Six week follow-up of metabolic effects induced by a high-fat diet and streptozotocin in a rodent model of type 2 diabetes mellitus.

This study was initiated to refine and characterize a nongenetic experimental model of type 2 diabetes mellitus and to follow up various metabolic parameters up to six weeks after diabetes induction. Male Wistar rats were divided into 4 groups: CON group--consumed standard rat chow and served as control; HFD group--consumed high-fat diet (45% calories as fat); STZ group-was injected once intraperitoneally with streptozotocin (35 mg/kg) on day 14, and DM-2 group--consumed high-fat diet and was injected with streptozotocin. The metabolic parameters were measured one week after streptozotocin injection (week 3) and at the end of the study (week 9). Our results confirm that HFD-group developed dyslipidaemia, obesity and insulin resistance. All metabolic parameters remained largely unaltered in STZ-group during the study. Only the combination of high-fat diet and streptozotocin (DM-2 group) induced type 2 diabetes that was characterized with moderate hyperglycaemia, insulin resistance, hypertriglyceridaemia, elevated free fatty acids, hypercholesterolaemia and increased plasma glucagon levels at the time of diabetes onset (week 3). The observed changes of the metabolic parameters after six additional weeks demonstrated an aggravated diabetic state, as confirmed from significantly increased fasting plasma glucose values, insufficient insulin secretion, severe hyperlipidaemia, increased glucagon levels, decreased serum adiponectin concentrations and significantly elevated urinary protein excretion. These results indicate that apart from its utility as a model of diabetes aetiology, this model could also be used for elucidating the role of the hormones adiponectin and glucagon in the progression of type 2 diabetes, as well as for investigating the diabetic complications.

Hyperhomocysteinemia and of Methylenetetrahydrofolate Reductase (C677T) Genetic Polymorphism in Patients with Deep Vein Thrombosis.

AIM: To determine the concentration of total plasma homocysteine (tHcy) as well as different genotypes of methylenetetrahydrofolate reductase MTHFR (C677T) in healthy subjects and patients with deep vein thrombosis (DVT). MATERIAL AND METHODS: The investigation comprised a total of 160 subjects divided in two main groups: 80 healthy subjects (control group) and 80 patients with deep vein thrombosis. Concentration of tHcy was determined by spectrophotometric cyclic enzymatic method and mutation of MTHFR (C677T) gene was examined by polymerase chain reaction according to Schneider. RESULTS: The results obtained for plasma tHcy in the control group were 11.62±3.43 µmol/L, while tHcy level was significantly higher in patients with deep vein thrombosis as compared to the control group, 15.19±3.63 µmol/L (р<0.001). The analysis of the results has shown that MTHFR (C677T) genetic polymorphism was responsible for mild to moderate hyperhomocysteinemia in the majority of subjects. CONCLUSION: The level of tHcy in the examined patients was significantly higher in comparison with the control group. Multiple regression analysis has shown that tHcy level in CT and TT genotypes of MTHFR (C677T) was statistically higher in comparison with CC genotype of MTHFR (C677T) in both, the control group and the DVT patients.

Measles outbreak in Macedonia: epidemiological, clinical and laboratory findings and identification of susceptible cohorts.

OBJECTIVES: Despite a 92-99% national vaccination coverage since 2000, the former Yugoslav Republic of Macedonia experienced a large measles outbreak between 2010 and 2011. Here we investigate the characteristics of patients hospitalized during this outbreak at the Clinic of Infectious Diseases in Skopje. METHODS: Epidemiological, clinical and laboratory data of 284 measles patients, including 251 from Skopje (43.80% of the 573 reported cases) and 33 from elsewhere in Macedonia were collected. RESULTS: The most affected age groups were children up to 4 years of age and adolescents/adults of 15 years and older. Most patients were unvaccinated (n=263, 92.61%) and many had non-Macedonian nationalities (n=156, 54.93%) or belonged to the Roma ethnicity (n=73, 25.70%). Bronchopneumonia and diarrhea were the most common complications. Eighty-two out of 86 tested patients (95.35%) had measles-specific IgM antibodies. The outbreak was caused by the measles variant D4-Hamburg. CONCLUSIONS: The epidemic identified pockets of susceptibles in Skopje and indicated that additional vaccination opportunities in particular for people with non-Macedonian nationality and traveler communities are warranted to ensure efficient measles control in Macedonia. The high attack rate among children of less than 1 year suggests that vaccination before 12 months of age should be considered in high risk settings.

