point in the methylation pathway.
Our results can partially support this view since the pa-tients with higher Hcy values on day  also had significantly higher CK-MB activity on day  compared to other group of patients.CK-MB activity refl ects the cardiac tissue damage and increased Hcy concentration might be a marker of reparatory processes in cardiac tissue.Lower serum Hcy levels on day  might correspond to patients with no previous ischemic heart disease, whose myocardial cells are not already aff ected.Th e rise in Hcy levels on days after the infarction would then be explained by the post infarction presence of a "frontier area" of cardiac cells between infracted and healthy tissue.It may be that these cells are not necrotized but remain in a situation of hypoxemia, with a reduced capacity to metabolize Hcy.In contrast, patients with elevated Hcy levels on day  of the infarction may have a history of coronary atherosclerosis with asymptomatic myocardial ischemia that had already aff ected their myocardial cells ().Th e subsequent decrease in Hcy levels on days after infarction could be explained by a reduction in total number of ischemic myocardial cells due to post MI necrosis.Our results showed that patients with an increase in Hcy levels during early post myocardial infarction also had a signifi cant increase in platelets count during the study period.Although, the mechanism responsible for this is not clear Li et al. () showed that the L-arginine/NO pathway is one of the various targets of Hcy in human platelets which could be disturbed by Hcy.Authors suggested that Hcy diminishes NO production through decreased uptake of L-arginine resulting in increased platelet reactivity.In this context increased Hcy concentration might be responsible for increased platelets count during post infarction period and not the other way around.It is known that Hcy levels are elevated in patients with impaired folate and vitamin B status and hence the diff erent models of Hcy changes might be due to vitamin defi ciency in our AMI patients.Osorio et al. ()

Conclusion
Our study of serial Hcy changes in patients with AMI revealed two diff erent patterns of Hcy changes in early post infarction period which might refl ect two distinct populations of AMI patients.Although further research is necessary, possible explanation for the observed fi ndings could be a diff erent genetic background, vitamin and oxidative status of patients with AMI.
KEY WORDS: homocysteine, acute myocardial infarction, post myocardial period, risk factor AMINA VALJEVAC ET AL.: CHANGES IN SERUM HOMOCYSTEINE LEVEL FOLLOW TWO DIFFERENT TRENDS IN PATIENTS DURING EARLY POST MYOCARDIAL INFARCTION PERIOD were drawn from antecubital vein into siliconized tubes.Tubes were immediately put on ice and transferred to Central Laboratory for clinical biochemistry at University of Sarajevo Clinics Centre.Blood samples were centrifuged within  minutes at room temperature at  g and kept at -°C until analysis.Cluster analysis revealed two different trends of serum Hcy changes from day  nd to day  th .For analysis AIM patients were divided into two subgroups: AIM  group comprised of  () AIM patients with increase in serum Hcy and in AIM  group there were  patients () with the decrease in Hcy levels from day  nd to day  th .

Data are presented as mean±SEM. LDH-lactate dehydrogenase, CK- creatine kinase; CK-MB-izoform creatine kinase
creased antioxidant capacity ().Considering the fact that Hcy is metabolized into glutathione, main intracellular antioxidant, the decrease in Hcy levels during this period might be refl ective of patients with preserved capacity for transsulfuration pathway with glutathione as fi nal product.Other group of AMI patients might be the