Determination of the diagnostic values of asymmetric dimethylarginine as an indicator for evaluation of the endothelial dysfunction in patients with rheumatoid arthritis.

Introduction. To compare the diagnostic values of laboratory variables, to present evaluations of the diagnostic test for asymmetric dimethyl arginine (ADMA), rheumatoid factor (RF), C-reactive protein (CRP), and DAS28 index, and to define the effect of untreated rheumatoid arthritis on endothelial function. In order to determine whether ADMA changes depending on the disease evolution, ADMA was used as an indicator for endothelial dysfunction. Methods. Using an ELISA technology of DLD-Diagnostika-GMBH for the detection of ADMA, the samples of serum and urine have been examined in 70 participants (35 RA who were not treated, 35 healthy controls). RF was defined with the test for agglutination (Latex RF test) in the same participants. Results. Out of 35 examined patients with RA, RF appeared in 17 patients (sensitivity of the test, 51.42%). In 20 of the 35 examined patients with RA, we found the presence of ADMA (sensitivity of the test, 57.14%). Anti-CCP antibody was present in 24 examined patients with RA (sensitivity of the test, 68.57%). Conclusion. ADMA has equal or very similar sensitivity and specificity to RF in untreated RA (sensitivity of 57.14% versus 48.57%, specificity of 88.57% versus 91.42%) in the detection of asymptomatic endothelial dysfunction in untreated RA.

Symmetric dimethyl arginine and N-acetyl-ß-D-glucosaminidase lysozimuria of proximal renal tubules as a target for nephrotoxicity in patients with rheumatoid arthritis treated with disease modifying antirheumatic drugs.

BACKGROUND: The aim of this study was to determine the effect of initial therapy with some disease modifying antirheumatic drugs (DMARDs) (Methotrexate and Ketoprofen) on glomerular and tubular integrity in patients with Rheumatoid arthritis (RA). OBJECTIVES: To determine whether there is a change in clinical and laboratory indicators of renal function in course of the follow up of treatment and whether that change correlates with the dynamics of the quantity of enzymes excreted in urine and reactants of the acute phase. MATERIALS AND METHODS: Using colorimetric method for determination of NAG, samples of 70 participants were examined (35 RA patients treated with Ketoprofen only, 35 RA patients treated with combined use of Methotrexate and Ketoprofen). The follow up was 5 time-intervals in the course of 24 weeks. RESULTS: There was moderate correlation between NAG and microalbuminuria (r=0,34) in the group of patients treated with Ketoprofen only, while statistically significant correlation (r=0,21) was seen in group of patients with combined use of Methotrexate and Ketoprofen. NAG enzymuria in size, number of patients registered, and time of appearance were greater and appears earlier in the group with the combined use of Methotrexate and Ketoprofen compared with the mono-therapy with Ketoprofen. Mean urinary NAG induction was increasing with the concomitant use of Methotrexate and Ketoprofen. CONCLUSIONS: Methotrexate is more potent NAG inductor than Ketoprofen and provokes greater tubular enzymuria than Ketoprofen.

Cholesteryl ester transfer protein, low density lipoprotein particle size and intima media thickness in patients with coronary heart disease.

Cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport and high density lipoprotein (HDL) metabolism. Predominance of small, dense LDL particles is associated with an increased risk of atherosclerosis and coronary heart disease (CHD).The aim of the study was to determine the potential relationship between the CETP concentration and low density lipoprotein (LDL) particle size and their association with intima media thickness (IMT) in patients with CHD. Lipid parameters, CETP concentration and LDL particle size were determined in 100 healthy subjects (control group) and in 100 patients with CHD, aged 43 to 77 years. Plasma CETP concentrations were measured by an enzyme-linked immuno-sorbent assay with two different monoclonal antibodies. LDL subclasses were separated by nondenaturing polyacrilamide 3-31% gradient gel electrophoresis. CETP concentration was higher in patients compared to controls (2.02 ± 0.75 mg/ml vs. 1.74 ± 0.63 mg/ml, p<0.01). Mean LDL particle size (nm) was significantly smaller in patients than in controls (24.5 ± 1.1 vs. 26.1 ± 0.9; p<0.001). There was no relation between LDL particle size and CETP concentration (r=-0.1807, p=0.072). Age, diastolic blood pressure, CETP concentration and LDL particle size were independent factors for determing IMT by multiple linear regression analysis. They accounted for 35.2 % of the observed variability in IMT. CETP is not an independent contributor of LDL particle size. CETP might play a role in determining lipoprotein distributions, but did not seem to be the sole factor in the formation of small LDL particles.

Distribution of low density lipoprotein subclasses in Macedonian children.

BACKGROUND: Current reports claim that small and dense LDL particles are more atherogenic than larger LDL particles. There are many studies presenting LDL subclass distribution in adults, but there is not enough data regarding children in the literature on this problem. The aim of our study was to examine LDL subclass distribution in healthy children in the Republic of Macedonia. MATERIALS/METHODS: Plasma LDL subclasses in 100 children aged 9-18 years were analyzed using non-denaturing polyacrilamide gradient (3-31%) gel electrophoresis. Conventional plasma lipid and apoprotein parameters thought to be related to LDL size were determined as well. RESULTS: The results obtained showed the prevalence of large LDL subclasses (phenotype A) in 89% of the children, whereas small LDL subclasses (phenotype B) were observed in 11%. The mean LDL size was 26.37 +/- 0.68 nm, and there was no difference between gender groups. No association was noted between LDL size and plasma lipid and apoprotein levels, age, or BMI. CONCLUSIONS: LDL size and distribution is not gender- or age-dependent, or influenced by plasma lipid and apoprotein concentrations in childhood. This suggests that analysis of LDL subclass phenotype may provide better information on the risk of atherosclerosis development in adulthood.

Apoprotein(a) phenotypes and plasma lipoprotein(a) concentration in patients with diabetes mellitus.

OBJECTIVES: To determine whether apo(a) isoforms and plasma Lp(a) concentrations in association with some lipid parameters increase the relative risk for the development of atherosclerosis in patients with diabetes mellitus (IDDM and NIDDM). DESIGN AND METHODS: Apo (a) isoforms, Lp(a) and plasma lipids were determined in 40 IDDM and 65 NIDDM patients and in 182 healthy individuals. Apo(a) isoforms were separated by 3 to 15% gradient SDS-PAGE followed by immunoblotting. RESULTS: Logistical analysis showed that: Lp(a) levels >30 mg/dL (RR = 0.25, p < 0.000001; RR = 0.18, p < 0.00002), HTA (RR = 0.212, p < 0.00001; RR = 0.30, p < 0.00001), LMW-S1 apo(a) isoform (RR = 6.86, p < 0.0131; RR = 7.04, p < 0.0057) play a significant role in aterogenecity in both groups of patients with DM (IDDM and NIDDM). The 6.50-fold increase in risk was found in NIDDM patients with high Lp(a) levels (>30 mg/dL) and plasma total/HDL cholesterol ratio (4.5-5.8). CONCLUSION: Elevated Lp(a) levels, LMW S1 apo(a) isoform, HTA and combination of increased Lp(a) levels and total/HDL cholesterol ratio increase the risk for the development of atherosclerosis in patients with DM (IDDM and NIDDM).

Frequency distribution of apoprotein(a) isoforms in patients with diabetes mellitus and healthy subjects.

AIM: To determine the frequency distribution of apoprotein(a) isoforms in patients with insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus and healthy subjects. METHOD: We separated and visualized 5 apo(a) isoforms in 40 patients with IDDM (12 men aged 48.00-/+4.59 and 28 women aged 52.37-/+8.21), 65 patients with NIDDM (26 men aged 61.88-/+9.25 and 39 women aged 60.15-/+7.98), and 182 healthy subjects, using 3-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting. RESULTS: The frequency distribution of apo(a) isoforms was very similar in patients with diabetes mellitus and the control group. Atherogenic low molecular weight (LMW) S1 apo(a) isoform was more frequent in patients with IDDM (7.5%) and NIDDM (6.15%) than in the control group (0.78%). LMW S1 apo(a) isoform in patients with IDDM (relative risk [RR], 6.86; 95% confidence interval [CI], 1.19-25.21; p<0.001) and patients with NIDDM (RR, 7.04; 95% CI, 1.40-35.40; p=0.0057) as well as high molecular weight >S4 apo(a) isoform in patients with NIDDM (RR, 2.39; 95% CI, 1.28-5.21; p=0.0067) significantly increased the risk for the development of atherosclerosis. Mean molecular weight of S3, S1, and B apo(a) isoforms was higher in patients with IDDM and NIDDM than in the healthy subjects carriers of the same isoforms, but this difference was not statistically significant. We estimated high inverse statistical correlation between apo(a) size (kDa) and plasma lipoprotein(a) concentration in all study groups, patients with IDDM (p<0.001), patients with NIDDM (p<0.001), and healthy subjects (p<0.01). CONCLUSION: Not only the increased plasma Lp(a) levels, but also apoprotein(a) isoforms may play an important role as a risk factor for the development of atherosclerosis in patients with diabetes mellitus.

LDL and HDL subclass distribution in patients with end-stage renal diseases.

OBJECTIVES: To examine the alterations in LDL and HDL subclass distribution in ESRD patients compared with a control group and to investigate the relationship of LDL particle size to the other plasma lipoproteins levels. DESIGN AND METHODS: Plasma lipids, LDL and HDL subclasses were determined in 63 hemodialysis patients (HD), 42 predialysis patients and 345 control subjects. Lipoprotein subclasses were separated by polyacrylamide 3 to 31% gradient gel electrophoresis. RESULTS: In predialysis group, 88% subjects had small LDL particles compared with 58.5% of hemodialysis patients and 16.5% of control subjects. Mean LDL size particle diameter was significantly smaller in HD and predialysis patients in comparison with controls (p < 0,0005, p < 0,0001; respectively). Significant inverse correlation between LDL particle size and triglyceride level was observed for both patient groups. Decreased levels of the largest HDL2b subclass was found in both predialysis (16.5%) and in HD patients (30%) as compared with controls (50%), and increased levels of the small HDL3a subclass was found only in predialysis group (21%) in comparison with controls (4.5%). CONCLUSIONS: Alterations in LDL and HDL subclass distribution toward smaller particles is the main lipid abnormality associated with atherogensis found in ESRD. ESRD is associated with reduced levels of HDL2b subclass and increased levels of HDL3c subclass, which occurs in coronary artery disease (CAD) as well.

Low density lipoprotein particle size phenotyping in healthy persons and patients with myocardial infarction.

AIM: To determine distribution, size, and phenotype of low density lipoprotein (LDL) subclasses and examine the influence of plasma lipid concentrations on lipoprotein particle size in both healthy population and patients with myocardial infarction. METHOD: Nondenaturing gradient (3-31%) gel electrophoresis for lipoprotein separation was used to determine the distribution and size of LDL subclasses in 132 patients with myocardial infarction and 334 healthy control subjects. RESULTS: Large LDL subclasses (LDL1, LDL2, phenotype A) were dominant in 88.5% of the healthy population, whereas in most patients with myocardial infarction (81%) the dominant subclasses were LDL3 and LDL4 (phenotype B). Only 19% of the patients belonged to the phenotype A (LDL1 and LDL2). Mean LDL subclass size (nm) was significantly smaller in patients with myocardial infarction than in controls (24.381.07 nm vs 25.940.89 nm; p<0.001). In both groups, LDL size was independent of LDL plasma cholesterol but associated with high triglyceride plasma concentrations. CONCLUSION: Coronary artery disease is associated with the predominance of small LDL particles and high plasma triglyceride concentrations. The risk of development of cardiovascular disease can be assessed more accurately by determining lipoprotein subclasses.

Gradient gel electrophoretic separation of LDL and HDL subclasses on BioRad Mini Protean II and size phenotyping in healthy Macedonians.

BACKGROUND: Lipoprotein subclass determinations provide a more detailed reflection of lipoprotein metabolism and an accurate prediction for risk of cardiovascular disease. Gradient gel electrophoresis for lipoprotein separation on Pharmacia electrophoretic apparatus has been most commonly used for many years. METHODS: In this paper, we describe a new method for separating LDL and HDL subclasses by nondenaturing polyacrylamide gradient (3-31%) gel electrophoresis, using BioRad Mini Protean II electrophoretic cells. RESULTS: The mean particle diameters of cholesterol-stained LDL and HDL lipoproteins were estimated after calibrating the gels with size standards, using fractional absorbance profiles. For the first time in the Republic of Macedonia, lipoprotein distribution and size phenotyping were studied in 345 healthy individuals. Large LDL subclasses (phenotype A) were dominant in 88.5% of the population, whereas small LDL subclasses (phenotype B) were dominant in 11.5%. The mean dominant LDL size was 26.08+/-0.8 nm. Five HDL subclasses were separated on the same gels, and HDL2b and HDL2a (larger) were dominant in healthy Macedonians. CONCLUSION: Antiatherogenic, larger LDL and HDL particles are most commonly found in healthy populations in the Republic of Macedonia.